NZ729317B2 - Method for measuring reactivity of fviii - Google Patents
Method for measuring reactivity of fviii Download PDFInfo
- Publication number
- NZ729317B2 NZ729317B2 NZ729317A NZ72931715A NZ729317B2 NZ 729317 B2 NZ729317 B2 NZ 729317B2 NZ 729317 A NZ729317 A NZ 729317A NZ 72931715 A NZ72931715 A NZ 72931715A NZ 729317 B2 NZ729317 B2 NZ 729317B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- coagulation factor
- binds
- fviii
- activity
- antibody
- Prior art date
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Abstract
was attempted to produce a substance that neutralizes the activity of a bispecific antibody having FVIII function-mimicking activity, and to devise a method for measuring FVIII reactivity whereby accuracy is maintained even in the presence of this bispecific antibody. As a result, it was discovered that FVIII activity in the plasma of a hemophilia A patient can be accurately evaluated by an APTT-based one-stage clotting assay, and further discovered that the FVIII inhibitor titer in the plasma of a hemophilia A patient having an inhibitor to FVIII can be accurately evaluated by an APTT-based Bethesda assay. In particular, the present invention relates to measuring the reactivity of coagulation factor VIII through contacting (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated coagulation factor X, which functionally substitutes for coagulation VIII with (2) one or more combinations of antibodies that neutralize this bispecific antibody. ed that FVIII activity in the plasma of a hemophilia A patient can be accurately evaluated by an APTT-based one-stage clotting assay, and further discovered that the FVIII inhibitor titer in the plasma of a hemophilia A patient having an inhibitor to FVIII can be accurately evaluated by an APTT-based Bethesda assay. In particular, the present invention relates to measuring the reactivity of coagulation factor VIII through contacting (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated coagulation factor X, which functionally substitutes for coagulation VIII with (2) one or more combinations of antibodies that neutralize this bispecific antibody.
Description
METHOD FOR MEASURING REACTIVITY OF FVIII
Technical Field
The present invention broadly relates to methods for measuring the reactivity of FVIII
in the presence of a substance having an activity of functionally substituting for coagulation
factor VIII (FVIII) (for example, methods for measuring FVIII activity or FVIII inhibitor titer).
The present invention also broadly relates to kits and such for measuring the reactivity of FVIII
in the presence of a substance having an activity of functionally substituting for FVIII.
Background Art
Hemophilia is a hemorrhagic disease 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 of these genes are located on the X chromosome; and since they are
X-chromosome-linked recessive genetic abnormalities, 99% or more of those who develop the
disease are men. It is known that the prevalence rate is approximately one in 10,000 male
births, and the ratio between hemophilia A and hemophilia B is approximately 5:1.
The main bleeding sites in hemophilia patients include intraarticular, intramuscular,
subcutaneous, intraoral, intracranial, digestive tract, intranasal, and such. Among them,
repeated intraarticular bleeding can develop into hemophilic arthropathy accompanied by
articular disorders and difficulty in walking, which eventually may require joint replacement.
Therefore, it is a major factor that lowers the QOL of hemophilia patients.
The severity of hemophilia correlates well with the FVIII activity or FIX activity in
blood. Patients with a coagulation factor activity of less than 1% are classified as severe,
patients with an activity of 1% or more to less than 5% are classified as moderate, and patients
with an activity of 5% or more and less than 40% are classified as mild. Patients with severe
symptoms, accounting for approximately half of hemophilia patients, exhibit bleeding symptoms
several times a month if they do not receive the later-described preventive replacement therapy,
and this frequency is markedly high compared to those of moderately symptomatic patients and
mildly symptomatic patients.
In addition to hemophilia and acquired hemophilia, von Willebrand’s disease caused by
functional abnormality or deficiency of von Willebrand factor (vWF) is known to be a related
bleeding abnormality. vWF is not only necessary for platelets to undergo normal adhesion to
the subendothelial tissues at lesion sites of vascular walls, but it is also necessary for forming a
complex with FVIII and keeping FVIII in the blood at a normal level. In von Willebrand's
disease patients, these functions are decreased, leading to hemostasis dysfunction.
For prevention and/or treatment of bleeding in hemophilia patients, blood coagulation
factors purified from plasma or those produced by genetic engineering techniques are mainly
used. In severe hemophilia patients, maintaining the FVIII activity or FIX activity in the blood
at 1% or more by FVIII or FIX replacement therapy are considered to be effective for preventing
manifestation of bleeding symptoms (Non-patent Documents 1 and 2). On the other hand, in
hemophilia patients, particularly severe hemophilia patients, antibodies against FVIII or FIX
which are called inhibitors may be generated. When such inhibitors are generated, the effect of
the coagulation factor formulation is blocked by the inhibitors. As a result, neutralization
treatment using large amounts of the coagulation factor formulation, or bypass treatment using a
complex concentrate or an activated coagulation factor VII formulation (FVIIa formulation) is
carried out.
Measurement of the FVIII activity in hemophilia A is carried out mainly by one-stage
clotting assay based on activated partial thromboplastin time (APTT) (Non-patent Document 3)
and chromogenic assay which is a system reconstructed using a purified coagulation factor
(Non-patent Document 4).
Measurement of the FVIII inhibitor titer in hemophilia A is carried out mainly by
Bethesda assay or Nijmegen Bethesda assay (Non-patent Documents 5 and 6).
Recently, a bispecific antibody that binds to both FIX and/or activated coagulation
factor IX (FIXa) and coagulation factor X (FX) and/or activated blood coagulation factor X
(FXa), and substitutes for the cofactor function of FVIII or more specifically, the function of
promoting FX activation by FIXa, was found (Non-patent Documents 7 and 8; Patent
Documents 1, 2, and 3). The bispecific antibody functionally substitutes for FVIII to improve
the decrease in coagulation reaction due to FVIII deficiency or functional abnormality. For
example, with respect to thrombin production and APTT which are indicators of the coagulation
reaction, the bispecific antibody shortens the APTT of plasma derived from a hemophilia A
patient regardless of the presence of an FVIII inhibitor, and increases the production of thrombin.
The APTT-shortening effect of the bispecific antibody was remarkable in comparison to FVIII.
This is because FVIII in plasma shows cofactor activity only after activation by activated factor
X (FXa) or thrombin, whereas the above-mentioned bispecific antibody does not need such
activation process, and for that reason, exhibits the cofactor function more quickly.
Furthermore, antibodies against FIXa Fab and against FX Fab of the bispecific antibody
were obtained, and the concentrations of the bispecific antibody in plasma samples from animal
testing were determined (Non-patent Document 9).
The bispecific antibody substitutes for the cofactor function of FVIII, thus affecting the
assay system that measures the reactivity of FVIII itself. For example, when measuring the
plasma FVIII activity by APTT-based one-stage clotting assay to diagnose the severity of
hemophilia A or to monitor the pharmacological activity of an FVIII formulation in an FVIII
formulation-administered patient, the action of promoting the shortening of coagulation time of
the bispecific antibody strongly interferes in the presence of the bispecific antibody, which
greatly impairs the accuracy of measurement. Furthermore, when determining the plasma
FVIII inhibitor titer by APTT-based Bethesda assay, the action of promoting the shortening of
coagulation time of the bispecific antibody strongly interferes in the presence of the bispecific
antibody, which greatly impairs the accuracy of measurement. That is, in patients administered
with the bispecific antibody, the FVIII activity and FVIII inhibitor titer cannot be accurately
measured. Therefore, methods that enable measurement of the FVIII activity and FVIII
inhibitor titer even in the presence of a bispecific antibody are desired.
Citation List
[Non-patent Documents]
Non-patent Document 1: N Engl J Med. 2007; 357(6): 535-44
Non-patent Document 2: Thromb Res. 2011; 127 (suppl1):S14-7
Non-patent Document 3: Thromb Diath Haemorrh. 1962 May 15; 7: 215-28
Non-patent Document 4: Haemostasis. 1989 19: 196-204.
Non-patent Document 5: Thromb Diath Haemorrh. 1975; 34(3): 869-72
Non-patent Document 6: Thromb Haemost. 1995 Feb; 73(2): 247-51.
Non-patent Document 7: Nat Med. 2012; 18(10): 1570-74
Non-patent Document 8: PLoS One. 2013; 8(2): e57479.
Non-patent Document 9: J Thromb Haemost. 2014; 12(2): 206-13 Supporting Information
[Patent Documents]
Patent Document 1: WO2005/035756
Patent Document 2: WO2006/109592
Patent Document 3: WO2012/067176
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such documents,
or such sources of information, in any jurisdiction, are prior art, or form part of the common
general knowledge in the art.
[Summary of the Invention]
In a first aspect, the invention relates to a method for measuring reactivity of
coagulation factor VIII, wherein the method comprises the following steps:
A) contacting (1) a blood-derived sample containing a substance that has an activity of
functionally substituting for coagulation factor VIII, wherein the substance having an activity of
functionally substituting for coagulation factor VIII is a bispecific antibody that binds to
coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or
activated blood coagulation factor X, with (2) one or more combinations of antibodies that
neutralize the bispecific antibody; and
B) measuring reactivity of coagulation factor VIII in the sample,
wherein the one or more combinations of antibodies are selected from the group consisting of:
(a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X;
(b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated
coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that
binds to coagulation factor X; and
(c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that
binds to activated coagulation factor IX.
In a second aspect, the invention relates to a kit when used for measuring reactivity of
coagulation factor VIII according to the first aspect, wherein the kit comprises one or more
combinations of antibodies selected from the group consisting of:
(a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X;
(b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated
coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that
binds to coagulation factor X; and
(c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that
binds to activated coagulation factor IX.
In a third aspect, the invention relates to a method for diagnosing the disease severity of
a patient administered with a substance having an activity of functionally substituting for
coagulation factor VIII, wherein the substance having an activity of functionally substituting for
coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or
activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation
factor X, and wherein the method comprises the following steps:
1) measuring the reactivity of coagulation factor VIII in a blood-derived sample of the patient
according to the method according to the first aspect; and
2) diagnosing the disease severity of the patient based on the measurement results.
In a fourth aspect, the invention relates to a method for measuring inhibitor titer in a
blood-derived sample of a patient administered with a substance having an activity of
functionally substituting for coagulation factor VIII, wherein the substance having an activity of
functionally substituting for coagulation factor VIII is a bispecific antibody that binds to
coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or
activated blood coagulation factor X, and wherein the method comprises measuring inhibitor
titer in a blood-derived sample of the patient according to the method according to the first
aspect, wherein the method for measuring reactivity of coagulation factor VIII is a method for
measuring the coagulation factor VIII activity or a method for measuring the coagulation factor
VIII inhibitor titer.
In a fifth aspect, the invention relates to a method for monitoring pharmacological
activity of an FVIII formulation in a patient administered with the FVIII formulation and a
substance having an activity of functionally substituting for coagulation factor VIII, wherein the
substance having an activity of functionally substituting for coagulation factor VIII is a
bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and
to coagulation factor X and/or activated blood coagulation factor X, and wherein the method
comprises the following steps:
1) measuring the reactivity of coagulation factor VIII in a blood-derived sample of the patient
according to the method according to the first aspect; and
2) monitoring pharmacological activity of the FVIII formulation in the patient based on the
measurement results.
[Problems to be Solved by the Invention]
The present invention broadly relates to methods for measuring the reactivity of FVIII
in the presence of a substance having an activity of functionally substituting for FVIII, for
example, methods for measuring FVIII activity or FVIII inhibitor titer. Furthermore, an
objective of the present invention is to provide methods and/or kits for measuring the reactivity
of FVIII, such as FVIII activity and FVIII inhibitor titer, in the presence of a substance having an
activity of functionally substituting for FVIII; and/or to at least provide the public with a useful
choice.
[Means for Solving the Problems]
To solve the above-mentioned problems, the present inventors produced substances that
neutralize the activity of the bispecific antibody and by targeting the test items that measure the
reactivity of FVIII, searched for measurement conditions that ensure accuracy even in the
presence of the bispecific antibody. As a result, the present inventors found out that by using
neutralizing antibodies against the bispecific antibody at appropriate concentrations (for example,
concentrations at which the bispecific antibody can be sufficiently neutralized), the FVIII
activity in the plasma of hemophilia A patients can be evaluated accurately by APTT-based
one-stage clotting assay, and also found out that the FVIII inhibitor titer in the plasma of a
hemophilia A patient carrying the FVIII inhibitor can be evaluated accurately by APTT-based
Bethesda assay. Furthermore, the present inventors successfully discovered kits containing
neutralizing antibodies against the bispecific antibody having an FVIII-substituting activity for
use in the measurement. The present description is based on these findings and includes the
following:
a method for measuring reactivity of coagulation factor VIII, wherein the method comprises
the step of contacting
(1) a blood-derived sample containing a substance that has an activity of functionally
substituting for coagulation factor VIII, with
(2) one or more substances that neutralize the substance having an activity of functionally
substituting for coagulation factor VIII;
[2] the method of [1], wherein the substance having an activity of functionally substituting for
coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or
activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation
factor X;
the method of [1] or [2], wherein the bispecific antibody is any one of the antibodies
described below, in which a first polypeptide is associated with a third polypeptide and a second
polypeptide is associated with a fourth polypeptide:
a bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 9, the second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are common L
chains of SEQ ID NO: 10 (Q499-z121/J327-z119/L404-k); or
a bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 36, the second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 37, and the third polypeptide and the fourth polypeptide are common L
chains of SEQ ID NO: 38 (Q153-G4k/J142-G4h/L180-k);
[4] the method of any one of [1] to [3], wherein the neutralizing substance is one or more
substances selected from the group consisting of peptides, polypeptides, organic compounds,
aptamers, and antibodies that neutralize the substance having an activity of functionally
substituting for coagulation factor VIII;
the method of any one of [2] to [4], wherein the neutralizing substance is one or more
antibodies selected from the group consisting of an antibody that binds to Fab comprising an
antigen-binding site that binds to coagulation factor IX, an antibody that binds to Fab comprising
an antigen-binding site that binds to activated coagulation factor IX, an antibody that binds to
Fab comprising an antigen-binding site that binds to coagulation factor X, an antibody that binds
to Fab comprising an antigen-binding site that binds to activated coagulation factor X, and a
bispecific antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and/or activated coagulation factor IX and Fab comprising an antigen-binding site that
binds to coagulation factor X and/or activated coagulation factor X;
the method of any one of [1] to [5], wherein the neutralizing substance is one or more
combinations selected from the group consisting of the following antibody combinations:
(a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X;
(b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated
coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that
binds to coagulation factor X;
(c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
activated coagulation factor IX; and
(d) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that
binds to activated coagulation factor IX;
the method of any one of [1] to [6], wherein the method for measuring reactivity of
coagulation factor VIII is a method for measuring the coagulation factor VIII activity or a
method for measuring the coagulation factor VIII inhibitor titer;
[8] a kit for use in the method of any one of [1] to [7], wherein the kit comprises one or more
antibodies selected from the group consisting of an antibody that binds to Fab comprising an
antigen-binding site that binds to coagulation factor IX, an antibody that binds to Fab comprising
an antigen-binding site that binds to activated coagulation factor IX, an antibody that binds to
Fab comprising an antigen-binding site that binds to coagulation factor X, an antibody that binds
to Fab comprising an antigen-binding site that binds to activated coagulation factor X, and a
bispecific antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and/or activated coagulation factor IX and Fab comprising an antigen-binding site that
binds to coagulation factor X and/or activated coagulation factor X;
the kit of [8], wherein the kit comprises one or more combinations selected from the group
consisting of the following antibody combinations:
(a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X;
(b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated
coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that
binds to coagulation factor X;
(c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to
activated coagulation factor IX; and
(d) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation
factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to
coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that
binds to activated coagulation factor IX;
a method for diagnosing the disease severity of a patient administered with a substance
having an activity of functionally substituting for coagulation factor VIII, wherein the method
uses the method of any one of [1] to [7];
a method for diagnosing inhibitor titer in a patient administered with a substance having an
activity of functionally substituting for coagulation factor VIII, wherein the method uses the
method of any one of [1] to [7];
a method for monitoring pharmacological activity of an FVIII formulation in a patient
administered with the FVIII formulation and a substance having an activity of functionally
substituting for coagulation factor VIII, wherein the method uses the method of any one of [1] to
the method of any one of [10] to [12], wherein the patient is a patient selected from the
group consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand
disease patient, and a patient with hemophilia A in which an inhibitor against blood coagulation
factor VIII and/or activated blood coagulation factor VIII emerges;
the kit of [8] or [9], wherein the kit is for diagnosing the disease severity of a patient
administered with a substance having an activity of functionally substituting for coagulation
factor VIII;
[15] the kit of [8] or [9], wherein the kit is for diagnosing inhibitor titer in a patient administered
with a substance having an activity of functionally substituting for coagulation factor VIII;
the kit of [8] or [9], wherein the kit is for monitoring pharmacological activity of an FVIII
formulation in a patient administered with the FVIII formulation and a substance having an
activity of functionally substituting for coagulation factor VIII; and
the kit of any one of [14] to [16], wherein the patient is a patient selected from the group
consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand disease
patient, and with a patient with hemophilia A in which an inhibitor against blood coagulation
factor VIII and/or activated blood coagulation factor VIII emerges.
[Effects of the Invention]
Described herein are methods that can measure FVIII activity and FVIII inhibitor titer
without being influenced by the activity of a substance having an FVIII-substituting activity. A
substance having an FVIII-substituting activity includes a bispecific antibody that binds to FIX
and/or FIXa and FX and/or FXa.
Brief Description of the Drawings
Fig. 1 shows the results of one-stage clotting assay performed under neutralization of
the anti-FIXa/FX bispecific antibody using rAQ8-mIgG2b and rAJ540-rbtIgG. When
FVIII-deficient plasma containing 10 U/dL or 100 U/dL recombinant FVIII supplemented with
an anti-FIXa/FX bispecific antibody, ACE910, was diluted with a buffer (#3 and #7), the FVIII
activities were shown to be above the range of the calibration curve. On the other hand, when
FVIII-deficient plasma containing 10 U/dL or 100 U/dL recombinant FVIII supplemented with
the anti-FIXa/FX bispecific antibody ACE910 was diluted with a buffer containing the two types
of antibodies against the anti-FIXa/FX bispecific antibody (#4 and #8), the FVIII activities were
shown to be similar to those of the groups without addition of the anti-FIXa/FX bispecific
antibody (#1 and #5). When FVIII-deficient plasma containing 10 U/dL or 100 U/dL
recombinant FVIII was diluted with a buffer containing only the two types of antibodies against
the anti-FIXa/FX bispecific antibody (#2 and #6), the FVIII activities were shown to be similar
to those of the groups without addition of the anti-FIXa/FX bispecific antibody (#1 and #5).
Fig. 2 shows the results of one-stage clotting assay performed under neutralization of
the anti-FIXa/FX bispecific antibody with AQ1 and AJ541 or AQ1 and AJ522. When
FVIII-deficient plasma containing 10 U/dL recombinant FVIII supplemented with an
anti-FIXa/FX bispecific antibody, ACE910, was diluted with a buffer (#4), the FVIII activities
were shown to be above the range of the calibration curve. On the other hand, when
FVIII-deficient plasma containing 10 U/dL recombinant FVIII supplemented with the
anti-FIXa/FX bispecific antibody ACE910 was diluted with a buffer containing two types of
antibodies,AQ1 and AJ541, against the anti-FIXa/FX bispecific antibody (#5), or a buffer
containing two types of antibodies, AQ1 and AJ522, against the anti-FIXa/FX bispecific
antibody (#6), the FVIII activities were shown to be similar to those of the groups without
addition of the anti-FIXa/FX bispecific antibody (#1). When FVIII-deficient plasma containing
U/dL recombinant FVIII was diluted with a buffer containing only the two types of antibodies,
AQ1 and AJ541, against the anti-FIXa/FX bispecific antibody (#2), or a buffer containing only
the two types of antibodies, AQ1 and AJ522 (#3), the FVIII activities were shown to be similar
to those of the groups without addition of the anti-FIXa/FX bispecific antibody (#1).
Fig. 3 shows the results of one-stage clotting assay performed under neutralization of
the anti-FIXa/FX bispecific antibody with AQ512 and AJ114 or AQ512 and AJ521. When
FVIII-deficient plasma containing 10 U/dL recombinant FVIII supplemented with an
anti-FIXa/FX bispecific antibody, hBS23, was diluted with a buffer (#4), the FVIII activities
were shown to be above the range of the calibration curve. On the other hand, when
FVIII-deficient plasma containing 10 U/dL recombinant FVIII supplemented with the
anti-FIXa/FX bispecific antibody hBS23 was diluted with a buffer containing two types of
antibodies, AQ512 and AJ114, against the anti-FIXa/FX bispecific antibody (#5), or a buffer
containing two types of antibodies, AQ512 and AJ521, against the anti-FIXa/FX bispecific
antibody (#6), the FVIII activities were shown to be similar to those of the groups without
addition of the anti-FIXa/FX bispecific antibody (#1). When FVIII-deficient plasma containing
U/dL recombinant FVIII was diluted with a buffer containing only the two types of antibodies,
AQ512 and AJ114, against the anti-FIXa/FX bispecific antibody (#2), or a buffer containing only
the two types of antibodies, AQ512 and AJ521 (#3), the FVIII activities were shown to be
similar to those of the groups without addition of the anti-FIXa/FX bispecific antibody (#1).
Fig. 4 shows the results of Bethesda assay performed under neutralization of the
anti-FIXa/FX bispecific antibody using rAQ8-mIgG2b and rAJ540-rbtIgG. FVIII inhibitor
plasma containing only the anti-FIXa/FX bispecific antibody ACE910 (#3) showed an activity
equivalent to 100% or more of FVIII of the calibration curve. On the other hand, FVIII
inhibitor plasma containing the anti-FIXa/FX bispecific antibody and the two types of antibodies
against the anti-FIXa/FX bispecific antibody (#4) showed an FVIII inhibitor titer similar to that
of the inhibitor plasma without additives (#1). FVIII inhibitor plasma containing only the two
types of antibodies against the anti- FIXa/FX bispecific antibody (#2) showed results similar to
that of #1.
Mode for Carrying Out the Invention
The method for measuring FVIII activity described comprises the step of contacting (1)
and (2) described below. Otherwise, the method can be carried out according to methods
generally used for measuring FVIII activity. Details will be explained in the Examples as well.
(1) a blood-derived sample containing a substance having an activity of functionally
substituting for FVIII
(2) a substance that neutralizes the substance having an activity of functionally substituting for
FVIII
Methods for measuring FVIII activity
The FVIII activity measurement methods that are generally used and known to those
skilled in the art can be used, and for example, one can use a one-stage clotting assay (Casillas et
al., (1971) Coagulation 4: 107-11) that uses factor VIII-deficient plasma (Sysmex, Kobe, Japan),
which is based on coagulation time (aPTT measurements). One-stage clotting assay is carried
out, for example, by the following method. Three solutions, 50 μL of ten-fold diluted test
plasma, 50 μL of FVIII-deficient plasma, and 50 μL of an APTT reagent are mixed; and this is
incubated at 37°C for five minutes, followed by addition of 50 μL of a calcium solution to
initiate the coagulation reaction, and then the time to coagulation is measured. Furthermore,
instead of the test plasma, serially diluted samples of normal plasma (FVIII activity in a ten-fold
diluted normal plasma is specified as 100%) are measured, and a calibration curve is produced
by plotting the FVIII activity on the horizontal axis and coagulation time on the vertical axis.
The coagulation time of the test plasma is converted to FVIII activity using the calibration curve,
and FVIII activity in the test plasma is calculated. Herein, unless stated otherwise, the phrase
“measurement of FVIII activity” is used as a phrase that may include “measurement of activated
coagulation factor VIII (FVIIIa) activity”.
In addition to one-stage clotting assay, thrombin generation assay (TGA), measurement
methods that use rotation thromboelastometry, FVIII chromogenic assay, coagulation waveform
analysis, thrombin and activated factor X production assay, and such may be used as the method
for measuring FVIII activity. The method for measuring FVIII inhibitor titer described includes
the step of contacting (1) and (2) described below. Otherwise, the method can be carried out
according to generally used methods for measuring FVIII inhibitor titer. Details will be
explained in the Examples as well.
(1) a blood-derived sample containing a substance having an activity of functionally
substituting for FVIII
(2) a substance that neutralizes the substance having an activity of functionally substituting for
FVIII
Methods for measuring FVIII inhibitor titer
The FVIII inhibitor titer measurement methods that are generally used and known to
those skilled in the art can be used, and for example, one can use Bethesda assay (Kasper et al.,
(1975) Thrombos Diath Haemorrh 34: 869-872), ELISA method, and Nijmegen Bethesda assay
(Nijmegen modification assay) (Verbruggen et al., (1995) Thromb Haemost 73: 247-251).
Bethesda assay is carried out, for example, by the following method. A solution produced by
mixing equal amounts of normal plasma and test plasma is incubated at 37°C for two hours, and
then the residual factor VIII activity in normal plasma is measured by one-stage clotting assay
based on activated partial thromboplastin time (APTT). The action of inhibiting 50% of the
factor VIII activity in normal plasma is specified as 1 Bethesda (1BU), and therefore the FVIII
inhibitor titer is calculated in units of Bethesda. When the FVIII inhibitor titer in the test
plasma is high and the residual FVIII activity does not lie within the range of 25% to 75%, test
plasma suitably diluted with a buffer is used to recalculate the Bethesda units, and subsequently,
the value is multiplied by the dilution ratio to calculate the FVIII inhibitor titer in the test plasma.
FVIII
FVIII is one of a series of molecules involved in blood coagulation, which demonstrates
cofactor activity when it is activated by thrombin or FXa and promotes the FX activation
reaction by FIXa.
FVIII inhibitor
The FVIII inhibitor is an isoantibody against foreign FVIII and is emerged in 20% to
30% of hemophilia A patients. An individual who is originally normal may produce
autoantibodies against FVIII posteriori. Generally, most FVIII inhibitor isoantibodies and
autoantibodies function as anti-FVIII neutralizing antibodies, and decrease or eliminate FVIII
activity.
Activity of substituting for FVIII
A substance having an activity of functionally substituting for FVIII described herein
can be rephrased as a substance having an FVIII-like activity. In the description, the phrase
“functionally substitute/substituting for FVIII” means that FX activation by FIXa is promoted
(FXa generation by FIXa is promoted). More specifically, in the description, the phrase
“functionally substitute/substituting for FVIII” means recognizing FIX and/or FIXa, and FX
and/or FXa, and promoting activation of FX by FIXa (promoting FXa generation by FIXa).
The activity of promoting FXa generation can be evaluated using, for example, a measurement
system comprising FIXa, FX, synthetic substrate S-2222 (synthetic substrate of FXa), and
phospholipids. Such measurement system shows correlation between the severity of the disease
and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomba¨ck M et al.
Clinical applications of a chromogenic substrate method for determination of FVIII activity.
Thromb Haemost 1985; 54: 811-23).
A preferred embodiment of a substance having an activity of functionally substituting
for FVIII described herein includes, for example, a bispecific antibody that binds to FIX and/or
FIXa, and to FX and/or FXa. Such an antibody can be obtained according to methods
described, for example, in WO2005/035756, WO2006/109592, and WO2012/067176. The
bispecific antibody described herein includes antibodies described in these documents.
A preferred bispecific antibody includes, for example, ACE910
(Q499-z121/J327-z119/L404-k) (a bispecific antibody in which the H chain consisting of the
amino acid sequence of SEQ ID NO: 9 and the L chain of SEQ ID NO: 10 are associated, and
the H chain consisting of the amino acid sequence of SEQ ID NO: 11 and the L chain of SEQ ID
NO: 10 are associated) and hBS23 (Q153-G4k/J142-G4h/L180-k) (a bispecific antibody in
which the H chain consisting of the amino acid sequence of SEQ ID NO: 36 and the L chain of
SEQ ID NO: 38 are associated, and the H chain consisting of the amino acid sequence of SEQ
ID NO: 37 and the L chain of SEQ ID NO: 38 are associated), which are bispecific antibodies
described in a patent document (WO2012/067176).
Neutralization
“Neutralization” in a substance that neutralizes the substance having an activity of
functionally substituting for FVIII described herein refers to, for example, complete or partial
inhibition of the activity of functionally substituting for FVIII of a substance that has an activity
of functionally substituting for FVIII. For example, when the substance having the activity of
functionally substituting for FVIII is an antibody, complete or partial inhibition of the activity of
functionally substituting for FVIII may be accomplished by completely or partially inhibiting
binding of the antibody to the antigen, but is not limited thereto
Neutralizing substances
The term “substance” of the neutralizing substance in the substance that neutralizes the
substance having an activity of functionally substituting for FVIII described herein refers to, for
example, peptides, polypeptides, organic compounds, aptamers, antibodies, and such that bind to
the substance having an activity of functionally substituting for FVIII.
A plurality of neutralizing substances can be used in combination, and for example,
antibodies and aptamers can be used in combination.
Polypeptides
Polypeptides in the description normally refer to proteins and peptides having a length
of approximately ten amino acids or longer. Generally, they are biologically derived
polypeptides, but are not particularly limited to such polypeptides, and may be, for example,
polypeptides comprising an artificially designed sequence. Furthermore, they may be any
native polypeptides, or synthetic polypeptides, recombinant polypeptides, or such. Additionally,
the fragments of the above-mentioned polypeptide are also included in the polypeptides as
described herein.
Organic compounds
Organic compounds in the description are, for example, low-molecular-weight
compounds, preferably with a molecular weight of 1000 or less.
Aptamers
The term “aptamer” refers to a nucleic acid molecule that binds specifically to a target
molecule such as a polypeptide. For example, aptamers of the description can be RNA
aptamers capable of binding specifically to substances having an FVIII-substituting activity.
Production and therapeutic use of aptamers are well established in this field. For example,
aptamers can be obtained by using the SELEX method (see U.S. Patent Nos. 5475096, 5580737,
5567588, 5707796, 5763177, 6699843, and such).
Antibodies
When the substance having an activity of functionally substituting for FVIII is a
bispecific antibody that binds to FIX and/or FIXa and to FX and/or FXa, examples of antibodies
that bind to the substance having an activity of functionally substituting for FVIII include
antibodies selected from the group consisting of antibodies that bind to Fab containing an
antigen-binding site that binds to FIX, antibodies that bind to Fab containing an antigen-binding
site that binds to FIXa, antibodies that bind to Fab containing an antigen-binding site that binds
to FX, antibodies that bind to Fab containing an antigen-binding site that binds to FXa, and
bispecific antibodies that bind to Fab containing an antigen-binding site that binds to FIX and/or
FIXa and to Fab containing an antigen-binding site that binds to FX and/or FXa. The
above-mentioned antibodies can be used separately or in multi-combinations. For example, it is
possible to use multiple antibodies that bind to Fab containing an antigen-binding site that binds
to one type of antigen, for example, multiple types of antibodies that bind to Fab containing an
antigen-binding site that binds to FIX. For example, when the substance having an activity of
functionally substituting for FVIII is a bispecific antibody that binds to FIX and/or FIXa and to
FX and/or FXa, the following antibody combinations can be used:
(a) an antibody that binds to Fab containing an antigen-binding site that binds to FIX and an
antibody that binds to Fab containing an antigen-binding site that binds to FX;
(b) an antibody that binds to Fab containing an antigen-binding site that binds to FIXa and an
antibody that binds to Fab containing an antigen-binding site that binds to FX;
(c) an antibody that binds to Fab containing an antigen-binding site that binds to FIX and an
antibody that binds to Fab containing an antigen-binding site that binds to FIXa; and
(d) an antibody that binds to Fab containing an antigen-binding site that binds to FIX, an
antibody that binds to Fab containing an antigen-binding site that binds to FX, and an antibody
that binds to Fab containing an antigen-binding site that binds to FIXa.
An example of an antibody that binds to Fab containing an antigen-binding site that
binds to FIX and/or FIXa includes the AQ8, AQ1, and AQ512 antibodies. The nucleotide
sequences of the variable regions and the amino acid sequences predicted therefrom were
analyzed by GENETYX Ver. 9 (GENETYX CORPORATION).
The amino acid sequence and the nucleotide sequence of the H chain variable region of AQ8
are indicated by the following SEQ ID NOs:
amino acid sequence: SEQ ID NO: 1; and
nucleotide sequence: SEQ ID NO: 5.
The amino acid sequence and the nucleotide sequence of the L chain variable region of AQ8 are
indicated by the following SEQ ID NOs:
amino acid sequence: SEQ ID NO: 2; and
nucleotide sequence: SEQ ID NO: 6.
The amino acid sequences and the nucleotide sequences of the H-chain CDRs 1 to 3 of AQ8 are
indicated by the following SEQ ID NOs:
CDR1 amino acid sequence: SEQ ID NO: 12;
CDR2 amino acid sequence: SEQ ID NO: 13;
CDR3 amino acid sequence: SEQ ID NO: 14;
CDR1 nucleotide sequence: SEQ ID NO: 15;
CDR2 nucleotide sequence: SEQ ID NO: 16; and
CDR3 nucleotide sequence: SEQ ID NO: 17.
The amino acid sequences and the nucleotide sequences of the L-chain CDRs 1 to 3 of AQ8 are
indicated by the following SEQ ID NOs:
CDR1 amino acid sequence: SEQ ID NO: 18;
CDR2 amino acid sequence: SEQ ID NO: 19;
CDR3 amino acid sequence: SEQ ID NO: 20;
CDR1 nucleotide sequence: SEQ ID NO: 21;
CDR2 nucleotide sequence: SEQ ID NO: 22; and
CDR3 nucleotide sequence: SEQ ID NO: 23.
An example of an antibody that binds to Fab containing an antigen-binding site that
binds to FX and/or FXa includes the AJ540, AJ541, AJ522, AJ114, and AJ521 antibodies. The
nucleotide sequences of the variable regions and the amino acid sequences predicted therefrom
were analyzed by GENETYX Ver. 9 (GENETYX CORPORATION).
The amino acid sequence and the nucleotide sequence of the H chain variable region of AJ540
are indicated by the following SEQ ID NOs:
amino acid sequence: SEQ ID NO: 3; and
nucleotide sequence: SEQ ID NO: 7.
The amino acid sequence and the nucleotide sequence of the L chain variable region of AJ540
are indicated by the following SEQ ID NOs:
amino acid sequence: SEQ ID NO: 4; and
nucleotide sequence: SEQ ID NO: 8.
The amino acid sequences and the nucleotide sequences of the H-chain CDRs 1 to 3 of AJ540
are indicated by the following SEQ ID NOs:
CDR1 amino acid sequence: SEQ ID NO: 24;
CDR2 amino acid sequence: SEQ ID NO: 25;
CDR3 amino acid sequence: SEQ ID NO: 26;
CDR1 nucleotide sequence: SEQ ID NO: 27;
CDR2 nucleotide sequence: SEQ ID NO: 28; and
CDR3 nucleotide sequence: SEQ ID NO: 29.
The amino acid sequences and the nucleotide sequences of the L-chain CDRs 1 to 3 of AJ540
are indicated by the following SEQ ID NOs:
CDR1 amino acid sequence: SEQ ID NO: 30;
CDR2 amino acid sequence: SEQ ID NO: 31;
CDR3 amino acid sequence: SEQ ID NO: 32;
CDR1 nucleotide sequence: SEQ ID NO: 33;
CDR2 nucleotide sequence: SEQ ID NO: 34; and
CDR3 nucleotide sequence: SEQ ID NO: 35.
The term “antibody” is used in the broadest sense, and may be monoclonal antibodies,
polyclonal antibodies, dimers, multimers, multispecific antibodies (for example, bispecific
antibodies), antibody derivatives, and modified antibody products (Miller K et al. J Immunol.
2003, 170(9), 4854-61) as long as they display a desired biological activity. The antibodies
may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or
those derived from another species, or they may be artificially synthesized antibodies. The
antibodies disclosed herein can be of any type (for example, IgG, IgE, IgM, IgD, and IgA), class
(for example, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecules.
The immunoglobulins can be derived from any species (for example, human, mouse, or rabbit).
The terms “antibody”, “immune globulin” and “immunoglobulin” are used interchangeably in a
broad sense.
The term “antibody derivative” includes a portion of an antibody, preferably an antibody
variable region, or at least an antigen-binding region of an antibody. Antibody derivatives
include, for example, Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, and single-chain
antibodies (scFv), sc(Fv)2, Fab3, domain antibodies (dAb) (WO2004/058821, WO2003/002609),
diabodies, triabodies, tetrabodies, minibodies, and multispecific antibodies formed from antibody
derivatives, but are not limited thereto. Here, “Fab” is constructed from a single light chain and
the CH1 domain and variable region of a single heavy chain. Furthermore, “Fv” is the smallest
antibody derivative, and includes a complete antigen-recognizing region and an antigen-binding
region. The antibody derivative may be, for example, a fusion between an IgG antibody and Fc.
For example, one can refer to Example 2 in U.S. Patent No. 5641870 specification; Zapata G et
al. Protein Eng. 1995, 8(10), 1057-1062; Olafsen T et al. Protein Eng. Design & Sel. 2004,
17(4): 315-323; Holliger P et al. Nat. Biotechnol. 2005, 23(9): 1126-36; Fischer N et al.
Pathobiology. 2007, 74(1): 3-14; Shen J et al. J Immunol Methods. 2007, 318, 65-74; and Wu et
al. Nat Biotechnol. 2007, 25(11), 1290-7.
Examples of modified antibody products may include antibodies linked to various
molecules such as polyethylene glycol (PEG). Antibodies as described herein n include such
modified antibody products. The substance to be linked is not limited in the modified antibody
products described herein . To yield such modified antibody products, chemical modifications
can be made to the obtained antibodies. Such methods are already established in this field.
“Bispecific” antibodies refer to antibodies having variable regions that recognize
different epitopes, where the regions are within the same antibody molecule. Bispecific
antibodies may be antibodies that recognize two or more different antigens or antibodies that
recognize two or more different epitopes on the same antigen. Bispecific antibodies may
include not only whole antibodies but antibody derivatives. Antibodies of the description also
include bispecific antibodies. Herein, anti-FIXa/FX bispecific antibody and bispecific antibody
that binds to FIXa and FX are used synonymously.
Methods for producing genetically engineered antibodies
Recombinant antibodies produced by using genetic engineering techniques can be used
as the antibodies. Recombinant antibodies can be obtained by cloning DNAs encoding the
antibodies from hybridomas or antibody-producing cells such as sensitized lymphocytes that
produce antibodies, inserting them into vectors, and then introducing them into hosts (host cells)
to produce the antibodies.
The antibodies include human antibodies, mouse antibodies, and rat antibodies, and
their origin is not limited. They may also be genetically modified antibodies such as chimeric
antibodies and humanized antibodies.
Methods for obtaining human antibodies are known. For example, transgenic animals
carrying the entire repertoire of human antibody genes can be immunized with antigens of
interest to obtain human antibodies of interest (see International Publication WO 93/12227, WO
92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).
Genetically modified antibodies can be produced using known methods. Specifically,
for example, chimeric antibodies comprise H chain and L chain variable regions of an
immunized animal antibody, and H chain and L chain constant regions of a human antibody.
Chimeric antibodies can be obtained by linking DNAs encoding the variable regions of the
antibody derived from the immunized animal, with DNAs encoding the constant regions of a
human antibody, inserting this into an expression vector, and then introducing it into host to
produce the antibodies.
Humanized antibodies are modified antibodies that are also referred to as reshaped
human antibodies. A humanized antibody is constructed by transferring the CDRs of an
antibody derived from an immunized animal to the complementarity determining regions of a
human antibody. Conventional genetic recombination techniques for such purposes are known
(see European Patent Application Publication No. EP 239400; International Publication No. WO
96/02576; Sato K et al., Cancer Research 1993, 53: 851-856; International Publication No. WO
99/51743).
Bispecific antibodies are antibodies that have specificity to two different antigens.
While bispecific antibodies are not limited to those of the IgG type, for example,
IgG-type bispecific antibodies can be secreted from a hybrid hybridoma (quadroma) produced by
fusing two types of hybridomas that produce IgG antibodies (Milstein C. et al., Nature 1983,
305: 537-540). They can also be secreted by introducing the L chain and H chain genes
constituting the two types of IgGs of interest, a total of four types of genes, into cells to
co-express the genes.
In this case, by introducing suitable amino acid substitutions to the CH3 regions of the
H chains, IgGs having a heterogeneous combination of H chains can be preferentially secreted
(Ridgway JB et al. Protein Engineering 1996, 9: 617-621; Merchant AM et al. Nature
Biotechnology 1998, 16: 677-681; ; Davis JH et al. Protein Eng Des Sel. 2010,
4: 195-202).
Regarding the L chains, since the diversity of L chain variable regions is lower than that
of H chain variable regions, one can expect to obtain common L chain that can confer binding
ability to both H chains. The antibodies described herein may be antibodies comprising
common L chains. Bispecific IgGs can be efficiently expressed by introducing the gene of the
common L chain and both H chains into cells.
Epitopes
Antibodies which are an embodiment of substances that neutralize the substance having
an activity of functionally substituting for FVIII described herein include antibodies that bind
to an epitope overlapping with an epitope bound by the antibodies described above, and
preferably antibodies that bind to the same epitope.
Whether an antibody recognizes the same epitope as or an epitope overlapping with an
epitope that is recognized by another antibody can be confirmed by competition between the two
antibodies against the epitope. Competition between the antibodies can be evaluated by
competitive binding assays using means such as enzyme-linked immunosorbent assay (ELISA),
fluorescence energy transfer method (FRET), and fluorometric microvolume assay technology
(FMAT (Registered trademark)). The amount of antibodies bound to an antigen indirectly
correlate with the binding ability of candidate competitor antibodies (test antibodies) that
competitively bind to the same or overlapping epitope. In other words, as the amount of or the
affinity of test antibodies against the same or overlapping epitope increases, the amount of
antibodies bound to the antigen decreases, and the amount of test antibodies bound to the antigen
increases. Specifically, the appropriately labeled antibodies and test antibodies are
simultaneously added to the antigens, and then the bound antibodies are detected using the label.
The amount of the antibodies bound to the antigen can be easily determined by labeling the
antibodies in advance. This label is not particularly limited, and the labeling method is selected
according to the assay technique used. Specific examples of the labeling method include
fluorescent labeling, radiolabeling, and enzyme labeling.
Herein, the “antibody that binds to the overlapping epitope” or “antibody that binds to
the same epitope” refers to a test antibody that can reduce the amount of binding of the labeled
antibody by at least 50% at a concentration that is usually 100 times higher, preferably 80 times
higher, more preferably 50 times higher, even more preferably 30 times higher, and still more
preferably 10 times higher than a concentration of the non-labeled antibody at which binding of
the non-labeled antibody reduces the amount of binding of the labeled antibody by 50% (IC50).
The epitope recognized by the antibody can be analyzed by methods known to those skilled in
the art, and for example, it can be performed by Western blotting and such.
Antibody production methods
Antibodies of the description can be produced by methods known to those skilled in the
art. Specifically, DNA encoding the antibody of interest is inserted into an expression vector.
Insertion into an expression vector is carried out such that the expression will take place under
the control of expression regulatory regions such as enhancers and promoters. Next, host cells
are transformed using this expression vector to express the antibodies. Appropriate
combinations of the host and expression vector can be used in this step.
Examples of the vectors include M13 series vectors, pUC series vectors, pBR322,
pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or
pT7 can also be used for the purpose of cDNA subcloning and excision.
Particularly, expression vectors are useful for using the vectors for the purpose of
producing the antibody. For example, when the host is E. coli such as JM109, DH5 α, HB101,
or XL1-Blue, the expression vectors indispensably have a promoter that permits efficient
expression in E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341, 544-546; and
FASEB J (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or
T7 promoter. Examples of such vectors include the vectors mentioned above as well as
pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system” (manufactured by QIAGEN),
pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).
The vectors may contain a signal sequence for polypeptide secretion. In the case of
production in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987)
169, 4397) can be used as the signal sequence for polypeptide secretion. The vectors can be
transferred to the host cells using, for example, calcium chloride methods or electroporation
methods.
In addition to the E. coli expression vectors, examples of the vectors for producing the
antibody of the description include mammal-derived expression vectors (e.g., pcDNA3
(manufactured by Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF,
and pCDM8), insect cell-derived expression vectors (e.g., “Bac-to-BAC baculovirus expression
system” (manufactured by GIBCO BRL), and pBacPAK8), plant-derived expression vectors (e.g.,
pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw),
retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g.,
“Pichia Expression Kit” (manufactured by Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus
subtilis-derived expression vectors (e.g., pPL608 and pKTH50).
For the purpose of expression in animal cells such as CHO cells, COS cells, or NIH3T3
cells, the vectors indispensably have a promoter necessary for intracellular expression, for
example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMTV-LTR promoter,
EF1 α promoter (Mizushima et al., Nucleic Acids Res (1990) 18, 5322), CAG promoter (Gene
(1991) 108, 193), or CMV promoter and, more preferably, have a gene for screening for
transformed cells (e.g., a drug resistance gene that can work as a marker by a drug (neomycin,
G418, etc.)). Examples of the vectors having such properties include pMAM, pDR2, pBK-RSV,
pBK-CMV, pOPRSV, and pOP13.
An exemplary method intended to stably express the gene and increase the number of
intracellular gene copies involves transfecting CHO cells deficient in nucleic acid synthesis
pathway with vectors having a DHFR gene serving as a complement thereto (e.g., pCHOI) and
using methotrexate (MTX) in the gene amplification. An exemplary method intended to
transiently express the gene involves using COS cells having a gene which expresses an SV40 T
antigen on their chromosomes to transform the cells with vectors having a replication origin of
SV40 (pcD, etc.). Also, a replication origin derived from polyomavirus, adenovirus, bovine
papillomavirus (BPV), or the like may be used. The expression vectors for increasing the
number of gene copies in a host cell system can additionally contain a selection marker such as
an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine
guanine phosphoribosyltransferase (Ecogpt) gene, or a dihydrofolate reductase (dhfr) gene.
The antibodies of the description obtained by the methods described above can be
isolated from inside host cells or from outside of the cells (the medium, or such), and purified to
practically pure and homogeneous antibodies. The antibodies can be separated and purified by
methods routinely used for separating and purifying antibodies, and the type of method is not
limited. For example, the antibodies can be separated and purified by appropriately selecting
and combining column chromatography, filtration, ultrafiltration, salting-out, solvent
precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, isoelectrofocusing, dialysis, recrystallization, and such.
The chromatographies include, for example, affinity chromatography, ion exchange
chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and
adsorption chromatography (Strategies for Protein Purification and Characterization: A
Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1996). The chromatographic methods described above can be conducted using
liquid-chromatography, for example, HPLC and FPLC. Columns used for affinity
chromatography include protein A columns and protein G columns. Columns using protein A
include, for example, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The
description includes antibodies that are highly purified using these purification methods.
The obtained antibodies can be purified to homogeneity. Separation and purification
of the antibodies can be performed using separation and purification methods generally used for
protein separation and purification. For example, the antibodies can be separated and purified
by appropriately selecting and combining column chromatography such as affinity
chromatography, filtration, ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel
electrophoresis, isoelectric focusing, and such, without limitation (Antibodies: A Laboratory
Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns used for
affinity chromatography include, for example, protein A columns and protein G columns.
Methods for obtaining samples
As described herein, blood-derived samples are preferably blood-derived samples
collected from a test subject. Such blood-derived samples can be obtained from test subjects
administered with a substance having an FVIII-substituting activity. A test subject includes, for
example, a patient with hemorrhagic symptoms at any part in the body (hemorrhagic disease
patient). The main bleeding sites are intraarticular, intramuscular, subcutaneous, intraoral,
intracranial, digestive tract, intranasal, and such, but are not limited thereto. The hemorrhagic
disease patient is preferably a patient with hemorrhagic disease caused by decrease or deficiency
in an FVIII activity and/or FVIIIa activity. The patient with hemorrhagic disease caused by
decrease or deficiency in the FVIII activity and/or FVIIIa activity is a patient with hemorrhagic
symptoms, and examples include patients with a priori or posteriori decrease or deficiency in
either or both of the FVIII activity and FVIIIa activity. Decrease in the activities of FVIII and
FVIIIa means that in comparison to those of healthy individuals, these activities are preferably
less than 40% (for example, less than 40%, less than 30%, less than 20%, or less than 10%),
more preferably less than 10% (for example, less than 10%, less than 9%, less than 8%, less than
7%, or less than 6%), even more preferably less than 5% (for example, less than 5%, less than
4%, less than 3%, or less than 2%), and particularly preferably less than 1% in a patient, without
being limited thereto.
More specifically, examples of such diseases include diseases selected from among
hemophilia (hemophilia A and hemophilia B), acquired hemophilia, and von Willebrand’s
disease caused by functional abnormality or deficiency of von Willebrand factor (vWF), but are
not limited thereto. Blood-derived samples include serum, plasma, or whole blood. In theUse
of plasma samples is preferred. Methods for obtaining blood-derived samples from test
subjects are well known to those skilled in the art.
Kits
Various types of reagents such as buffers required for the method for measuring the
reactivity of FVIII of the description can be packaged in advance as a kit. The kit described
may include in addition to the buffer, plasma samples isolated from a human whose FVIII
activity and FIX activity in the blood are normal, a substance having an FVIII-substituting
activity, and anything that can be used in FVIII activity measurement, or anything that can be
used in FVIII inhibitor titer measurement. Furthermore, the various types of reagents included
in the kit can be made into a powder or liquid form according to their mode of use.
Furthermore, they can be stored in appropriate containers and used when suitable.
The disease severity of a patient administered with the substance having an activity of
functionally substituting for FVIII, for example, can be diagnosed by using the described
method . Reactivity of FVIII can be measured using the described method, and the disease
severity and/or inhibitor titer for the patient can be diagnosed/assessed based on the
measurement results. The diagnosis and assessment methods can be performed by methods
known to those skilled in the art.
The pharmacological activity of an FVIII formulation in patients administered with the
FVIII formulation and a substance having an activity of functionally substituting for FVIII, for
example, can be monitored by using the described methods. Monitoring can be carried out by
methods known to those skilled in the art.
The described kit can be used as a kit for diagnosing the disease severity of a patient
administered with a substance having an activity of functionally substituting for FVIII.
Reactivity of FVIII can be measured using the kit of this description, and the disease severity of
the patient can be diagnosed/assessed based on the measurement results. The diagnosis and
assessment methods can be performed by methods known to those skilled in the art.
The described kit can be used, for example, as a kit for monitoring the pharmacological
activity of an FVIII formulation in a patient administered with the FVIII formulation and a
substance having an activity of functionally substituting for FVIII. Monitoring can be carried
out by methods known to those skilled in the art.
For example, one may use a method for treating a patient, which comprises the steps of:
(a) administering a first dose of a substance having an activity of functionally substituting for
FVIII;
(b) monitoring the reactivity of FVIII in the patient;
(c) determining a second dose of the substance having an activity of functionally substituting
for FVIII based on the observed reactivity of FVIII; and
(d) administering to the patient the second dose of the substance having an activity of
functionally substituting for FVIII.
Furthermore, one may use, for example, a method for treating a patient, which
comprises the steps of:
(a) administering a substance having an activity of functionally substituting for FVIII following
a first administration interval;
(b) monitoring the reactivity of FVIII in the patient;
(c) determining a second administration interval for the substance having an activity of
functionally substituting for FVIII based on the observed reactivity of FVIII; and
(d) administering to the patient the substance having an activity of functionally substituting for
FVIII following the second administration interval.
One may also use, for example, a method for treating a patient, which comprises
monitoring the reactivity of FVIII, and changing the administration dose and/or the
administration interval of the substance having an activity of functionally substituting for
coagulation factor VIII depending on the reactivity of FVIII.
The substance having an activity of functionally substituting for FVIII is preferably a
bispecific antibody that binds to FIX and/or FIXa and to FX and/or FXa. It is more preferably
the antibody described below, which 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
bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 9, the second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are common L
chains of SEQ ID NO: 10 (Q499-z121/J327-z119/L404-k), or
bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 36, the second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 37, and the third polypeptide and the fourth polypeptide are common L
chains of SEQ ID NO: 38 (Q153-G4k/J142-G4h/L180-k).
The dose is, for example, 0.001 mg/kg to 100 mg/kg for the aforementioned bispecific
antibody. It is preferably approximately 0.001 mg/kg, approximately 0.003 mg/kg,
approximately 0.005 mg/kg, approximately 0.01 mg/kg, approximately 0.03 mg/kg,
approximately 0.05 mg/kg, approximately 0.1 mg/kg, approximately 0.3 mg/kg, approximately
0.5 mg/kg, approximately 1 mg/kg, approximately 3 mg/kg, approximately 5 mg/kg,
approximately 10 mg/kg, approximately 20 mg/kg, approximately 30 mg/kg, approximately 40
mg/kg, approximately 50 mg/kg, approximately 60 mg/kg, approximately 70 mg/kg,
approximately 80 mg/kg, approximately 90 mg/kg, and approximately 100 mg/kg. The doses
before and after the monitoring step may be the same or different. In the case of the
aforementioned bispecific antibody, the administration interval is, for example, at least one day
or more. The interval is preferably 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2
weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12
weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21
weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 1 month, 2 months, 3 months, 4 months, 5
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year. The dose
intervals before and after the monitoring step may be the same or different.
The target patients for the methods or kits of the description are, for example,
hemophilia A patients, acquired hemophilia A patients, von Willebrand disease patients, and
hemophilia A patients with emergence of an inhibitor against FVIII and/or FVIIIa.
As used herein, embodiments represented by the expression “comprising …” include
embodiments represented by the expression “essentially consisting of …” and embodiments
represented by the expression “consisting of …”.
The term “comprising” as used in this specification and claims also means “consisting at
least in part of”. When interpreting statements in this specification, and claims which include the
term “comprising”, it is to be understood that other features that are additional to the features
prefaced by this term in each statement or claim may also be present. Related terms such as
“comprise” and “comprised” are to be interpreted in similar manner.
All patents and reference documents explicitly cited herein are incorporated by
reference into this description in their entirety.
The present invention will be further illustrated by the Examples, but it is not to be
construed as being limited thereto.
[Examples]
Herein below, the present invention will be specifically described by the Examples, but
it is not to be construed as being limited thereto.
[Example 1] Production of antibodies against the anti-FIXa/FX bispecific antibody and sequence
determination of the variable region
An attempt was made to generate antibodies against ACE910
(Q499-z121/J327-z119/L404-k) (bispecific antibody in which an H chain consisting of the amino
acid sequence of SEQ ID NO: 9 is associated with the L chain of SEQ ID NO: 10, and an H
chain consisting of the amino acid sequence of SEQ ID NO: 11 is associated with the L chain of
SEQ ID NO: 10), which is a bispecific antibody described in the Patent Document 3 (WO
2012/067176). Gene recombination techniques and pepsin digestion were used to produce
F(ab')2 composed from the respective Fabs of the anti-FIXa side and the anti-FX side.
Mice and rats were immunized with anti-FIXa-F(ab')2 or anti-FX-F(ab')2. Cells
obtained from the spleen removed from the mice or rats or from rat lymph nodes were subjected
to cell fusion with mouse myeloma cells by following general methods to produce the
hybridomas. The culture supernatants of the hybridomas were evaluated by ELISA which
detects the binding of ACE910 to the anti-FIXa-arm or the anti-FX-arm, and ultimately, mouse
antibodies, AQ8 and AQ1, and rat antibody, AQ512, which bind only to the anti-FIXa-arm but
not to the anti-FX-arm of ACE910, and rat antibodies, AJ540, AJ114, AJ521, AJ522, and AJ541,
which bind only to the anti-FX-arm but not to the anti-FIXa-arm of ACE910 were selected. In
addition, the nucleotide sequences of the variable regions of the AQ8 antibody or the AJ540
antibody were analyzed. The nucleotide sequences of the variable regions of AQ8 and AJ540,
and amino acid sequences predicted therefrom were analyzed using GENETYX Ver.9
(GENETYX CORPORATION).
[Example 2] Production of expression vectors for recombinant mouse antibody AQ8 and
recombinant rat-rabbit chimeric antibody AJ540.
Recombinant mouse antibody AQ8 was prepared by combining the variable region
sequences of the AQ8 antibody obtained in Example 1 with a known mouse IgG2b constant
region sequences (heavy chain: EMBL accession No. J00461; light chain: EMBL accession No.
V00807) to produce the full-length antibody gene, and then inserting it into an expression vector.
Similarly, a recombinant rat-rabbit chimeric antibody AJ540 was produced by combining a
known rabbit IgG (heavy chain: EMBL accession No. L29172, light chain: EMBL accession No.
X00231) with the variable regions of the AJ540 antibody. The produced expression clone
plasmids were introduced into HEK293 cells, large-scale culturing and purification with Protein
A and gel filtration were performed, and recombinant mouse antibody AQ8 (rAQ8-mIgG2b) and
recombinant rat-rabbit chimeric antibody AJ540 (rAJ540-rbtIgG) were produced.
[Example 3] One-stage clotting assay carried out under neutralization of the anti-FIXa/FX
bispecific antibody using rAQ8-mIgG2b and rAJ540-rbtIgG
To FVIII-deficient plasma (George King) containing 10 U/dL or 100 U/dL recombinant
FVIII (Kogenate FS, Bayer Yakuhin, Ltd.), the anti-FIXa/FX bispecific antibody ACE910 was
added at 0 μg/mL or 300 μg/mL. Furthermore, each of the prepared plasma samples was
divided into the following two groups to prepare measurement sample solutions: a group
subjected to ten-fold dilution using an imidazole buffer (Kyowa Medex); and a group subjected
to ten-fold dilution using an imidazole buffer supplemented with 300 μg/mL each of
rAQ8-mIgG2b and rAJ540-rbtIgG. Amounts of rAQ8-mIgG2b and rAJ540-rbtIgG required to
sufficiently neutralize ACE910 were added. Details of the combinations are shown below.
[Table 1]
Sample Plasma Dilution buffer
No. Type Dilution
rate
#1 FVIII deficient plasma containing 10 10-fold Imidazole buffer
#2 U/dL recombinant FVIII 10-fold Imidazole buffer supplemented
with rAQ8-mIgG2b and
rAJ540-rbtIgG
#3 FVIII deficient plasma containing 10 10-fold Imidazole buffer
#4 U/dL recombinant FVIII 10-fold Imidazole buffer supplemented
with rAQ8-mIgG2b and
supplemented with 300 μg/mL
anti-FIXa/FX bispecific antibody rAJ540-rbtIgG
#5 FVIII deficient plasma containing 10-fold Imidazole buffer
#6 100 U/dL recombinant FVIII 10-fold Imidazole buffer supplemented
with rAQ8-mIgG2b and
rAJ540-rbtIgG
#7 FVIII deficient plasma containing 10-fold Imidazole buffer
100 U/dL recombinant FVIII
#8 10-fold Imidazole buffer supplemented
supplemented with 300 μg/mL with rAQ8-mIgG2b and
anti-FIXa/FX bispecific antibody rAJ540-rbtIgG
Furthermore, to produce a calibration curve for conversion of coagulation time to FVIII
activity, solutions of standard plasma, Coagtrol N (Sysmex), were prepared by performing
-fold, 20-fold, 40-fold, 80-fold, and 160-fold dilutions using an imidazole buffer (FVIII
activities for the respective calibration curve solutions were specified as 93%, 46.5%, 23.3%,
11.6%, and 5.81%). Fifty microliters of a measurement sample solution or calibration curve
solution, 50 μL of factor VIII-deficient human plasma (Sysmex), and 50 μL of Thrombocheck
APTT-SLA (Sysmex) were mixed and incubated at 37°C for five minutes. After incubation, 50
μL of 0.02 mol/L calcium chloride solution (Sysmex) was added to initiate coagulation, and the
coagulation time was measured using automatic blood coagulation analyzer KC4 Delta (Stago).
Coagulation time of a measurement sample was converted to FVIII activity according to
the coagulation time at each FVIII activity of the calibration curve solution.
Results
The results are shown in Fig. 1. When FVIII-deficient plasma containing 10 U/dL or
100 U/dL recombinant FVIII supplemented with an anti-FIXa/FX bispecific antibody was
diluted with a buffer (#3, #7), the FVIII activities were shown to be above the range of the
calibration curve, and could not be accurately measured. On the other hand, when
FVIII-deficient plasma containing 10 U/dL or 100 U/dL recombinant FVIII supplemented with
an anti-FIXa/FX bispecific antibody was diluted with a buffer containing two types of antibodies
against the anti-FIXa/FX bispecific antibody (#4, #8), the FVIII activities were shown to be
similar to those of the groups without addition of the anti-FIXa/FX bispecific antibody (#1, #5).
Therefore, this shows that the antibodies against the anti-FIXa/FX bispecific antibody
completely neutralized the activity of the bispecific antibody to enable accurate measurement of
the FVIII activity in plasma even in the presence of the bispecific antibody. When
FVIII-deficient plasma containing 10 U/dL or 100 U/dL recombinant FVIII was diluted with a
buffer containing only the two types of antibodies against the anti-FIXa/FX bispecific antibody
(#2, #6), the FVIII activities were shown to be similar to those of the groups without addition of
the anti-FIXa/FX bispecific antibody (#1, #5); therefore, antibodies against the anti-FIXa/FX
bispecific antibody were found to have neutralizing effects specific to the bispecific antibody.
[Example 4] One-stage clotting assay carried out under neutralization of the anti-FIXa/FX
bispecific antibody using AQ1 and AJ541 or AQ1 and AJ522
To FVIII-deficient plasma (George King) containing 10 U/dL recombinant FVIII
(Kogenate FS, Bayer Yakuhin, Ltd.), the anti-FIXa/FX bispecific antibody ACE910 was added at
0 μg/mL or 10 μg/mL. Furthermore, each of the prepared plasma was divided into three groups
to prepare measurement sample solutions: a group subjected to ten-fold dilution using an
imidazole buffer (Kyowa Medex); a group subjected to ten-fold dilution using an imidazole
buffer supplemented with 100 μg/mL each of AQ1 and AJ541; and a group subjected to ten-fold
dilution using an imidazole buffer supplemented with 100 μg/mL each of AQ1 and AJ522.
Amounts of AQ1, AJ541 and AJ522 required to sufficiently neutralize ACE910 were added.
Details of the combinations are shown below.
[Table 2]
Sample Plasma Dilution buffer
Type Dilution
rate
#1 FVIII deficient plasma 10-fold Imidazole buffer
containing 10 U/dL recombinant
#2 10-fold Imidazole buffer supplemented
FVIII
with AQ1 and AJ541
#3 10-fold Imidazole buffer supplemented
with AQ1 and AJ522
#4 FVIII-deficient plasma 10-fold Imidazole buffer
containing 10 U/dL recombinant
#5 10-fold Imidazole buffer supplemented
FVIII supplemented with 10 with AQ1 and AJ541
#6 μg/mL ACE910 10-fold Imidazole buffer supplemented
with AQ1 and AJ522
Furthermore, to produce a calibration curve for conversion of coagulation time to FVIII
activity, solutions of standard plasma, Coagtrol N (Sysmex), were prepared by performing
-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold and 640-fold dilutions using an imidazole
buffer (FVIII activities for the respective calibration curve solutions were specified as 102%,
51.0%, 25.5%, 12.8%, 6.38%, 3.19% and 1.59%). Fifty microliters of a measurement sample
solution or calibration curve solution, 50 μL of factor VIII-deficient human plasma (Sysmex),
and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated at 37°C for five
minutes. After incubation, 50 μL of 0.02 mol/L calcium chloride solution (Sysmex) was added
to initiate coagulation, and the coagulation time was measured using automatic blood
coagulation analyzer KC4 Delta (Stago).
Coagulation time of a measurement sample was converted to FVIII activity according to
the coagulation time at each FVIII activity of the calibration curve solution.
Results
The results are shown in Fig. 2. When FVIII-deficient plasma containing 10 U/dL
recombinant FVIII supplemented with an anti-FIXa/FX bispecific antibody, ACE910, was
diluted with a buffer (#4), the FVIII activity was shown to be above the range of the calibration
curve, and could not be accurately measured. On the other hand, when FVIII-deficient plasma
containing 10 U/dL recombinant FVIII supplemented with an anti-FIXa/FX bispecific antibody,
ACE910, was diluted with a buffer containing two types of antibodies, AQ1 and AJ541, against
the anti-FIXa/FX bispecific antibody (#5) or a buffer containing two types of antibodies, AQ1
and AJ522, against the anti-FIXa/FX bispecific antibody (#6), the FVIII activity was shown to be
similar to that of the group without addition of the anti-FIXa/FX bispecific antibody (#1).
Therefore, this shows that not only rAQ8-mIgG2b and rAJ540-rbtIgG, but other antibody
combinations are also effective as antibodies against the anti-FIXa/FX bispecific antibody to
completely neutralize the activity of the bispecific antibody ACE910.
[Example 5] One-stage clotting assay carried out under neutralization of the anti-FIXa/FX
bispecific antibody using AQ512 and AJ114 or AQ512 and AJ521
To FVIII-deficient plasma (George King) containing 10 U/dL recombinant FVIII
(Kogenate FS, Bayer Yakuhin, Ltd.), the anti-FIXa/FX bispecific antibody hBS23 was added at 0
μg/mL or 10 μg/mL. Furthermore, each of the prepared plasma was divided into three groups
to prepare measurement sample solutions: a group subjected to ten-fold dilution using an
imidazole buffer (Kyowa Medex); a group subjected to ten-fold dilution using an imidazole
buffer supplemented with 100 μg/mL each of AQ512 and AJ114; and a group subjected to
ten-fold dilution using an imidazole buffer supplemented with 100 μg/mL each of AQ512 and
AJ521. Amounts of AQ512, AJ114, and AJ521 required to sufficiently neutralize hBS23 were
added. Details of the combinations are shown below.
[Table 3]
Sample Plasma Dilution buffer
Type Dilution
rate
#1 FVIII-deficient plasma 10-fold Imidazole buffer
containing 10 U/dL recombinant
#2 10-fold Imidazole buffer supplemented
FVIII with AQ512 and AJ114
#3 10-fold Imidazole buffer supplemented
with AQ512 and AJ521
#4 FVIII-deficient plasma 10-fold Imidazole buffer
containing 10 U/dL recombinant
#5 10-fold Imidazole buffer supplemented
FVIII supplemented with 10 with AQ512 and AJ114
#6 μg/mL hBS23 10-fold Imidazole buffer supplemented
with AQ512 and AJ521
Furthermore, to produce a calibration curve for conversion of coagulation time to FVIII
activity, solutions of standard plasma, Coagtrol N (Sysmex), were prepared by performing
-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold and 640-fold dilutions using an imidazole
buffer (FVIII activities for the respective calibration curve solutions were specified as 102%,
51.0%, 25.5%, 12.8%, 6.38%, 3.19% and 1.59%). Fifty microliters of a measurement sample
solution or calibration curve solution, 50 μL of factor VIII-deficient human plasma (Sysmex),
and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated at 37°C for five
minutes. After incubation, 50 μL of 0.02 mol/L calcium chloride solution (Sysmex) was added
to initiate coagulation, and the coagulation time was measured using automatic blood
coagulation analyzer KC4 Delta (Stago).
Coagulation time of a measurement sample was converted to FVIII activity according to
the coagulation time at each FVIII activity of the calibration curve solution.
Results
The results are shown in Fig. 3. When FVIII-deficient plasma containing 10 U/dL
recombinant FVIII supplemented with an anti-FIXa/FX bispecific antibody, hBS23, was diluted
with a buffer (#4), the FVIII activity was shown to be above the range of the calibration curve,
and could not be accurately measured. On the other hand, when FVIII-deficient plasma
containing 10 U/dL recombinant FVIII supplemented with an anti-FIXa/FX bispecific antibody,
hBS23, was diluted with a buffer containing two types of antibodies, AQ512 and AJ114, against
the anti-FIXa/FX bispecific antibody (#5) or a buffer containing two types of antibodies, AQ512
and AJ521, against the anti-FIXa/FX bispecific antibody (#6), the FVIII activity was shown to be
similar to that of the group without addition of the anti-FIXa/FX bispecific antibody (#1).
These show that even with hBS23, a bispecific antibody different from ACE910, the FVIII
activity in plasma can be accurately measured despite the presence of the bispecific antibody by
completely neutralizing its activity, and therefore the present approach is effective for various
bispecific antibodies that have FVIII-substituting activity.
[Example 6] Bethesda assay carried out under neutralization of the anti-FIXa/FX bispecific
antibody using rAQ8-mIgG2b and rAJ540-rbtIgG
To factor VIII-deficient human plasma (containing FVIII inhibitors) (George King
Bio-Medical), anti-FIXa/FX bispecific antibody ACE910 was added at 0 μg/mL or 300 μg/mL.
Furthermore, each of the prepared plasma samples was subjected to 25-fold dilution or 30-fold
dilution using a 0.25% (w/v) bovine serum albumin (Sigma-Aldrich)-containing imidazole buffer
(Kyowa Medex) (hereinafter referred to as BSA-imidazole). To Coagtrol N (Sysmex) which is
standard plasma, rAQ8-mIgG2b and rAJ540-rbtIgG were either not added, or they were added at
300 μg/mL each.
Two types of the prepared plasma samples were mixed in equal amounts in the
following combinations (a total of 8 types), and then subjected to incubation at 37°C for two
hours.
[Table 4]
Sample Plasma 1 Plasma 2
Type Dilution rate
#1 Factor VIII-deficient human 25-fold Coagtrol N without addition of
plasma (containing inhibitors) rAQ8-mIgG2b and rAJ540-rbtIgG
-fold
without addition of the
#2 25-fold
Coagtrol N containing 300 μg/mL
anti-FIXa/FX bispecific
-fold rAQ8-mIgG2b and 300 μg/mL
antibody
rAJ540-rbtIgG
#3 Factor VIII-deficient human 25-fold Coagtrol N without addition of
plasma (containing inhibitors) rAQ8-mIgG2b and rAJ540-rbtIgG
-fold
containing 300 μg/mL
#4 25-fold Coagtrol N containing 300 μg/mL
anti-FIXa/FX bispecific
-fold rAQ8-mIgG2b and 300 μg/mL
antibody rAJ540-rbtIgG
After incubation, the mixed solutions were further diluted ten-fold with BSA-imidazole
to prepare measurement sample solutions. Furthermore, to prepare a calibration curve for
conversion of coagulation time to FVIII activity values, solutions were prepared by diluting
Coagtrol N with BSA-imidazole at 20-fold, 40-fold, 80-fold, 160-fold, and 320-fold dilution
(FVIII activities of the respective calibration curve solutions were specified as 100%, 50%, 25%,
12.5%, and 6.25%).
Fifty microliters of a measurement sample solution or calibration curve solution, 50 μL
of factor VIII-deficient human plasma (Sysmex), and 50 μL of Thrombocheck APTT-SLA
(Sysmex) were mixed and incubated at 37°C for three minutes. After incubation, 50 μL of 0.02
mol/L calcium chloride solution (Sysmex) was added to initiate coagulation, and the coagulation
time was measured using automatic blood coagulation analyzer KC4 Delta (Stago).
Coagulation time of a measurement sample was converted to FVIII activity according to
the coagulation time at each FVIII activity of the calibration curve solution. Furthermore, when
the residual FVIII activity was 50%, this was specified as 1 Bethesda, and after calculating the
Bethesda values in the measurement sample, mean value calculated by multiplying the value by
or 30 was determined as the inhibitor titer in each of the original sample solutions.
Results
The results are shown in Fig. 4. The FVIII inhibitor plasma containing only the
anti-FIXa/FX bispecific antibody (#3) showed an activity that was 100% or more of FVIII of the
calibration curve; therefore, the FVIII inhibitor titer could not be determined.
On the other hand, FVIII inhibitor plasma containing the anti-FIXa/FX bispecific
antibody and the two types of antibodies against the anti-FIXa/FX bispecific antibody (#4)
showed an FVIII inhibitor titer similar to that of the inhibitor plasma without additives (#1).
Therefore, this shows that the antibodies against the anti-FIXa/FX bispecific antibody
completely neutralized the activity of the bispecific antibody to enable accurate measurement of
the FVIII inhibitor titer in plasma even in the presence of the bispecific antibody. FVIII
inhibitor plasma containing only the two types of antibodies against the anti-FIXa/FX bispecific
antibody (#2) showed similar results to that of #1; therefore, antibodies against the anti-FIXa/FX
bispecific antibody were found to have neutralizing effects specific to the bispecific antibody.
Industrial Applicability
Described herein are methods for measuring the reactivity of FVIII in the presence of a
bispecific antibody having an activity of functionally substituting for FVIII, for example,
methods for measuring FVIII activity or FVIII inhibitor titer. Use of the described methods
enables accurate measurement of the reactivity of FVIII in patients during treatment of
hemorrhagic diseases, such as hemophilia, by using the bispecific antibody.
Certain statements that appear herein are broader than what appears in the statements of the
invention. These statements are provided in the interests of providing the reader with a better
understanding of the invention and its practice. The reader is directed to the accompanying claim
set which defines the scope of the invention.
Claims (14)
1. A method for measuring reactivity of coagulation factor VIII, wherein the method comprises 5 the following steps: A) contacting (1) a blood-derived sample containing a substance that has an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or 10 activated blood coagulation factor X, with (2) one or more combinations of antibodies that neutralize the bispecific antibody; and B) measuring reactivity of coagulation factor VIII in the sample, wherein the one or more combinations of antibodies are selected from the group consisting of: (a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation 15 factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor X; (b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor X; and 20 (c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that binds to activated coagulation factor IX. 25
2. The method of claim 1, wherein the bispecific antibody is any one of the antibodies described below, in which a first polypeptide is associated with a third polypeptide and a second polypeptide is associated with a fourth polypeptide: a bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 9, the second polypeptide is an H chain consisting of the amino acid 30 sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are common L chains of SEQ ID NO: 10 (Q499-z121/J327-z119/L404-k); or a bispecific antibody in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 36, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 37, and the third polypeptide and the fourth polypeptide are common L 35 chains of SEQ ID NO: 38 (Q153-G4k/J142-G4h/L180-k).
3. The method of any one of claims 1 to 2, wherein the method for measuring reactivity of coagulation factor VIII is a method for measuring the coagulation factor VIII activity or a method for measuring the coagulation factor VIII inhibitor titer. 5
4. A kit when used for measuring reactivity of coagulation factor VIII according to any one of claims 1 to 3, wherein the kit comprises one or more combinations of antibodies selected from the group consisting of: (a) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to 10 coagulation factor X; (b) an antibody that binds to Fab comprising an antigen-binding site that binds to activated coagulation factor IX and an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor X; and (c) an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation 15 factor IX, an antibody that binds to Fab comprising an antigen-binding site that binds to coagulation factor X, and an antibody that binds to Fab comprising an antigen-binding site that binds to activated coagulation factor IX.
5. A method for diagnosing the disease severity of a patient administered with a substance 20 having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X, and wherein the method comprises the following steps: 25 1) measuring the reactivity of coagulation factor VIII in a blood-derived sample of the patient according to the method of any one of claims 1 to 3; and 2) diagnosing the disease severity of the patient based on the measurement results.
6. A method for measuring inhibitor titer in a blood-derived sample of a patient administered 30 with a substance having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X, and wherein the method comprises measuring inhibitor titer in a blood-derived sample of the patient according to the 35 method of claim 3.
7. A method for monitoring pharmacological activity of an FVIII formulation in a patient administered with the FVIII formulation and a substance having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a bispecific antibody that binds to coagulation factor 5 IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X, and wherein the method comprises the following steps: 1) measuring the reactivity of coagulation factor VIII in a blood-derived sample of the patient according to the method of any one of claims 1 to 3; and 2) monitoring pharmacological activity of the FVIII formulation in the patient based on the 10 measurement results.
8. The method of any one of claims 5 to 7, wherein the patient is a patient selected from the group consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand disease patient, and a patient with hemophilia A in which an inhibitor against blood coagulation 15 factor VIII and/or activated blood coagulation factor VIII emerges.
9. The kit of claim 4, wherein the kit is a kit when used for diagnosing the disease severity of a patient administered with a substance having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for 20 coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X.
10. The kit of claim 4, wherein the kit is a kit when used for measuring inhibitor titer in a 25 patient administered with a substance having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X.
11. The kit of claim 4, wherein the kit is a kit when used for monitoring pharmacological activity of an FVIII formulation in a patient administered with the FVIII formulation and a substance having an activity of functionally substituting for coagulation factor VIII, wherein the substance having an activity of functionally substituting for coagulation factor VIII is a 35 bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and to coagulation factor X and/or activated blood coagulation factor X.
12. The kit of any one of claims 9 to 11, wherein the patient is a patient selected from the group consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand disease patient, and with a patient with hemophilia A in which an inhibitor against blood 5 coagulation factor VIII and/or activated blood coagulation factor VIII emerges.
13. A method of any one of claims 1-3 and 5-8, substantially as herein described with reference to any example thereof and with reference to the accompanying figures. 10
14. A kit of any one of claims 4 and 9-12, substantially as herein described with reference to any example thereof and with reference to the accompanying figures.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014-196974 | 2014-09-26 | ||
JP2014196974 | 2014-09-26 | ||
PCT/JP2015/076848 WO2016047652A1 (en) | 2014-09-26 | 2015-09-24 | Method for measuring reactivity of fviii |
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NZ729317A NZ729317A (en) | 2021-11-26 |
NZ729317B2 true NZ729317B2 (en) | 2022-03-01 |
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