Co-expression of Factor VIII with anti-FVIII Camelid antibody ligands: Effect on expression levels of bio-therapeutic FVIII

Tolley, Caroline (2015) Co-expression of Factor VIII with anti-FVIII Camelid antibody ligands: Effect on expression levels of bio-therapeutic FVIII. Doctor of Philosophy (PhD) thesis, University of Kent. (Full text available)

PDF
Download (1MB) Preview
[img]
Preview

Abstract

Production of recombinant FVIII, the protein that is missing or dysfunctional in haemophilia A patients, is highly inefficient compared to other recombinant clotting factors such as FIX. This is predominantly due to complex intracellular trafficking, short half-life and protein instability. This study aimed to increase the amount of functional FVIII produced in mammalian cells by co-expression with anti-FVIII Camelid antibody fragments (VHH). Three VHH ligands were supplied by BAC BV (as DNA constructs), two of which when expressed in yeast are known to bind recombinant FVIII (ligands 2 and 7) and are used commercially as FVIII purification tools. From these three constructs, nine new VHH plasmids constructs were designed and transiently expressed in a stable BHK-human FVIII-expressing cell line. Of the nine VHH fragments that were co-expressed in the BHK FVIII cell line, four of these had a statistically significant impact on the ‘clotting time’ of the cell media as demonstrated by the activated partial thromboplastin time assay (aPTT). Two ligand 2 constructs (L2C1 and L2C2) prolonged the coagulation time by 4 seconds (P-value 0.0001, 95% confidence intervals 38.5-43.5), and 3.4 seconds (P-value 0.0072, 95% CI 36.5-40.3) respectively, indicating a decrease in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Two ligand 7 constructs (L7C1 and L7C3) caused a decrease in coagulation time of 3.2 seconds (P=0.0057, 95% CI 30.5-33.3), and 4 seconds (P=0.0002, 95% CI 29.1-32.9) respectively, indicating an increase in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Ligand 7 and ligand 2 both bind to the FVIII light chain, albeit in different regions and with different affinities (data confidential to BAC BV). BAC studies showed that ligand 7 competes with vWF on the FVIII light chain, which is known to increase stability of FVIII in vivo, whereas ligand 2 does not compete for this binding site. The opposing effects of ligand 7 and ligand 2 on FVIII clotting times seen in this study could be due to their differences in FVIII binding properties, since it is known that binding location of FVIII ligands can have an impact on FVIII clotting activity.

Item Type: Thesis (Doctor of Philosophy (PhD))
Uncontrolled keywords: FVIII haemophilia protein co-expression camelid antibody
Subjects: R Medicine
T Technology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Users 1 not found.
Date Deposited: 31 Mar 2016 09:26 UTC
Last Modified: 01 Apr 2016 10:54 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/54757 (The current URI for this page, for reference purposes)
  • Depositors only (login required):

Downloads

Downloads per month over past year