Factor viii variants having a decreased cellular uptake
a technology of coagulation factor and fviii, which is applied in the direction of drug composition, extracellular fluid disorder, peptide/protein ingredient, etc., can solve the problems of affecting the survival rate of patients, affecting the effect of fviii treatment, and causing major complications in haemophilia care, so as to reduce the cellular uptake and reduce the lrp binding effect, the effect of increasing the half life of the circulatory system
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example 1
Generation of FVIII Variants
[0095]A fragment encoding the cMyc tag was inserted in the C-terminus of the heavy chain in the expression construct encoding FVIII with a 21 amino acid B domain linker (Haemophilia 2010; 16: 349-48). The expression level and activity of this FVIII-cMyc2 were similar to untagged FVIII. FVIII-cMyc2 was used as template for the variants and as control in the assays described in example 3-5. Additional restriction sites were added to the FVIII-cMyc2 expression construct to ease swapping of domains among variants. The A1 domain is flanked by SalI and PshAI / MfeI, A2 is flanked by PshAI / MfeI and AvrII / NruI, A3 is flanked by AgeI / MluI and BstZ171IBstAPI, C1 by BstZ171I / BstAPI and SwaI / SphI and C2 by SwaI / SphI and SfiI. The site-directed mutagenesis of exposed basic amino acids (lysine or arginine) was conducted by Geneart AG (Regensburg, Germany).
[0096]Mutants with reduced affinity to LRP1 were localized to especially the feets of the C1 and C2 domains (see FIG....
example 2
Expression of the FVIII Mutants
[0097]Serum free transfection was performed using HKB11 cells (Cho M-S et al. J Biomed Sci 2002; 9: 631-63) and 293fectin (Invitrogen) following the manufacturer's recommendations. HKB11 suspension cells were grown in commercial FREESTYLE™ 293 Expression Medium (Invitrogen #. 12338-018) supplemented with 50 U mL−1 penicillin and 50 ug mL−1 streptomycin. Cells were grown as suspension cells in shakers and incubated at 37° C. under 5% CO2 and 95% relative humidity. Cells were seeded at a density of 3×105 cells mL−1 and passaged every 3 to 4 days. Viable and total cell concentrations were evaluated by Cedex (Innovatis) analysis using image analysis software for automated cell counting. Viable cells were highlighted by their ability to exclude the dye trypan blue. Cells were harvested 96 hours after transfection and the cell pellet isolated by gentle centrifugation. Afterwards, the cell pellet was re-suspended in the FREESTYLE™ 293 Expression medium contai...
example 3
FVIII:C Measured in Chromogenic Assay
[0098]The FVIII activity (FVIII:C) of the rFVIII compound was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: FVIII samples and a FVIII standard (human calibration plasma, Chromogenix) were diluted in Coatest assay buffer (50 mM TRIS, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty μL of samples (culture supernatant or purified FVIII variants) pre-diluted 100-, 400-, 1600- and 6400-fold, standards and buffer negative control were added to 96-well Spectramax® microtiter plates in duplicates. The factor IXa / factor X reagent, the phospholipid reagent and CaCl2 from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 μL of this added to the wells. After 15 min incubation at room temperature 50 μL of the factor Xa substrate 5-2765 / thrombin inhibitor I-2581 mix was added and the reactions incubated 5 min at room temperature before 25 μL 1 M citric acid, pH 3, was added. The absorbance at 405 nm was...
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