U.S. patent application number 13/000284 was filed with the patent office on 2011-06-30 for use of anti-factor xi antibodies for prevention of thrombus formation.
Invention is credited to Erik Hack.
Application Number | 20110159006 13/000284 |
Document ID | / |
Family ID | 40933725 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110159006 |
Kind Code |
A1 |
Hack; Erik |
June 30, 2011 |
USE OF ANTI-FACTOR XI ANTIBODIES FOR PREVENTION OF THROMBUS
FORMATION
Abstract
The present invention relates to binding molecules such as
antibodies that specifically bind plasma coagulation factor XI and
that inhibit factor XI activation and/or activity. The factor
XI-binding molecules of the invention may used in methods for
preventing or treating diseases, disorders and/or conditions that
are mediated by factor XI activation and/or wherein inhibition of
factor XI has a beneficial effect.
Inventors: |
Hack; Erik; (Diemen,
NL) |
Family ID: |
40933725 |
Appl. No.: |
13/000284 |
Filed: |
June 19, 2009 |
PCT Filed: |
June 19, 2009 |
PCT NO: |
PCT/NL09/50361 |
371 Date: |
March 10, 2011 |
Current U.S.
Class: |
424/158.1 ;
530/389.3 |
Current CPC
Class: |
A61P 7/02 20180101; C07K
2317/76 20130101; A61K 2039/505 20130101; C07K 16/36 20130101; A61P
9/10 20180101 |
Class at
Publication: |
424/158.1 ;
530/389.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/36 20060101 C07K016/36; A61P 9/10 20060101
A61P009/10; A61P 7/02 20060101 A61P007/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 19, 2008 |
US |
61073882 |
Claims
1. A binding molecule that specifically binds factor XI and
inhibits factor XI activity, for use in preventing or treating a
disease, disorder and/or condition that is mediated by factor XI
activation and/or wherein inhibition of factor XI has a beneficial
effect.
2. A binding molecule according to claim 1, wherein the binding
molecule inhibits at least one of: (a) binding at or near the
peptidyl bond Arg.sup.369-Ile.sup.370 in factor XI, thereby
preventing cleavage of the bond; (b) binding at or near the active
site located in the factor XI light chain region of the molecule;
(c) binding at or near sites in factor XI sites that are involved
in interactions with factor IX (d) binding to the factor XI A1
domain, thereby preventing the interaction of factor XI with high
molecular weight kininogen and/or thrombin; and (e) binding to the
facctor XI A2 or A3 domain, thereby preventing the interaction of
factor XI with factor IX, heparin and/or platelets.
3. A binding molecule according to claim 1, wherein the binding
molecule preferentially binds to the activated form of factor
XI.
4. A binding molecule according to claim 3, wherein the binding
molecule inhibits functional activity of the activated form of
factor XI by one or more of: (a) binding at or near to the active
site located in the factor XI light chain region; (b) binding at or
near sites factor XI sites that are involved in interactions with
factor IX; (c) binding to the factor XI A1 domain, thereby
preventing the interaction of factor XI with high molecular weight
kininogen and/or thrombin; and (d) binding to the factor XI A2 or
A3 domain, thereby preventing the interaction of factor XI with
factor IX, heparin and/or platelets.
5. A binding molecule according to claim 1, wherein the binding
molecule is an antibody or a factor XI-binding antibody
fragment.
6. A binding molecule according to claim 1, wherein the binding
molecule is administered at a dosage equivalent to a dosage of
0.5-20 mg of IgG per kg body weight.
7. A binding molecule according to claim 1, wherein the binding
molecule is administered at a dosage that yields a molar ratio in
serum that is equal to or less than 4 moles of factor XI binding
sites to 1 mole of factor XI, wherein the factor XI binding sites
of the binding molecule have a Kd that is equal to or less than 1
nM.
8. A binding molecule according to claim 1, for use in preventing
or treating a pathological thrombosis or in preventing thrombosis
in a subject who is at increased risk of developing thrombosis
resulting from a medical procedure.
9. A binding molecule according to claim 8, for use in preventing
or treating at least one of the following disorders, diseases or
conditions: myocardial infarction, ischemic stroke, cardio-embolism
due to atrial fibrillation, vascular access thrombosis, deep venous
thrombosis, arterial thrombosis, coronary artery thrombosis,
atherosclerosis, arthritis, vasculitis, respiratory distress
syndrome, ischemic heart disease, ischemic cerebral disease,
pulmonary embolism, venous thrombo-embolism resulting from surgery
or immobilization, thrombosis and occlusion of a synthetic graft,
stent or AV-fistula, prosthetic heart valve, diffuse intravascular
coagulation, hemodialysis, atrial fibrillation, sepsis, septic
shock, organ failure, kidney failure, toxicity induced by the in
vivo administration of a therapeutic protein, multiple trauma,
ischemia-reperfusion injury, local undesired fibrin deposition or
fibrin deposition in lung alveoli during adult respiratory
distress.
10. A binding molecule according to claim 1, wherein the binding
molecule is administered intravenously, intramuscularly or
subcutaneously.
11. A binding molecule according to claim 10, wherein the binding
molecule is administered intravenously as a bolus infusion or as a
continuous infusion over an interval of up to 24 hours.
12. A binding molecule according to claim 9, for use in preventing,
reducing or treating thrombosis and occlusion of a synthetic graft,
stent or AV-fistula in a kidney undergoing regular renal dialysis,
wherein the binding molecule is administered to the patient in
dialyzed body fluid that is returned to the patient.
13. A binding molecule according to claim 9, for use in preventing
or treating thrombosis in a patient with atrial fibrillation,
unstable angina pectoris, venous thrombo-embolism, prosthetic heart
valve, ischemic heart disease, ischemic cerebral disease, a
vascular graft, diffuse intravascular coagulation, sepsis, or a
patient undergoing prostate or orthopaedic surgery, wherein the
binding molecule is administered no more than once per 2 weeks.
14. A binding molecule according to claim 13, wherein the binding
molecule has a half life of at least 6 days and a Kd that is less
than 1 nM.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of haematology, in
particular the field of coagulation. The invention relates to
methods for inhibiting the formation of blood clots using binding
molecules that specifically bind and inhibit the activation and/or
activity of factor XI. The antigen binding molecule may or may not
be specific for the activated conformation of factor XI and is
preferably used in methods for reducing or preventing thrombus
formation on synthetic grafts, atherosclerotic plaques or in other
pathological thrombotic and thrombo-embolic processes.
BACKGROUND OF THE INVENTION
[0002] Coagulation consists of a humoral and a cellular response.
The former leads to the conversion of soluble fibrinogen into
insoluble fibrin, the latter consists of activation of platelets
leading to a platelet plug. Platelet plugs, fibrin threads and
included red blood cells together constitute a blood clot. A key
molecule in clot formation is thrombin. This protease converts
soluble fibrinogen into the insoluble fibrin, it activates
platelets, and converts a number of other factors including factors
XI, VIII and V, into active species.
[0003] Pathological thrombosis refers to clot formation that is not
part of a normal hemostatic process and that may result in disease
symptoms. For example, thrombosis on an atherosclerotic plaque in a
coronary artery may result in acute myocardial infarction and can
be considered as a type of pathological thrombosis. Deep venous
thrombosis and thrombosis on vascular grafts are other examples of
pathological thrombosis.
[0004] Current anticoagulant drugs inhibit the basal pathway of
coagulation: heparin (via antithrombin III) hits thrombin, factors
Xa and IXa, coumarins inhibit the synthesis of prothrombin, factors
VII, IX and X, whereas LMW heparin mainly inhibits factor Xa. The
therapeutic window of these drugs is narrow, requiring careful
monitoring of patients. Some of the newer anticoagulant drugs under
development target at the FVIII/FIX amplification loop.
[0005] Considering the severe bleeding tendency resulting from a
complete deficiency of factor VIII or IX, in hemophilia A or B,
respectively, targeting at this level likely will also require
careful monitoring to prevent the risk of bleeding side effects,
particularly at overdosing.
[0006] Factor XI is not a target for current anticoagulants. This
is mainly due to the fact that factor XI deficiency, in contrast to
a deficiency of factor VIII or IX, does not result in a severe
bleeding tendency. As a matter of fact many factor XI deficient
persons never experience a severe bleeding episode. Only two
studies have been performed with antibodies, in both cases
polyclonal, against factor XI that inhibit the function of factor
XI in vivo, either by inhibiting the activity of the molecule or by
preventing its activation. In a first study (Minnema et al., 1998,
J Clin Invest. 101:10-14) polyclonal antibodies against rabbit
factor XI were used to evaluate the effect of factor XI blockade on
clot formation in vivo in an experimental thrombosis model in
rabbits. Incorporation of anti-factor XI antibodies in jugular vein
thrombi resulted in an almost twofold increase in endogenous
thrombolysis compared with a control antibody. A similar effect was
observed when the anti-factor XI antibody was administered
systemically.
[0007] The effect of administration of neutralizing polyclonal goat
anti-factor XI-antibodies on the accumulation of platelets and
fibrin on arterio-venous grafts in baboons was topic of another
study to evaluate factor XI as target for antithrombotic therapy
(Gruber and Hanson, 2003, Blood 102:953-955). In that study the
role of factor XI-dependent thrombus propagation under arterial
flow conditions was investigated. Under the conditions used rapid
thrombus growth was produced on the grafts of Dacron or Teflon
deployed into arteriovenous shunts in baboons treated with
anti-human factor XI antibody. Administration of the polyclonal
antibodies against factor XI markedly reduced intraluminal thrombus
growth on both surfaces. The antithrombotic effect of the
polyclonal antibodies against factor XI was found to be comparable
with that of heparin at doses that significantly prolonged the
partial thromboplastin time, prothrombin time, and bleeding time,
whereas anti-FXI antibodies only affected the partial
thromboplastin time but not prothrombin time and bleeding time.
[0008] There is however still a need in the art for anti-coagulant
therapies based on antigen binding molecules that target factor
XI.
DESCRIPTION OF THE INVENTION
[0009] The present invention relates to means and methods for
anti-coagulant therapy that are based on the inhibition of factor
XI. The inventors have found that factor XI is a preferred target
for inhibition as this leaves coagulation via the "basal" pathway
and the FIX/VIII amplification loop intact, which is sufficient for
most hemostatic conditions. In other words, factor XI inhibitors
have less risk for bleeding side effects. Indeed, factor XI
deficiency or factor XI inhibition had no effect on the bleeding
time, in contrast to high dose heparin which strongly prolonged
bleeding time. These data make factor XI an attractive target for
anticoagulant therapy, the more since e.g. severe bleeding
complications such as intracerebral hemorrhage rarely occur in
persons with factor XI deficiency (F Peyvandi et al., Haematologica
2002; 87:512-514). Thus, because of its active role in pathologic
thrombosis and a minor role in normal hemostasis, factor XI is an
attractive target for anti-coagulant therapy.
[0010] In a first aspect therefore, the present invention relates
to a binding molecule that specifically binds to factor XI and that
inhibits the functional activity of factor XI. Factor XI is herein
understood as the mammalian plasma coagulation factor XI. In a
preferred embodiment a binding molecule of the invention
specifically binds to and inhibits the functional activity of at
least human factor XI. Plasma coagulation factor XI is a
glycoprotein present in human plasma at a concentration of 25-30 nM
as a zymogen that when converted by limited proteolysis to an
active serine protease, participates in the contact phase of blood
coagulation. The sequence of the human factor XI gene has been
identified and the deduced amino acid sequence is depicted in SEQ
ID NO: 1. The gene for human factor XI is 23 kilobases (kb) in
length and consists of 15 exons (I-XV) and 14 introns. Exon I
encodes the 5'-untranslated region, and exon II encodes a signal
peptide. The next eight exons (III-X) encode four tandem repeat
sequences of 90 or 91 amino acids (Apple domains) that are present
in the amino-terminal region of the mature protein. The
carboxyl-terminal region of the protein, which contains the
catalytic serine protease domain, is encoded by five exons (XI-XV)
that are interrupted by four introns. Factor XI is a zymogen, which
is unique among the other clotting factors in that it exists as a
homo-dimer consisting of two identical subunits of 80 kDa connected
by a single disulfide bond. Conversion of zymogen factor XI to the
active form, factor XIa, is accomplished by a single cleavage at
Arg.sub.369-Ile.sub.370 (SEQ ID NO: 1), upon which cleavage each
subunit is converted into a 35 kDa light chain and a 50 kDa heavy
chain linked by disulfide bonds. The light chain contains a
catalytic site. Each heavy chain is composed of four 90-91 amino
acid repeats called apple domains (A1-A4) with A1 located at the
aminoterminal end of the heavy chain. Thus fully activated factor
XI consists of two light chains of 35 kDa and two heavy chains of
50 kDa, and contains two active sites. The A1 domain contains the
binding site for high molecular weight kininogen and thrombin. The
function of the A2 domain is not clear, initially it was reported
to contain an interaction site with factor IX, but later it was
shown that this site was located in the A3 domain. The A3 domain
also contains a binding site for heparin, and for platelets. The
binding sites for factor IX and platelets are close to each other
and it has been postulated by Gailani et al. (D Gailani et al.,
Blood 2001; 97:3117-3122) that this is the reason for the dimeric
structure: due to its dimeric structure factor XI can bind via the
A3 domain of one subunit to platelets and use its other A3 domain
to interact with factor IX. In this way, factor XI can target
activation of coagulation to the platelet surface. Finally, the A4
domain of factor XI is involved in dimer formation, contains a free
cystein of unknown significance, and an interaction site with
factor XIIa. Factor XI can be activated by at least three
coagulation proteases, factor XIIa, factor XIa (autoactivation) and
thrombin.
[0011] A binding molecule of the invention, that can bind to, that
has affinity for and/or that has specificity for a factor XI target
molecule (or an epitope on the factor XI target molecule) may be
said to be "against" or "directed against" said target molecule or
antigen. The term "specificity" refers to the number of different
types of epitopes or antigenic targets on factor XI to which a
particular (antigen-) binding molecule can bind. The specificity of
an antigen-binding molecule can be determined based on affinity
and/or avidity. The affinity, represented by the equilibrium
constant for the dissociation of an antigen with an antigen-binding
protein (K.sub.D), is a measure for the binding strength between an
antigenic determinant and an antigen-binding site on the
antigen-binding protein. Alternatively, the affinity can also be
expressed as the affinity constant (K.sub.A), which is 1/K.sub.D.
Affinity can be determined in a manner known per se, depending on
the specific combination of antigen-binding protein and antigen of
interest.
[0012] Avidity is herein understood to refer to the strength of
binding of a target molecule with multiple binding sites by a
larger complex of binding agents, i.e. the strength of binding of
multivalent binding. Avidity is related to both the affinity
between an antigenic determinant and its antigen-binding site on
the antigen-binding molecule and the number of binding sites
present on the antigen-binding molecule. Affinity, on the other
hand refers to simple monovalent receptor ligand systems.
[0013] Typically, factor XI-binding molecules of the invention will
bind the target molecule with a dissociation constant (K.sub.0) of
about 10.sup.-7 to 10.sup.-12 M or less, and preferably 10.sup.-8
to 10.sup.-12 M or less, and/or with a binding affinity of at least
10.sup.-7 M, preferably at least 10.sup.-8 M, more preferably at
least 10.sup.-9 M, such as at least 10.sup.-10, 10.sup.-11,
10.sup.-12 M or more. Any K.sub.D value greater than 10.sup.-4 M
(i.e. less than 100 .mu.M) is generally considered to indicate
non-specific binding. Preferably, a binding molecule of the
invention will bind to factor XI with an affinity less than 50, 10
or 5 nM, more preferably less than 1 nM, such as less than 500,
200, 100, 50, 10 or 5 pM. Specific binding of a binding molecule to
factor XI can be determined in any suitable manner known per se,
including, for example, Scatchard analysis and/or competitive
binding assays, such as radioimmunoassays (RIA), enzyme
immunoassays (EIA) and sandwich competition assays, and the
different variants thereof known per se in the art.
[0014] The binding molecules of the invention inhibit at least one
of the functional activities of factor XI and the activation of
factor XI. Preferably, binding molecules of the invention inhibit
the functional activity of factor XI and/or inhibit the activation
of factor XI, independently of how factor XI is activated. A
preferred binding molecule of the invention, e.g. an antibody or
antibody fragment, has only one binding site that binds and
inhibits factor XI activation or factor XI activity.
[0015] In one embodiment, the binding molecule inhibits the
functional activity of factor XI by preventing it to become
activated.
[0016] In a further embodiment, the binding molecule inhibits the
activation of factor XI by binding to or near to the peptidyl bond
Arg.sub.369-Ile.sub.370, and preventing the cleavage of this bond
by thrombin, factor XIIa, factor XIa or any other protease.
Preferably, upon binding of the binding molecule to factor XI,
there is a reduction in the activation of factor XI by factor XIIa,
thrombin or factor XIa of at least 60, 70, 80, 90, 95, 99%. More
preferably no activation of factor XI by factor XIIa, thrombin or
factor XIa, is detectable upon binding of the binding molecule to
factor XI.
[0017] The amount of reduction in the activation of factor XI may
be determined in the various assays as described in the Examples
herein by comparing activation of factor XI in the presence of the
binding molecule with activation in the absence of the
molecule.
[0018] In a further embodiment, the binding molecule inhibits the
activation of factor XI by binding to the A1 domain of factor XI
thereby preventing the interaction of factor XI with high molecular
weight kininogen and/or thrombin (BN Bouma et al., Curr Opin
Hematol 2000, 7:266-272). Preferably, upon binding of the binding
molecule to factor XI, there is a reduction in the activation of
factor XI by factor XIIa, thrombin or factor XIa of at least 60,
70, 80, 90, 95, 99%. More preferably no activation of factor XI by
thrombin is detectable upon binding of the binding molecule to
factor XI. The amount of reduction in the activation of factor XI
may be determined in the various assays as described in the
Examples herein by comparing activation of factor XI in the
presence of the binding molecule with activation in the absence of
the molecule.
[0019] In a further embodiment, the binding molecule inhibits the
activity of factor XI by binding to or near to the active site
located in the light chain region of the molecule.
[0020] In a further embodiment, the binding molecule inhibits the
activity of factor XI by binding to the A2 or A3 domain of factor
XI, that are involved in the interaction with factor IX, heparin
and or platelets (BN Bouma et al., Curr Opin Hematol 2000,
7:266-272).
[0021] In a further embodiment, the binding molecule inhibits the
activation of factor XI by binding to or near to sites that are
involved in the interaction with its substrate factor IX.
[0022] In a further embodiment, the binding molecule preferentially
binds to the activated form of factor XI and inhibits the
activation of factor XI by binding to or near to sites that are
involved in the interaction with its substrate factor IX.
[0023] In a further embodiment, the binding molecule preferentially
binds to the activated form of factor XI and inhibits the
activation of factor XI by binding to the A1 domain of factor XI
thereby preventing the interaction of factor XI with high molecular
weight kininogen and/or thrombin.
[0024] In a further embodiment, the binding molecule preferentially
binds to the activated form of factor XI and inhibits the activity
of factor XI by binding to or near to sites that are involved in
the interaction with its substrate factor IX.
[0025] In a further embodiment, the binding molecule preferentially
binds to the activated form of factor XI and inhibits the activity
of factor XI by binding to or near to the active site located in
the light chain region of the molecule.
[0026] In a further embodiment, the binding molecule preferentially
binds to the activated form of factor XI and inhibits the activity
of factor XI by binding to the A2 or A3 domain of factor XI, that
are involved in the interaction with factor IX, heparin and or
platelets.
[0027] The binding molecules of the present invention are thus
characterised by their ability to prevent activation and/or inhibit
the activity of factor XI and/or to inhibit activation of factor
IX. Said inhibitors can be selected by the assessment of their
effect in various assays as described in the Examples herein. In
particular factor XI-binding molecules can be selected by the
assessment of their effect on the clotting activity of the
coagulation system as determined with an activated partial
thromboplastin time (APTT) in human plasma. The functional
properties of factor XI-binding molecules of the invention may be
tested by adding these to fresh human plasma, followed by
measurement of the APTT in a regular clotting assay. In case of an
inhibiting antibody, a prolongation of the APTT will be observed.
As controls normal plasma (normal APTT) and factor XI-deficient
plasma (prolonged APTT) are tested. These clotting assays are well
known in the art (see also Examples herein).
[0028] The effects of factor XI-binding molecules on the function
of the factor XI molecule can also be tested using chromogenic
substrates. Chromogenic substrates consist of small peptides
coupled to p-nitroanilide (pNA). Hydrolysis of the substrate
releases pNA which can be measured with a spectrophotometer.
Specificity of the substrate for certain proteases is dependent on
the precise sequence of the peptide linked to pNA. In case of
factor XIa, the substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O;
Chromogenix, Molndal, Sweden) is appropriate. Measurement of factor
XIa activity with this substrate can be done using the method
described by Minnema et al. (1998, Blood 92:3294-3301). Factor
XI-binding molecules that inhibit the catalytic center of factor
XIa can be identified by their inhibiting effect on the chromogenic
activity of factor XIa in such a chromogenic activity.
[0029] In an alternative method, purified factor XIIa can be used
to activate factor XI, which then can be monitored by measuring its
chromogenic activity. Factor XI-binding molecules that inhibit the
activation of factor XI by factor XIIa will manifest themselves by
decreasing the amount of factor XIa generated in this system.
[0030] In one embodiment, a factor XI-binding molecule of the
invention that inhibits the functional activity or activation of
factor XI preferably is a molecule that produces at least 90%
inhibition of factor XI activity at a concentration of about 50-80
nM in an activated partial thromboplastin time (APTT) assay. More
preferably the molecule produces at least 95% inhibition of factor
XI activity at a concentration of about 50-80 nM in an activated
partial thromboplastin time (APTT) assay. Most preferably the
molecule produces at least 99% inhibition of factor XI activity at
a concentration of about 50-80 nM in an activated partial
thromboplastin time (APTT) assay.
[0031] In a further embodiment, a factor XI-binding molecule of the
invention that inhibits the functional activity or activation of
factor XI is a molecule that produces at least 90% inhibition of
factor XI activity at a concentration of about 20-50 nM in an
activated partial thromboplastin time (APTT) assay. More preferably
the molecule produces at least 95% inhibition of factor XI activity
at a concentration of about 20-50 nM in an activated partial
thromboplastin time (APTT) assay. Most preferably the molecule
produces at least 99% inhibition of factor XI activity at a
concentration of about 20-50 nM in an activated partial
thromboplastin time (APTT) assay.
[0032] The term "activator" as used throughout the invention refers
to "a molecule capable of activating factor XI in such a way that
this subsequently activates factor IX, which in presence of its
cofactor FVIII in its turn activates factor X and the rest of the
coagulation cascade". Activator molecules include thrombin, factor
XIIIa and factor XIa.
[0033] In a preferred embodiment, a factor XI-binding molecule of
the invention is an antibody or a factor XI-binding fragment of an
antibody. As used herein, the term "binding molecule" thus
encompasses, but is not limited to, an antibody and fragments
thereof, a unibody, a diabody, a triabody, a tetravalent or other
multivalent antibody specifically binding factor XI and inhibiting
the functional activity of factor XI. The term "antibody" refers to
polyclonal antibodies, monoclonal antibodies, antibodies which are
derived from a phage library, humanised antibodies, human
antibodies, synthetic antibodies, chimeric antibodies, single
domain antigen binding proteins and antibody fragments such as, but
not limited to single-chain Fv's.
[0034] Also antibodies made in other animal species such as camelid
antibodies or fragments thereof ("Nanobodies") fall within the
scope of this application. Furthermore, molecules with
antibody-like binding properties such as Designed Repeat Proteins
like DARPins (Designed Ankyrin Repeat Proteins) are within the
scope of this application.
[0035] The factor XI-binding molecule of the invention is a
component that specifically binds to the target molecule with a
desired binding affinity (as herein defined). The factor XI-binding
protein of the invention preferably is a mono-specific
antigen-binding protein. A composition comprising a mono-specific
antigen-binding protein, is understood to mean a composition having
a homogeneous population of the factor XI-binding protein. It
follows that the mono-specific factor XI-binding protein is
specific for a single epitope on a factor XI monomer. It is however
expressly included in the invention that the compositions of the
invention may comprise more than one type of mono-specific factor
XI-binding protein, each consisting of a homogeneous population.
Usually, however, in the context of the present invention, a
composition of the invention will not comprise more than 4, 6, 8,
10 or 20 different mono-specific factor XI-binding proteins. The
factor XI-binding protein will usually be an antibody or fragment
thereof, in which case the mono-specific factor XI-binding protein
will thus be a monoclonal antibody or a fragment thereof, which may
be obtained from a cloned cell-line (e.g. hybridoma) or expressed
from a cloned coding sequence. The term "monoclonal antibody" is
thus not intended to be limited by the manner in which it is made.
The term mono-specific factor XI-binding protein as used herein
thus excludes polyclonal antibodies and antisera.
[0036] In addition, any construct of an antibody or a fragment is
also a subject of current invention. As used herein, the term
"construct" relates to diabodies, triabodies, tetravalent
antibodies, pepta- or hexabodies, and the like, that are derived
from an anti-human factor XI antibody according to the present
invention. Said multivalent antibodies comprising at least one
hypervariable domain from an anti-factor XI antibody according to
the present invention can be mono-, bi- or multispecific. A
preferred antibody or antibody fragment has only one binding site
that binds and inhibits factor XI activation or factor XI
activity.
[0037] As used herein, the term "human antibody", as used herein is
intended to include antibodies having variable and constant regions
derived from human germline immunoglobulin sequences. The human
antibodies may include amino acids 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.
[0038] As used herein, the term "humanised antibody" means that at
least a portion of the framework regions of an immunoglobulin or
engineered antibody construct is derived from human immunoglobulin
sequences. It should be clear that any method to humanise
antibodies or antibody constructs, as for example by variable
domain resurfacing (Roguska et al., 1994) or CDR grafting or
reshaping (Hurle et al., 1994), can be used.
[0039] As used herein, the term "chimeric antibody" refers to an
engineered antibody construct comprising of variable domains of one
species (such as mouse, rat, goat, sheep, cow, lama or camel
variable domains), which may be humanised or not, and constant
domains of another species (such as non-human primate or human
constant domains) (for review see Hurle et al., 1994, supra). It
should be clear that any method known in the art to develop
chimeric antibodies or antibody constructs can be used.
[0040] In one embodiment of the invention, the antibody is an
intact murine IgG1, an intact human IgG1 mutated in the constant
region to reduce or prevent complement activation or Fc receptor
interactions, or an intact human IgG4.
[0041] In one embodiment of the invention, the antibody or antibody
fragment is a monomeric IgM antibody subunit.
[0042] In one embodiment of the invention, the antibody or antibody
fragment does not activate the classical or lectin pathways of
complement, and/or does not interact with Fc-receptors.
[0043] In one embodiment of the invention, the binding molecule is
a binding-domain immunoglobulin fusion protein comprising (i) a
binding domain polypeptide in the form of a heavy chain variable
region or a light chain variable region that is fused to an
immunoglobulin hinge region polypeptide, (ii) an immunoglobulin
heavy chain CH2 constant region fused to the hinge region, and
(iii) an immunoglobulin heavy chain CH3 constant region fused to
the CH2 constant region.
[0044] As used herein, in connection with antibodies, the term
"fragment" or "fragments" refers to Fab, F(ab').sub.2, Fv, scFv, Fd
(consisting of the V.sub.H and C.sub.H1 domains), dAb (consisting
of a V.sub.H domain), a complementarity determining region (CDR),
e.g. V.sub.H CDR3, and other fragments which retain the antigen
binding function and specificity of the parent antibody. The
methods for producing said fragments are well known to a person
skilled in the art and can be found, for example, in Antibody
Engineering, Oxford University Press, Oxford (1995) (1996) and
Methods in Molecular Biology, Humana Press, New Jersey (1995).
[0045] As used herein, the term "single chain Fv", also termed
scFv, refers to engineered antibodies prepared by isolating the
binding domains (both heavy and light chains) of a binding
antibody, and supplying a linking moiety which permits preservation
of the binding function. This forms, in essence, a radically
abbreviated antibody, having only that part of the hyper-variable
domain necessary for binding the antigen. Determination and
construction of single chain antibodies are described in e.g. U.S.
Pat. No. 4,946,778 to Ladner et al.
[0046] As used herein the term "single domain antigen binding
protein" refers to antibodies or fragments thereof that are derived
from antibodies naturally devoid of light chains. Antibodies
naturally devoid of light chains may be obtained e.g. by
immunisation of camelids (e.g. llama's) or sharks (see further
below). These antibodies comprise heavy chains only and are devoid
of light chains. The advantage of use of such single domain heavy
chain antibodies is that they are exceptionally stable even at
higher temperatures, small and are easily produced in microbial
host organisms such as Saccharomyces cerevisiae. Thus, a factor
XI-binding protein of the invention preferably comprises an
immunoglobulin-derived variable domain that comprises a complete
antigen-binding site for an epitope on a target molecule in a
single polypeptide chain. Such factor XI-binding proteins
specifically include but are not limited to:
[0047] 1) antibodies obtainable from camelids and sharks that
consist of only heavy chains and that are naturally devoid of light
chains;
[0048] 2) variable domains of the antibodies defined in 1), usually
referred to as VHH domains (see WO2006/040153);
[0049] 3) engineered forms of the antibodies defined in 1) or
domains in 2) such as e.g. "camelidised" antibodies in which frame
work sequences of a camelid (or shark) VHH domain are grafted with
CDRs obtained from other sources;
[0050] 4) engineered forms of immunoglobuline-like variable domains
in which frame works sequences from a variety of
immunoglobuline-like molecules are combined with CDRs specific for
a given target molecule as e.g. described in WO 04/108749.
[0051] Monoclonal antibodies of the present invention can be
obtained by isolating immune cells from an animal immunised with
human factor XI or with activated human factor XI (factor XIa), or
with parts of these molecules, and immortalisation of these cells
to yield antibody secreting cell lines such as hybridomas. Cell
lines that produce the desired antibodies can be identified by
screening culture supernatants for the presence of antibody
activity, and by establishment of the effect of the selected
antibody on the functional activity of factor XI.
[0052] Human factor XI, factor XIa, fragments thereof and/or
synthetic peptides comprising factor XI amino acid sequences,
isolated according to a variety of purification methods may be used
to immunise an appropriate host animal.
[0053] A variety of immunization protocols may be employed, and may
consist of intravenous, subcutaneous, or intraperitoneal
immunization, followed by one or more boosts. A suitable adjuvant
is Freund's adjuvant. The precise schedule of administration of the
human factor XI, factor XIa, or fragments thereof to the host
animal in general is not well defined. The choice of the
immunization procedure is more dependent on host animal antibody
responses to the administered factor XI, as measured by a suitable
assay (vide supra). A suitable immunization procedure, however, is
hyperimmunization with human factor XI, or factor XIa, or fragments
thereof at a concentration that, dependent on the host animal, may
be in the range of 10 to 500 microgram, mixed with Freund's
complete adjuvant. The mix is injected subcutaneously. Injections
are repeated 2 to 5 times using the same factor XI preparation
mixed with Freund's incomplete adjuvant. Blood samples are taken
from the animal 1 week after the 3.sup.rd, 4.sup.th, 5.sup.th
injection, and screened for the presence of antibodies against
factor XI. In a scheme as indicated above, the length of the
interval between the injections can be varied. Alternative
adjuvants can also be used.
[0054] Alternatively, lymphocytes, human or murine or other, may be
immunised in vitro, as for example can be achieved via a procedure
outlined by Voss B, 1986 and in EPA 8610791.6. Other procedures
have also been described: (Luben et al., 1980; Reading, 1986;
Reading, 1982). As a source of human lymphocytes, those obtained
from patients with antibodies against human factor XI can be used,
for example from persons deficient for factor XI who have developed
antibody responses against administered exogenous factor XI (0
Salomon et al., Blood 2003; 101:4783-4788).
[0055] An alternative approach for immunization comprises the use
of synthetic peptides that mimic the sequence of functional sites
of factor XI, such as individual apple domains. The methods of
making antibodies against peptides are well-known in the art and
generally require coupling of the peptides to a suitable carrier
molecule, for example bovine serum albumin or keyhole limpet
hemocyanin. The peptides can be made according to procedures well
known in the art. The procedure also may use commercially available
peptide synthesiser machines.
[0056] Also the hybridoma cell line that produces the antibody may
be used as a source of DNA or mRNA encoding the desired antibody,
which may be isolated and transferred to cells by known genetic
techniques to produce genetically engineered antibody.
[0057] The initial screening step of culture supernatants of
hybridomas obtained by fusion of lymphocytes of mice immunised with
factor XI, factor XIa, parts thereof, or with factor XI peptides,
with an appropriate fusion partner, is preferably done by an
enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay
(RIA). Both assays are known to those skilled in the art, and
consist of coupling of human factor XI or factor XIa to a
solid-phase matrix, and assaying for antibody binding to factor XI
or factor XIa by a second, labelled antibody. In case peptides are
used for immunization, peptides coupled to a solid-phase matrix,
also can be used in these assays.
[0058] The preferred assay is an enzyme-linked sorbent assay in
which purified human factor XI or factor XIa (JF Tait et al., J
Biol Chem 1987; 262:11651-11656) is used for coating, and which is
further carried out according to the procedure described by (Smeenk
R T J, et al. 1987, Arthr Rheum 30: 607), which comprises an
incubation step with the hybridoma supernatants and an incubation
step with the labeled anti-mouse immunoglobulin reagent.
[0059] Subsequently, an alternative screening procedure may be used
to assess whether the selected antibody may bind factor XI or
factor XIa in solution. This is achieved by a method in which an
anti-immunoglobulin agent is coupled to a solid-phase matrix, and
bound antibodies against factor XI or factor XIa are specifically
detected using labelled purified factor XI or factor XIa. A
suitable radioimmunoassay procedure for screening anti-factor XI or
anti-factor XIa antibodies may be that described for detection of
anti-C3 antibodies by (Hack et al., J Immunol. 1988; 141:1602-9).
Alternatively, solutions containing human factor XI or factor XIa
may be incubated with the antibody coupled to a solid-phase matrix
via an anti-immunoglobulin reagent. The matrix is then washed, and
bound factor XI or factor XIa is dissociated from it. The eluted
factor XI or factor XIa is then detected by SDS-PAGE followed by
Western blotting.
[0060] Monoclonal antibodies can also be produced in various other
ways with techniques well understood by those having ordinary skill
in the art. Details of these techniques are described in
(Antibodies: A Laboratory Manual, Harlow et al., Cold Spring Harbor
Publications, p. 726, 1988), or are described by (Campbell, A. M.
"Monoclonal Antibody Technology Techniques in Biochemistry and
Molecular Biology," Elsevier Science Publishers, Amsterdam, The
Netherlands, 1984) or by (St. Groth et al., J. Immunol. Methods
35:1-21, 1980). These other techniques include, but are not limited
to techniques for recombinant production of monoclonal
antibodies.
[0061] Monoclonal antibodies of any mammalian species, including
humans, can be used in this invention. Accordingly, the antibodies
according to this embodiment may be human monoclonal antibodies.
Such human monoclonal antibodies may be prepared, for instance, by
the generation of hybridomas, derived from immunised transgenic
animals, containing large sections of the human immunoglobulin (Ig)
gene loci in the germline, integrated by the yeast artificial
chromosomal (YAC) technology (Mendez et al., 1997).
[0062] Furthermore, reference may be made to (Lonberg et al., 1995)
and U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0063] Suitable methods for the production of human monoclonal
antibodies have been described in WO 04/035607 (Genmab) and WO
04/043989 (Medarex). Further similar methods have been described in
WO 03/017935 (Genmab), WO 02/100348 (Genmab), WO 02/064634
(Medarex) and WO 03/040169 (Medarex).
[0064] Fully human antibodies which recognise a selected epitope
can also be generated using a technique referred to as "guided
selection". In this approach a selected non-human monoclonal
antibody, e.g. a mouse antibody, is used to guide the selection of
a completely human antibody recognising the same epitope (Jespers
et al., 1994).
[0065] Additionally, recombinant antibodies, such as chimeric and
humanised monoclonal antibodies comprising both human and non-human
portions, which can be made using standard recombinant DNA
techniques, are within the scope of the invention. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine mAb and a human immunoglobulin constant
region, see e.g. U.S. Pat. No. 4,816,567; and U.S. Pat. No.
4,816,397. Humanised antibodies are antibody molecules from
non-human species having one or more complementarily determining
regions from the non-human species and a framework region from a
human immunoglobulin molecule, see e.g. U.S. Pat. No. 5,585,089.
Such chimeric and humanised monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in WO 87/02671; EP 184 187; EP 171 496; EP
173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EP 125,023, (Better
et al., 1988); (Liu et al., 1987); (Liu et al., 1987); (Sun et al.,
1987); (Nishimura et al., 1987); (Wood et al., 1985); and (Shaw et
al., 1988); (Morrison, 1985); (Oi et al., 1986); U.S. Pat. No.
5,225,539; (Jones et al., 1986); (Verhoeyen et al., 1988); (Beidler
et al., 1988); and (Kwon et al., 2002).
[0066] Moreover, the present invention also comprises antibodies in
which one or more alterations have been made in the Fc region in
order to change functional or pharma-cokinetic properties of the
antibodies. Such alterations may result in a decrease or increase
of C1q binding and CDC (complement dependent cytotoxicity) or of
Fc.gamma.R binding and antibody-dependent cellular cytotoxicity
(ADCC). Substitutions can for example be made in one or more of the
amino acid positions 234, 235, 236, 237, 297, 318, 320, and 322 of
the heavy chain constant region, thereby causing an alteration in
an effector function while retaining binding to antigen as compared
with the unmodified antibody, cf. U.S. Pat. No. 5,624,821 and U.S.
Pat. No. 5,648,260. Further reference may be had to WO 00/42072
disclosing antibodies with altered Fc regions that increase ADCC,
and WO 94/29351 disclosing antibodies having mutations in the
N-terminal region of the CH2 domain that alter the ability of the
antibodies to bind to FcR and thereby decreases the ability of the
antibodies to bind to C1q which in turn decreases the ability of
the antibodies to fix complement. Alterations which prevent or
reduce the complement activation via the constant region of the
antibodies are especially preferred.
[0067] The antibodies of the present invention can be prepared by
using a technique which provides for the production of antibody
molecules by continuous cell lines in culture. These include but
are not limited to the hybridoma technique originally described by
Kohler and Milstein (Kohler et al., 1975).
[0068] Human antibodies of the invention also can be produced in a
host cell transfectoma using, for example, a combination of
recombinant DNA techniques and gene transfection methods as is well
known in the art, see e.g. (Morrison, 1985).
[0069] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification, site directed mutagenesis) and
can be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vectors or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the V.sub.H segment is
operatively linked to the C.sub.H segment(s) within the vector and
the V.sub.L segment is operatively linked to the C.sub.L segment
within the vector. Additionally or alternatively, the recombinant
expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell.
[0070] The antibody chain gene can be cloned into the vector such
that the signal peptide is linked in-frame to the amino terminus of
the antibody chain gene. The signal peptide can be an
immunoglobulin signal peptide or a heterologous signal peptide
(i.e., a signal peptide from a non-immunoglobulin protein).
[0071] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or beta-globin
promoter.
[0072] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. No. 4,399,216, U.S. Pat. No.
4,634,665 and U.S. Pat. No. 5,179,017, all by Axel et al.). For
example, typically the selectable marker gene confers resistance to
drugs, such as G418, hygromycin or methotrexate, on a host cell
into which the vector has been introduced. Preferred selectable
marker genes include the dihydrofolate reductase (DHFR) gene (for
use in dhfr-host cells with methotrexate selection/amplification)
and the neo gene (for G418 selection).
[0073] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection,
lipofectin transfection and the like.
[0074] In one embodiment the antibodies are expressed in eukaryotic
cells, such as mammalian host cells. Preferred mammalian host cells
for expressing the recombinant antibodies of the invention include
CHO cells (including dhfr-CHO cells, described in (Urlaub et al.,
1980), used with a DHFR selectable marker, e.g., as described in
(R. J. Kaufman et al., 1982), NS/0 myeloma cells, COS cells, HEK293
cells and SP2.0 cells. In particular for use with NS/0 myeloma
cells, another preferred expression system is the GS (glutamine
synthetase) gene expression system disclosed in WO 87/04462, WO
89/01036 and EP 338 841. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0075] Alternatively the cloned antibody genes can be expressed in
other expression systems, including prokaryotic cells, such as
microorganisms, e.g. E. coli for the production of scFv antibodies,
algi, as well as insect cells. Furthermore, the antibodies can be
produced in transgenic non-human animals, such as in milk from
sheep and rabbits or eggs from hens, or in transgenic plants. See
e.g. (Verma, et al., 1998); (Pollock et al., 1999); and (Fischer et
al., 1999).
[0076] Additional information concerning the generation, design and
expression of recombinant antibodies can be found in (Mayforth,
Designing Antibodies, Academic Press, San Diego (1993)).
[0077] Regardless of the nature of the antibody, polyclonal,
monoclonal, or recombinant, it may be purified by standard
techniques well known in the art. Most of these techniques use
affinity chromatography, often in combination with a precipitation
step.
[0078] Cell lines that secrete antibody against human factor XI or
factor XIa can be identified by assaying culture supernatants,
ascitic fluid, etc., for the presence of antibody. The preferred
screening procedure comprises two sequential steps, the first being
identification of hybridomas that secrete mAb against human factor
XI or factor XIa, the second being determination of the ability of
the mAb to inhibit the activation or the functional activity of
factor XI and to prevent or reduce activation of factor IX and the
rest of the coagulation system. Factor XI-binding diabodies,
triabodies, tetravalent antibodies of the invention can be produced
by the following methods: 1) chemical linkage of anti-factor XI
antibodies of the present invention or univalent fragments thereof
following a method as described by e.g. (Fanger M W, Morganelli P
M, Guyre P M. Bispecific antibodies. Crit Rev Immunol. 1992;
12:101-24); 2) genetically engineering of non-covalently-linked
diabodies as described by e.g. (Holliger P, Prospero T, Winter G.
"Diabodies": small bivalent and bispecific antibody fragments. Proc
Natl Acad Sci USA. 1993; 90:6444-8) and tetravalent antibodies as
described by e.g. (Pack P, Muller K, Zahn R, Pluckthun A.
Tetravalent miniantibodies with high avidity assembling in
Escherichia coli. J Mol Biol. 1995; 246:28-34); 3) single chain
antibodies fused to protein A or Streptavidin as described by e.g.
(Kipriyanov et al., Affinity enhancement of a recombinant antibody:
formation of complexes with multiple valency by a single-chain Fv
fragment-core streptavidin fusion. Protein Eng. 1996; 9(2):203-11)
and bispecific tetravalent antibodies as described in e.g. EP 0 517
024 to Bosslet and Seeman; 4) genetically engineering of triabodies
as described by e.g. (Kortt et al., Triabodies: single chain Fv
fragments without a linker form trivalent trimers. FEBS Lett. 1997;
409(3):437-41); 5) phage display of Ab combinatorial libraries
resulting in the production of high-affinity antibodies and
screening of random DNA sequence phage display libraries for small
antigen-binding peptides as described in e.g. U.S. Pat. Nos.
5,403,484 and 5,571,698 and 5,223,409, (McGuinness et al., Phage
diabody repertoires for selection of large numbers of bispecific
antibody fragments. Nat Biotechnol. 1996; 14(9):1149-54),
(Hoogenboom H R. Designing and optimizing library selection
strategies for generating high-affinity antibodies. Trends
Biotechnol. 1997; 15(2):62-70); and, 6) generation of hybridomas,
derived from immunised transgenic mice, containing large sections
of the human immunoglobulin (Ig) gene loci in the germ line,
integrated by the yeast artificial chromosomal (YAC) technology,
resulting in effective blocking antibodies as described by e.g.
(Mendez et al., 1997, Nat Genet. 15(2):146-56. Erratum in: Nat
Genet 1997, 16(4):410).
[0079] In a second aspect, the invention relates to methods of
preventing or treating a disease, disorder and/or condition that is
mediated by factor XI activation and/or wherein inhibition of
factor XI has a beneficial effect. The methods preferably comprise
the step of administering to a subject a binding molecule as
disclosed herein above, in an amount effective to treat or prevent
the disease, disorder and/or condition.
[0080] Also in this aspect, the invention also relates to the use
of a binding molecule as disclosed herein above for the preparation
of a medicament for preventing or treating a disease, disorder
and/or condition that is mediated by factor XI activation and/or
wherein inhibition of factor XI has a beneficial effect.
[0081] In this aspect the invention alternatively relates to a
binding molecule as disclosed herein above, for use in preventing
or treating a disease, disorder and/or condition that is mediated
by factor XI activation and/or wherein inhibition of factor XI has
a beneficial effect.
[0082] In one embodiment a disease, disorder and/or condition that
is mediated by factor XI activation and/or wherein inhibition of
factor XI has a beneficial effect is disease, disorder and/or
condition in which coagulation is involved, such as e.g. a
thromboembolic or inflammatory disease mediated by coagulation
activation via factor XI. Thus, in this embodiment the binding
molecules of the invention may be used in a treatment for reducing
or preventing thrombus formation and/or its complications.
[0083] According to another embodiment the binding molecules of the
present invention can be used for inhibiting coagulation in various
human diseases. As a result the inhibitors of the present invention
can be used for the preparation of a medicament for attenuating
thrombo-embolic disorders by inhibiting coagulation in vivo. The
binding molecules can be used alone or in combination with other
drugs.
[0084] In a further embodiment the binding molecules of the present
invention can be used alone or in combination with other drugs in
any suitable ratios, for the preparation of a medicament to treat a
subject suffering of a disease or disease symptoms resulting from
pathologic thrombosis and or embolism, or at risk with respect to
such a disease.
[0085] The present binding molecules inhibit factor XI-dependent
amplification of coagulation or factor XI-dependent coagulation by
inhibiting the activation and or activity of factor XI.
[0086] They are therefore suitable for the prevention or treatment
of disorders, diseases and conditions in which coagulation is
involved. These disorders, diseases and conditions include e.g.
(acute) myocardial infarction, ischemic stroke, cardio-embolism due
to atrial fibrillation, vascular access thrombosis, deep venous
thrombosis, arterial thrombosis, coronary artery thrombosis,
atherosclerosis, arthritis, vasculitis, respiratory distress
syndrome, pulmonary embolism, thrombo-embolism resulting from
surgery such as prostate surgery, orthopaedic surgery, such as e.g.
hip and knee-replacement, thrombo-embolism resulting from
immobilization, thrombosis and occlusion of synthetic grafts,
stents, or AV-fistula, diffuse intravascular coagulation (DIC),
hemodialysis, atrial fibrillation, sepsis, septic shock, organ
failure, kidney failure, toxicity induced by the in vivo
administration of therapeutic proteins (e.g. cytokines or mAbs),
multiple trauma, ischemia-reperfusion injuries and local undesired
fibrin deposition such as e.g. fibrin deposition in the lung
alveoli during adult respiratory distress.
[0087] Thus, in the present invention, patients suffering from a
disease involving coagulation-mediated damage can be administered
an effective amount of a monoclonal antibody against factor XI, or
fragments a monoclonal antibody against factor XI as described so
that activation of factor XI is inhibited. By "effective amount" is
meant a concentration of the binding molecule, which is capable of
inhibiting coagulation activation.
[0088] Treatment (prophylactic or therapeutic) will generally
consist of administering a binding molecule of the invention
parenterally, preferably intravenously, intraarterially,
intramuscularly or subcutaneously. Gruber and Hanson (Gruber and
Hanson, 2003, Blood 102:953-955) administered goat anti-factor XI
antibodies at a dose of 16-50 mg per kg to achieve sufficient
inhibition of factor XI in baboons. In contrast, the dose and
administration regimen of a binding molecule of the invention
preferably is in the range of a dosage that is equivalent to a
dosage of 0.5-20 mg of IgG per kg body weight per week. More
preferably, a binding molecule of the invention is administered at
a dosage that is equivalent to a dosage of less than 18, 16, 14,
12, 10 8, 6 or 4 mg of IgG per kg body weight per week and/or at a
dosage that is equivalent to a dosage of at least 0.6, 0.8, 1.0,
1.2, 1.5, 2, or 4 mg of IgG per kg body weight per week. It is
understood that in case of e.g. antibody fragments the dosage to be
used will be the molar equivalent of the corresponding amount of mg
of an IgG molecule per kg body weight as indicated.
[0089] It is further understood that the dosage regimes for the
binding molecules of the invention are based on the average serum
half-life of a human antibody of about 7 days. The skilled person
will know how to adjust the dosage regime of binding molecules with
a half-life that is shorter or longer than 7 days.
[0090] A further factor that influences the dosage regime for a
given binding molecule of the invention is its affinity for factor
XI. For example, a binding molecule having a Kd of about 1 nM
requires a serum level of about 100 nM to produce 99% factor XI
inhibition and a binding molecule having a Kd of about 0.1 nM
requires a serum level of about 35 nM to produce 99% factor XI
inhibition. On the other hand, 95% factor XI inhibition may be
achieved with a binding molecule having a Kd of about 1 nM at a
serum level of about 50 nM or at 27 nM with a binding molecule
having a Kd of about 0.1 nM. Table 1 shows dosage regimes for
anti-factor XI-binding molecule per kg of body weight as function
of Kd and interval between administrations.
TABLE-US-00001 TABLE 1 Dose of anti/FXI mAb (intact antibody) per
kg of body weight as function of Kd and interval between
administrations for intact human IgG with a half life of 7 days
required for 99% inhibition of circulating factor XI. Interval
between Dose (mg-kg) administrations Kd 0.1 nM Kd 1 nM Kd 10 nM
Minimal plasma level 35 nM (=0.005 mg/ml) 125 nM (=0.02 mg/ml) 1025
nM (=0.2 mg/ml) mAb to achieve 99% inhibition 1 week 0.5 mg/kg body
weight 2 mg/kg body weight 10 mg/kg body weight 2 weeks 1 mg/kg
body weight 4 mg/kg body weight 20 mg/kg body weight 3 weeks 2
mg/kg body weight 8 mg/kg body weight 40 mg/kg body weight 4 weeks
4 mg/kg body weight 16 mg/kg body weight 80 mg/kg body weight
[0091] In a further embodiment, the binding molecule is
administered at a dosage wherein the inhibition of factor XI
activation in a human body fluid is measurable as at least one of
(a) substantially complete blockade of factor IX activation in the
body fluid; and (b) a substantially complete blockade of thrombin
generation in the body fluid. A substantially complete blockade of
factor IX activation and/or of thrombin generation is herein
understood as factor IX activation and/or thrombin generation being
reduced to less than 1% of the amount of factor IX activation and
of thrombin generation in the absence of the binding molecule in
the body fluid.
[0092] An administered dosage that effects substantially complete
blockade of factor IX activation and/or of thrombin generation
preferably is a dosage that yields a molar ratio in the body fluid
that is equal to or less than 35, 4, or 1 moles of factor XI
binding sites of the binding molecule to 1 mole of factor XI,
wherein the factor XI binding sites of the binding molecule have a
Kd that is equal to or less than 10 nM, 1 nM, or 0.1 nM,
respectively. The body fluid is herein understood to be blood,
plasma or serum.
[0093] The skilled person will know how to adjust the dosage
regimes in Table 1 for binding molecules with a half-life that is
shorter or longer than 7 days, with a M.sub.w different from human
IgG, with a Kd different from 10 nM, 1 nM or 0.1 nM and/or to
achieve another percentage of inhibition of circulating factor
XI.
[0094] In a further embodiment, a binding molecule of the
invention, when administered to a human patient via intravenous
infusion, provides complete inhibition of factor XI at dosages
below 0.005 or 0.003 g/kg.
[0095] In a further embodiment, a binding molecule of the
invention, when administered to a human patient via intravenous
infusion, provides therapeutic benefits at dosages below 0.005 or
0.003 g/kg.
[0096] In a further embodiment a binding molecule of the invention
is modified to achieve a desired in vivo serum half life. For this
purpose a polyalkyleneglycol group (e.g. polyethyleneglycol (PEG)
group, polypropylene glycol, polybutylene glycol) or a serum
protein such as e.g. serum albumin or transferrin can be linked or
conjugated to the binding molecule and/or the amino acid sequence
of the binding molecule can be modified. In particular the amino
acid sequence of the constant domains of a binding molecule that is
an antibody can be modified (e.g. introducing amino acid
substitutions, deletions and/or insertions). These modifications be
thus be used to achieve an in vivo serum half life of the binding
molecule of more than 20 days, 10 to 20 days, 5 to 10 days, 1 to 5
days or less than 24 hours.
[0097] In one embodiment, the equivalent of the above-indicated
weekly dosages of the binding molecules according to the invention
can be administered by infusion. Such administration can be
repeated as many times as desired. The administration may be
performed by bolus injection or infusion or continuous infusion
over a period of from less than 2 hours to 24 hours, such as from 2
to 12 hours. In another embodiment, the binding molecules of the
invention can be administered by slow continuous infusion over a
long period, such as more than 24 hours.
[0098] Such regimen may be continued or repeated one or more times
as necessary, for example, after 6 months or 12 months. The dosage
can be determined or adjusted by measuring the amount of
circulating factor XI-binding molecules of the invention antibodies
upon administration in a biological sample by using anti-idiotypic
antibodies which target the factor XI-binding molecules of the
invention. In yet another embodiment, the antibodies can be
administered by maintenance therapy, such as, e.g., once a week for
a period of 6 months or more.
[0099] In one embodiment, the binding molecule of the invention is
used in the prevention or reduction of thrombosis (and occlusion)
of synthetic grafts, stents, or AV-fistula in e.g. kidney-patients
undergoing regular dialysis. In this group of patients the binding
molecule may be administered at least weekly or several times (e.g.
2, 3 or 4) per week, preferably when the patients are undergoing
dialysis. In a preferred embodiment, the binding molecule is
administered to the patient through the dialysis apparatus, e.g. in
the dialysed body fluid that is returned to the patient.
[0100] In another embodiment, the binding molecule of the invention
is administered in patient with no regular parenteral access that
require nonetheless continuous anticoagulant therapy, such as e.g.
in patients with atrial fibrillation, unstable angina pectoris,
deep venous thrombosis, diffuse intravascular coagulation, prostate
surgery, orthopaedic surgery, particularly of the hip, and other
thrombo-embolic disorders. In these patients preferably a certain
number of administrations of the binding molecule per time period
is applied, e.g. once per 2, 3, 4 or 6 weeks. In such instances the
binding molecule preferably has a half life of at least 6, 7, 8,
10, 12 or 14 days and a Kd that is less than 1 nM, preferably less
than 0.5 nM, and most preferably less than 0.1 nM.
[0101] For parenteral administration, the binding molecule will be
formulated in an injectable form combined with a pharmaceutically
acceptable parenteral vehicle. Such vehicles are well-known in the
art and examples include saline, dextrose solution, Ringer's
solution and solutions containing small amounts of human serum
albumin.
[0102] Typically, the monoclonal antibody or fragments thereof will
be formulated in such vehicles at a concentration of from about 20
mg to about 100 mg per ml. In one embodiment of this invention the
binding molecule is administered by intravenous injection.
[0103] It should be understood that intended to come within the
scope of this invention is virtually every method of administering
monoclonal antibodies or fragments thereof as described by the
present invention, to yield sufficiently high levels either in the
circulation or locally.
[0104] In a third aspect, the invention relates to a pharmaceutical
composition comprising a binding molecule as disclosed herein above
and a pharmaceutically acceptable carrier. The pharmaceutical
compositions may be formulated with pharmaceutically acceptable
carriers or diluents as well as any other known adjuvants and
excipients in accordance with conventional techniques such as those
disclosed in (Remington: The Science and Practice of Pharmacy,
19.sup.th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa.,
1995).
[0105] The pharmaceutical compositions according to the invention
may be formulated in accordance with routine procedures for
administration by any route, such as oral, topical, parenteral,
sublingual, transdermal or by inhalation. The compositions may be
in the form of tablets, capsules, powders, granules, lozenges,
creams or liquid preparations, such as oral or sterile parenteral
solutions or suspensions or in the form of a spray, aerosol or
other conventional method for inhalation.
[0106] The term "pharmaceutically acceptable carrier"` relates to
carriers or excipients, which are inherently nontoxic and
nontherapeutic. Examples of such excipients are, but are not
limited to, saline, Ringer's solution, dextrose solution and Hank's
solution.
[0107] Nonaqueous excipients such as fixed oils and ethyl oleate
may also be used. A preferred excipient is 5% dextrose in saline.
The excipient may contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability,
including buffers and preservatives.
[0108] The pharmaceutical composition may be administered by any
suitable route and mode. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. The pharmaceutical compositions
of the present invention include those suitable for oral, nasal,
topical (including buccal and sublingual), rectal, vaginal and/or
parenteral administration.
[0109] Formulations of the present invention which are suitable for
vaginal administration include pessaries, tampons, creams, gels,
pastes, foams or spray formulations containing such carriers as are
known in the art to be appropriate.
[0110] Dosage forms for the topical or transdermal administration
of compositions of this invention include powders, sprays,
ointments, pastes, creams, lotions, gels, solutions, patches and
inhalants.
[0111] The pharmaceutical composition is preferably administered
parenterally. The phrases "parenteral administration" and
"administered parenterally" as used herein means modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0112] In one embodiment that pharmaceutical composition is
administered by intravenous or subcutaneous injection or
infusion.
[0113] In one embodiment the antibody of the invention are
administered in crystalline form by subcutaneous injection, cf.
(Yang et al., 2003 PNAS, 100(12):6934-6939).
[0114] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in the form
of a pharmaceutically acceptable salt or in a suitable hydrated
form, and/or the pharmaceutical compositions of the present
invention, are formulated into pharmaceutically acceptable dosage
forms by conventional methods known to those of skill in the
art.
[0115] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonicity agents,
antioxidants and absorption delaying agents, and the like that are
physiologically compatible.
[0116] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
[0117] Preferably, the carrier is suitable for parenteral
administration, e.g. intravenous or subcutaneous injection or
infusion.
[0118] Pharmaceutical compositions typically must be sterile,
non-pyrogenic 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. Examples of suitable aqueous and
non-aqueous carriers which may be employed in the pharmaceutical
compositions of the invention include water, 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.
[0119] The pharmaceutical 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 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 isotonicity agents, such as sugars,
polyalcohols such as mannitol, sorbitol, glycerol or sodium
chloride in the compositions. Pharmaceutically-acceptable
antioxidants may also be included, for example (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0120] Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0121] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients e.g.
as enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients e.g. 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 ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0122] If appropriate, the binding molecule may be used in a
suitable hydrated form or in the form of a pharmaceutically
acceptable salt. A "pharmaceutically acceptable salt" refers to a
salt that retains the desired biological activity of the parent
compound and does not impart any undesired toxicological effects
(Berge, S. M., et al., (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0123] Depending on the route of administration, the active
compound, i.e., binding molecule, may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound. For example, the
compound may be administered to a subject in an appropriate
carrier, for example, liposomes. Liposomes include
water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan et al., J. Neuroimmunol. 1984; 7:27).
[0124] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for the preparation of such formulations
are generally known to those skilled in the art. See, e.g.,
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0125] The pharmaceutical compositions can be administered with
medical devices known in the art.
[0126] For example, in a preferred embodiment, a therapeutic
composition of the invention can be administered with a needleless
hypodermic injection device, such as the devices disclosed in U.S.
Pat. No. 5,399,163, U.S. Pat. No. 5,383,851, U.S. Pat. No.
5,312,335, U.S. Pat. No. 5,064,413, U.S. Pat. No. 4,941,880, U.S.
Pat. No. 4,790,824, or U.S. Pat. No. 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicaments through the skin; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which
discloses an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0127] In a further embodiment, the binding molecules of the
invention can be formulated to prevent or reduce their transport
across the placenta. This can be done by methods known in the art,
e.g., by PEGylation of the antibodies or by use of F(ab').sub.2
fragments. Further references can be made to (Cunningham-Rundles C
et al, 1992); and to (Landor, 1995).
[0128] Dosage regimens are 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. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0129] 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 compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, 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.
[0130] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a composition of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. It is
preferred that administration be intravenous, intramuscular,
intraperitoneal, or subcutaneous.
[0131] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
[0132] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
[0133] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
EXAMPLES
Materials
[0134] Human factor XI, factor XIa, factor XII, beta-factor XIIa,
factor IX were obtained from Kordia Life Science, Leiden, the
Netherlands. The purity was determined on 12% SDS-PAGE using silver
staining.
[0135] S299
(Methyl-sulfonyl-D-cyclo-hexylglycyl-glycyl-arginine-p-nitroanilide)
was from American Diagnostics (Greenwich, Conn.), and S2366
(L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide) was from
Diapharma (West Chester, Ohio).
[0136] Aprotinin and p-aminobenzamidine (pAB) were from Sigma (St.
Louis, Mo.).
Example 1
Chromogenic assay for factor XIa
[0137] A mixture of 75 .mu.L phosphate-buffered saline (PBS), pH
7.4, containing the chromogenic substrate S-2366 1 mmol/L (final
concentration), 5 .mu.L of factor XIa at different final
concentrations (range, 0.01-10 nmol/L) and 20 .mu.L, PBS are added
to wells of microtiter plates (Dynatech, Plochingen, Germany).
Conversion of the substrate is measured by using a
spectrophotometer. Microtiter plates are read on a Multiskan plate
reader (Labsystems, Helsinki, Finland) after incubation for various
time intervals at room temperature at 405 nm. The effect of
monoclonal antibodies on the chromogenic activity is achieved by
pre-incubation 5 .mu.L of factor XIa at a final concentration of 1
nmol/L with 20 .mu.L PBS containing various concentrations of
monoclonal antibody to be tested for 15 minutes at room
temperature. Seventy-five .mu.L of PBS containing S2366 is added
and the conversion of the substrate is measured as described above.
A reference consisting of various dilutions of factor XIa (0.1-1
nmol/L) is tested as control. In case of antibodies that inhibit
the active site of factor XIa, a decrease in the rate of conversion
by the standard amount of factor XIa is observed. This assay is
assay 1 described in example 8.
Example 2
Chromogenic assay for factor XI activation by factor XIIa
[0138] Five .mu.L of PBS containing 10 nm factor XI is incubated
with 5 .mu.L of 1 nMol/L factor XIIa for 30 minutes at room
temperature. Thereafter, 75 .mu.L phosphate-buffered saline (PBS),
pH 7.4, containing the chromogenic substrate S-2366 1 mmol/L (final
concentration), and 15 .mu.L of PBS are added to wells of
microtiter plates (Dynatech, Plochingen, Germany). Conversion of
the substrate is measured by using a spectrophotometer. Microtiter
plates are read on a Multiskan plate reader (Labsystems, Helsinki,
Finland) after incubation for various time intervals at room
temperature at 405 nm. The effect of monoclonal antibodies on the
activation is tested by adding 15 .mu.L of PBS containing the
antibody to be tested to the mixture of factor XI and factor XIIa,
in a way that first the antibody is added to factor XI, and after
15 minutes incubation factor XIIa is added. Dilutions of factor
XIIa are tested as control. Seventy-five .mu.L of PBS containing
S2366 are then added and the conversion of the substrate is
measured as described above. A reference consisting of various
dilutions of factor XIa (0.1-1 nmol/L) is also tested as control.
In case of antibodies that inhibit the activation of factor XI, a
decrease in the rate of conversion by the amount of factor XIa
generated by factor XIIa is observed. This assay is referred to as
assay 2 in Table 2.
Example 3
Chromogenic Assay for Factor IX Activation by Factor XIa
[0139] This assay is performed essentially as described by T Ogawa
et al., J Biol Chem 2005; 280:23523-30. Briefly, factor IX (1000
nM) in 50 mM Tris-HCl pH 7.5 buffer containing 0.1 mg per ml BSA
containing 5 mM CaCl.sub.2 was activated by addition of factor XIa
(1 nM active sites, 0.5 nM protein). At various time points (0-240
minutes), 50 .mu.l aliquots were removed and supplemented with
aprotinin (final concentration 15 .mu.M) to inhibit factor XIa. The
steady-state kinetics of hydrolysis of S299 (1 mM) by quenched
samples was studied in Tris-BSA buffer containing 5 mM CaCl.sub.2
and 33% ethylene glycol. Changes in absorbance at 405 nm were
measured. Duplicate assays were run. The factor IX concentration
tested in the assay was 1000 nM. Generation of factor IXa as a
function of time was determined by interpolation from the linear
dependence of the initial rate of 5299 hydrolysis on known
concentrations of factor IXa. Initial steady-state rates of factor
IXa formation were determined from slopes of plots documenting
linear appearance of factor IXa with time. The effect of monoclonal
antibodies in the assay is tested by pre-incubation of factor XIa
with an equal volume of monoclonal antibody.
[0140] This assay is referred to as assay 3 in Table 2.
Example 4
Clotting Assay for Factor XI
[0141] Effect of the monoclonal antibodies on factor XI or factor
XIa clotting activity is tested by incubating factor XI or factor
XIa with an equal volume of PBS containing 1 to 10 molar excess of
antibody versus factor XI. Factor XI clotting activity is then
tested with a one-stage clotting assay. These assays are well known
to those skilled in the art. These assays are referred to as assay
4 in Table 2 when performed with factor XI, and as assay 5 when
performed with factor XIa.
Example 5
Immunization with Human Factor XI or Factor XIa Immunogens and the
Production of Hybridomas
[0142] The following describes the immunization of mice with
purified human factor XI with the aim to isolate lymphocytes from
the immunised mice and producing murine hybridomas. It will be
further appreciated that the procedure can be employed to produce
antibodies against factor XI, factor XIa or fragments thereof.
[0143] A suitable procedure for immunisation is described by (Hack
C E, Paardekooper J, Smeenk R J, Abbink J, Eerenberg A J, Nuijens J
H. Disruption of the internal thioester bond in the third component
of complement (C3) results in the exposure of neodeterminants also
present on activation products of C3. An analysis with monoclonal
antibodies. J Immunol. 1988; 141:1602-9), for C3. A similar
procedure is used for factor XI or factor XIa. Briefly, mice are
immunised by repeated intraperitoneal injections of 25 .mu.g of
purified human factor XI or XIa given at three-week intervals. The
first factor XI or XIa gift is mixed with complete Freund's
adjuvant, the subsequent with incomplete Freund's adjuvant. Four
days after the final boost, spleens are removed from the immunised
mice and the splenocytes are fused with the murine myeloma
cell-line SP2/0-Ag14, according to the procedure first described by
(Kohler G, Milstein C. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature. 1975; 256:495-7),
except that feeder cells are replaced by IL-6. Immunised mice are
sacrificed and splenocytes teased from the spleens, and washed in
serum free Dulbecco's Modified Eagles medium. Similarly, SP2/0-Ag14
myeloma cells are washed, and added to the splenocytes yielding a
5:1 ratio of splenocytes to myeloma cells. The cells are then
pelleted, and the supernatant is removed. One ml of a 40% (v/v)
solution of polyethylene glycol 1500 is then added dropwise over a
60 sec period, after which the cells are incubated for another 60
sec at 37.degree. C. Nine ml of Dulbecco's Modified Eagles medium
is then added with gentle agitation. The cells are pelleted, washed
to remove residual polyethylene glycol, and finally plated at a
concentration of 10.sup.5 cellsper well in Dulbecco's Modified
Eagles medium containing 10% (v/v) fetal calf serum (100 .mu.l per
well). After 24 hours, 100 .mu.l of a 2.times. solution of
hypoxanthine/azaserine selection medium is added to each well. At
day 4 hypoxanthine/azaserine selection medium is replenished, at
day 7 it is replaced by Dulbecco's Modified Eagles medium
containing 10% (v/v) fetal calf serum. During all incubations
monocyte derived or recombinant human IL-6 is present in the
culture at concentrations of approximately 10 pg/ml. About 80% of
the wells exhibit cell growth at day 10.
Example 6
Enzyme Linked Immunosorbent Assay for the Detection of Factor XI
Antibodies
[0144] The wells are screened for the presence of antibody to
factor XI or XIa using an enzyme-linked immunosorbent assay, in
which purified human factor XI or XIa is used for coating (2
.mu.g/ml in phosphate buffered saline, pH 7.4 [PBS]; 100
.mu.l/well). Residual non-specific binding sites are then blocked
by 30 minutes incubation at room temperature with PBS/0.1% (w/v)
Tween 20 (PBS-T) containing 0.2% (w/v) gelatin (PBS-TG). Then,
after a washing procedure (5 times with PBS-T), the plates are
incubated for 60 minutes at 37.degree. C. with 20 .mu.l of
hybridoma supernatant together with 80 .mu.l of PBS-TG. Finally,
bound murine antibodies are detected by incubation with
peroxidase-conjugated polyclonal goat anti-mouse immunoglobulin
antibodies for 120 minutes at 37.degree. C. Finally, the plates are
washed with distilled water (5 times), and developed with
3,5,3',5'-tetramethyl benzidine. Anti-factor XI antibodies obtained
in this way are further analyzed. This assay is referred to as
assay 6 in Table 2, the denotation is FXI/FXIa when a mAb binds to
factor XI and factor XIa when tested in these ELISAs; FXI when only
binding to factor XI and not to factor XIa; and FXIa when the
monoclonal antibody only binds to factor XIa and not to factor
XI.
Example 7
Preparation of Purified Monoclonal Antibody Anti-Factor XI
[0145] Antibody is produced in vitro from the hybridoma anti-factor
XI or XIa by culturing the cells in 1 liter roller-bottles in
Iscove's Modified Dulbecco medium supplemented with 2% (v/v) fetal
calf serum, 10 pg/ml IL-6, 50 .mu.M 2-mercaptoethanol, and
penicillin and streptomycin. The cells are grown to a density of
>10.sup.6 cells per ml, and one to two weeks later the
supernatants are collected. Solid ammonium sulphate is added to
yield 50% saturation (i.e., approximately 2M), and an
antibody-enriched fraction is obtained by centrifugation for 30
minutes at 1,300 g. The precipitate is dissolved in 1.5 M NaCl/0.75
M glycine, pH 8.9, and put onto a protein A-Sepharose column
(Pharmacia). The column is washed with PBS, and then anti-factor XI
monoclonal antibody is eluted off with glycine-HCl, pH 2.5.
Fractions are neutralised instantaneously with 2M TRIS, pH 8.0, and
those containing protein are pooled and dialysed against PBS.
Example 8
Functional Characterization of Anti-Factor XI or XIa Monoclonal
Antibodies
[0146] Antibodies against factor XI are characterized by testing
them in the assays described above. Outcome of these experiments is
as shown in Table 2.
TABLE-US-00002 TABLE 2 Examples of characterization of monoclonal
antibodies against factor XI mAb Assay 1 Assay 2 Assay 3 Assay 4
Assay 5 Assay 6 Interpretation 1 No effect No effect No effect No
effect No effect FXI/FXIa MAB binds to all forms of FXI; not
suitable as inhibitor 2 No effect No effect No effect No effect No
effect FXI MAB binds to native FXI; not suitable as inhibitor 3 No
effect No effect No effect No effect No effect FXIa MAB binds to
neo- epitope on FXIa; not suitable as inhibitor 4 Inhibits Inhibits
Inhibits Inhibits Inhibits FXI/FXIa MAb binds to active site of
FXI(a); inhibitor of FXI 5 No effect Inhibits No effect Inhibits No
effect FXI MAb binds native FXI near to Arg.sub.369-Ile.sub.370;
inhibitor of FXI 6 No effect No effect Inhibits Inhibits Inhibits
FXI/FXIa MAb binds to FIX binding site of FXI; inhibitor 7 No
effect No effect No effect Inhibits Inhibits FXI/FXIa MAb binds to
HK binding site of FXI; potential inhibitor 8 No effect Inhibits
Inhibits Inhibits Inhibits FXIa MAb binds to neo- epitope near to
active site; inhibitor
[0147] For application as inhibitor several types of antibody are
selected, referred to in the table as "inhibitor". Note that this
table is not complete, other functional patterns may be
encountered.
Example 9
Effects of Anti-Factor XI Monoclonal Antibody on Experimental
Thrombosis in Mouse Model
[0148] Efficacy of anti-factor XI monoclonal antibody to attenuate
or prevent thrombosis in vivo is studied in factor XI knock-out
mice (D Gailani et al., Blood Coagul. Fibrinolysis. 1997;
8:134-144), which are supplemented with human factor XI. The
animals are then subjected to experimental thrombus formation
induced by FeCl.sub.3-induced injury, for example as described by T
Renne et al., J Exp Med 2005; 202:271-281. Briefly, 4-5-wk-old mice
are anesthetized by i.p. injection of 2,2,2-tribromoethanol and
2-methyl-2-butanol (0.15 ml/10 g of body weight from a 2.5%
solution; Sigma-Aldrich). Human factor XI (for example 0.2 mg/kg)
is injected via the tail vein, shortly before the experiment is
started. 10e8 CFSE-labeled platelets per mouse are injected via the
tail vein as a second injection. Monoclonal antibody against human
factor XI is administered either together with, before or after the
injection of human factor XI at a dose of 0.5-10 mg/kg. The
mesentery is externalized through a midline abdominal incision.
35-60-.mu.m-diameter arterioles are visualized at 10.times. with an
inverted microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.)
equipped with a 100-W fluorescent lamp source (HBO) and a CCD
camera (CV-M300; Visitron Systems GmbH) connected to an S-VHS video
recorder (AG-7355; Panasonic). After topical application of a
filter paper (2.times.1 mm) saturated with 20% FeCl.sub.3 for 1
min, arterioles are monitored for 40 min or until complete
occlusion (blood flow stopped for >1 min) occurs. Platelet
adhesion is defined as the number of fluorescently labeled
platelets bound to the vessel wall 5 min after injury. A thrombus
was defined as a platelet aggregate >20 .mu.m in diameter.
Efficacy of the monoclonal antibody to inhibit thrombus formation
is shown by comparing results of mice supplemented with human
factor XI only (thrombus formation; occlusion), human factor XI and
anti-factor XI monoclonal antibody (no thrombus formation; no
occlusion), or not supplemented at all (no thrombus formation; no
occlusion), and wild-type mice (thrombus formation; occlusion).
Example 10
Effects of Anti-Factor XI Monoclonal Antibody on a Primate Model
for Experimental Thrombosis
[0149] Efficacy of anti-factor XI monoclonal antibody to attenuate
or prevent thrombosis in vivo is also studied in non-human primate
models for thrombosis, for example the models as described by A
Gruber et al. (Blood. 2003; 102:953-955). Briefly, baboons are
appropriately anaestesized and injected intravenously with
intravenous porcine heparin (1000 U/mL; Wyeth-Ayerst, Pearl River,
N.Y.) as an anti-coagulant control or affinity-purified monoclonal
antibody against factor XI at a dose of 0.5-10 mg per kg. In
addition, some animals do not receive heparin or anti-factor XI
monoclonal antibody. The antithrombotic effects of heparin or
anti-factor XI monoclonal antibody are studied using thrombogenic
devices deployed for 60 minutes into surgically placed high flow
chronic arteriovenous (AV) shunts. The device to be used can be a
20-mm long dacron graft segment (0.25 mL) and a silicone extension
chamber (1.3 mL), or a 20-mm long (0.25 mL) ringed expanded
polytetrafluoroethylene (ePTFE, teflon) graft segments (WL Gore,
Newark, Del.) in the shunt. The hypothrombogenic ePTFE graft is
converted into an acutely thrombogenic tissue factor-dependent
device as described by Gruber et al. Prior to deployment, the graft
is rinsed by passing 50 mL saline through the lumen. Blood flow is
kept at 100 mL/min by proximal clamping. Thrombogenesis is assessed
by measuring radiolabeled fibrin and platelet contents of the
thrombogenic devices. In brief, the terminal fibrin content of
graft/chamber thrombi (fibrin deposition) was determined by direct
.sup.125Iodine-labeled fibrin counting. The number of deposited
platelets in 35-cm long 4-mm internal diameter shunt segments that
incorporate the devices and associated thrombi is quantified by
.sup.111Indium-labeled platelet imaging with 5-minute data
acquisition periods. Net platelet accumulation rate (NPAR) is
calculated as the change in the platelet content of the device
within one period.
[0150] Those skilled in the art will recognize or be able to
ascertain, using routine experimentation, many equivalents of the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
Any combination of the embodiments disclosed in the dependent
claims is also contemplated to be within the scope of the
invention.
Sequence CWU 1
1
11607PRTHomo sapiens 1Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys
Phe Glu Gly Gly Asp1 5 10 15Ile Thr Thr Val Phe Thr Pro Ser Ala Lys
Tyr Cys Gln Val Val Cys 20 25 30Thr Tyr His Pro Arg Cys Leu Leu Phe
Thr Phe Thr Ala Glu Ser Pro 35 40 45Ser Glu Asp Pro Thr Arg Trp Phe
Thr Cys Val Leu Lys Asp Ser Val 50 55 60Thr Glu Thr Leu Pro Arg Val
Asn Arg Thr Ala Ala Ile Ser Gly Tyr65 70 75 80Ser Phe Lys Gln Cys
Ser His Gln Ile Ser Ala Cys Asn Lys Asp Ile 85 90 95Tyr Val Asp Leu
Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser Val Ala 100 105 110Lys Ser
Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val His Cys 115 120
125His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu His Arg
130 135 140Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr
Arg Ile145 150 155 160Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser
Leu Lys Ser Cys Ala 165 170 175Leu Ser Asn Leu Ala Cys Ile Arg Asp
Ile Phe Pro Asn Thr Val Phe 180 185 190Ala Asp Ser Asn Ile Asp Ser
Val Met Ala Pro Asp Ala Phe Val Cys 195 200 205Gly Arg Ile Cys Thr
His His Pro Gly Cys Leu Phe Phe Thr Phe Phe 210 215 220Ser Gln Glu
Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu Leu Lys225 230 235
240Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg Ile Lys Lys Ser Lys Ala
245 250 255Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro
Val Phe 260 265 270Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu
Gly Glu Glu Leu 275 280 285Asp Ile Val Ala Ala Lys Ser His Glu Ala
Cys Gln Lys Leu Cys Thr 290 295 300Asn Ala Val Arg Cys Gln Phe Phe
Thr Tyr Thr Pro Ala Gln Ala Ser305 310 315 320Cys Asn Glu Gly Lys
Gly Lys Cys Tyr Leu Lys Leu Ser Ser Asn Gly 325 330 335Ser Pro Thr
Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly Tyr Thr 340 345 350Leu
Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile Lys Pro 355 360
365Arg Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro Trp Gln
370 375 380Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys
Gly Gly385 390 395 400Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala
Ala His Cys Phe Tyr 405 410 415Gly Val Glu Ser Pro Lys Ile Leu Arg
Val Tyr Ser Gly Ile Leu Asn 420 425 430Gln Ser Glu Ile Lys Glu Asp
Thr Ser Phe Phe Gly Val Gln Glu Ile 435 440 445Ile Ile His Asp Gln
Tyr Lys Met Ala Glu Ser Gly Tyr Asp Ile Ala 450 455 460Leu Leu Lys
Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln Arg Pro465 470 475
480Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr Asp Cys
485 490 495Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile
Gln Asn 500 505 510Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn
Glu Glu Cys Gln 515 520 525Lys Arg Tyr Arg Gly His Lys Ile Thr His
Lys Met Ile Cys Ala Gly 530 535 540Tyr Arg Glu Gly Gly Lys Asp Ala
Cys Lys Gly Asp Ser Gly Gly Pro545 550 555 560Leu Ser Cys Lys His
Asn Glu Val Trp His Leu Val Gly Ile Thr Ser 565 570 575Trp Gly Glu
Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr Thr Asn 580 585 590Val
Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala Val 595 600
605
* * * * *