Antibodies which bind and inhibit blood factor VIII

#The present invention relates to the modification of inhibitory antibodies in order to achieve a variable maximal inhibitory activity and its application in the development of antithrombotic agents as well as to pharmaceutical compositions and mixtures including such antibodies.

#The formation of blood clots does not only limit bleeding in case of injury (haemostasis) but can occlude important arteries or veins, leading to serious organ damage and death. Thrombosis is thus blood clot formation at the wrong time and place.

#Upon damage of a vessel, the coagulation (clotting) system is immediately initiated producing thrombin and blood platelets adhering to matrix proteins, which in turn leads to the aggregation of additional platelets into a growing platelet plug in concert with the conversion of fibrinogen in the blood to the insoluble fibrin.

#The above detailed description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:

#FIG. 1: Schematic representation of the biantennary structure most commonly found in the antigen binding part of antibodies. NeuAc=N-acetylneuramic acid (sialic acid); Gal=galactose; GlcNac=N-acetylglucosamine; Man=mannose; Fuc=fucose; Asn=asparagine.

#FIG. 2: Schematic representation of the removal of glycan by sequential enzymatic treatment.

#The present invention will now be described with reference to the following specific, non-limiting examples.

#KRIX-1 (0.5 mg/ml in PBS) was mixed with N-glycosidase-F (roche diagnostics Gmbh, Mannheim, Germany) at final concentration of 2 U/ml. The mixture was incubated at 37° C. during 72 hours under gentle stirring.

#The inhibitory activity of native and deglycosylated KRIX-1 was assessed in a Bethesda assay (Kasper et al. (1975), cited supra). Therefore, one volume of antibody at various dilutions in TBS (Tris 20 mM, NaCl 0.15 M, pH 7.4) was mixed with one volume of a pool of normal human plasma and incubated for 2 h at 37° C. The pool of normal plasma had been constituted by mixing plasma from 10 normal individual and buffered by addition of Hepes (100 mM) to a final concentration of 10 mM. The residual FVIII activity was then measured using a modification of the DADE FVIII chromogenic assay (Dade A G, Marburg, Germany). In this assay, thrombin-activated FVIII accelerates the conversion of factor X into factor Xa in the presence of factor IXa, PL and calcium ions; factor Xa activity is then assessed by hydrolysis of a p-nitroanilide substrate. Reagents, which were reconstituted according to the manufacturer's instruction, comprised bovine factor X (1 mM), factor IXa (0.3 mM) and thrombin (0.3 mM); CaCl2 (30 mM), PL (60 mM), a chromogenic factor Xa substrate (CH3OCO-D-CHG-Gly-Arg-pNA.AcOH; 3.4 mM), and a thrombin inhibitor (L-amidinophenylalanine piperidine). Aliquots of 30 μl of plasma/antibody mixture were retrieved at the end of the 2 h incubation period and displayed in microtitration plates; 30 μl of the factor X and factor IXa/thrombin reagents were added sequentially. After 90 sec, 60 μl of the chromogenic substrate were added and the incubation extended for 10 min at 37° C. The reaction was then blocked by addition of 30 μl citric acid (1 M), and OD was measured at 405 nm. The residual FVIII activity was determined by comparing the OD405 nm of test samples with that obtained with FVIII solutions of known concentrations. The residual FVIII activity was expressed as the percentage of activity measured in plasma aliquots handled and diluted exactly as test samples throughout the entire experiment.