Figure 1.
Figure 1. Anti-VWF nanobodies in a FVIII-nanobody fusion protein. (A) Immobilized nanobodies (5 μg/mL) were incubated with humanVWF, murineVWF, proteolytic fragment SpII (ie, VWF residues 2129-2813), fragment SpIII (residues 764-2128), D′-D3-HPC4 (residues 764-1247) and A1-A2-A3-HPC4 (residues 1260-1874). Bound proteins were probed using polyclonal anti-VWF antibodies or with monoclonal antibody HPC4 and detected via peroxidase-mediated hydrolysis of 3-3′-5-5′-tetramethylbenzidine. Plotted are optical density values corrected for binding of the proteins to albumin-coated wells (mean ± SD; n = 3). (B) Association and dissociation of humanVWF (25 μg/mL, gray lines; or 250 μg/mL, black lines) to immobilized nanobodies (10 μg/mL) was analyzed via BLI-analysis using Octet-QK equipment. Association was allowed for 600 seconds, and dissociation was monitored for 900 seconds. Representative sensorgrams for each nanobody are presented. (C) Schematic representation of FVIII-KB013bv, in which FVIII B-domain residues Gln744-Arg1648 are replaced by 2 copies of nanobody KB-VWF-013. Original thrombin activation sites and VWF binding sites are conserved in this protein, whereas Arg1648 is no longer present. (D) Purified FVIII-KB013bv and BDD-FVIII (1 μg/mL) were incubated in the absence or presence of thrombin (10 nM) for 30 minutes at 37°C and analyzed via western blotting using polyclonal anti-FVIII and anti-nanobody antibodies. Sc-FVIII, single chain FVIII; FVIII-HC, FVIII heavy chain; FVIII-LC, FVIII light chain. (E) Wells coated with purified VWF (10 μg/mL) were incubated with FVIII-KB013bv (0-25 ng/mL; red symbols) or BDD-FVIII (0-1 μg/mL; blue symbols). Bound FVIII was probed using peroxidase-labeled monoclonal anti-FVIII antibody 833 and detected via peroxidase-mediated hydrolysis of 3-3′-5-5′-tetramethylbenzidine. Plotted are typical binding curves of an experiment performed in duplicate. Half-maximal binding values were calculated from 3 independent experiments and were 4.0 ± 2.1 ng/mL and 170 ± 22 ng/mL for FVIII-KB013bv and BDD-FVIII, respectively.

Anti-VWF nanobodies in a FVIII-nanobody fusion protein. (A) Immobilized nanobodies (5 μg/mL) were incubated with humanVWF, murineVWF, proteolytic fragment SpII (ie, VWF residues 2129-2813), fragment SpIII (residues 764-2128), D′-D3-HPC4 (residues 764-1247) and A1-A2-A3-HPC4 (residues 1260-1874). Bound proteins were probed using polyclonal anti-VWF antibodies or with monoclonal antibody HPC4 and detected via peroxidase-mediated hydrolysis of 3-3′-5-5′-tetramethylbenzidine. Plotted are optical density values corrected for binding of the proteins to albumin-coated wells (mean ± SD; n = 3). (B) Association and dissociation of humanVWF (25 μg/mL, gray lines; or 250 μg/mL, black lines) to immobilized nanobodies (10 μg/mL) was analyzed via BLI-analysis using Octet-QK equipment. Association was allowed for 600 seconds, and dissociation was monitored for 900 seconds. Representative sensorgrams for each nanobody are presented. (C) Schematic representation of FVIII-KB013bv, in which FVIII B-domain residues Gln744-Arg1648 are replaced by 2 copies of nanobody KB-VWF-013. Original thrombin activation sites and VWF binding sites are conserved in this protein, whereas Arg1648 is no longer present. (D) Purified FVIII-KB013bv and BDD-FVIII (1 μg/mL) were incubated in the absence or presence of thrombin (10 nM) for 30 minutes at 37°C and analyzed via western blotting using polyclonal anti-FVIII and anti-nanobody antibodies. Sc-FVIII, single chain FVIII; FVIII-HC, FVIII heavy chain; FVIII-LC, FVIII light chain. (E) Wells coated with purified VWF (10 μg/mL) were incubated with FVIII-KB013bv (0-25 ng/mL; red symbols) or BDD-FVIII (0-1 μg/mL; blue symbols). Bound FVIII was probed using peroxidase-labeled monoclonal anti-FVIII antibody 833 and detected via peroxidase-mediated hydrolysis of 3-3′-5-5′-tetramethylbenzidine. Plotted are typical binding curves of an experiment performed in duplicate. Half-maximal binding values were calculated from 3 independent experiments and were 4.0 ± 2.1 ng/mL and 170 ± 22 ng/mL for FVIII-KB013bv and BDD-FVIII, respectively.

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