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Antibody capable of neutralizing substance having activity alternative to function of coagulation factor VIII (FVIII)

一种凝固因子、双特异性抗体的技术，应用在抗凝血因子免疫球蛋白、引入外来遗传物质而修饰的细胞、抗体等方向，能够解决凝固时间缩短、不可准确地测量FVIII活性和FVIII抑制剂滴度、影响测量FVIII测定系统等问题

Active Publication Date: 2017-08-29
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The bispecific antibody replaces the cofactor function of FVIII, thus affecting the assay system that measures the reactivity of FVIII itself
For example, when measuring plasma FVIII activity by an APTT-based one-step coagulation assay to diagnose the severity of hemophilia A or to monitor the pharmacological activity of a FVIII preparation in a patient administered a FVIII preparation, in the presence of said bispecific antibody The effect of promoting shortening of the clotting time of the bispecific antibody strongly interferes, which greatly impairs the accuracy of the measurement
Furthermore, when plasma FVIII inhibitor titers were determined by the APTT-based Bethesda assay, the effect of promoting a shortened clotting time of the bispecific antibody in its presence strongly interfered, which would greatly Accuracy of damage measurement
That is, FVIII activity and FVIII inhibitor titers cannot be accurately measured in patients administered bispecific antibodies

Method used

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  • Antibody capable of neutralizing substance having activity alternative to function of coagulation factor VIII (FVIII)
  • Antibody capable of neutralizing substance having activity alternative to function of coagulation factor VIII (FVIII)
  • Antibody capable of neutralizing substance having activity alternative to function of coagulation factor VIII (FVIII)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0210] [Example 1] Production of Antibodies against Anti-FIXa / FX Bispecific Antibodies and Sequence Determination of Variable Regions

[0211] An attempt was made to generate an antibody against ACE910 (Q499-z121 / J327-z119 / L404-k) (bispecific antibody in which the H chain consisting of the amino acid sequence of SEQ ID NO: 9 is combined with the L chain of SEQ ID NO: 10, and the H chain consisting of the amino acid sequence of SEQ ID NO: 11 combined with the L chain of SEQ ID NO: 10), which is a bispecific antibody described in the patent document (WO 2012 / 067176). F(ab')2 consisting of individual Fabs on the anti-FIXa side and anti-FX side was generated using genetic recombination technology and pepsin digestion.

[0212] Mice and rats were immunized with anti-FIXa-F(ab')2 or anti-FX-F(ab')2. Cell fusion of cells obtained from spleens removed from mice or rats or from lymph nodes of rats with mouse myeloma cells was performed according to general methods to produce hybridoma...

Embodiment 2

[0213] [Example 2] Production of expression vectors for recombinant mouse antibody AQ8 and recombinant rat-rabbit chimeric antibody AJ540.

[0214]The recombinant mouse antibody AQ8 was prepared as follows: The variable region sequence of the AQ8 antibody obtained in Example 1 was combined with the known mouse IgG2b constant region sequence (heavy chain: EMBL Accession No. J00461; light chain: EMBL Accession No. V00807) combined to produce a full-length antibody gene, which is then inserted into an expression vector. Similarly, recombinant rat-rabbit chimeric antibody AJ540 was generated by combining known rabbit IgG (heavy chain: EMBL Accession No. L29172, light chain: EMBL Accession No. X00231) with the variable region of the AJ540 antibody. The resulting expression clone plasmids were introduced into HEK293 cells, large-scale culture and purification using protein A and gel filtration were performed, and recombinant mouse antibody AQ8 (rAQ8-mIgG2b) and recombinant rat-rabbi...

Embodiment 3

[0215] [Example 3] One-step coagulation assay under neutralization of anti-FIXa / FX bispecific antibody using rAQ8-mIgG2b and rAJ540-rbtIgG

[0216] To FVIII-deficient plasma (George King) containing 10 U / dL or 100 U / dL recombinant FVIII (Kogenate FS, Bayer Yakuhin, Ltd.), 0 μg / mL or 300 μg / mL of anti-FIXa / FX bispecific Antibody ACE910. In addition, each of the prepared plasma samples was divided into the following two groups to prepare measurement sample solutions: one group was diluted 10 times using imidazole buffer (Kyowa Medex); -RbtIgG was diluted 10-fold in imidazole buffer. The amount of rAQ8-mIgG2b and rAJ540-rbtIgG required to sufficiently neutralize ACE910 was added. Details of the combination are shown below.

[0217] [Table 1]

[0218]

[0219] Furthermore, in order to generate a calibration curve for converting clotting time into FVIII activity, 10-fold, 20-fold, 40-fold, 80-fold, and 160-fold dilutions were performed by using an imidazole buffer (the FVIII...

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Abstract

It is tried to produce an antibody capable of neutralizing the activity of a substance having an activity alternative to the function of FVIII, for the purpose of using the antibody in a method for measuring the reactivity of FVIII in the presence of the substance having the activity alternative to the function of FVIII. As the result, it is found that, when the produced antibody is used, the activity of FVIII in plasma from a hemophilia A patient can be evaluated accurately in an APTT-based one-stage clotting assay. It is also found that, when the produced antibody is used, the titer of an FVIII inhibitor in plasma from an FVIII inhibitor-carrying hemophilia A patient can be evaluated accurately in an APTT-based Bethesda assay.

Description

technical field [0001] The present invention relates to antibodies for use in a method for measuring the reactivity of FVIII in the presence of a substance having an activity to functionally replace coagulation factor VIII (FVIII). Background technique [0002] Hemophilia is a bleeding disorder caused by a congenital defect or dysfunction of FVIII or coagulation factor IX (FIX). The former is called hemophilia A and the latter is called hemophilia B. Both genes are located on the X chromosome; and because they are X-chromosome-linked recessively inherited genetic abnormalities, 99% or more of those who develop the disease are males. It is known that the prevalence is about 1 in 10,000 male births and the ratio between hemophilia A and hemophilia B is about 5:1. [0003] The main bleeding sites in hemophilia patients include intra-articular, intramuscular, subcutaneous, intraoral, intracranial, gastrointestinal, intranasal, etc. In them, repeated intra-articular bleeding c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C07K16/36C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12P21/08
CPCC07K16/4241A61K2039/507C07K2317/76C07K16/00C07K16/36C07K16/42C07K2317/31C07K2317/55C12N5/10C12N15/09
Inventor 井川智之武藤厚北泽刚久铃木司
Owner CHUGAI PHARMA CO LTD
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