U.S. patent application number 14/012204 was filed with the patent office on 2013-12-26 for antibodies against tissue factor pathway inhibitor (tfpi).
This patent application is currently assigned to NOVO NORDISK A/S. The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Jes Thorn Clausen, Ida Hilden.
Application Number | 20130344090 14/012204 |
Document ID | / |
Family ID | 42104649 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344090 |
Kind Code |
A1 |
Hilden; Ida ; et
al. |
December 26, 2013 |
Antibodies Against Tissue Factor Pathway Inhibitor (TFPI)
Abstract
The invention relates to antibodies that specifically bind to
tissue factor pathway inhibitor (TFPI) and that reduce clotting
time in (a) human FVIII-deficient plasma and/or (b) human whole
blood. Such antibodies have utility in the treatment of bleeding
disorders and in the stimulation of blood clotting.
Inventors: |
Hilden; Ida; (Vanloese,
DK) ; Clausen; Jes Thorn; (Hoeng, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVO NORDISK A/S
Bagsvaerd
DK
|
Family ID: |
42104649 |
Appl. No.: |
14/012204 |
Filed: |
August 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13140840 |
Jul 13, 2011 |
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PCT/EP2009/067566 |
Dec 18, 2009 |
|
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14012204 |
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61203512 |
Dec 23, 2008 |
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Current U.S.
Class: |
424/172.1 ;
530/389.1 |
Current CPC
Class: |
A61P 7/04 20180101; C07K
16/38 20130101; C07K 2317/565 20130101; C07K 16/18 20130101; C07K
2317/76 20130101 |
Class at
Publication: |
424/172.1 ;
530/389.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2008 |
EP |
08172522.8 |
Claims
1. An isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) comprising a heavy chain CDR1, a heavy
chain CDR2, and a heavy chain CDR3 and a light chain comprising a
light chain CDR1, a light chain CDR2, and a light chain CDR3,
wherein the heavy chain CDR comprises an amino acid residues 98 to
107 of SEQ ID NO: 6.
2. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 1, wherein a heavy chain
comprises an amino acid sequence having 90% identity to SEQ ID NO:
6.
3. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 2, wherein a light chain
comprises an amino acid sequence having 90% identity to SEQ ID NO:
3.
4. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 3 that reduces clotting time in
vivo without significantly reducing platelet levels.
5. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 3, wherein the antibody is a
monoclonal antibody.
6. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 3, wherein the antibody reduces
clotting time in human FVIII-deficient plasma.
7. The isolated antibody that specifically binds to tissue factor
pathway inhibitor (TFPI) of claim 3, wherein the antibody reduces
clotting time in human FVIII-deficient whole blood.
8. A pharmaceutical composition comprising the isolated antibody
that specifically binds to tissue factor pathway inhibitor (TFPI)
of claim 1 and a pharmaceutically acceptable carrier or
diluent.
9. A method of treating a bleeding disorder in a subject or for
stimulating blood clotting in a subject, the method comprising
administering to said subject the isolated antibody according to
claim 1 for treatment of a bleeding disorder or the stimulation of
blood clotting.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/140,840, filed Jul. 13, 2011 (Allowed), which is a 35 U.S.C.
.sctn.371 national stage application of International Patent
Application PCT/EP2009/067566 (published as WO 2010/072687), filed
Dec. 18, 2009, which claimed priority of European Patent
Application 08172522.8, filed Dec. 22, 2008; this application
further claims priority under 35 U.S.C. .sctn.119 of U.S.
Provisional Application 61/203,512, filed Dec. 23, 2008.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies that
specifically bind to tissue factor pathway inhibitor (TFPI).
[0003] Background to the Invention
[0004] Vessel wall injury exposes tissue factor (TF) to the blood
circulation and TF forms a complex with Factor VII/activated Factor
VII (FVII/FVIIa) on the surface of TF-bearing cells. This leads to
the activation of Factor X (FX) to FXa which together with FVa
generates a limited amount of thrombin (FIIa). Small amounts of
thrombin activate platelets, and this results in surface exposure
of phospholipids that supports the binding of the tenase complex
consisting of FVIIIa/FIXa
[0005] The tenase complex produces large amounts of FXa, which
subsequently facilitates a full thrombin burst. A full thrombin
burst is needed for the formation of a mechanically strong fibrin
structure and stabilization of the haemostatic plug. FVIII or FIX
is missing or present at low levels in haemophilia patients, and
due to the lack of tenase activity, the capacity to generate FXa is
low and insufficient to support the propagation phase of the
coagulation. In contrast, the TF-mediated initiation phase is not
dependent on the formation of the tenase complex. However, the
TF-pathway will, shortly after an initial FXa generation, be
blocked by plasma inhibitors.
[0006] Tissue factor pathway inhibitor (TFPI) downregulates ongoing
coagulation by neutralizing the catalytic activity of FXa and by
inhibition of the TF-FVIIa complex in the presence of FXa. TFPI
either inhibits the TF/FVIIa/FXa complex on the cellular surface or
inhibits released FXa followed by FVIIa/TF inhibition.
SUMMARY OF THE INVENTION
[0007] The Inventors have identified antibodies which specifically
bind to TFPI and thereby modulate its activity. The present
invention relates to these antibodies and to other related
antibodies that are derived from these antibodies or have similar
binding properties to these antibodies.
[0008] Accordingly, the present invention relates to antibodies
that specifically bind to tissue factor pathway inhibitor (TFPI)
and that reduce clotting time in (a) human FVIII-deficient plasma
and/or (b) human whole blood.
[0009] Preferred antibodies comprise the light chain variable
region of SEQ ID NO: 3 and the heavy chain variable region of SEQ
ID NO: 6.
[0010] The invention also provides polynucleotides which encode an
antibody of the invention, such as polynucleotides which encode an
antibody light chain and/or an antibody heavy chain of the
invention.
[0011] The invention also provides pharmaceutical compositions
comprising an antibody or polynucleotide of the invention and a
pharmaceutically acceptable carrier or diluent.
[0012] The antibodies, polynucleotides and compositions of the
invention are also provided for use in (a) the treatment or
prevention of a bleeding disorder or (b) the stimulation of blood
clotting. That is, the invention provides a method for (a) the
treatment or prevention of a bleeding disorder or (b) the
stimulation of blood clotting, the method comprising administering
to a patient in need thereof a therapeutically or prophylactically
effective amount of an antibody, polynucleotide or composition of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A and 1B show the cuticle bleeding time measured in
transient haemophiliac rabbits following treatment with control IgG
(Haemophilia) or the anti-TFPI antibody TFPI-3F18A2 (3F18).
[0014] FIG. 2 shows the nucleotide sequences and translated
polypeptide sequences for the VH and VL sequences of the antibody
TFPI-3F18A4B1.
[0015] FIG. 3 shows the amino acid sequences of the VL (A) and VH
(B) of the 3F18 antibody. Numbering above the sequences is shown
according to Kabat. Positions corresponding to CDR loops are
highlighted in bold underlined text in the Kabat numbering.
[0016] FIG. 4 shows the sequence of TFPI1. The Kunitz domains are
shown in bold: TFPI1 Kunitz domain 1=amino acids 26 to 76; TFPI1
Kunitz domain 2=amino acids 97-147; TFPI1 Kunitz domain 3=amino
acids 188-238. The C-terminal part of TFPI1 is shown in italics at
amino acids 240 to 276.
[0017] FIG. 5 shows the platelet number following stimulation with
anti-FVIII antibody, anti-TFPI-antibody and then bleed. This was
carried out in a control haemophilia model and in the presence of
the anti-TFPI antibody 3F18 as described herein.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0018] SEQ ID NO: 1 gives the amino acid sequence of human
TFPI1.
[0019] SEQ ID Nos 2 to 4 give the polynucleotide (coding and
complement) and polypeptide sequences for the light chain variable
domain (VL) of the TFPI-3F18A4B1 monoclonal antibody.
[0020] SEQ ID Nos 5 to 7 give the polynucleotide (coding and
complement) and polypeptide sequences for the heavy chain variable
domain (VH) of the TFPI-3F18A4B1 monoclonal antibody.
[0021] SEQ ID NO: 8 gives the sequence of a reverse primer used for
heavy chain variable domain amplification and SEQ ID NO: 9 gives
the sequence of a reverse primer used for light chain
amplification.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to antibodies that bind to
TFPI. The antibodies preferably specifically bind to TFPI, i.e.
they bind to TFPI but they do not bind, or bind at a lower
affinity, to other molecules. In particular, the invention relates
to antibodies that bind to TFPI and that modulate its activity.
Antibodies of the invention may thus possess the ability to shorten
clotting time. For example, an antibody of the invention may have
the ability to shorten clotting time in human FVIII-deficient
plasma or to reduce time to clot by thromboelastography (TEG)
analysis of human whole blood. The invention also relates to uses
for such antibodies, such as therapeutic and pharmaceutical
uses.
[0023] The term TFPI as used herein encompasses any naturally
occurring form of TFPI which may be derived from any suitable
organism. For example, TFPI for use as described herein may be a
mammalian TFPI, such as human, mouse, rat, primate, bovine, ovine,
or porcine TFPI. Preferably the TFPI is human TFPI. The TFPI may be
a mature form of TFPI such as a TFPI protein that has undergone
post-translational processing within a suitable cell. Such a mature
TFPI protein may, for example, be glycosylated. The TFPI may be a
full length TFPI protein. The term TFPI also encompasses variants,
isoforms and other homologs of such TFPI molecules. Variant TFPI
molecules will generally be characterised by having the same type
of activity as naturally occurring TFPI, such as the ability to
convert neutralize the catalytic activity of FXa, or the ability to
inhibit a complex of TF-FVIIa/FXa.
[0024] An antibody of the invention will have the ability to bind
to TFPI. Preferably, an antibody of the invention will bind
specifically to TFPI. That is, an antibody of the invention will
preferably bind to TFPI with greater binding affinity than that at
which it binds to another molecule. An antibody of the invention
may have the ability to bind or specifically bind to a TFPI
molecule as described herein such as any target molecule as
described herein.
[0025] The terms "binding activity" and "binding affinity" are
intended to refer to the tendency of an antibody molecule to bind
or not to bind to a target. Binding affinity may be quantified by
determining the dissociation constant (Kd) for an antibody and its
target. Similarly, the specificity of binding of an antibody to its
target may be defined in terms of the comparative dissociation
constants (Kd) of the antibody for its target as compared to the
dissociation constant with respect to the antibody and another,
non-target molecule.
[0026] Typically, the Kd for the antibody with respect to the
target will be 2-fold, preferably 5-fold, more preferably 10-fold
less than Kd with respect to the other, non-target molecule such as
unrelated material or accompanying material in the environment.
More preferably, the Kd will be 50-fold less, even more preferably
100-fold less, and yet more preferably 200-fold less.
[0027] The value of this dissociation constant can be determined
directly by well-known methods, and can be computed even for
complex mixtures by methods such as those, for example, set forth
in Caceci et al. (Byte 9:340-362, 1984). For example, the Kd may be
established using a double-filter nitrocellulose filter binding
assay such as that disclosed by Wong & Lohman (Proc. Natl.
Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays to
evaluate the binding ability of ligands such as antibodies towards
targets are known in the art, including for example, ELISAs,
Western blots, RIAs, and flow cytometry analysis. The binding
kinetics (e.g., binding affinity) of the antibody also can be
assessed by standard assays known in the art, such as by
Biacore.TM. system analysis.
[0028] A competitive binding assay can be conducted in which the
binding of the antibody to the target is compared to the binding of
the target by another, known ligand of that target, such as another
antibody. The concentration at which 50% inhibition occurs is known
as the Ki. Under ideal conditions, the Ki is equivalent to Kd. The
Ki value will never be less than the Kd, so measurement of Ki can
conveniently be substituted to provide an upper limit for Kd.
[0029] Preferred Kd (or Ki) values for an antibody of the invention
may be at least 1.times.10.sup.-11M, at least 1.times.10.sup.-10M,
at least 1.times.10.sup.-9M or 1.times.10.sup.-8M. An antibody of
the invention may have a Kd (or Ki) for its target of
1.times.10.sup.-7M or less, 1.times.10.sup.-8M or less or
1.times.10.sup.-9M or less.
[0030] An antibody that specifically binds its target may bind its
target with a high affinity, such as a Kd (or Ki) as discussed
above, and may bind to other, non-target molecules with a lower
affinity. For example, the antibody may bind to a non-target
molecules with a Kd (or Ki) of 1.times.10.sup.-6M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more. An antibody
of the invention is preferably capable of binding to its target
with an affinity that is at least two-fold, 10-fold, 50-fold,
100-fold or greater than its affinity for binding to an other
non-target molecule.
[0031] The target molecule may be any TFPI molecule as described
herein, such as a naturally occurring TFPI molecule, a fully mature
TFPI molecule or a full-length TFPI molecule.
[0032] Preferred TFPI molecules are fully mature, naturally
occurring, full length mammalian TFPI molecules. For example, the
TFPI molecule may consist of, or may comprise, the amino acid
sequence of SEQ ID NO: 1 or a fragment or other variant thereof as
described herein.
[0033] The target molecule may be a variant of a TFPI molecule such
as a fragment of a TFPI molecule. For example, the target molecule
may be a fragment or other variant of TFPI which maintains a
suitable epitope for antibody binding. For example, the target
molecule may be a fragment or other variant of TFPI which retains
an epitope as described herein. The target molecule may comprise
such an epitope.
[0034] In one embodiment, the target molecule is a full length TFPI
molecule. The full length TFPI molecule may comprise a first,
second and third Kunitz domain as described herein. The full length
TFPI molecule may comprise a first, second and third Kunitz domain
as described herein and also a carboxy terminal region as described
herein. The full length TFPI molecule may be a naturally occurring
TFPI molecule such as a full length TFPI polypeptide as expressed
from a TFPI gene, or as secreted by TFPI expressing cells. The full
length TFPI molecule may be a naturally occurring TFPI molecule as
found circulating in free form in plasma. The full length TFPI
molecule is not a truncated TFPI molecule such as a
naturally-occurring truncated TFPI molecule as described
herein.
[0035] In one embodiment, the target molecule is a truncated TFPI
molecule. For example, the truncated TFPI molecule may comprise a
carboxy terminal truncation. For example, a number of
naturally-occurring truncated forms of TFPI are known. These may
comprise a truncation of part or all of the carboxy terminal part
of TFPI. They may further comprise truncation of part or all of one
or more of the Kunitz domains. For example, a truncated form of
TFPI may comprise the deletion of the carboxy terminal part and
part or all of the third Kunitz domain.
[0036] Truncated TFPI is preferably used as a target molecule when
antibodies are desired to be directed against specific truncated
forms of TFPI such as naturally occurring truncated TFPI. For
example, one naturally occurring truncated form of TFPI comprises
only amino acids 1 to 161 of the full length TFPI molecule
(referred to herein as TFPI (1-161)). TFPI (1-161) is an active
form of TFPI that has reduced activity compared with the full
length molecule. TFPI (1-161) differs in structure from full length
TFPI and antibodies generated against TFPI (1-161) as a target
molecule may therefore differ from antibodies generated against
full length TFPI
[0037] In one embodiment the target molecule is a
naturally-occurring form of TFPI. This may be used in a form in
which it is present in vivo. For example, the target molecule may
be a full length naturally-occurring TFPI as discussed above. The
target molecule may be a truncated naturally-occurring TFPI as
discussed above. The target molecule may be TFPI in a form in which
it is present in plasma in vivo. The target molecule may be TFPI
that is bound to lipoprotein in the same way as is present in
plasma in vivo. The target molecule may be TFPI that is bound to
cells in the same way as occurs in vivo, such as TFPI that is bound
to endothelial cells. An antibody of the invention may bind to any
one or more of these naturally occurring forms of TFPI. The
antibody of the invention may be able to bind to all of these
naturally occurring forms of TFPI, or may be able to discriminate
between these different forms, binding to some but not others.
[0038] The target molecule may be or may comprise a Kunitz domain
of TFPI. Such a target molecule may comprise amino acids 26-76 of
SEQ ID NO: 1 or an equivalent Kunitz domain 1 region from another
TFPI polypeptide. Such a target molecule may comprise amino acids
97 to 147 of SEQ ID NO: 1 or amino acids 91 to 150 of SEQ ID NO: 1
or an equivalent Kunitz domain 2 region from another TFPI
polypeptide. Such a target molecule may comprise amino acids 188 to
138 of SEQ ID NO: 1 or an equivalent Kunitz domain 3 region from
another TFPI polypeptide. The target molecule may be, or may
comprise, a fragment of a Kunitz domain of TFPI. For example, the
target molecule may comprise five or more, eight or more, ten or
more, twelve or more or fifteen or more amino acids from a Kunitz
domain.
[0039] The target molecule may be or may comprise the carboxy
terminal part of TFPI. The carboxy terminal part of TFPI may be
defined as that part of the amino acid sequence of TFPI which lies
between the third Kunitz domain and the C terminal of the protein.
Such a target molecule may comprise amino acids 240-276 of SEQ ID
NO: 1 or an equivalent carboxy terminal region from another TFPI
polypeptide. The target molecule may be, or may comprise, a
fragment of the carboxy terminal part of TFPI. For example, the
target molecule may comprise five or more, eight or more, ten or
more, twelve or more or fifteen or more amino acids from the
carboxy terminal part of TFPI.
[0040] The target molecule may comprise five or more, eight or
more, ten or more, twelve or more or fifteen or more surface
accessible residues of TFPI or of a particular region of TFPI such
as a particular Kunitz domain or the C terminal part of TFPI. A
surface accessible residue is a residue having more than 40%
relative accessibility
[0041] For example, for the Kunitz 2 domain of TFPI1 (see SEQ ID
NO: 1), the following amino acids have a greater than 40% relative
accessibility: 94-95, 98, 100-110, 118-121, 123-124, 131, 134,
138-142 and 144-145. The target molecule may comprise five or more,
eight or more, ten or more, twelve or more or fifteen or more of
these residues, such as a fragment of TFPI that includes five or
more, eight or more, ten or more, twelve or more or fifteen or more
of these residues. For the Kunitz 1 domain of TFPI1 (see SEQ ID NO:
1), the following amino acids are expected to have a greater than
40% relative accessibility: 23-24, 27, 28-41, 47-50, 52-53, 60-65,
67-71 and 73-74. The target molecule may comprise five or more,
eight or more, ten or more, twelve or more or fifteen or more of
these residues, such as a fragment of TFPI that includes five or
more, eight or more, ten or more, twelve or more or fifteen or more
of these residues. For the Kunitz 3 domain of TFPI1 (see SEQ ID NO:
1), the following amino acids are expected to have a greater than
40% relative accessibility: 186-187, 190, 192-208, 213, 215-220,
225-228, 230-234 and 236-237. The target molecule may comprise five
or more, eight or more, ten or more, twelve or more or fifteen or
more of these residues, such as a fragment of TFPI that includes
five or more, eight or more, ten or more, twelve or more or fifteen
or more of these residues.
[0042] The target molecule may comprise a known epitope from TFPI.
The antibody may thus bind to the same epitope as another known
antibody of the invention.
[0043] As used herein, the term "epitope" generally refers to the
site on a target antigen which is recognised by an immune receptor
such as an antibody. Preferably it is a short peptide derived from
or as part of a protein. However the term is also intended to
include peptides with glycopeptides and carbohydrate epitopes. A
single antigenic molecule, such as a target protein as described
herein, may comprise several different epitopes. Epitopes can be
identified from knowledge of the amino acid and corresponding DNA
sequences of the peptide, as well as from the nature of particular
amino acids (e.g., size, charge, etc.) and the codon dictionary,
without undue experimentation. See, e.g., Ivan Roitt, Essential
Immunology, 1988; Janis Kuby, Immunology, 1992 e.g., pp. 79-81.
[0044] The location of an epitope may be identified by routine
methods. For example, the general location of an epitope may be
determined by assessing the ability of an antibody to bind to
different fragments or variant TFPI polypeptides. The specific
amino acids within TFPI that make contact with an antibody may also
be determined using routine methods, such as that described in the
Examples. For example, the antibody and target molecule may be
combined and the antibody/target complex may be crystallised. The
crystal structure of the complex may be determined and used to
identify specific sites of interaction between the antibody and its
target.
[0045] An antibody of the invention may bind to the same epitope or
region as another antibody of the invention. For example, where an
antibody of the invention is known, other antibodies of the
invention may be identified by comparing their binding to TFPI with
that of the known antibody. An antibody of the invention may bind
to the same epitope or region as the 3F18 antibody described
herein.
[0046] An antibody of the invention may have the ability to
cross-compete with another antibody of the invention for binding to
TFPI or another appropriate target as described herein. For
example, an antibody of the invention may cross-compete with the
3F18 antibody described herein for binding to TFPI or to a suitable
fragment or variant of TFPI that is bound by the 3F18 antibody,
such as a target molecule as described herein. Such cross-competing
antibodies can be identified based on their ability to
cross-compete with a known antibody of the invention in standard
binding assays. For example, BIAcore analysis, ELISA assays or flow
cytometry may be used to demonstrate cross-competition. Such
cross-competition may suggest that the two antibodies bind to the
same or similar epitopes.
[0047] An antibody of the invention may therefore be identified by
a method that comprises such a binding assay which assesses whether
or not a test antibody is able to cross-compete with a known
antibody of the invention for a binding site on the target
molecule. Methods for carrying out competitive binding assays are
well known in the art. For example they may involve contacting
together a known antibody of the invention and a target molecule
under conditions under which the antibody can bind to the target
molecule. The antibody/target complex may then be contacted with a
test antibody and the extent to which the test antibody is able to
displace the antibody of the invention from antibody/target
complexes may be assessed. An alternative method may involve
contacting a test antibody with a target molecule under conditions
that allow for antibody binding, then adding an antibody of the
invention that is capable of binding that target molecule and
assessing the extent to which the antibody of the invention is able
to displace the test antibody from antibody/target complexes.
[0048] The ability of a test antibody to inhibit the binding of an
antibody of the invention to the target demonstrates that the test
compound can compete with an antibody of the invention for binding
to the target and thus that the test antibody binds to the same
epitope or region on the TFPI protein as the known antibody of the
invention. A test antibody that is identified as cross-competing
with a known antibody of the invention in such a method is also a
potential antibody according to the present invention. The fact
that the test antibody can bind TFPI in the same region as a known
antibody of the invention and cross-compete with the known antibody
of the invention suggests that the test antibody may act as a
ligand at the same binding site as the known antibody and that the
test antibody may therefore mimic the action of the known antibody.
This can be confirmed by assessing the activity of TFPI in the
presence of the test compound as described herein.
[0049] The known antibody of the invention may be an antibody as
described herein, such as the TFPI-3F18A4B1 (3F18) antibody as
described herein or any variant or fragment thereof as described
herein that retains the ability to bind to TFPI.
[0050] An antibody of the invention may bind to the same epitope as
the 3F18 antibody, the as described herein or any variant or
fragment thereof as described herein that retains the ability to
bind to TFPI.
[0051] Specific binding may be assessed with reference to binding
of the antibody to a molecule that is not the target. This
comparison may be made by comparing the ability of an antibody to
bind to the target and to another molecule. This comparison may be
made as described above in an assessment of Kd or Ki. The other
molecule used in such a comparison may be any molecule that is not
the target molecule. Preferably the other molecule is not identical
to the target molecule. Preferably the target molecule is not a
fragment of the target molecule.
[0052] An antibody of the invention may bind to its target but not
bind to a peptide or protein that (a) does not have an identical
amino acid sequence to the target and (b) is not a fragment of the
target. For example, where the target is TFPI or a specific
fragment or epitope thereof, an antibody of the invention may bind
to the TFPI, fragment or epitope, but does not bind to a peptide or
protein that (a) has an identical sequence to the TFPI, fragment or
epitope and (b) is not a fragment of said TFPI, fragment or
epitope.
[0053] The other molecule used to determine specific binding may be
unrelated in structure or function to the target. For example, the
other molecule may be an unrelated material or accompanying
material in the environment.
[0054] The other molecule used to determine specific binding may be
another molecule involved in the same in vivo pathway as the target
molecule. For example, where the target is TFPI or a fragment or
variant thereof, the other molecule used for comparison may be a
protein that forms part of the blood coagulation cascade. By
ensuring that the antibody of the invention has specificity for
TFPI over another such molecule, unwanted in vivo cross-reactivity
may be avoided.
[0055] The other molecule used for comparison may be related to the
target molecule. For example, where it is desired to identify an
antibody that binds only to a specific epitope, the other molecule
for comparison may be a TFPI molecule in which that epitope is
lacking or disrupted. The other molecule used for comparison may
thus be another target molecule that is different to the target
molecule bound by the antibody in question.
[0056] The antibody of the invention may retain the ability to bind
to some molecules that are related to the target molecule. For
example, a full-length mature human TFPI may be used as the target,
but the antibody may also be able to bind to, e.g. immature forms
of human TFPI, fragments or truncated forms of human TFPI, TFPI
that is bound to lipoprotein or to a cell or TFPI from other
species, such as other mammalian TFPI.
[0057] Alternatively, the antibody of the invention may have
specificity for a particular target molecule. For example, it may
bind to one target molecule as described herein, but may not bind,
or may bind with significantly reduced affinity to a different
target molecule as described herein. For example, a full length
mature human TFPI may be used as the target, but the antibody that
binds to that target may be unable to bind to or may bind with
lesser affinity to, e.g. immature forms of human TFPI, fragments or
truncated forms of human TFPI, TFPI that is bound to lipoprotein or
to a cell or TFPI from other species, such as other mammalian
TFPI.
[0058] An antibody of the invention may bind to TFPI and in doing
so may inhibit an activity of TFPI.
[0059] As explained above, TFPI downregulates blood coagulation. It
does this by inhibiting the activity of FXa and by inhibiting the
TF-FVIIa complex in the presence of FXa. The activity of TFPI that
is inhibited by an antibody of the invention may be any of these
activities or any downstream effect thereof. For example, an
antibody of the invention may lead to an increase in blood
coagulation, an increase in the presence or levels of FXa or an
increased activity of TF-FVIIa. Preferably, an antibody of the
invention reduces clotting time when contacted with (a) human FVIII
deficient plasma or (b) human whole blood.
[0060] The measurement of TFPI activity may comprise assessing the
activity of the TFPI in inhibiting coagulation or reducing clotting
time in a blood sample. For example, such a method may comprise
contacting TFPI with a sample of blood or a blood product such as
plasma or serum that comprises blood coagulation factors under
conditions in which coagulation should occur, and determining
whether coagulation of the blood is inhibited or clotting time is
reduced by the presence of the TFPI. The level of blood coagulation
or clotting time in such a sample may then be compared to that in
an equivalent sample in which a test antibody is also present. If
the level of coagulation is increased or clotting time is reduced
in the antibody sample, this suggests that the antibody is
inhibiting the activity of TFPI in the sample.
[0061] Blood coagulation may be detected by looking for coagulation
of the blood itself, or one or more characteristics of the
coagulation cascade that lie downstream to the point of action of
TFPI. For example, the method may assess levels of FXa or
activation of TF-FVIIa in the sample.
[0062] Various other methods for assessing blood coagulation and
clotting time are well known in the art. For example, any effect of
an antibody on blood clotting time may be assessed using a dilute
prothrombin time analysis as described in the Examples. Briefly,
human plasma is contacted with human thromboplastin. The time taken
for the plasma to clot is measured in the presence and absence of
the test antibody. A positive control may be used in such an
analysis, such as addition of FVIIa which would be expected to
reduce clotting time. An antibody of the invention should be
capable of reducing clotting time in such a method. Preferably, an
antibody of the invention should be capable of reducing clotting
time in a dose-dependent manner.
[0063] Thromboelastography may be used to assess the kinetics of
clot formation and fibrinolysis in samples of whole blood. The
ability of an antibody to reduce clotting time or to stimulate
blood coagulation may thus be similarly assessed in a whole blood
sample by comparing the time taken for clot formation in the
presence and absence of the antibody.
[0064] Methods to assess the functional effects of an antibody of
the invention may thus be carried out in vitro. Such methods are
preferably carried out on samples of human blood or plasma. Such
samples may be normal human blood or plasma or may be deficient in,
or supplemented with, one or more factors involved in blood
coagulation. For example, these methods may be carried out using
normal human whole blood, normal human plasma or FVIII-deficient
plasma or whole blood. FVIII-deficient blood or plasma may be
generated by contacting a suitable blood or plasma sample with
neutralising anti-FVIII antibody.
[0065] Preferably, an antibody of the invention is capable of
reducing clotting time and/or stimulating blood coagulation in a
sample of (a) human whole blood, (b) human plasma, (c)
FVIII-deficient human whole blood or (d) FVIII-deficient human
plasma.
[0066] Methods to determine the ability of an antibody to stimulate
blood coagulation or reduce clotting time may also be carried out
in vivo. For example, in vivo studies may be carried out in
transient haemophilic rabbits as described in the Examples.
Briefly, rabbits may be made transient haemophilic by
administration of anti-FVIII antibody. The test antibody may then
be administered and cuticle bleed time and/or platelet number
assessed. A reduction in cuticle bleed time in the presence of a
test antibody indicates that the antibody is capable of reducing
clotting time and stimulating blood coagulation. An antibody having
such an effect may therefore be an antibody of the present
invention.
[0067] An antibody of the present invention may also lead to no
significant decrease in platelet numbers. In particular, an
antibody of the invention may be capable of reducing clotting time
and/or stimulating blood coagulation in a sample of (a) human whole
blood, (b) human plasma, (c) FVIII-deficient human whole blood or
(d) FVIII-deficient human plasma or in an animal in vivo without
leading to any significant decrease in platelet numbers. Platelet
numbers can be assessed in the same sample or animal as the other
effects discussed above, or can be assessed separately. For
example, platelet numbers can be assessed in a blood sample such as
a sample of blood obtained from a patient or experimental animal.
Platelet numbers may be assessed following administration of the
antibody to a transient haemophilic rabbit as described above.
Preferred antibodies of the invention are capable of reducing
cuticle bleed time in a transient haemophilic rabbit without
leading to a concurrent decrease in platelet numbers. A change in
platelet numbers may be assessed by comparing platelet numbers
before and after administration of the antibody or by comparing
platelet numbers between a sample or animal treated with the
antibody of interest and a control sample or animal not treated
with that antibody. Preferably, there will be no difference or no
statistically significant difference in platelet numbers when
making such comparisons. That is, the antibody of the invention
will not have caused any decrease in platelet numbers.
[0068] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An antibody refers to a
glycoprotein comprising at least two heavy (H) chains and two light
(L) chains inter-connected by disulfide bonds, or an antigen
binding portion thereof. Each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as VH) and a heavy chain
constant region. Each light chain is comprised of a light chain
variable region (abbreviated herein as VL) and a light chain
constant region. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The VH and
VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). The constant regions of the antibodies may mediate
the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (e.g., effector cells)
and the first component (Clq) of the classical complement
system.
[0069] An antibody of the invention may be a monoclonal antibody or
a polyclonal antibody. In one embodiment, an antibody of the
invention is a monoclonal antibody. An antibody of the invention
may be a chimeric antibody, a CDR-grafted antibody, a human or
humanised antibody or an antigen binding portion of any thereof.
For the production of both monoclonal and polyclonal antibodies,
the experimental animal is suitably a mammal such as a goat,
rabbit, rat or mouse.
[0070] Polyclonal antibodies are antibodies that are derived from
different B cell lines. A polyclonal antibody may comprise a
mixture of different immunoglobulin molecules that are directed
against a specific antigen. The polyclonal antibody may comprise a
mixture of different immunoglobulin molecules that bind to one or
more different epitopes within an antigen molecule. Polyclonal
antibodies may be produced by routine methods such as immunisation
of a suitable animal, with the antigen of interest. Blood may be
subsequently removed from the animal and the IgI fraction
purified.
[0071] Monoclonal antibodies are immunoglobulin molecules that are
identical to each other and have a single binding specificity and
affinity for a particular epitope. Monoclonal antibodies (mAbs) of
the present invention can be produced by a variety of techniques,
including conventional monoclonal antibody methodology e.g., the
standard somatic cell hybridization technique of Kohler and
Milstein (1975) Nature 256: 495, or viral or oncogenic
transformation of B lymphocytes. The preferred animal system for
preparing hybridomas is the murine system. Hybridoma production in
the mouse is a very well-established procedure. Immunization
protocols and techniques for isolation of immunized splenocytes for
fusion are known in the art. Fusion partners (e.g., murine myeloma
cells) and fusion procedures are also known.
[0072] To generate hybridomas producing monoclonal antibodies of
the invention, splenocytes and/or lymph node cells from immunized
mice can be isolated and fused to an appropriate immortalized cell
line, such as a mouse myeloma cell line. The resulting hybridomas
can be screened for the production of antigen-specific antibodies.
The antibody secreting hybridomas can be replated, screened again,
and if still positive for suitable IgG, the monoclonal antibodies
can be subcloned at least twice by limiting dilution. The stable
subclones can then be cultured in vitro to generate small amounts
of antibody in tissue culture medium for characterization.
[0073] The term "antigen-binding portion" of an antibody refers to
one or more fragments of an antibody that retain the ability to
specifically bind to an antigen, such as TFPI or another target
protein as described herein. It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include a Fab fragment, a F(ab').sub.2 fragment, a Fab'
fragment, a Fd fragment, a Fv fragment, a dAb fragment and an
isolated complementarity determining region (CDR). Single chain
antibodies such as scFv and heavy chain antibodies such as VHH and
camel antibodies are also intended to be encompassed within the
term "antigen-binding portion" of an antibody. These antibody
fragments may be obtained using conventional techniques known to
those of skill in the art, and the fragments may be screened for
utility in the same manner as intact antibodies.
[0074] An antibody of the invention may be prepared, expressed,
created or isolated by recombinant means, such as (a) antibodies
isolated from an animal (e.g., a mouse) that is transgenic or
transchromosomal for the immunoglobulin genes of interest or a
hybridoma prepared therefrom, (b) antibodies isolated from a host
cell transformed to express the antibody of interest, e.g., from a
transfectoma, (c) antibodies isolated from a recombinant,
combinatorial antibody library, and (d) antibodies prepared,
expressed, created or isolated by any other means that involve
splicing of immunoglobulin gene sequences to other DNA
sequences.
[0075] An antibody of the invention may be a human antibody or a
humanised antibody. The term "human antibody", as used herein, is
intended to include antibodies having variable regions in which
both the framework and CDR regions are derived from human germline
immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0076] Such a human antibody may be a human monoclonal antibody.
Such a human monoclonal antibody may be produced by a hybridoma
which includes a B cell obtained from a transgenic nonhuman animal,
e.g., a transgenic mouse, having a genome comprising a human heavy
chain transgene and a light chain transgene fused to an
immortalized cell.
[0077] Human antibodies may be prepared by in vitro immunisation of
human lymphocytes followed by transformation of the lymphocytes
with Epstein-Barr virus.
[0078] The term "human antibody derivatives" refers to any modified
form of the human antibody, e.g., a conjugate of the antibody and
another agent or antibody.
[0079] The term "humanized antibody" is intended to refer to
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences. Additional framework region
modifications may be made within the human framework sequences.
[0080] Antibodies of the invention can be tested for binding to the
target protein by, for example, standard ELISA or Western blotting.
An ELISA assay can also be used to screen for hybridomas that show
positive reactivity with the target protein. The binding
specificity of an antibody may also be determined by monitoring
binding of the antibody to cells expressing the target protein, for
example by flow cytometry.
[0081] The specificity of an antibody of the invention for target
protein may be further studied by determining whether or not the
antibody binds to other proteins. For example, where it is desired
to produce an antibody that specifically binds TFPI or a particular
part, e.g. epitope, of TFPI, the specificity of the antibody may be
assessed by determining whether or not the antibody also binds to
other molecules or modified forms of TFPI that lack the part of
interest.
[0082] As explained above, antibodies of the invention may modify
the activity of TFPI. Antibodies having the required binding
properties may thus be further tested to determine their effects on
the activity of TFPI. Thus, methods may be used to identify
suitable antibodies that are capable of binding to TFPI and that
are capable of modifying, and in particular reducing, its
activity.
[0083] Once a suitable antibody has been identified and selected,
the amino acid sequence of the antibody may be identified by
methods known in the art. The genes encoding the antibody can be
cloned using degenerate primers. The antibody may be recombinantly
produced by routine methods.
[0084] A "polypeptide" is used herein in its broadest sense to
refer to a compound of two or more subunit amino acids, amino acid
analogs, or other peptidomimetics. The term "polypeptide" thus
includes short peptide sequences and also longer polypeptides and
proteins. As used herein, the term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including
glycine and both the D or L optical isomers, and amino acid analogs
and peptidomimetics.
[0085] The present inventors have identified an antibody as
described in the examples. This antibody is referred to herein as
TFPI-3F18A4B1 or 3F18. The present invention encompasses this
antibody and variants and fragments thereof which retain one or
more activities of this antibody. The activities of these antibody
include the ability to bind to TFPI, the ability to bind to
specific locations in the TFPI molecule and the ability to inhibit
the activity of TFPI, optionally without leading to a drop in
platelet numbers.
[0086] A suitable fragment or variant of any of these antibodies
will retain the ability to bind to TFPI. It will preferably retain
the ability to specifically bind to TFPI. It will preferably retain
the ability to specifically bind to the same epitope or region of
the TFPI molecule as the antibody (3F18) from which it is derived.
It will preferably retain one or more additional functions of the
antibody from which it is derived, such as the ability to inhibit
TFPI activity or the ability to reduce clotting time, optionally
without leading to a drop in platelet numbers.
[0087] Polypeptide or antibody "fragments" according to the
invention may be made by truncation, e.g. by removal of one or more
amino acids from the N and/or C-terminal ends of a polypeptide. Up
to 10, up to 20, up to 30, up to 40 or more amino acids may be
removed from the N and/or C terminal in this way. Fragments may
also be generated by one or more internal deletions.
[0088] An antibody of the invention may be, or may comprise, a
fragment of the 3F18 antibody or a variant thereof. The antibody of
the invention may be or may comprise an antigen binding portion of
this antibody or a variant thereof as discussed further above. For
example, the antibody of the invention may be an Fab fragment of
this antibody or a variant thereof or may be a single chain
antibody derived from this antibody or a variant thereof.
[0089] The amino acid sequences for the VL and VH chains of the
3F18 antibody are given in SEQ ID Nos: 3 and 6 respectively.
[0090] An antibody of the invention may comprise the 3F18 VL amino
acid sequence shown in SEQ ID NO: 3 or a fragment or variant
thereof. An antibody may additionally or alternatively comprise the
3F18 VL amino acid sequence shown in SEQ ID NO: 6 or a fragment or
variant thereof as described herein.
[0091] An antibody of the invention may comprise a fragment of one
of the VL or VH amino acid sequences shown in FIG. 2. For example,
an antibody of the invention may comprise a fragment of at least 7,
at least 8, at least 9, at least 10, at least 12, at least 15, at
least 18, at least 20 or at least 25 consecutive amino acids from
SEQ ID Nos 3 or 6. Such a fragment will preferably retain one or
more of the functions discussed above, such as the ability to bind
to TFPI.
[0092] A suitable fragment or variant of any of these VH or VL
sequences will retain the ability to bind to TFPI. It will
preferably retain the ability to specifically bind to TFPI. It will
preferably retain the ability to specifically bind to the same
epitope or region of the TFPI molecule as the antibody (3F18) from
which it is derived. It will preferably retain one or more
additional functions of the antibody from which it is derived, such
as the ability to inhibit TFPI activity or the ability to reduce
clotting time.
[0093] An antibody of the invention may comprise a CDR region from
one of the three specific antibodies identified herein such as a
CDR region from within SEQ ID NO: 3 or 6. Methods for identifying
CDR regions are known in the art. The location of CDRs may be
assessed in silico, for example by assigning numbering according to
Kabat. The locations of amino acid residues involved in antibody
binding, either in the antibody molecule or in the epitope, may be
assessed experimentally by X ray crystallisation of the complex
formed between an antibody and its epitope. Distances between the
two molecules may be assessed. For example, the paratope in the
antibody and the epitope in the antigen may be identified as
comprising those amino acids having a heavy atom within a distance
of 4 A from a heavy atom in the other molecule.
[0094] For example, as shown in FIG. 3, using Kabat numbering, the
CDR sequences within the light chain of 3F18 may be identified at
amino acids 24 to 39, 55 to 61 and 94 to 102 of SEQ ID NO: 3. The
CDR sequences within the heavy chain of 3F18 may be identified at
amino acids 31 to 35, 50 to 65 and 98 to 107 of SEQ ID NO: 6. An
antibody of the invention may comprise any one or more of the CDR
sequences shown in FIG. 3. For example, an antibody of the
invention may comprise one, two or all three of the amino acid
sequences shown at residues 24 to 39, 55 to 61 and 94 to 102 of SEQ
ID NO: 3. An antibody of the invention may alternatively or
additionally comprise one, two or all three of the amino acid
sequences shown at residues 31 to 35, 50 to 65 and 98 to 107 of SEQ
ID NO: 6. An antibody of the invention may comprise all six amino
acid sequences shown at residues 24 to 39, 55 to 61 and 94 to 102
of SEQ ID NO: 3 and 31 to 35, 50 to 65 and 98 to 107 of SEQ ID NO:
6.
[0095] An antibody of the invention may alternatively be or may
comprise a variant of one of these specific sequences such a
variant of the 3F18 antibody. For example, a variant may be a
substitution, deletion or addition variant of any of the above
amino acid sequences.
[0096] A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up
to 20, up to 30 or more amino acid substitutions and/or deletions
from the specific sequences and fragments discussed above.
"Deletion" variants may comprise the deletion of individual amino
acids, deletion of small groups of amino acids such as 2, 3, 4 or 5
amino acids, or deletion of larger amino acid regions, such as the
deletion of specific amino acid domains or other features.
"Substitution" variants preferably involve the replacement of one
or more amino acids with the same number of amino acids and making
conservative amino acid substitutions. For example, an amino acid
may be substituted with an alternative amino acid having similar
properties, for example, another basic amino acid, another acidic
amino acid, another neutral amino acid, another charged amino acid,
another hydrophilic amino acid, another hydrophobic amino acid,
another polar amino acid, another aromatic amino acid or another
aliphatic amino acid. Some properties of the 20 main amino acids
which can be used to select suitable substituents are as
follows:
TABLE-US-00001 Ala aliphatic, hydrophobic, neutral Cys polar,
hydrophobic, neutral Asp polar, hydrophilic, charged (-) Glu polar,
hydrophilic, charged (-) Phe aromatic, hydrophobic, neutral Gly
aliphatic, neutral His aromatic, polar, hydrophilic, charged (+)
Ile aliphatic, hydrophobic, neutral Lys polar, hydrophilic,
charged(+) Leu aliphatic, hydrophobic, neutral Met hydrophobic,
neutral Asn polar, hydrophilic, neutral Pro hydrophobic, neutral
Gln polar, hydrophilic, neutral Arg polar, hydrophilic, charged (+)
Ser polar, hydrophilic, neutral Thr polar, hydrophilic, neutral Val
aliphatic, hydrophobic, neutral Trp aromatic, hydrophobic, neutral
Tyr aromatic, polar, hydrophobic
[0097] Preferred "derivatives" or "variants" include those in which
instead of the naturally occurring amino acid the amino acid which
appears in the sequence is a structural analog thereof. Amino acids
used in the sequences may also be derivatized or modified, e.g.
labelled, providing the function of the antibody is not
significantly adversely affected.
[0098] Derivatives and variants as described above may be prepared
during synthesis of the antibody or by post-production
modification, or when the antibody is in recombinant form using the
known techniques of site-directed mutagenesis, random mutagenesis,
or enzymatic cleavage and/or ligation of nucleic acids.
[0099] Preferably variant antibodies according to the invention
have an amino acid sequence which has more than 60%, or more than
70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or
95% amino acid identity to SEQ ID Nos 3 or 6, or a fragment
thereof. This level of amino acid identity may be seen across the
full length of the relevant SEQ ID NO sequence or over a part of
the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more
amino acids, depending on the size of the full length
polypeptide.
[0100] In connection with amino acid sequences, "sequence identity"
refers to sequences which have the stated value when assessed using
ClustalW (Thompson et al., 1994, supra) with the following
parameters:
[0101] Pairwise alignment parameters--Method: accurate, Matrix:
PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;
[0102] Multiple alignment parameters--Matrix: PAM, Gap open
penalty: 10.00, % identity for delay: 30, Penalize end gaps: on,
Gap separation distance: O, Negative matrix: no, Gap extension
penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap
penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity
at a particular residue is intended to include identical residues
which have simply been derivatized.
[0103] The present invention thus provides antibodies having
specific VH and VL amino acid sequences and variants and fragments
thereof which maintain the function or activity of these VH and VL
domains.
[0104] Accordingly, an antibody of the invention may comprise:
[0105] (a) a light chain variable region amino acid sequence of SEQ
ID NO: 3;
[0106] (b) a fragment of at least 7 amino acids of (a) which
retains the ability to specifically bind to TFPI; or
[0107] (c) a variant of (a) having at least 70% amino acid sequence
identity to a sequence of (a) and retaining the ability to
specifically bind to TFPI.
[0108] An antibody of the invention may comprise:
[0109] (a) a heavy chain variable region amino acid sequence of SEQ
ID NO: 6;
[0110] (b) a fragment of at least 7 amino acids of (a) which
retains the ability to specifically bind to TFPI; or
[0111] (c) a variant of (a) having at least 70% amino acid sequence
identity to a sequence of (a) and retaining the ability to
specifically bind to TFPI.
[0112] An antibody of the invention may comprise the light chain
variable region of SEQ ID NO: 3 and the heavy chain variable region
of SEQ ID NO: 6.
[0113] An antibody of the invention may comprise
[0114] (a) the light chain variable region of SEQ ID NO: 3 and the
heavy chain variable region of SEQ ID NO: 6;
[0115] (b) a variant of (a) in which one or both of the heavy chain
and light chain sequences is modified such that it comprises a
fragment of at least 7 amino acids of the sequence specified in
(a); or
[0116] (c) a variant of any of (a) or (b) in which one or both of
the heavy and light chain sequences is modified such that it has at
least 70% amino acid sequence identity to a sequence of (a) or
(b)
[0117] wherein the antibody retains the ability to specifically
bind to TFPI. The antibody may also retain one or more additional
functions or activities of an antibody of the invention as
described herein such as the ability to inhibit TFPI or the ability
to shorten clotting time, optionally without leading to a drop in
platelet numbers.
[0118] Preferred fragments and variants of SEQ ID NO: 3 will
comprise (i) amino acids 24 to 39 of SEQ ID NO: 3; and/or (ii)
amino acids 55 to 61 of SEQ ID NO: 3; and/or (iii) amino acids 94
to 102 of SEQ ID NO: 3. Preferred fragments and variants of SEQ ID
NO: 6 will comprise (i) amino acids 31 to 35 of SEQ ID NO: 6;
and/or (ii) amino acids 50 to 65 of SEQ ID NO: 6; and/or (iii)
amino acids 98 to 107 of SEQ ID NO: 6.
[0119] As explained above, an antibody of the invention may bind to
the same epitope or region as another antibody of the invention.
Thus it will be seen that such an antibody may bind to the same
epitope or region of TFPI as any of the specific antibodies,
fragments and variants described herein.
[0120] The invention also relates to polynucleotides that encode
antibodies of the invention. Thus, a polynucleotide of the
invention may encode any antibody as described herein. The terms
"nucleic acid molecule" and "polynucleotide" are used
interchangeably herein and refer to a polymeric form of nucleotides
of any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Non-limiting examples of polynucleotides include a
gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide of the invention may be provided in isolated or
purified form.
[0121] A nucleic acid sequence which "encodes" a selected
polypeptide is a nucleic acid molecule which is transcribed (in the
case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus. For the
purposes of the invention, such nucleic acid sequences can include,
but are not limited to, cDNA from viral, prokaryotic or eukaryotic
mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and
even synthetic DNA sequences. A transcription termination sequence
may be located 3' to the coding sequence.
[0122] In one embodiment, a polynucleotide of the invention
comprises a sequence which encodes a VH or VL amino acid sequence
as described above. For example, a polynucleotide of the invention
may encode a polypeptide comprising the sequence of SEQ ID No: 3 or
6 or a variant or fragment thereof as described above. Such a
polynucleotide may consist of or comprise a nucleic acid sequence
of any one of SEQ ID NOs: 2, 4, 5 and 7. A suitable polynucleotide
sequence may alternatively be a variant of one of these specific
polynucleotide sequences. For example, a variant may be a
substitution, deletion or addition variant of any of the above
nucleic acid sequences. A variant polynucleotide may comprise 1, 2,
3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75
or more nucleic acid substitutions and/or deletions from the
sequences given in the sequence listing.
[0123] Suitable variants may be at least 70% homologous to a
polynucleotide of any one of SEQ ID NOs: 2, 4, 5 and 7. preferably
at least 80 or 90% and more preferably at least 95%, 97% or 99%
homologous thereto. Methods of measuring homology are well known in
the art and it will be understood by those of skill in the art that
in the present context, homology is calculated on the basis of
nucleic acid identity. Such homology may exist over a region of at
least 15, preferably at least 30, for instance at least 40, 60,
100, 200 or more contiguous nucleotides. Such homology may exist
over the entire length of the unmodified polynucleotide
sequence.
[0124] Methods of measuring polynucleotide homology or identity are
known in the art. For example the UWGCG Package provides the
BESTFIT program which can be used to calculate homology (e.g. used
on its default settings) (Devereux et al (1984) Nucleic Acids
Research 12, p 387-395).
[0125] The PILEUP and BLAST algorithms can also be used to
calculate homology or line up sequences (typically on their default
settings), for example as described in Altschul S. F. (1993) J Mol
Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol
215:403-10.
[0126] Software for performing BLAST analysis is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al, supra).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score goes
to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment. The BLAST program uses as
defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see
Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA
89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5,
N=4, and a comparison of both strands.
[0127] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0128] The homologue may differ from a sequence in the relevant
polynucleotide by less than 3, 5, 10, 15, 20 or more mutations
(each of which may be a substitution, deletion or insertion). These
mutations may be measured over a region of at least 30, for
instance at least 40, 60 or 100 or more contiguous nucleotides of
the homologue.
[0129] In one embodiment, a variant sequence may vary from the
specific sequences given in the sequence listing by virtue of the
redundancy in the genetic code. The DNA code has 4 primary nucleic
acid residues (A, T, C and G) and uses these to "spell" three
letter codons which represent the amino acids the proteins encoded
in an organism's genes. The linear sequence of codons along the DNA
molecule is translated into the linear sequence of amino acids in
the protein(s) encoded by those genes. The code is highly
degenerate, with 61 codons coding for the natural amino acids and 3
codons representing "stop" signals. Thus, most amino acids are
coded for by more than one codon--in fact several are coded for by
four or more different codons. A variant polynucleotide of the
invention may therefore encode the same polypeptide sequence as
another polynucleotide of the invention, but may have a different
nucleic acid sequence due to the use of different codons to encode
the same amino acids.
[0130] Polynucleotide "fragments" according to the invention may be
made by truncation, e.g. by removal of one or more nucleotides from
one or both ends of a polynucleotide. Up to 10, up to 20, up to 30,
up to 40, up to 50, up to 75, up to 100, up to 200 or more amino
acids may be removed from the 3' and/or 5' end of the
polynucleotide in this way. Fragments may also be generated by one
or more internal deletions. Such fragments may be derived from a
sequence of SEQ ID NOs: 2, 4, 5 or 7 or may be derived from a
variant polynucleotide as described herein. Preferably such
fragments are between 30 and 300 residues in length, e.g. 30 to
300, 30 to 200, to 100, 100 to 200 or 200 to 300 residues.
Alternatively, fragments of the invention may be longer sequences,
for example comprising at least 50%, at least 60%, at least 70%, at
least 80% or at least 90% of a full length polynucleotide of the
invention.
[0131] An antibody of the invention may thus be produced from or
delivered in the form of a polynucleotide which encodes, and is
capable of expressing, it. Where the antibody comprises two or more
chains, a polynucleotide of the invention may encode one or more
antibody chains.
[0132] For example, a polynucleotide of the invention may encode an
antibody light chain, an antibody heavy chain or both. Two
polynucleotides may be provided, one of which encodes an antibody
light chain and the other of which encodes the corresponding
antibody heavy chain. Such a polynucleotide or pair of
polynucleotides may be expressed together such that an antibody of
the invention is generated.
[0133] Polynucleotides of the invention can be synthesised
according to methods well known in the art, as described by way of
example in Sambrook et al (1989, Molecular Cloning--a laboratory
manual; Cold Spring Harbor Press).
[0134] The nucleic acid molecules of the present invention may be
provided in the form of an expression cassette which includes
control sequences operably linked to the inserted sequence, thus
allowing for expression of the antibody of the invention in vivo.
These expression cassettes, in turn, are typically provided within
vectors (e.g., plasmids or recombinant viral vectors). Such an
expression cassette may be administered directly to a host subject.
Alternatively, a vector comprising a polynucleotide of the
invention may be administered to a host subject. Preferably the
polynucleotide is prepared and/or administered using a genetic
vector. A suitable vector may be any vector which is capable of
carrying a sufficient amount of genetic information, and allowing
expression of a polypeptide of the invention.
[0135] The present invention thus includes expression vectors that
comprise such polynucleotide sequences. Such expression vectors are
routinely constructed in the art of molecular biology and may for
example involve the use of plasmid DNA and appropriate initiators,
promoters, enhancers and other elements, such as for example
polyadenylation signals which may be necessary, and which are
positioned in the correct orientation, in order to allow for
expression of a peptide of the invention. Other suitable vectors
would be apparent to persons skilled in the art. By way of further
example in this regard we refer to Sambrook et al.
[0136] The invention also includes cells that have been modified to
express an antibody of the invention. Such cells include transient,
or preferably stable higher eukaryotic cell lines, such as
mammalian cells or insect cells, lower eukaryotic cells, such as
yeast or prokaryotic cells such as bacterial cells. Particular
examples of cells which may be modified by insertion of vectors or
expression cassettes encoding for an antibody of the invention
include mammalian HEK293T, CHO, HeLa and COS cells. Preferably the
cell line selected will be one which is not only stable, but also
allows for mature glycosylation and cell surface expression of a
polypeptide.
[0137] Such cell lines of the invention may be cultured using
routine methods to produce an antibody of the invention, or may be
used therapeutically or prophylactically to deliver antibodies of
the invention to a subject. Alternatively, polynucleotides,
expression cassettes or vectors of the invention may be
administered to a cell from a subject ex vivo and the cell then
returned to the body of the subject.
[0138] In another aspect, the present invention provides
compositions and formulations comprising molecules of the
invention, such as the antibodies, polynucleotides, vectors and
cells described herein. For example, the invention provides a
pharmaceutical composition comprising one or more molecules of the
invention, such as one or more antibodies of the invention,
formulated together with a pharmaceutically acceptable carrier.
[0139] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for parenteral, e.g.
intravenous, intramuscular or subcutaneous administration (e.g., by
injection or infusion). Depending on the route of administration,
the antibody may be coated in a material to protect the antibody
from the action of acids and other natural conditions that may
inactivate or denature the antibody.
[0140] Preferred pharmaceutically acceptable carriers comprise
aqueous carriers or diluents. Examples of suitable aqueous carriers
that may be employed in the pharmaceutical compositions of the
invention include water, buffered water and saline. Examples of
other carriers include ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants. In many cases,
it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the composition.
[0141] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. These
compositions may also contain adjuvants such as preservatives,
wetting agents, emulsifying agents and dispersing agents.
Prevention of presence of microorganisms may be ensured both by
sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0142] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration.
[0143] Sterile injectable solutions can be prepared by
incorporating the active agent (e.g. antibody) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by
sterilization microfiltration. Generally, dispersions are prepared
by incorporating the active agent into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
agent plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0144] Pharmaceutical compositions of the invention may comprise
additional active ingredients as well as an antibody of the
invention. As mentioned above, compositions of the invention may
comprise one or more antibodies of the invention. They may also
comprise additional therapeutic or prophylactic agents. For
example, where a pharmaceutical composition of the invention is
intended for use in the treatment of a bleeding disorder, it may
additionally comprise one or more agents intended to reduce the
symptoms of the bleeding disorder. For example, the composition may
comprise one or more clotting factors. The composition may comprise
one or more other components intended to improve the condition of
the patient. For example, where the composition is intended for use
in the treatment of patients suffering from unwanted bleeding such
as patients undergoing surgery or patients suffering from trauma,
the composition may comprise one or more analgesic, anaesthetic,
immunosuppressant or anti-inflammatory agents.
[0145] Also within the scope of the present invention are kits
comprising antibodies or other compositions of the invention and
instructions for use. The kit may further contain one ore more
additional reagents, such as an additional therapeutic or
prophylactic agent as discussed above.
[0146] The antibodies, other molecules and compositions of the
present invention have numerous in vitro and in vivo therapeutic
utilities involving the treatment and prevention of clotting
related disorders. For example, these antibodies and compositions
can be administered to cells in culture, in vitro or ex vivo, or to
human subjects, e.g., in vivo, to prevent or treat a variety of
disorders.
[0147] In particular, the present invention provides methods for
the treatment of bleeding disorders or for the enhancement of blood
clotting comprising administering to a patient in need thereof an
effective amount of an antibody or other molecule or composition of
the invention. For example, such methods may be for the treatment
of clotting factor deficiencies such as haemophilia A, haemophilia
B, Factor XI deficiency, Factor VII deficiency, thrombocytopenia or
von Willebrand's disease. Such methods may be for the treatment of
conditions accompanied by the presence of a clotting factor
inhibitor. Such methods may be for the treatment of excessive
bleeding. The antibodies and compositions of the invention may be
used to treat patients before, during, or after surgery or
anticoagulant therapy or after trauma. The antibodies and
compositions described herein may be used in any such treatment or
may be used in the manufacture of a medicament for use in any such
treatment.
[0148] The antibodies and compositions of the present invention may
be administered for prophylactic/preventative and/or therapeutic
treatments.
[0149] In therapeutic applications, antibodies or compositions are
administered to a subject already suffering from a disorder or
condition as described above, in an amount sufficient to cure,
alleviate or partially arrest the condition or one or more of its
symptoms. Such therapeutic treatment may result in a decrease in
severity of disease symptoms, or an increase in frequency or
duration of symptom-free periods. An amount adequate to accomplish
this is defined as"therapeutically effective amount". For example,
where the treatment is for unwanted bleeding, therapy may be
defined as a decrease in the amount of bleeding or suitable
coagulation to stop the bleeding altogether.
[0150] In prophylactic or preventative applications, antibodies or
compositions are administered to a subject at risk of a disorder or
condition as described above, in an amount sufficient to prevent or
reduce the subsequent effects of the condition or one or more of
its symptoms. An amount adequate to accomplish this is defined as a
"prophylactically effective amount". For example, where the
treatment is to prevent unwanted bleeding, a prophylactic effect
may be defined as the prevention of bleeding or a reduced period or
quantity of bleeding compared to that that would be seen in the
absence of the modulator.
[0151] Effective amounts for each purpose will depend on the
severity of the disease or injury as well as the weight and general
state of the subject.
[0152] As used herein, the term "subject" includes any human or
non-human animal. The term "non-human animal" includes all
vertebrates, e.g., mammals and non-mammals, such as non-human
primates, sheep, dogs, cats, horses, cows, chickens, amphibians,
reptiles, etc.
[0153] An antibody or composition of the present invention may be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Preferred routes of administration for antibodies or compositions
of the invention include intravenous, intramuscular, intradermal,
intraperitoneal, subcutaneous, spinal or other parenteral routes of
administration, for example by injection or infusion. The phrase
"parenteral administration" as used herein means modes of
administration other than enteral and topical administration,
usually by injection. Alternatively, an antibody or composition of
the invention can be administered via a non-parenteral route, such
as a topical, epidermal or mucosal route of administration.
[0154] A suitable dosage of an antibody of the invention may be
determined by a skilled medical practitioner. Actual dosage levels
of the active ingredients in the pharmaceutical compositions of the
present invention may be varied so as to obtain an amount of the
active ingredient which is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic
factors including the activity of the particular antibody employed,
the route of administration, the time of administration, the rate
of excretion of the antibody, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0155] A suitable dose of an antibody of the invention may be, for
example, in the range of from about 0.1 .mu.g/kg to about 100 mg/kg
body weight of the patient to be treated. For example, a suitable
dosage may be from about 1 .mu.g/kg to about 10 mg/kg body weight
per day or from about 10 g/kg to about 5 mg/kg body weight per
day.
[0156] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic response). For example, a
single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0157] Antibodies may be administered in a single dose or in
multiple doses. The multiple doses may be administered via the same
or different routes and to the same or different locations.
Alternatively, antibodies can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency may vary depending on the half-life
of the antibody in the patient and the duration of treatment that
is desired. The dosage and frequency of administration can also
vary depending on whether the treatment is prophylactic or
therapeutic. In prophylactic applications, a relatively low dosage
may be administered at relatively infrequent intervals over a long
period of time. In therapeutic applications, a relatively high
dosage may be administered, for example until the patient shows
partial or complete amelioration of symptoms of disease.
[0158] As mentioned above, antibodies of the invention may be
co-administered with one or other more other therapeutic agents.
The other agent may be an agent that will enhance the effects of
the modulator. The other agent may be an agent that acts to enhance
blood coagulation, such as a blood coagulation factor. In
particular, the modulators of the invention may be co-administered
with Factor VII or Factor VIIa. The other agent may be intended to
treat other symptoms or conditions of the patient. For example, the
other agent may be an analgesic, anaesthetic, immunosuppressant or
anti-inflammatory agent.
[0159] Combined administration of two or more agents may be
achieved in a number of different ways. In one embodiment, the
antibody and the other agent may be administered together in a
single composition. In another embodiment, the antibody and the
other agent may be administered in separate compositions as part of
a combined therapy. For example, the modulator may be administered
before, after or concurrently with the other agent.
[0160] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of all figures and all references, patents
and published patent applications cited throughout this application
are expressly incorporated herein by reference.
EXAMPLES
Example 1
Production and Characterisation of a Monoclonal Antibody Directed
Against TFPI
Summary
[0161] Monoclonal antibodies were generated against tissue factor
pathway inhibitor (TFPI). A monoclonal antibody having the desired
binding specificity was identified, cloned and sequenced. This
antibody was found to significantly reduce cuticle bleeding time in
vivo, but to lead to no significant drop in platelet number.
Methods and Results
[0162] All kits were used according to the manufacturers'
instructions. Abbreviations: HC: heavy chain; LC: light chain; VH:
variable domain--heavy chain; VL: variable domain--light chain;
PCR: polymerase chain reaction.
Immunisation and Fusion
[0163] Mice were immunized with both full length TFPI and the short
version TFPIB161B which contains only the first two Kunitz domains.
RBF mice were used for immunizations and production of mouse
monoclonal antibodies. Injections were made subcutaneously in the
back of the mice. 20 .mu.g protein was mixed with complete Freund's
adjuvant for the first injection. In the subsequent immunizations,
incomplete Freund's adjuvant was used with same concentration of
the antigen. Ten days after the last immunization, eye-blood from
mice was screened by ELISA for TFPI specific antibodies. Mice with
positive serum titres were boosted with 10 .mu.g of TFPI by
intravenous injection, and sacrificed after three days. The spleens
were removed aseptically and dispersed to a single cell suspension.
Fusion of spleen cells and myeloma cells was done by the PEG-method
or by electrofusion.
Binding Assay: ELISA
[0164] Immunoplates were coated with anti-mouse IgG. Culture
supernatants from the hybridoma cells were added to the plates and,
after washing, soluble biotinylated human TFPI or TFPIB161B was
added to test for specific binding.
Neutralizing Assays: FXa Assay and TF/FVIIa/FXa Assay
[0165] FXa inhibition assay: a fixed concentration of TFPI giving
rise to 90% inhibition of FXa was pre-incubated with culture
supernatants from hybridoma cells containing anti TFPI monoclonal
antibodies and added to FXa plus FXa-specific chromogenic
substrate. This assay addresses TFPI binding to FXa (the K2
domain)
[0166] FVIIa/TF/FXa inhibition assay: 1) Incubation of culture
supernatants from hybridoma cells containing anti TFPI monoclonal
antibodies anti and fixed TFPI (90% inhibition of FVIIa/TF); 2)
Incubation of TFPI+FVIIa+TF+FXa; 3) Addition of FX (FX>>FXa)
followed by incubation with FXa chromogenic substrate.
Dilute Prothrombin Time (dPT)
[0167] A dilute Prothrombin (PT) analysis: human plasma in
combination with diluted human thromboplastin (TF source). Clot
time in the plasma was measured upon addition of increasing protein
A purified TFPI monoclonal antibody concentrations to look for dose
dependent reduction of clotting time. FVIIa (25 nM) was the
positive control and must shorten this clot time.
Binding Interaction Analysis
[0168] Binding interaction analysis was obtained by Surface Plasmon
Resonance in a Biacore 3000. Capture of the relevant monoclonal
antibody at a fixed concentration was obtained with immobilised
mouse anti-IgG. Different concentrations of TFPI were tested.
Determination of binding constants (k.sub.on, k.sub.off, K.sub.D)
was obtained assuming a 1:1 interaction of TFPI and the antibody of
interest.
Thrombelastography
[0169] Records the kinetic of clot formation and fibrinolysis in
whole blood. Haemophilia A like condition is induced by
pre-incubating the blood with neutralizing anti-FVIII IgG.
In Vivo Studies
[0170] Rabbits were made transient haemophilic by iv.
administration of 2000 RBU/kg of monoclonal anti-FVIII-antibodies.
After 10 minutes, the rabbits received 12000 U/kg of
anti-TFPI-antibody (3F18; 1.93 mg/kg). Cuticle bleeding was induced
45 minutes after anti-FVIII-antibody administration.
[0171] The 3F18 antibody caused a significant reduction in cuticle
bleeding time, comparable with the effect of 10 mg/kg rFVIIa (FIG.
1). Administration of the 3F18 antibody led to no significant drop
in platelet number (FIG. 5).
Antibody Cloning and Sequencing
[0172] Murine heavy chain and light chain sequence for anti-TFPI
antibodies were cloned from a hybridoma: TFPI-3F18A4B1 (abbreviated
herein to 3F18). Total RNA, extracted from hybridoma cells using
the RNeasy-Mini Kit from Qiagen, was used as templates for cDNA
synthesis. cDNA was synthesized in a 5'-RACE reaction using the
SMART.TM. RACE cDNA amplification kit from Clontech. Subsequent
target amplification of HC and LC sequences was performed by PCR
using Phusion Hot Star polymerase (Finnzymes) and the universal
primer mix (UPM) included in the SMART.TM. RACE kit as a forward
primer. A reverse primer with the following sequence was used for
HC (VH domain) amplification: 5'-CCCTTGACCAGGCATCCCAG-3' (primer
#129). A reverse primer with the following sequence was used for LC
amplification: 5'-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3' (primer
#69).
[0173] PCR products were separated by gel electrophoresis,
extracted using the GFX PCR DNA and Gel Band Purification Kit from
GE Healthcare Bio-Sciences and cloned for sequencing using a Zero
Blunt TOPO PCR Cloning Kit and chemically competent TOP10 E. coli
from Invitrogen. Colony PCR was performed on selected colonies
using an AmpliTaq Gold Mas-ter Mix from Applied Biosystems and
M13uni/M13rev primers. Colony PCR clean-up was performed using the
ExoSAP-IT enzyme mix (usb). Sequencing was performed at MWG
Biotech, Martinsried Germany using either M13uni(-21)/M13rev(-29)
or T3/T7 sequencing primers. Sequences were analyzed and annotated
using the Vector NTI program.
[0174] From the hybridoma TFPI-3F18A4B1 a single unique murine
kappa type LC was identified and a single unique murine HC,
subclass IgG1. VH & VL Sequences are shown in FIG. 2, leader
peptide sequences are not included.
Sequence CWU 1
1
91276PRTHomo sapiensDOMAIN(26)..(76)Kunitz domain 1 1Asp Ser Glu
Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu 1 5 10 15 Pro
Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp 20 25
30 Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr
35 40 45 Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn
Gln Asn 50 55 60 Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp Asn 65 70 75 80 Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln
Gln Glu Lys Pro Asp Phe 85 90 95 Cys Phe Leu Glu Glu Asp Pro Gly
Ile Cys Arg Gly Tyr Ile Thr Arg 100 105 110 Tyr Phe Tyr Asn Asn Gln
Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly 115 120 125 Gly Cys Leu Gly
Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys 130 135 140 Asn Ile
Cys Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly 145 150 155
160 Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys
165 170 175 Val Pro Ser Leu Phe Glu Phe His Gly Pro Ser Trp Cys Leu
Thr Pro 180 185 190 Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg
Phe Tyr Tyr Asn 195 200 205 Ser Val Ile Gly Lys Cys Arg Pro Phe Lys
Tyr Ser Gly Cys Gly Gly 210 215 220 Asn Glu Asn Asn Phe Thr Ser Lys
Gln Glu Cys Leu Arg Ala Cys Lys 225 230 235 240 Lys Gly Phe Ile Gln
Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys 245 250 255 Arg Lys Arg
Lys Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe 260 265 270 Val
Lys Asn Met 275 2339DNAMus musculusCDS(1)..(339) 2gat gtt gtg atg
acc cag act cca ctc act ttg tcg gtt acc att gga 48Asp Val Val Met
Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 caa cca
gct tcc atc tct tgc aag tca agt cag agc ctc tta gat agt 96Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30
gat gga aaa acc tat tta aat tgg tta tta cag agg cca ggc gag tct
144Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser
35 40 45 cca aag ctc ctt atc tat ctg gtg tct aaa ctg gac tct gga
gtc cct 192Pro Lys Leu Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly
Val Pro 50 55 60 gac agg ttc act ggc agt gga tca ggg aca gat ttc
aca ctg aaa atc 240Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ttg gga gtt
tat tac tgc tta caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Leu Gln Gly 85 90 95 aca cat ttt cct cac acg ttc gga
ggg ggg acc aag ctg gaa ata aaa 336Thr His Phe Pro His Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 cgg 339Arg 3113PRTMus
musculusDOMAIN(24)..(39)CDR 3Asp Val Val Met Thr Gln Thr Pro Leu
Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr
Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser 35 40 45 Pro Lys Leu
Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln
Gly 85 90 95 Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg 4339DNAMus musculus 4ccgttttatt
tccagcttgg tcccccctcc gaacgtgtga ggaaaatgtg taccttgtaa 60gcagtaataa
actcccaaat cctcagcctc cactctgctg attttcagtg tgaaatctgt
120ccctgatcca ctgccagtga acctgtcagg gactccagag tccagtttag
acaccagata 180gataaggagc tttggagact cgcctggcct ctgtaataac
caatttaaat aggtttttcc 240atcactatct aagaggctct gacttgactt
gcaagagatg gaagctggtt gtccaatggt 300aaccgacaaa gtgagtggag
tctgggtcat cacaacatc 3395354DNAMus musculusCDS(1)..(354) 5gaa gtg
aag ctg gta gag tct ggg gga ggc ttg gtg aag cct gga ggg 48Glu Val
Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
tcc ctg aga ctc tcc tgt gca gcc tct gga ttc act ttc agt aac tat
96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30 gcc ctg tct tgg gtt cgc cag act cca gac aag agg ctg gag
tgg gtc 144Ala Leu Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu
Trp Val 35 40 45 gca tcc att agt agt ggt ggt gcc acc tac tat cca
gac agt gtg gag 192Ala Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro
Asp Ser Val Glu 50 55 60 ggc cga ttc acc atc tcc aga gat aat gtc
agg aac atc ctg tac ctg 240Gly Arg Phe Thr Ile Ser Arg Asp Asn Val
Arg Asn Ile Leu Tyr Leu 65 70 75 80 caa atg agc agt ctg cag tct gag
gac acg gcc atg tat tac tgt aca 288Gln Met Ser Ser Leu Gln Ser Glu
Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 aga gga gcc tac ggc tcg
gac tac ttt gac tac tgg ggc caa ggc acc 336Arg Gly Ala Tyr Gly Ser
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 act ctc aca gtc
tcc tca 354Thr Leu Thr Val Ser Ser 115 6118PRTMus
musculusDOMAIN(31)..(35)CDR 6Glu Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu Ser Trp Val
Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile
Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Glu 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu 65 70
75 80 Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Thr 85 90 95 Arg Gly Ala Tyr Gly Ser Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 7354DNAMus
musculus 7tgaggagact gtgagagtgg tgccttggcc ccagtagtca aagtagtccg
agccgtaggc 60tcctcttgta cagtaataca tggccgtgtc ctcagactgc agactgctca
tttgcaggta 120caggatgttc ctgacattat ctctggagat ggtgaatcgg
ccctccacac tgtctggata 180gtaggtggca ccaccactac taatggatgc
gacccactcc agcctcttgt ctggagtctg 240gcgaacccaa gacagggcat
agttactgaa agtgaatcca gaggctgcac aggagagtct 300cagggaccct
ccaggcttca ccaagcctcc cccagactct accagcttca cttc
354820DNAArtificialprimer 8cccttgacca ggcatcccag
20936DNAArtificialprimer 9gctctagact aacactcatt cctgttgaag ctcttg
36
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References