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

Doctoral thesis English OPEN
Tolley, Caroline (2015)
  • Subject: R | T
    mesheuropmc: hemic and lymphatic diseases | congenital, hereditary, and neonatal diseases and abnormalities | animal diseases

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.\ud 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.\ud 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.
  • References (20)
    20 references, page 1 of 2

    Braakman, I. and N. J. Bulleid (2011). Protein Folding and Modification in the Mammalian Endoplasmic Reticulum. Annual Review of Biochemistry, Vol 80. R. D. Kornberg, C. R. H.

    Raetz, J. E. Rothman and J. W. Thorner. 80: 71-99.

    Brandizzi, F, and Barlowe, C. (2013). Organization of the ER Golgi interface for membrane traffic control. Nature Reviews Molecular Cell Biology 14, 382-392.

    Burnouf, T. and M. Radosevich (2001). "Affinity chromatography in the industrial purification of plasma proteins for therapeutic use." Journal of Biochemical and Biophysical Methods 49(1-3): 575-586.

    Bustin, S. (2002). "Quantification of mRNA using real-time reverse transcription PCR (RTPCR): trends and problems." Journal of Molecular Endocrinology 29(1): 23-39.

    Butash, K. A., et al. (2000). "Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency." Biotechniques 29(3): 610-614, 616, 618-619.

    Cao, W., et al. (2008). "Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13." Proc Natl Acad Sci U S A 105(21): 7416-7421.

    Carr, M.E. et al (2015). Emerging and future therapies for hemophilia. Journal of Blood Medicine. Volume 6. Pages 245-255.

    Castaman, G., et al. (2010). "F8 mRNA studies in haemophilia A patients with different splice site mutations." Haemophilia 16(5): 786-790.

    Lenting, P. J., et al. (2010). "The disappearing act of factor VIII." Haemophilia 16: 6-15.

  • Related Research Results (2)
  • Metrics
    0
    views in OpenAIRE
    0
    views in local repository
    52
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Kent Academic Repository - IRUS-UK 0 52
Share - Bookmark