Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Abstract

Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) 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 VL and VH 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 VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH 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.

Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor

“… 3 Recent pull-down assays illustrated that the KM33 V H domain, but not the V L domain, retained its affinity toward FVIII. 19 However, several interactions are observed in the ET3i:NB33 cryo-EM structure at lower map contour levels between FVIII residues Y2043-Q2045 and the NB33 V L domain, as previously suggested by HDX-MS experiments. 5 Together, the structure that we report is in agreement with previous studies demonstrating that the KM33 epitope overlaps with amino acids in the C1 domain, previously shown to bind VWF and/or phospholipid membranes.…”

Plasma Clearance of Coagulation Factor VIII and Extension of Its Half-Life for the Therapy of Hemophilia A: A Critical Review of the Current State of Research and Practice

“…In our recent study, we investigated the possibility of an FVIII fusion with a singlechain variable fragment (scFv), recognizing FVIII C1-domain with high affinity and inhibiting FVIII binding to LRP1 and VWF, with the idea to affect both pathways of FVIII plasma clearance. However, upon thrombin-mediated disintegration of this scFv, its subunits still remained bound to FVIIIa and interfered with its cofactor function [122]. Lessons learned from this study indicate that affinity of such intramolecular ligand to FVIII should be reasonably lower to allow its dissociation upon thrombin cleavage.…”