Mutations of six amino acid residues in a B domain-deleted blood coagulation factor VIII have a cumulative effect on increasing its secretion.

BACKGROUND: High levels of factor VIII (FVIII) expression are needed for various applications to treat Hemophilia A. Besides deletion of the B-domain (BDD) and codon-optimization, F309S mutation, and other 5 mutations (X5) in FVIII were previously described to increase its expression. OBJECTIVES: To investigate whether combining the 6 aforementioned mutations (X6) results in a further increase of FVIII expression. METHODS: The 5 (X5) and 6 (X6) mutations were introduced into a BDD-FVIII (wild-type [WT]), and proteins were expressed in cell cultures with different transgene copy numbers, purified, and tested for specific activity, binding to von Willebrand factor and a low-density lipoprotein receptor-related protein fragment, tyrosine sulfation levels and immunogenicity in silico and in human T-cell culture. The 6 mutations were also reproduced in full-length FVIII (FL-FVIII) and tested for secretion levels. RESULTS: From the single-copy transgene cell lines, secretion levels of X5 and X6 increased 1.6-fold and 2.3-fold, respectively, compared with WT. These levels increased proportionally with increasing transgene copy number, approaching saturation. The specific activity, binding to von Willebrand factor and lipoprotein receptor-related protein fragment, and assessments of immunogenicity of X6 in model systems were similar to WT, while tyrosine sulfation levels, were moderately lower at the highest gene dose. However, the 6 mutations reproduced in FL-FVIII did not result in increased secretion. CONCLUSION: Combining the 6 mutations in BDD-FVIII improved its expression and did not affect general protein properties, making it promising for future product development. The data also indicate that BDD-FVIII and FL-FVIII have different expression mechanisms.