IN VITRO METHOD FOR MEASURING THE REACTIVITY OF COAGULATION FACTOR VIII (FVIII) IN A BLOOD SAMPLE

Abstract

As a result of the discoveries, it is possible to accurately measure FVIII activity in the plasma of a patient with hemophilia A by means of a one-step coagulation assay based on APTT, and it is also possible to accurately measure the titer of FVIII inhibitors in the plasma of a patient with hemophilia A who has undergone treatment with FVIII inhibitors by means of a Bethesda assay based on APTT.

Claims

1. An in vitro method for measuring the reactivity of coagulation factor VIII (FVIII) in a blood sample from a patient who has been administered a bispecific antibody that has activity to functionally substitute for coagulation factor VIII and binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated coagulation factor X, characterized in that the method comprises the steps of: (1) contacting the sample containing the bispecific antibody with the antibody that neutralizes the bispecific antibody; and (2) measuring the reactivity of coagulation factor VIII, wherein the bispecific antibody is a bispecific antibody in which a first polypeptide is associated with a third polypeptide and a second polypeptide is associated with a fourth polypeptide, wherein the first polypeptide is an H chain consisting of an amino acid sequence of SEQ ID NO: 9,The second polypeptide is an H chain consisting of an amino acid sequence from SEQ ID NO: 11, and the third and fourth polypeptides are L chains from SEQ ID NO: 10 (Q499-z121 / J327-z119 / L404-k), wherein the neutralizing antibodies are a combination of (a) a Fab-binding antibody comprising an antigen-binding site that binds to coagulation factor IX and / or activated coagulation factor IX, and (b) a Fab-binding antibody comprising an antigen-binding site that binds to coagulation factor X and / or activated coagulation factor X, wherein the Fab-binding antibody comprising an antigen-binding site that binds to coagulation factor IX and / or activated coagulation factor IX comprises (i) an antibody heavy chain variable region having the amino acid sequence CDR 1 of the heavy chain from SEQ ID NO: 12,the amino acid sequence CDR 2 of the heavy chain of SEQ ID NO: 13 and the amino acid sequence CDR 3 of the heavy chain of SEQ ID NO: 14, or a variable region of the antibody heavy chain having the amino acid sequence of SEQ ID NO: 1 and (ii) a variable region of the antibody light chain having the amino acid sequence CDR 1 of the light chain of SEQ ID NO: 18, the amino acid sequence CDR 2 of the light chain of SEQ ID NO: 19 and the amino acid sequence CDR 3 of the light chain of SEQ ID NO: 20, or a variable region of the antibody light chain having the amino acid sequence of SEQ ID NO: 2, and wherein the Fab-binding antibody comprising an antigen-binding site that binds to coagulation factor X and / or activated coagulation factor X comprises (i) a variable region of the antibody heavy chain having the amino acid sequence CDR 1 of the heavy chain of SEQ ID NO: 24,the amino acid sequence CDR 2 of the heavy chain of SEQ ID NO: 25 and the amino acid sequence CDR 3 of the heavy chain of SEQ ID NO: 26, or a variable region of the antibody heavy chain having the amino acid sequence of SEQ ID NO: 3 and (ii) a variable region of the antibody light chain having the amino acid sequence CDR 1 of the light chain of SEQ ID NO: 30, the amino acid sequence CDR 2 of the light chain of SEQ ID NO: 31 and the amino acid sequence CDR 3 of the light chain of SEQ ID NO: 32, or a variable region of the antibody light chain having the 3 amino acid sequence of SEQ ID NO:

4. 1 Claim follows,