Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-FACTOR XI ANTIBODIES
TECHNICAL FIELD
[0001] This disclosure relates to antibodies capable of binding to the
coagulation factor XI
(FXI) and/or its activated form factor Xla (FXIa), and to fragments of FXI
and/or FXIa, and uses
thereof, including uses as anticoagulation agents for treating thrombosis that
do not compromise
hemostasis.
BACKGROUND
[0002] Thrombosis is a condition that involves blood clotting in a blood
vessel, thereby
blocking or obstructing blood flow in the affected area. This condition can
lead to serious
complications if the blood clots travel along the circulatory system to a
crucial body part such as
heart, brain, and lungs, causing heart attack, stroke, pulmonary embolism,
etc. Thrombosis is the
major cause of most strokes and myocardial infarctions, deep vein thrombosis
(DVT), pulmonary
embolism, and other cardiovascular events.1'2 Thrombosis can be treated or
prevented by
anticoagulants such as low-molecular-weight heparin, warfarin, and Factor Xa
direct inhibitors.
The most common adverse effect of these currently available therapies is
impairing
haemostasis.3-5 Therefore, these therapies are limited by the dose and patient
compliance because
patients are required to be closely monitored after the treatment.
[0003] There is a need for an effective thrombosis therapy or prophylaxis
with minimal side
effects. This disclosure satisfies the need in the art.
SUMMARY
[0004] Provided herein in certain embodiments are antibodies that bind to
coagulation factor
XI (FXI) and/or its activated form factor XIa (FXIa), and to fragments of FXI
and/or FXIa. In
some embodiments, the antibodies are monoclonal antibodies. In some
embodiments, the
antibodies are recombinant antibodies. In some embodiments, the antibodies are
humanized
antibodies. In some embodiments, the antibodies are immunologically active
portions of
immunoglobulin molecules, e.g., Fabs, Fvs, or scFvs. In some embodiments, the
antibodies bind
to the A3 domain of FXI and/or FXIa. In some embodiments, the antibodies
include one or more
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CDRs consisting of or comprising the amino acid sequences of SEQ ID NOs: 11-
16, 27-32, 43-
48, 59-64, 75-80, 91-96, 107-112, 123-128, 139-144, 155-160, 171-176, and 187-
192.
[0005] Provided herein is a pharmaceutical composition for treating and/or
preventing
thrombosis and/or complications or conditions associated with thrombosis. The
pharmaceutical
composition comprises one or more anti-FXI and/or anti-FXIa antibodies as
disclosed herein. In
some embodiments, the pharmaceutical composition further comprises one or more
pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or
a combination
thereof.
[0006] Provided herein is a nucleic acid encoding an anti-FXI and/or anti-
FXIa antibody as
disclosed herein, or a functional fragment of either antibody, as well as a
vector comprising the
nucleic acid, and a host cell comprising the vector. In some embodiments, the
vector is an
expression vector that is capable of producing the antibody or a functional
fragment thereof
encoded by the nucleic acid in a host cell.
[0007] Provided herein is a kit comprising one or more anti-FXI and/or anti-
FXIa antibodies
as disclosed herein for use in treating and/or preventing thrombosis and/or
complications or
conditions associated with thrombosis. Alternatively, the kit comprises a
pharmaceutical
composition comprising one or more anti-FXI and/or anti-FXIa antibodies as
disclosed herein for
use in treating and/or preventing thrombosis and/or complications or
conditions associated with
thrombosis. In certain embodiments, the kit further comprises instructions for
use.
[0008] Provided herein is a method of treating and/or preventing thrombosis
and/or
complications or conditions associated with thrombosis. The method includes
administering to a
subject in need thereof a therapeutically effective amount of one or more anti-
FXI and/or anti-
FXIa antibodies as disclosed herein. Alternatively, the method includes
administering to a
subject in need thereof a therapeutically effective amount of a pharmaceutical
composition
containing an anti-FXI antibody, an anti-FXIa antibody, or a functional
fragment of either
antibody.
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[0009] Provided herein is a use of an anti-FXI and/or anti-FXIa antibody as
disclosed herein
formulating a medicament for treating and/or preventing thrombosis and/or
complications or
conditions associated with thrombosis.
[0010] Provided herein is a method of producing an anti-FXI and/or anti-
FXIa antibody as
disclosed herein. The method entails the steps of transforming a host cell
with a vector
comprising a nucleic acid encoding the antibody, and expressing the antibody
in the host cell.
The method can further include purifying the expressed antibody from the host
cell. Additionally,
the purified antibody can be subjected to modifications such that the modified
recombinant
antibody retains the activity of the corresponding human antibody.
Alternatively, an antibody
disclosed herein can be produced from culturing a hybridoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figures 1A-1E illustrate the effects of five anti-FXI antibodies via
APTT assay in
human plasma. Human plasma supplemented with five different antibodies at a
concentration
ranging from 0 to 400 nM were tested in an APTT assay as described in Example
3. The five
antibodies tested included 19F6 (A), 34F8 (B), 42A5 (C), 1A6 (D), and 14E11
(E). Antibodies
1A6 and 14E11 were used as positive controls in this experiment.
[0012] Figures 2A-2C illustrate the effects of antibodies 19F6 (A), 34F8
(B), and 42A5 (C)
on the APTT assay in monkey plasma. The monkey plasma supplemented with three
different
antibodies at a concentration ranging from 0 to 400 nIVI were tested in an
APTT assay as
described in Example 4.
[0013] Figures 3A-3F illustrates SPR sensorgrams for FXI binding to
immobilized h-19F6
(A), h-34F8 (B), and h-42A5 (C), as well as SPR sensorgrams for FXIa binding
to immobilized
h-19F6 (D), h-34F8 (E), and h-42A5 (F). Data were fit with 1:1 binding model,
and curve fits at
test concentrations of FXI (0.005 - 1 ng/mL) are shown overlaid on the
sensorgrams. Each curve
indicates a different test concentration of FXI or FXIa.
[0014] Figures 4A-4C illustrate the concentration-response curves of
antibodies h-19F6 (A),
h-34F8 (B), and h-42A5 (C) inhibiting human FXIa from hydrolyzing S-2366.
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[0015] Figures 5A-5B illustrate the inhibitory effects of antibodies h-19F6
(A) and h-42A5
(B) on FXIa-mediated activation of FIX to FIXa. Human FIX (200 nM) was
incubated with
FXIa (5 nM) in PBS with 5 mM CaCl2 at room temperature with 1 uM h-19F6 or h-
42A5. At the
indicated intervals, samples were collected and the FIX as well as FIXa was
determined by
Western blots using goat anti-human FIX IgG (Affinity Biologicals). Figures 5C-
5D illustrate
the inhibitory effects of antibodies h-19F6 (C) and h-42A5 (D) on FXIIa-
mediated activation of
FXI to FXIa. Human FXI (500 nM) was incubated with FXlla (50 nM) in the
presence of 1 uM
of h-1 9F6 or h-42A5. FXI, as well as FXIa light chain, which represents FXIa
production, at
indicated time points was determined by Western blots. A human IgG4 (1 uM) was
used as the
control.
[0016] Figures 6A-6C illustrate the effects of antibodies h-34F8, h-19F6,
and h-42A5 on
APTT in cynomolgus monkeys. The monkeys were intravenously administered with
indicated
doses of h-34F8 (A), h-19F6 (B), and h-42A5 (C). Ex vivo clotting time APTT
was determined
at pre-dose (time 0), and 0.5, 1, 3, 6, 12, and 24 hours post-dose.
[0017] Figures 7A-7C illustrate the effects of antibodies h-34F8, h-19F6,
and h-42A5 on PT
in cynomolgus monkeys. Monkeys were intravenously administered with the
indicated doses of
h-34F8 (A), h-19F6 (B), and h-42A5 (C). Ex vivo clotting time PT was
determined at pre-dose
(time 0), and 0.5, 1, 3, 6, 12, and 24 hours post-dose.
[0018] Figures 8A-8C illustrate the effects of antibodies h-34F8, h-19F6,
and h-42A5 on AV
shunt thrombosis in cynomolgus monkeys. Escalating levels of h-34F8 (A), h-
19F6 (B), or h-
42A5 (C) were intravenously administered to monkeys (n=3 for h-34F8 and h-
19F6; n=4 for h-
42A5), changes of clot weight from pre-dose were determined in monkey model of
AV shunt
thrombosis. *P <0.05, **P < 0.01 and * **P <0.001 vs. Vehicle.
[0019] Figures 9A-9C illustrate the effects of antibodies h-34F8, h-19F6,
and h-42A5 on
bleeding time in cynomolgus monkeys. Escalating levels of h-34F8 (A), h-19F6
(B), or h-42A5
(C) were intravenously administered to monkeys (n=3 for 34F8 and h-19F6; n=4
for h-42A5),
bleeding time was assessed at pre-dose and at 30 min post each dose.
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[0020] Figures 10A-10B illustrate the antithrombotic effects of antibodies
h-34F8, h-19F6,
and h-42A5. Four groups of monkeys (n = 5) were intravenously administered
with the vehicle,
0.3 mg/kg of h-34F8, h-19F6, or h-42A5, for 2 hours, and FeCl3 was applied on
the left femoral
artery of each animal to induce thrombosis. The time to 80% thrombotic
occlusion (A) and to
100% thrombotic occlusion (B) were determined by monitoring the blood flow
velocity. *P <
0.05 and **P < 0.01 vs. vehicle.
[0021] Figures 11A-11D illustrate that the treatment with antibodies h-
34F8, h-19F6, or h-
42A5 did not prolong the bleeding time in monkeys. Four groups of monkeys (n =
5) were
intravenously administered with the vehicle, 0.3 mg/kg of h-34F8, h-19F6, or h-
42A5, and
template bleeding time was measured pre-dose and 1 hour post-dose. The
individual bleeding
time in h-34F8, h-19F6, and h-42A5 treated group is shown in (A), (B) and (C),
respectively.
The bleed time change upon vehicle, h-34F8, h-19F6, or h-42A5 treatment is
shown in (D).
[0022] Figures 12A-12B illustrate the effects of antibodies h-34F8, h-19F6,
and h-42A5 on
clotting times of monkey plasma. Four groups of monkeys (n = 5) were
intravenously
administered with the vehicle, 0.3 mg/kg of h-34F8, h-19F6, and h-42A5,
respectively, and blood
was collected pre-dose and about 3 hours post-dose for plasma preparation and
clotting time
APTT and PT determination. The APTT changes and PT changes are shown in (A)
and (B),
respectively. **P < 0.01 and ***P < 0.001 vs. vehicle.
[0023] Figure 13 illustrates the amino acid sequence of human FXI (SEQ ID
NO: 203).
[0024] Figures 14A-14B illustrate the effects of modified antibodies h-1
9F6 (A), and h-42A5
(B) on APTT in cynomolgus monkeys. The monkeys were intravenously administered
with
indicated doses of modified h-1 9F6 and h-42A5. Ex vivo clotting time APTT was
determined at
pre-dose (time 0), and 0.5, 2, 6, 12, 24, 48, 96, 168, 240, and 336 hours post-
dose.
[0025] Figures 15A-15B illustrate the effects of modified antibodies h-1
9F6 (A), and h-42A5
(B) on PT in cynomolgus monkeys. Monkeys were intravenously administered with
the
indicated doses of modified h-1 9F6 and h-42A5. Ex vivo clotting time PT was
determined at pre-
dose (time 0), and 0.5, 2, 6, 12, 24, 48, 96, 168, 240, and 336 hours post-
dose.
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[0026] Figures 16A-16B illustrate the effects of h-1 9F 6 and h-42A5 on
APTT and PT in
human plasma. Figure 16A shows the effects of h-19F6 and h-42A5 on APTT in
human
plasma. Figure 16B shows the effects of h-19F 6 and h-42A5 on PT in human
plasma.
[0027] Figure 17 shows the binding specificity of test antibodies to human
FXI. In
Western blotting, 10 [11_, of human standard plasma or FXI-deficient plasma
were served as
F XI-positive and F XI-negative controls.
[0028] Figure 18 shows the effects of h-19F6 and h-42A5 in AV shunt
thrombosis
models on bleeding times recorded at pre-dose and 1-hour post-dose.
[0029] Figures 19A-19D show the binding properties of h-19F6 and h-42A5 to
human
FXI. Figure 19A shows sensorgrams for h-19F6 captured on a sensor chip
subjected to
flows of indicated concentrations of FXI. Figure 19B shows sensorgrams for h-
42A5
captured on a sensor chip subjected to flows of indicated concentrations of
FXI. Figure 19C
shows antibodies captured when test antibodies (5 p.g/mL) flew through a
sensor chip
immobilized with equal amounts of 4 mutant FXIs in which the Al, A2, A3, or A4
domain
was replaced with the corresponding domain from prekallikrein. A reported anti-
FXI
antibody, 01A6, was also tested a positive control. Figure 19D shows that FXI
was
immobilized on a sensor chip. H-19F6 and h-42A5 (5 Kg/m1) were successively
injected
into flow cells on the sensor surface at a flow rate of 30 p1/minute, and the
response change
was monitored. The experiment was performed twice, and a representative result
is depicted.
[0030] Figures 20A-20B show the binding properties of h-1 9F 6 and h-42A5
to human
FXIa. Figure 20A shows sensorgrams for h-19F6 captured on a sensor chip
subjected to
flows of indicated concentrations of FXIa. Figure 20B shows sensorgrams for h-
42A5
captured on a sensor chip subjected to flows of indicated concentrations of
FXIa.
DETAILED DESCRIPTION
[0031] The following description of the invention is merely intended to
illustrate various
embodiments of the invention. As such, the specific modifications discussed
are not to be
construed as limitations on the scope of the invention. It will be apparent to
one skilled in the art
that various equivalents, changes, and modifications may be made without
departing from the
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scope of the invention, and it is understood that such equivalent embodiments
are to be included
herein.
[0032] Both intrinsic pathway and extrinsic pathway are involved in in vivo
blood
coagulation cascades. The intrinsic pathway, also called the contact
activation pathway, is
initiated by contact with a surface interface and results in activation of
FXH. The intrinsic
pathway also involves FXI, FIX and FVIII. The extrinsic pathway, also called
the tissue factor
(TF) pathway, is initiated by vascular injury and results in the formation of
an activated complex
of TF-FVIIa. These two pathways meet and activate the common pathway, leading
to conversion
of prothrombin to thrombin and eventually the formation of cross-linked fibrin
clot. An ideal
anticoagulant should be efficacious in preventing thrombosis without
compromising haemostasis.
Several lines of evidence suggest that the intrinsic coagulation pathway is
important for the
amplification phase of coagulation, whereas extrinsic and common pathways are
more
heavily involved in the initiation and propagation phases.5-8 These findings
indicate that the
intrinsic pathway plays a minor role during normal haemostasis and that the
inhibition of
intrinsic pathway may provide antithrombotic benefits with low bleeding risk.
FXI, a
component of the intrinsic pathway, has recently become an attractive target,
as it may have the
potential to elicit anti-thrombosis effects without affecting bleeding.3'5'6
[0033] FXI can be activated by factor XIIa via the intrinsic pathway to
FXIa, which in
turn activates factor IX. Epidemiological studies have suggested that FXI
deficiency in
humans is associated with decreased risk of venous thromboembolism and stroke,
whereas
increased FXI levels are associated with increased risk.9-11 In addition, FXI-
deficient
humans show a very low bleeding tendency.12,13 Furthermore, mice deficient in
FXI are
protected against many types of thrombosis without increased bleeding.14
Moreover, small-
molecule inhibitors, antibodies and antisense oligonucleotides that inhibit
FXI have
demonstrated antithrombotic properties with no bleeding risk in many animal
models of
thrombosis.
[0034] The antibodies disclosed herein binds to FXI and/or FXIa and target
the intrinsic
pathway of blood coagulation. The structure of FXI and FXI's involvement in
blood coagulation
have been reported in various publications.33
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[0035] Animal and clinical studies have suggested a robust association
between FXI and
thrombosis. FXI-deficient mice have been studied by many research teams and
have
displayed remarkable antithrombotic phenotypes in several models, including
FeCl3-induced
arterial and deep vein thrombosis models, a pulmonary embolism model, and a
cerebral artery
occlusion model.14,17,22,23 In human epidemiological studies, patients with
congenital FXI
deficiency are insusceptible to venous thromboembolism (VTE) orischaemic
stroke, and
subjects with higher levels of FXI are at greater risk for VTE and ischaemic
stroke than
those with lower levels.9-11 For physiological haemostasis, the role of FXI
appears
dispensable. FXI-deficient mice do not show excessive bleeding, as their tail-
bleeding times
are comparable to those of wild-type animals.23'24 In addition, severely FXI-
deficient
patients do not exhibit spontaneous bleeding, although they may display a
variable bleeding
tendency during surgical operations.12' 13 Combination of two or more anti-
thrombotics are
widely used clinically. A previous study showed that aspirin caused a similar
bleeding
tendency in wild-type and FXI-deficient mice, suggesting that targeting FXI
might still be
safe even in the presence of other anti-thrombotic therapies.14
[0036] All of the above-mentioned findings indicate that FXI/FXIa is a safe
drug target
for treating thrombosis-related diseases without compromising haemostasis.
Thus, many
approaches have been applied to target FXI/FXIa for developing therapeutics
for treating
thrombosis, such as antibodies, oligonucleotides, and small-molecule
inhibitors.5 As
described herein, antibody-type blockers of FXI/FXIa were generated. The
advantages of
antibodies include fast-acting properties and a low frequency of dosing, and a
major weakness
of antibodies is their potential immunogenicity.25 At least two test
antibodies were
humanized before conducting in vivo studies. Two humanized antibodies, h-19F6
and h-
42A5, demonstrated very high affinity to human FXI/FXIa. Interestingly, they
bind different
regions but the same domain (A3) of FXI. Without bound by any theory, the
antibodies might
both inhibit FXIa activity but have no effect on FXI activation mediated by
either FXIIa or
thrombin.
[0037] Various types of FXI/FXIa inhibitors have prolonged APTT and
exhibited
antithrombotic effects in different models. Anti-FXI antibody 14E11 increased
APTT by
approximately 1.3-fold and reduced thrombosis in exteriorized femoral
arteriovenous shunts
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in baboons.17 An antisense oligonucleotide inhibiting FXI expression reduced
plasma FXI
levels by approximately 50% and decreased thrombus formation in baboons.26'27
In addition,
an orally bioavailable small-molecule FXIa inhibitor, ONO-5450598,
significantly inhibited
thrombosis formation in monkey models of thrombosis.28 Furthermore, the
antithrombotic
effects of therapeutics targeting FXI/FXIa have also been confirmed in many
non-primate
animal models, such as mouse and rabbit thrombosis models.19'29-31 A recent
clinical trial
showed that an antisense oligonucleotide targeting FXI prevented venous
thrombosis in
patients undergoing knee arthroplasty.32 As demonstrated in the working
examples, two
distinctive primate thrombosis models were used to evaluate the antithrombotic
effects of h-
19F6 and h-42A5. In the AV shunt thrombosis models, both antibodies decreased
thrombosis formation in a dose-dependent manner. In FeCl3-induced thrombosis
models,
both antibodies extended the time for thrombosis-led vessel occlusion. These
results provide
further evidence of the anti-thrombotic roles of FXI/FXIa inhibitors. The dose-
dependent
reduction of thrombosis formation for h-19F6 and h-42A5 in AV shunt thrombosis
models
suggest that thrombosis formation may negatively correlate with the degree of
FXI inhibition,
which can be indicated by APTT prolongation. Because h-42A5 is more potent
than h-19F 6 in
prolonging APTT, the comparison of antithrombotic effects between h-42A5 and h-
19F6 in
the FeCl3-induced thrombosis models could also lead to such an indication.
Thus, more
intense inhibition of FXI/FXIa, as indicated by a longer APTT, by FXI/FXIa
inhibitors may
result in better anti-thrombotic outcomes.
[0038] Bleeding risk is the most concerning issue in developing
antithrombotic agents.
As previously mentioned, FXI-deficient patients may show a bleeding tendency
under
surgical settings. It is unclear to what extent plasma FXI activity inhibition
is still safe in
terms of bleeding risk. As demonstrated in the working examples, the bleeding
risk of
intensive inhibition of FXI/FXIa by h-19F6 and h-42A5 was tested in the same
monkeys
used in thrombosis experiments. In AV shunt thrombosis animals, no bleeding
tendency was
observed as the treating dose of h-19F6 or h-42A5 escalated, suggesting that
bleeding risk
may be independent of the extent of FXI inhibition. In FeCl3-induced
thrombosis animals,
neither h-19F6 nor h-42A5 treatment caused excessive bleeding. h-42A5
treatment resulted
in an approximately 2-fold elevation of plasma APTT, which indicated more than
99% FXI
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inhibition. Previous studies have never evaluated bleeding risk under such
intensive APTT-
prolongation and high-FXI-inhibition conditions. The antisense oligonucleotide
ISIS416856
only caused 30% elevation of APTT when its bleeding risk was evaluated.26 In
other
bleeding risk-evaluation studies in primates, a high potent anti-FXI antibody,
aXIMab,
caused an approximatelyl-fold increase in APTT (from 30.5 s to 65.6 5).26
Thus, the results
described herein demonstrate that intensive inhibition of FXI/FXIa does not
increase bleeding
risk in primates. Thus, FXI can be used as a drug target for thrombosis
treatment.
Anti-FXI or Anti-FXIa Antibodies
[0039] Provided herein are antibodies that bind to FXI, FXIa, and/or a
fragment of FXI or
FXIa and inhibit the formation of blood clot. These antibodies are capable of
binding to FXI,
FXIa, and/or a fragment of FXI or FXIa (e.g., a fragment comprising the A3
domain) and
exhibiting an inhibitory effect at a concentration that is much lower than the
maximum safety
dose. For example, in some embodiments a dose of the antibody between 0.1
mg/kg i.v. and 3
mg/kg i.v. exhibits an inhibitory effect on conversion of FXI to FXIa in
cynomolgus monkeys.
Moreover, the antibodies disclosed herein can be used as anticoagulation
agents with superior
safety due to their minimal risk of causing bleeding versus conventional
anticoagulation agents
such as heparin.
[0040] As demonstrated in the working examples, many anti-human FXI
antibodies were
generated by immunizing rats with human FXI to identify antibodies with
anticoagulation
properties. A dozen such antibodies were identified, and some of which were
humanized for
further development. The humanized rat anti-human FXI antibodies, such as h-
19F6 and h-
42A5 antibodies, were characterized in vitro and in vivo. In the in vitro
studies, the humanized
antibodies inhibited activated FXI (FXIa)-mediated hydrolysis of factor IX but
not factor XlIa-
induced FXI activation. The binding properties of the antibodies to FXI were
determined, and
the dissociation constants (KD) for h-19F6 and h-42A5 were 22 pM and 35 pM,
respectively.
These two antibodies bind different sites in the A3 domain of FXI. In the in
vivo studies, two
distinct primate thrombosis models were used to evaluate the anti-thrombotic
effects and
bleeding risks of the humanized antibodies. In arteriovenous (AV) shunt
thrombosis models,
both antibodies dose-dependently decreased thrombus formation without causing
bleeding. In
FeCl3-induced thrombosis models, both antibodies extended the time to
thrombosis-mediated
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vessel occlusion, and neither antibody increased bleeding. The two antibodies
showed anti-
thrombotic efficacy without compromising haemostasis in primates, further
confirming that
targeting FXI can be used for treating thrombosis.
[0041] As used herein, the term "comprising" with regard to a composition
or method means
that the composition or method includes at least the recited elements. The
term "consisting
essentially of' means that the composition or method includes the recited
elements, and may
further include one or more additional elements that do not materially affect
the novel and basic
characteristics of the composition or method. For example, a composition
consisting essentially
of recited elements may include those recited elements plus one or more trace
contaminants from
the isolation and purification method, pharmaceutically acceptable carriers
such as phosphate
buffered saline, preservatives, and the like. The term "consisting of' means
the composition or
method includes only the recited elements. Embodiments defined by each of the
transitional
terms are within the scope of this invention.
[0042] The term "antibody" as used herein refers to an immunoglobulin
molecule or an
immunologically active portion thereof that specifically binds to, or is
immunologically reactive
with a particular antigen, for example, FXI, FXIa, or a particular domain or
fragment of FXI or
FX1a, e.g., the A3 domain. In certain embodiments an antibody for use in the
present methods,
compositions, and kits is a full-length immunoglobulin molecule, which
comprises two heavy
chains and two light chains, with each heavy and light chain containing three
complementary
determining regions (CDRs). The term "antibody," in addition to natural
antibodies, also
includes genetically engineered or otherwise modified forms of
immunoglobulins, such as
synthetic antibodies, intrabodies, chimeric antibodies, fully human
antibodies, humanized
antibodies, peptibodies and heteroconjugate antibodies (e.g., bispecific
antibodies, multispecific
antibodies, dual-specific antibodies, anti-idiotypic antibodies, diabodies,
triabodies, and
tetrabodies). The antibodies disclosed herein can be monoclonal antibodies or
polyclonal
antibodies. In those embodiments where an antibody is an immunologically
active portion of an
immunoglobulin molecule, the antibody may be, for example, a Fab, Fab', Fv,
Fab' F(ab')2,
disulfide-linked Fv, single chain Fv antibody (scFv), single domain antibody
(dAb), or diabody.
The antibodies disclosed herein, including those that are immunologically
active portion of an
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immunoglobulin molecule, retain the ability to bind a specific antigen, for
example FXI or FX1a,
or to bind a specific fragment of FXI or FXIa such as the A3 domain.
[0043] In some embodiments, the anti-FXI and/or anti-FXIa antibodies
disclosed herein have
undergone post-translational modifications such as phosphorylation,
methylation, acetylation,
ubiquitination, nitrosylation, glycosylation, or lipidation associated with
expression in a
mammalian cell line, including a human or a non-human host cell. Techniques
for producing
recombinant antibodies and for in vitro and in vivo modifications of
recombinant antibodies are
known in the art. See, e.g., Liu et al., mAbs 6(5): 1145-1154 (2014), the
content of which is
incorporated by reference.
[0044] Also disclosed are polynucleotides or nucleic acids encoding the
anti-FXI and/or anti-
FXIa antibodies disclosed herein. In some embodiments, the polynucleotide or
nucleic acid
includes DNA, mRNA, cDNA, plasmid DNA. The nucleic acid encoding the antibody
or a
functional fragment thereof disclosed herein can be cloned into a vector, such
as a pTT5
mammalian expression vector, which may further include a promoter and/or other
transcriptional
or translational control elements such that the nucleic acid can be expressed
to produce the
antibody or the functional fragment thereof.
[0045] The nucleic acid (DNA) and/or amino acid (PRT) sequences, including
the sequences
of the VH and VL and CDRs, of some examples of the antibodies disclosed herein
are listed in
Table 1 below.
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Table 1: Antibody Sequences
SEQ
Description Type Sequence
ID NO
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCT
GTGTCTCTAGGGCAGAGGGC CAC CATCTCC TGCAG
AGCCAGCGAAAGTGTTGATAATTATGCCATTAGTTT
TATGAACTGGTTCCAACAGAAACCAGGACAGCCAC
3G12 1
CCAAACTCCTCATCTATGCTGCATCCAACCTAGGAT
-VL DNA
C CGGGGTC CC TGC CAGGTTTAGTGGCAGTGGGTC T
GGGACAGACTTCAGCCTCAACATCCATCCTATGGA
GGAGGATGATACTGCAATGTATTTCTGTCAGCAAG
ATAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACC
GAGCTGGAAATCAAA
CAGGTCACTCTGAAAGAGTCTGGCCCTGGGATATT
GCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTT
CTCTGGGTTTTCACTGAACACTCCTGGTATGGGTGT
GAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGG
3G12 2
AATGGCTGGCACACATTTACTGGGATGATGACAAG
-VH DNA
C GCTTTAAC CCATC CC TGAAGAGC CGAC TCACAAT
CTCCAAGGATACCTCCAGAGATCAGGTATTCCTCAT
GATCACCAGTGTGGACACTGCAGATTCTGCCACAT
AC TTC TGTGCTCGAAAAGGCC GCGGGCCC TTTAC TT
AC TGGGGCCAAGGGACTCTGGTCAC TGTCTC TTCA
3G12 CDR L1 3 D AGAGCCAGCGAAAGTGTTGATAATTATGCCATTAG
- - NA
TTTTATGAAC
3G12-CDR-L2 4 DNA GCTGCATCCAACCTAGGATCC
3G12-CDR-L3 5 DNA CAGCAAGATAAGGAGGTTCCGTGGACG
3G12-CDR-H1 6 DNA AC TC CTGGTATGGGTGTGAGC
3G12 CDR H2 7 D CACATTTAC TGGGATGATGACAAGC GCTTTAAC CC
- - NA
ATCCCTGAAGAGC
3G12-CDR-H3 8 DNA AAAGGCCGCGGGC CC TTTACTTAC
DIVLTQSPASLAVSLGQRATISCRASESVDNYAISFMN
3 G12-VL 9 PRT WF Q QKPGQPPKLLIYAASNLGS GVPARF S GS GS GTDF
SLNIEIPMEEDDTAMYFCQQDKEVPWTFGGG IELEIK
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QVTLKESGPGILQP S Q TL SLTC SF S GF SLNTPGMGVSW
3G12 10 PRT IRQPSGKGLEWLAEITYWDDDKRFNF'SLKSRLTISKDT
-VH
SRD QVFLMIT SVD TAD S ATYF'CARKGRGPF TWGQ G
TLVTVS S
3G12-CDR-L1 11 PRT RA SESVDNYAISFMN
3G12-CDR-L2 12 PRT AASNLGS
3G12-CDR-L3 13 PRT QQDKEVF'WT
3G12-CDR-H1 14 PRT TPGMGVS
3G12-CDR-H2 15 PRT HIYWDDDKRFNF'SLKS
3G12-CDR-H3 16 PRT KGRGPF TY
GACATTGTGCTGACCCAATCTCCAGCCTCTTTGGCT
GTGTCTCTAGGGCAGAGGGC CAC CATCTCC TGCAG
AGCCAGCGAAAGTGTTGATAATTATGGCATTAGTT
TTCTGAACTGGTTCCAACAGAAACCAGGACAGCCA
5B2 17 CC CAAACTCC TCATCTATGC TGCATCCAATCTAGGA
-VL DNA
TCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCT
GGGACAGACTTCAGCCTCAACATCCATCCTATGGA
GGAGGATGATACTGCAATGTATTTCTGTCAGCAAG
ATAAGGGGGTTCCGTGGACGTTCGGTGGAGGCACC
AAGCTGGAAATGAAA
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATT
GCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTT
CTCTGGGTTTTCACTGAACACTTCTGGTATGGGTGT
GAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGG
AGTGGCTGGCACACATTTACTGGGATGATGACAAG
5B2-VH 18 DNA CGCTATAAACCATCCCTGAAGAGCCGGCTCACAAT
CTCCAAGGATACCTCCAGAAACCAGGTATTCCTCA
TGATCACCAGTGTGGACACTGCAGATACTGCCACA
TACTACTGTGTTCGAAAAGGCCGCGGGCCCTTTGCT
AACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGC
A
5B2 CDR L1 19 D AGAGCCAGCGAAAGTGTTGATAATTATGGCATTAG
- - NA
TTTTCTGAAC
5B2-CDR-L2 20 DNA GCTGCATCCAATCTAGGATCC
5B2-CDR-L3 21 DNA CAGCAAGATAAGGGGGTTCCGTGGACG
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5B2-CDR-H1 22 DNA AC TTC TGGTATGGGTGTGAGC
5B2 CDR H2 23 D
CACATTTACTGGGATGATGACAAGCGCTATAAACC
- - NA
ATCCCTGAAGAGC
5B2-CDR-H3 24 DNA AAAGGCCGCGGGCCCTTTGCTAAC
DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFLN
5B2 VL 25 PRT WF Q QKF'GQPPKLLIYAASNLGS GVF'ARF S GS GS GTDF
- SLNIEIPMEEDDTAMYFCQQDKGVF'WTFGGGTKLEM
QVTLKESGPGILQPS QTLSLTC SF S GF SLNTS GMGVSW
5B2 26 PRT IRQPS GKGLEWLAHIYWDDDKRYKP SLK SRL TI SKD T
-VH
SRNQVFLMIT SVD TAD TATYYCVRKGRGPFANWGQ G
TLVTVS A
5B2-CDR-L1 27 PRT RASESVDNYGISFLN
5B2-CDR-L2 28 PRT AASNLGS
5B2-CDR-L3 29 PRT QQDKGVF'WT
5B2-CDR-H1 30 PRT TS GMGVS
5B2-CDR-H2 31 PRT HIYWDDDKRYKPSLKS
5B2-CDR-H3 32 PRT KGRGPFAN
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCT
GC CTCTCTGGGAGAAAGAGTCAGTCTCAC TTGTCG
GGCAAGTCAGGACATTGATATTCGCTTAAACTGGC
TTCGACAGGAACCAGATGGAACTATTAAACGCCTG
7C9 33 ATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCC
-VL DNA
AAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTA
TTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTT
TGTTGACTATTACTGTCTACAATATGCTAGTTCTCC
ATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAA
AA
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CAGATCCAGTTGGTGCAGTCTGGACCTGAACTGAA
GAAGCCTGGAGAGACCGTCAAGATCTCCTGCAAGG
CTTCTGGGTATATTTTCACAGACTATGGAATGAACT
GGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGG
ATGGGCTGGATAAACACCTACACTGGAGAGCCAAC
7C9-VH 34 DNA ATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTC
TTTGGAAACCTCTGCCAGCACTGCCTATTTACAGAT
CAACAACCTCAAAAATGAGGACACGGCTACATTTT
TCTGTGCAAGAAGGAGGATGGGTTATGCTGTGGAC
TACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTC
A
7C9-CDR-L1 35 DNA CGGGCAAGTCAGGACATTGATATTCGCTTAAAC
7C9-CDR-L2 36 DNA GCCACATCCAGTTTAGATTCT
7C9-CDR-L3 37 DNA CTACAATATGCTAGTTCTCCATTCACG
7C9-CDR-H1 38 DNA GACTATGGAATGAAC
7C9 CDR H2 39 D TGGATAAACACCTACACTGGAGAGCCAACATATGC
- - NA
TGATGACTTCAAGGGA
7C9-CDR-H3 40 DNA AGGAGGATGGGTTATGCTGTGGACTAC
DIQMTQ SPSSLSASLGERVSLTCRASQDIDIRLNWLRQ
7C9-VL 41 PRT EPDGTIKRLIYATSSLDSGVF'KRFSGSRSGSDYSLTISS
LESEDFVDYYCLQYAS SPF TF GS GTKLEIK
QIQLVQSGPELKKPGETVKISCKASGYIFTDYGMNWV
7C9 42 PRT KQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLE
-VH
T S AS TAYLQINNLKNED TATFF CARRRMGYAVDWG
QGTSVTVSS
7C9-CDR-L1 43 PRT RAS QDIDIRLN
7C9-CDR-L2 44 PRT ATSSLDS
7C9-CDR-L3 45 PRT LQYASSPFT
7C9-CDR-H1 46 PRT DYGMN
7C9-CDR-H2 47 PRT WINTYTGEPTYADDFKG
7C9-CDR-H3 48 PRT RRMGYAVDY
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GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCT
GTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAG
AGCCAGCGAAAGTGTTGATAATTATGCCATTAGTTT
TATGAATTGGTTCCAACAGAAACCAGGACAGCCAC
7F1 49
CCAAACTCCTCATCTATGCTGCATCCAACCTAGGAT
-VL DNA
CCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCT
GGGACAGACTTCAGCCTCAACATCCATCCTATGGA
GGAGGATGATACTGCAATGTATTTCTGTCAGCAAG
ATAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACC
AAGCTGGAGCTGAAA
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATAGT
GCAGCCCTCCCAGACCCTCAATCTGACTTGTTCTTT
CTCTGGATTTTCACTGAGCACTTCTGGTATGGGTGT
GAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGG
DNA 7F1 50
ATTGGCTGGCACACATTTACTGGGATGATGACAAG
-VEI
CGCTATAACCCATCCCTGATGAGCCGGCTCACAAT
CTCCAAGGATACCTCCAGAAACCAGGTATTCCTCA
TGATCACCAGTGTGGACACTGCAGATACTGCCACA
TACTACTGTGCTCGAAAAGGCCGCGGGCCCTTTGCT
TACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA
7F1 CDR L1 SiD
AGAGCCAGCGAAAGTGTTGATAATTATGCCATTAG
- - NA
TTTTATGAAT
7F1-CDR-L2 52 DNA GCTGCATCCAACCTAGGATCC
7F1-CDR-L3 53 DNA CAGCAAGATAAGGAGGTTCCGTGGACG
7F1-CDR-H1 54 DNA ACTTCTGGTATGGGTGTGAGC
7F1 CDR H2 55 D CACATTTACTGGGATGATGACAAGCGCTATAACCC
- - NA
ATCCCTGATGAGC
7F1-CDR-H3 56 DNA AAAGGCCGCGGGCCCTTTGCTTAC
DIVLTQSPASLAYSLGQRATISCRASESYDNYAISFMN
7F1-VL 57 PRT
WFQQKF'GQPPKLLIYAASNLGSGYF'ARFSGSGSGTDF
SLNIEIPMEEDDTAMYFCQQDKEVPWTFGGGTKLELK
QVTLKESGPGIVQPSQTLNLTCSFSGFSLSTSGMGYSW
7F1 58 PRT
IRQPSGKGLDWLAHIYWDDDKRYNF'SLMSRLTISKDT
-VEI
SRNQVFLMITSVDTADTATYYCARKGRGPFAWGQG
TLVTVSS
7F1-CDR-L1 59 PRT RASESYDNYAISFMN
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7F1-CDR-L2 60 PRT AA SNLGS
7F1-CDR-L3 61 PRT QQDKEVF'WT
7F1-CDR-H1 62 PRT TSGMGVS
7F1-CDR-H2 63 PRT HIYWDDDKRYNPSLMS
7F1-CDR-H3 64 PRT KGRGPFAY
CAGTTCACGCTGACTCAACCAAAGTCCGTGTCAGG
ATCTTTAAGAAGCACTATCACCATTCCCTGTGAGCG
CAGCAGTGGTGACATTGGAGATAGCTATGTGAGCT
GGTACCAACAACACTTGGGAAGACCCCCCATCAAT
13F4
GTGATCTATGCTGATGATCAAAGACCATCTGAAGT
-VL 65 DNA
GTCTGCTCGGTTCTCGGGCTCCATCGACAGCTCCTC
TAACTCAGCCTCACTGACCATCACTAATCTACAGAT
GGATGATGAGGCCGACTACTTCTGTCAGTCTTACG
ATACTTATATGGATGTTGTGTTCGGTGGTGGAACCA
AGC TCAATGTCC TA
GAGGTGCAGCTGAAGGAATCAGGACCTGGTCTGGT
GCAGCCCTCACAGACCCTGTCCCTCACCTGCACTGT
CTCTGGATTCTCATTAACGGACTACAGTGTACACTG
GGTTCGCCAGCCTCCAGGAAAAGGTCTGGAGTGGA
TGGGAGTAATGTGGAGTGGTGGAAGCACAGCATAT
13F4-VH 66 DNA
AATCCAGCTCTCACATCCCGACTGACCATTAGCAG
GGACACCTCCAAGAGCCAAGTTTTCTTAAAAATGA
ACAGTCTGCAAACTGAAGATACAGCCATTTACTAC
TGTACCAGAGCACCTTTTAACAACTGGGGCAATTG
GC TTCC TTAC TGGGGCCAAGGCAC TC TGGTCACTGT
CTCTTCA
13F4 CDR L1 67 D
GAGCGCAGCAGTGGTGACATTGGAGATAGCTATGT
- - NA
GAGC
13F4-CDR-L2 68 DNA GC TGATGATCAAAGACCATC T
13F4-CDR-L3 69 DNA CAGTCTTACGATACTTATATGGATGTTGTG
13F4-CDR-H1 70 DNA GACTACAGTGTACAC
13F4 CDR H2 71 D
GTAATGTGGAGTGGTGGAAGCACAGCATATAATCC
- - NA
AGCTCTCACATCC
13F4-CDR-H3 72 DNA GCACCTTTTAACAACTGGGGCAATTGGCTTCCTTAC
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QFTLTQPKSVSGSLRSTITIPCERSSGDIGDSYVSWYQ
13F4-VL 73 PRT QHLGRPPINVIYADDQRPSEVSARFSGSIDSSSNSASLT
ITNLQMDDEADYF'CQSYDTYMIDVVFGGGTKINVL
EVQLKESGPGLVQPSQTLSLTCTVSGFSLTDYSVHWV
13F4 74 PRT RQPPGKGLEWMGVMWSGGSTAYNF'ALTSRLTISRDT
-VH
SKSQVFLKIVINSLQTEDTAIYYCTRAPFNNWGNWITY
WGQGTLVTVSS
13F4-CDR-L1 75 PRT ERSSGDIGDSYVS
13F4-CDR-L2 76 PRT ADDQRPS
13F4-CDR-L3 77 PRT QSYDTYMDVV
13F4-CDR-H1 78 PRT DYSVH
13F4-CDR-H2 79 PRT VMWSGGSTAYNF'ALTS
13F4-CDR-H3 80 PRT APFNNWGNWLPY
CAATTCACGCTGACTCAACCAAAGTCCGTGTCAGG
CTCTTTAAGAAGCACTATCACCATTCCCTGTGAGCG
CAGCAGTGGTGACATTGGAGATAGCTATGTGAGCT
GGTACCAGCAACACTTGGGAAGACCCCCCATCAAT
19F6 GTGATCTATGCTGATGATCAAAGACCATCTGAAGT
-VL 81 DNA
GTCTGATCGGTTCTCGGGCTCCATCGACACCTCCTC
TAACTCAGCCTCACTGACCATCACTAATCTGCAGAT
GGATGATGCGGCCGACTACTTCTGTCAGTCTTACGA
TAGTAATATTGATTTTAACCCTGTTTTCGGTGGTGG
AACCAAGCTCACTGTCCTA
GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGT
GCAGCCTGGAAGGTCTCTGAGACTCTCCTGTACAG
CCTCAGGATTCACTTTCAGTAAATATGTCATGGCCT
GGGTCCGCCAGGCTCCAACGAAGGGGCTGGAGTGG
GTCGCATCCATTAATTATGATGGTAGTACCACTTAC
19F6-VH 82 DNA TATCGAGACTCCGTGCAGGGCCGGTTCACTCTCTCC
AGAGATAATGCAAAAACCACCCTATACCTGCAAAT
GGACAGTCTGAGGTCTGAGGACACGGCCACTTATT
ACTGTGCAAGGCACCCTTTTAACAACTTCGGGATTT
GGTTTGCTTACTGGGGCCAAGGCACTCTGGTCACTG
TCTCTTCA
GAGCGCAGCAGTGGTGACATTGGAGATAGCTATGT
19F6-CDR-L1 83 DNA
GAGC
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19F6-CDR-L2 84 DNA GCTGATGATCAAAGACCATCT
19F6-CDR-L3 85 DNA CAGTCTTACGATAGTAATATTGATTTTAACCCTGTT
19F6-CDR-H1 86 DNA AAATATGTCATGGCC
19F6 CDR H2 87 D
TCCATTAATTATGATGGTAGTACCACTTACTATCGA
- - NA
GACTCCGTGCAGGGC
19F6-CDR-H3 88 DNA CACCCTTTTAACAACTTCGGGATTTGGTTTGCTTAC
QFTLTQPKSVSGSLRSTMPCERSSGDIGDSYVSWYQ
19F6-VL 89 PRT QHLGRPPINVIYADDQRPSEVSDRE'SGSIDTSSNSASLT
ITNLQMDDAADYFCQSYDSNIDFNF'VFGGGTKLTVL
EVQLVESGGGLVQPGRSLRLSCTASGFTFSKYVMAW
19F6 90
PRT VRQAPTKGLEWVASINYDGSTTYYRDSVQGRFTLSR
-VH
DNAKTTLYLQMDSLRSEDTATYYCARHPFNNFGIWF
AYWGQGTLVTVSS
19F6-CDR-L1 91 PRT ERSSGDIGDSYVS
19F6-CDR-L2 92 PRT ADDQRPS
19F6-CDR-L3 93 PRT QSYDSNIDFNPV
19F6-CDR-H1 94 PRT KYVMA
19F6-CDR-H2 95 PRT SINYDGSTTYYRDSVQG
19F6-CDR-H3 96 PRT HPFNNFGIWFAY
GATGTCCGGATGACACAGTCTCCAGCTTCCCTGTCT
GCATCTCTGGGAGAAACTGTCAACATCGAATGTCT
AGCAAGTGAGGACATTTACAGTGATTTAGCATGGT
ATCAGCAGAAGCCAGGGAAATCTCCTCAGCTCCTG
21F12 97
ATCTATAATGCAAATAGTCTACAAAATGGGGTCCC
-VL DNA
TTCACGGTTTAGTGGCAGTGGTTCTGGCACGCAGTA
TTCTCTAAAAATATCCACCCTGCAATCTGAAGATGT
CGCGACTTATTTCTGTCAACAATATAGCAATTATCG
TCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCA
AT
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GAGGTGCAACTGGTGGAGTCTGGGGGAGGCCTAGT
GCAGCCTGGAAGGTCTCTGAAACTATCCTGTGTAG
CC TC TGGATTCACATTCAACAACCACTGGATGACC T
GGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG
GTTGCATCCATTACTGATAATGGTGGTAGCACTTAC
21F12-VEI 98 DNA
TATCCAGACTCTGTGAAGGGCCGATTCACTATCTCC
AGAGATAATGCAAAAAGCAC CC TATAC CTGCACAT
GAACAGTCTGAGGTCTGAGGACACGGCCACTTATT
AC TGTACAAGAGATC GGTATGAC TC TGATGGTTATT
ATTACGTGAGGTACTATGTTGTGGACGCCTGGGGT
CAAGGAGCTTCAGTCACTGTCTCCTCA
21F12-CDR-L1 99 DNA CTAGCAAGTGAGGACATTTACAGTGATTTAGCA
21F12-CDR-L2 100 DNA AATGCAAATAGTCTACAAAAT
21F12-CDR-L3 101 DNA CAACAATATAGCAATTATCGTCGGACG
21F12-CDR-H1 102 DNA AAC CAC TGGATGACC
21F12 CDR H2 103 D
TCCATTACTGATAATGGTGGTAGCACTTACTATCCA
- - NA
GACTCTGTGAAGGGC
21F12 CDR H3 104 D
GATCGGTATGACTCTGATGGTTATTATTACGTGAGG
- - NA
TACTATGTTGTGGACGCC
DVRMTQ SPASLS A SLGETVNIECLASEDIYSDLAWYQ
21F12-VI, 105 PRT
QKF'GK SPQLLIYNANSLQNGVF'SRE' S GS GS GTQY SLKI
STLQ SEDVATYF'CQQYSNYRRTFGGGTKLEIN
EVQLVESGGGLVQPGRSLKLSCVASGFTFNNHWMT
WIRQAF'GKGLEWVASITDNGGSTYYPDSVKGRFTISR
21F12-VEI 106 PRT DNAK
S TLYLEIMNSLRSED TATYYCTRDRYD SD GYYY
VRYYVVDAWGQGASVTVS S
21F12-CDR-L1 107 PRT LASEDIYSDLA
21F12-CDR-L2 108 PRT NANSLQN
21F12-CDR-L3 109 PRT QQYSNYRRT
21F12-CDR-H1 110 PRT NHWMT
21F12-CDR-H2 111 PRT SITDNGGS TYYF'D SVKG
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21F12-CDR-H3 112 PRT DRYDSDGYYYVRYYVVDA
GATGTTGTGTTGACACAGACTCCAGGTTCCCTGTCT
GTCACACTTGGACAGCAAGTTTCTATATCCTGTAGG
TCTAGTCAGAGCCTGGAAAGTCGTGATGGGAACAC
TTATTTGGAATGGTACCTACAGAAGCCAGGCCAGT
34F8
CTCCACAGGTCCTCCTCTATGGAGTTTCCAACCGAT
-VL 113 DNA
TGTCTGGGGTCCCAGACAGGTTCCTTGGCAGAGGG
TCAGGGGCAGATTTCACCCTCAAGATCAGCAGAGT
AGAGCCTGAGGACTTGGGAGTTTATTACTGCTTCCA
AGCTACACATGGTCCATTCACGTTCGGCTCAGGGA
CGAAGTTGGAAATGAAA
CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGT
GCAGCCCTCACAGACCCTGTCTCTCACCTGCACTGT
CTCTGGGTTCTCATTAACCACCTATCATGTGCACTG
GGTTCGACAGCCTCCAGGAAAAGGTCTGGAGTGGA
DNA 34F8 114
TGGGAATAATGTGGAGAGATGGAGACACATCATAT
-VEI
AATTCAGTTCTCAAATCTCGACTGAGCATCAGCAG
GGACATCTCCAAGAGCCAAGTTTTCTTAAAAATGA
GCAGTC TGCAAAC TGAAGACACAGC CAC TTAC TTC
TGTGCCAGAGGGGGGACTCTTACAACTCCCTTTACT
TACTGGGGCCAAGGCACTCTGGTCACTGTCTCTTCA
34F8 CDR L1 115 D
AGGTCTAGTCAGAGCCTGGAAAGTCGTGATGGGAA
- - NA
CACTTATTTGGAA
34F8-CDR-L2 116 DNA GGAGTTTCCAACCGATTGTCT
34F8-CDR-L3 117 DNA TTCCAAGCTACACATGGTCCATTCACG
34F8-CDR-H1 118 DNA AC CTATCATGTGCAC
34F8 CDR H2 11 9 D
ATAATGTGGAGAGATGGAGACACATCATATAATTC
- - NA
AGTTCTCAAATCT
34F8-CDR-H3 120 DNA GGGGGGACTCTTACAACTCCCTTTACTTAC
DVVLTQTPGSLSVTLGQQVSISCRSS QSLESRDGNTYL
34F8 VL 121
PRT EWYLQKPGQ SPQVLLYGV SNRL S GVF'DRFLGRGS GA
- DFTLKISRVEPEDLGVYYCFQATHGPFTE'GSGTKLEM
QVQLKESGPGLVQPSQTLSLTCTVSGFSLTTYHVHWV
34F8 122
PRT RQPPGKGLEWMGIMWRDGD TS YNS VLKSRLSI SRDIS
-VH
KS QVFLKNIS SLQ TED TATYFCARGGTLTTPF TWGQ
GTLVTVSS
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34F8-CDR-L1 123 PRT RS SQ SLESRDGNTYLE
34F8-CDR-L2 124 PRT GVSNRLS
34F8-CDR-L3 125 PRT FQATHGPFT
34F8-CDR-H1 126 PRT TYHVH
34F8-CDR-H2 127 PRT IMWRDGDTSYNSVLKS
34F8-CDR-H3 128 PRT GGTLTTF'F TY
GATATCCGGATGACACAGTCTCCAGCTTCCCTGTCT
GCATCTCTGGGAGAAACTGTCAACATCGAATGTCT
AGCAAGTGAGGACATTTACAGTGATTTAGCATGGT
ATCAGCAGAAGCCAGGGAAATCTCCACAACTCCTG
DNA 38E4 129 ATCTATAATGCAAATAGCGTGCAAAATGGGGTCCC
-VI,
TTCACGGTTTAGTGGCAGTGGATCTGGCACACAGT
ATTCTCTAAAAATAAACAGCCTGCAATCTGAAGAT
GTCGCGACTTATTTCTGTCAACAGTTTAACAGTTAT
CCGAACACGTTTGGAGCTGGGACCAAGCTGGAAAT
CAAA
GAGGTGCAACTTCAGGAGTCAGGACCTGGCCTTGT
GAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGT
CTCTGGTTTCTCCATCACTAATAATTACTGGGGCTG
GATCCGGAAGTTCCCAAGAAATAAAATGGAGTGGA
TTGGACACATAAGCTACAGTGGTAGCACTAACTAC
38E4-VE1 130 DNA AACCCATCTCTCAAAAGTCGCATCTCCATTACTAGA
GACTCATCGAAGAGTCAGTTCTTCCTGCAGTTGAAC
TCTTTAACTACTGAGGACACAGCCACATATTACTGT
GCAAGAGGATCTTATTACTATAGCGCATCGGGC TA
CTTTGATTATTGGGGCCAAGGAATCACGGTCACAG
TCTCCTCA
38E4-CDR-L1 131 DNA CTAGCAAGTGAGGACATTTACAGTGATTTAGCA
38E4-CDR-L2 132 DNA AATGCAAATAGCGTGCAAAAT
38E4-CDR-L3 133 DNA CAACAGTTTAACAGTTATCCGAACACG
38E4-CDR-H1 134 DNA AATAATTACTGGGGC
CACATAAGCTACAGTGGTAGCACTAACTACAACCC
38E4-CDR-H2 135 DNA
ATCTCTCAAAAGT
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38E4-CDR-H3 136 DNA GGATCTTATTACTATAGCGCATCGGGCTACTTTGAT
TAT
DIRMTQSPASLSASLGETVNIECLASEDIYSDLAWYQQ
38E4-VI, 137 PRT KF'GKSPQLLIYNANSVQNGVP SRF S GS GS GTQYSLKIN
SLQSEDVATYFCQQFNSYF'NTFGAGM,EIK
EVQLQESGPGLVKF'S Q SLSLTCSVSGESITNNWGW1
38E4 VEI 138 PRT RKFPRNKMEWIGHISY S GS TNYNF'SLKSRISI TRD S SKS
- QFFLQLNSLTTEDTATYYCARGSYYYSASGYEDWG
QGITVTVSS
38E4-CDR-L1 139 .. PRT LA SEDIYSDLA
38E4-CDR-L2 140 PRT NANSVQN
38E4-CDR-L3 141 PRT Q QFNS YF'NT
38E4-CDR-H1 142 PRT NI\TWG
38E4-CDR-H2 143 PRT HISYSGS TNYNF' SLKS
38E4-CDR-H3 144 PRT GS YYY S AS GYFDY
GAC GTGGTC TTGAC CCAAACC CC TGGATCAC TTAG
CGTGACACTGGGCGATCCAGCATCAATGTCCTGCA
GAAGCTCCCAGTCCTTGGAGAGTAGCGACGGCAAC
ACATACCTCGAGTGGTATCTGCAGAAATCCGGGCA
DNA 42A5 .. 145
GTCCCCACAGCTGCTGATCTACGGCGTGAGTAACA
-VI,
GGTTCAGCGGGGTGCCTGATAGGTTCGCCGGCAGC
GGGTCCGGGACAGATTTTACTCTCAAGATTAGCCG
C GTC GAAC CC GAGGAC CTGGGC GTGTACTAC TGTT
TTCAGGC CAC TCGGGAC CCC TTTACTTTC GGGAGC G
GGACAAAGCTGGAGATTAAT
CAGGTCCAGCTTAAAGAGTCCGGACCTGGACTTGT
GCAGCCATCCCAGACCTTGTCCTTGACCTGCACCGT
GTCAGGGTTCTCTCTCACCAGTTACCACCTGCATTG
GATCAGGCAGCCTCCCGGCAAGGGGCTGGAATGGA
DNA 42A5 146 TGGGGCTGATGTGGAGAGATGGGGATACATCTTAC
-VEI
AACAGCAGGCTGAAGAGCCGGCTGAGCATTACACG
GGACACCAGCAAGTCCCAGGTGTTCCTCAAGATGA
GC GGGCTCCAAACTGAGGACACAGC TACATACTAC
TGTGCACGCGGCATGACACTCGCCACTCCCTTTCTG
TATTGGGGCCAGGGCACTCTGGTCACTGTGTCCTCA
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42A5 CDR L1 147 D
AGAAGCTCCCAGTCCTTGGAGAGTAGCGACGGCAA
- - NA
CACATACCTCGAG
42A5-CDR-L2 148 DNA GGCGTGAGTAACAGGTTCAGC
42A5-CDR-L3 149 DNA TTTCAGGCCACTCGGGACCCCTTTACT
42A5-CDR-H1 150 DNA AGTTACCACCTGCAT
42A5 CDR H2 151 D
CTGATGTGGAGAGATGGGGATACATCTTACAACAG
- - NA
CAGGCTGAAGAGC
42A5-CDR-H3 152 DNA GGCATGACACTCGCCACTCCCTTTCTGTAT
DVVLTQTF'GSLSVTLGDPASMSCRSSQSLESSDGNTY
42A5-VI, 153 PRT LEWYLQKS GQ SPQLLIYGVSNRF S GVPDRFAGS GS GT
DFTLKISRVEPEDLGVYYCFQATRDPFTFGSGTKLEIN
QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYEILHWI
42A5 154
PRT RQPPGKGLEWMGLMWRDGDTSYNSRLKSRLSITRDT
-VEI
SKS QVFLKMS GLQTEDTATYYCARGMTLATPFLWG
QGTLVTVSS
42A5-CDR-L1 155 PRT RS SQSLES SDGNTYLE
42A5-CDR-L2 156 PRT GVSNRFS
42A5-CDR-L3 157 PRT FQATRDPFT
42A5-CDR-H1 158 PRT SYEILH
42A5-CDR-H2 159 PRT LMWRDGDTSYNSRLKS
42A5-CDR-H3 160 PRT GMTL,ATPFLY
GATATCCGGATGACACAGTCGCCAGCTTCCCTGTCT
GCATCTCTGGGAGAAACTGTCAACATCGAATGTCT
AGCAAGTGAGGACATTCACAGTGATTTAGCATGGT
ATCAGCAGAAGCCAGGGAAATCTCCTCAGCTCCTG
42F4 ATCTATAATGCAAATAGCTTGCAAAATGGGGTCCC
-VL 161 DNA
TTCACGGTTCAGTGGCAGTGGATCTGGCACACAGT
ATTCTCTAAAAATAACCAGCCTGCAATCTGAAGAT
GTCGCGACTTATTTCTGTCAACAATATACCAACTAT
CCGAACACGTTTGGAGCGGGGACCAAGCTGGAAAT
CAAT
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GAGGTGCAGCTTCAGGAGTCAGGACCTGGCCTTGT
GAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGT
CACTGGTTACTCCATCACTAATCATTACTGGGGCTG
GATCCGGAAATTCCCAGGAAATAAAATGGAGTGGA
TTGGACACATAAGCAACAGTGGTGGCACTAACTAC
42F4-VII 162 DNA AACCCATCACTCAAAAGTCGAATCTCCATTACTAG
AGACACATCGAAGAATCAGTTCTTCCTGCAGTTGA
AGTCTGTAACTACTGAGGACACAGCCACATATTAC
TGTACAAGAGGATCTTATTACTATAGCGCATCGGG
CTACTTTGATTACTGGGGCCAAGGAGTCCTGGTCAC
AGTCTCCTCC
42F4-CDR-L1 163 DNA CTAGCAAGTGAGGACATTCACAGTGATTTAGCA
42F4-CDR-L2 164 DNA AATGCAAATAGCTTGCAAAAT
42F4-CDR-L3 165 DNA CAACAATATACCAACTATCCGAACACG
42F4-CDR-H1 166 DNA AATCATTACTGGGGC
CACATAAGCAACAGTGGTGGCACTAACTACAACCC
42F4-CDR-H2 167 DNA
ATCACTCAAAAGT
42F4-CDR-H3 168 DNA GGATCTTATTACTATAGCGCATCGGGCTACTTTGAT
TAC
DIRMTQSPASLSASLGETVNIECLASEDIHSDLAWYQQ
42F4-VL 169 PRT KF'GKSPQLLIYI\TANSLQNGVF'SRFSGSGSGTQYSLKIT
SLQSEDVATYF'CQQYTNYF'NTFGAGTKLEIN
EVQLQESGPGLVKPSQSLSLTCSVTGYSITNHYWGWI
42F4 VII 170
PRT RKFPGNKMEWIGHISNSGGTNYNF'SLKSRISITRDTSK
-
NQFFLQLKSVTTEDTATYYCTRGSYYYSASGYF'DW
GQGVLVTVSS
42F4-CDR-L1 171 PRT LASEDIHSDLA
42F4-CDR-L2 172 PRT NANSLQN
42F4-CDR-L3 173 PRT QQYTNYF'NT
42F4-CDR-H1 174 PRT NETYWG
42F4-CDR-H2 175 PRT HISNSGGTNYNPSLKS
42F4-CDR-H3 176 PRT GSYYYSASGYF'DY
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GATATCCGGATGACACAGTCTCCAGCTTCCCTGTCT
GCATCTCTGGGAGAAACTGTCAACATCGGATGTCT
AGCAAGTGAGGACATTTACAGTGATTTAGCATGGT
ATCAGCAGAAGCCAGGGAAGTCTCCTCAGCTCCTG
DNA 45H1 177
ATCTATAATGCAAATAACTTGCAAAATGGGGTCCC
-VI,
TTCACGGTTTAGTGGCAGTGGATCTGGCACACAAT
ATTCTCTAAAAATAAACAGCCTGCAATCTGAAGAT
GTCGCGACTTATTTCTGTCAACAATATAACAGTTAT
CC GAACACGTTTGGAGCTGGGACCAAGC TGGAAAT
AAAA
GAGGTGCAGCTTCAGGAGTCAGGACCTGGCCTTGT
GAAACCCTCACAGTCACTCTCCCTCATTTGTTCTGT
CACTGGTTACTCCATCACTACAACTTACTGGGGCTG
GATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGA
TTGGACACATAAGTAACAGTGGTAGTACTAATTAC
45H1-VEI 178 DNA AACCCATCTCTCAAAAGTCGAATCTCCGTTACTAGA
GACACATCGACGAATCAGTTCTTCCTGCAGTTGAA
CTCTGTAACTACTGAGGACACAGCCACATATTACT
GTGCAAGAGGATCTTATTACTATAGCGCGTCGGGC
TACTTTGATTACTGGGGCCACGGAGTCATGGTCAC
AGTCTCCTCA
45H1-CDR-L1 179 DNA CTAGCAAGTGAGGACATTTACAGTGATTTAGCA
45H1-CDR-L2 180 DNA AATGCAAATAACTTGCAAAAT
45H1-CDR-L3 181 DNA CAACAATATAACAGTTATCCGAACACG
45H1-CDR-H1 182 DNA ACAACTTACTGGGGC
45H1 CDR H2 183 D CACATAAGTAACAGTGGTAGTACTAATTACAACCC
- - NA
ATCTCTCAAAAGT
45H1-CDR-H3 184 DNA GGATCTTATTACTATAGCGCGTCGGGCTACTTTGAT
TAC
DIRMTQ SPASLS A SLGETVNIGCLA SEDIYSDLAWYQ
45H1 -VI, 185 PRT QKF'GKSPQLLIYNANNLQNGVF' SRF S GS GS GTQYSLKI
NSLQSEDVATYFCQQYNSYF'NTFGAGTKLEIK
EVQLQESGPGLVKPSQSLSLICSVTGYSITTTWGWIR
45H1 VEI 186 PRT KFPGNKMEWIGHISNS GS TNYNF'SLKSRISVTRD TS TN
- QFFLQLNSVT'IED TATYYCARGS YYY SAS GYFDWG
HGVMVTVSS
45H1-CDR-L1 187 PRT LASEDIYSDLA
45H1-CDR-L2 188 PRT NANNLQN
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45H1-CDR-L3 189 PRT QQYNSYPNT
45H1-CDR-H1 190 PRT TTYVVG
45H1 - CDR-H2 191 PRT HISNSGS TNYNPSLKS
45H1-CDR-H3 192 PRT GS YYYSAS GYFDY
14E11
DIVMTQSFIKFMSTSVGDRVSITCKAS QDVSTAVAWY
-VL
193 PRT QQKPGQSPKLLIYLTSYRNTGVPDRFTGSGSGTDFTFT
(control)
IS SVQAEDLAVYYCQQHYKTPYSFGGGTKLERLR
QVQLEES GPGLVAPSQ SLSI TC TV S GF SLTGYGIYVVVR
14E11-VH 194 PRT QPPGKGLEWLGMIWGDGRTDYNSALKSRLSISKDNS
(control) KSQVFLKMNSLQTDDTARYYCARDYYGSKDYVVGQG
TTLTVS
DIVLTQ SPA SLAVSLGQRATI S CKA S Q SVDYD GD S YL
1A6 -VL
195 PRT NVVYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTD
(control)
FTLNIEIPVEEEDAATYYCQQSNGDPWTFGGGTKLEIK
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGW
1A6-VH
196 PRT IRQPSGKGLEWLABIWWDDDKYYNPSLKSQLTISKDT
(control)
SRNQVFLKITSVDAADTATYYCARKRSSVVAHYYAM
DYWGQGTSVTVS
[0046]
Provided in certain embodiments herein are humanized anti-FXI and/or anti-FXIa
antibodies. Various techniques are known in the art for humanizing antibodies
from non-human
species such that the antibodies are modified to increase their similarity to
antibodies naturally
occurring in humans. Six CDRs are present in each antigen binding domain of a
natural antibody.
These CDRs are short, non-contiguous sequences of amino acids that are
specifically positioned
to form the antigen binding domain as the antibody assumes its three
dimensional configuration.
The remainder of the amino acids in the antigen binding domains, referred to
as "framework"
regions, show less inter-molecular variability and form a scaffold to allow
correct positioning of
the CDRs. In some embodiments, the antibodies or fragments disclosed herein
have conserved
sequences for CDR3 regions.
[0047]
For example, humanization of the antibodies disclosed herein can be
accomplished by
CDR grafting of monoclonal antibodies produced by immunizing mice or rats. The
CDRs of a
mouse monoclonal antibody can be grafted into a human framework, which is
subsequently
joined to a human constant region to obtain a humanized antibody. Briefly, the
human germline
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antibody sequence database, the protein data bank (PDB), the INN
(International Nonproprietary
Names) database, and other suitable databases can be searched and the most
similar frameworks
to the antibodies can be identified by the search. In addition, some back
mutations to the donor
residues are made in the human acceptor frameworks. In some embodiments, the
variable
regions are linked to a human IgG constant region. For example, human IgGl,
IgG2, IgG3 and
IgG4 Fc domains can be used. It is within the purview of one of ordinary skill
in the art to
humanize a monoclonal antibody produced by a non-human species based on the
existing
technology.
[0048]
The sequences of the variable regions of several example humanized antibodies
are
shown in Table 2 below.
Table 2: Sequences of humanized antibodies
D E. S Q ID T Sequence (with mutations
highlighted in bold and
escriptmn ype
NO underlined, and CDRs highlighted in italic)
QFQLTQPSSVSASVGDRVTITCERSSGDIGDSYVSVVYQQ
h-19F 6-VL 197 PRT KPGI2PPKNVIYADDQRPSGVPDRFSGSIDGSGNSASLTI
SSLQAEDAADYFCQSYDSNIDFNPVFGGGTKLEVK
EVQLVESGGGLVQPGRSLRLSCTASGFTFSKYAMSWVR
h 19F6
198 PRT QAPGKGLEWVSA/NYDGSTTYYADSVKGRFTLSRDNSKN
- -VH
TLYLQMNSLRAEDTAVYYCARHPFNNFGIWFAYVVGQG
TLVTVS
DVVLTQTPLSLPVTPGEPA SI S CRSSQSLESRDGNTYLEW
h-34F8-VL 199 PRT YLQKPGQ S PQVLLYG VSNRLSGVPDRF S GS GS GTDF TLK
ISRVEAEDVGVYYCFQATHGPFTFGQGTKLEIKRT
QVQLjlESGPGLVKPSETLSLTCTVSGFSLTTYHVHWVR
h 34F8 200 PRT QPPGKGLEWIG/MWRDGDTYYNSSLKSRVTISRDTSKNQ
- -VH
VSLKLS SVTAADTAVYFCARGGTLTTPFTYWGQGTLVT
VS S
DVVLTQTPLSLSVTPG12PASISCRSSQSLESSDGNTYLEW
h-42A5-VL 201 PRT YLQKPGQ SPQLLIYG VSNRFSGVPDRF S GS GS GTDF TLKI
SRVEAEDLGVYYCFQATRDPFTFGQGTKLEIKRT
QVTLKESGPVLVKPTETLTLTCTVSGFSLTSYHLHWIRQ
h 42A5
202 PRT PPGKALEWMGLMWRDGDTSYNSRLKSRLTISRDTSKSQV
- -VH
VLTMTNMDPVDTATYYCARGMTLATPFLYVVGQGTLVT
VS S
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[0049] The antibodies provided herein include variants of the sequences
disclosed herein that
contain one or more mutations in their amino acid sequences while retaining
binding affinity for
FXI, FXIa, and/or a fragment thereof (e.g., a fragment comprising the A3
domain). In some
embodiments, the antibodies include a variable region having an amino acid
sequence that is at
least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical
to a sequence
selected from the group consisting of SEQ ID NOs: 9, 10, 25, 26, 41, 42, 57,
58, 73, 74, 89, 90,
105, 106, 121, 122, 137, 138, 153, 154, 169, 170, 185, 186, and 197-209, or a
fragment thereof
that retains binding affinity for FXI, FX1a, and/or a fragment thereof.
[0050] Also included in this disclosure are variants of nucleic acids
encoding antibodies that
bind to FXI, FX1a, and/or a fragment thereof (e.g., a fragment comprising the
A3 domain). In
some embodiments, the nucleic acids encoding the antibodies include a variable
region having a
nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at
least 98%, or at least 99%
identical to a sequence selected from the group consisting of SEQ ID NOs: 1,
2, 17, 18, 33, 34,
49, 50, 65, 66, 81, 82, 97, 98, 113, 114, 129, 130, 145, 146, 161, 162, 177,
and 178, or a fragment
thereof that encodes a polypeptide with binding affinity for FXI, FX1a, and/or
a fragment thereof
[0051] In some embodiments, the antibodies are further subjected to a
strategic Chemistry,
Manufacturing, and Control (CMC) development such that the novel antibodies
such as
monoclonal antibodies or humanized monoclonal antibodies disclosed herein are
advanced from
discovery to human clinical trials, and then to the pharmaceutical market. The
modifications
further improve the properties of the antibodies without compromising the
immunological
functions of the antibodies. In certain embodiments, a CMC modified antibody
is more stable
under various temperatures (e.g., 4 C, 25 C, and 37 C) for an extended period
of time (e.g., 3
days, 7 days, 14 days and 28 days) and under repeated freeze/thaw cycles
(e.g., -40 C/25 C for up
to 5 cycles) comparing to the unmodified antibody. Additionally, the CMC
modified antibodies
have an acceptable solubility. For example, for a given sequence of a light
chain or a heavy
chain, certain amino acids can be potential oxidation and glycosylation sites.
These amino acid
residues at the potential oxidation, deamidation, or glycosylation sites may
be mutated and
additional residues in the proximity can also be mutated and/or optimized to
maintain the 3D
structure and function of a particular antibody. In some embodiments, one or
more amino acid
residues in a CDR region having the potential of oxidation, deamidation, or
glycosylation are
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mutated to improve the stability of the antibody or a fragment thereof without
compromising the
immunological functions. In some embodiments, one or more Met residues in a
CDR region
having the potential of oxidation are mutated. In some embodiments, one or
more Asn residues
in a CDR region having the potential of deamidation are mutated.
[0052]
The sequences of the variable regions of several example CMC optimized,
humanized
antibodies are shown in Table 3 below.
Table 3: Sequences of CMC optimized, humanized antibodies
D E. S Q ID T Sequence (with mutations
highlighted in bold and
escriptmn ype
NO underlined, and with CDRs highlighted in
italic)
DIVMTQTPLSLSVTPGQPASISCRSSQSLESSDGNTYLEW
h-42A5-VL 204 PRT YLQKPGQSPQLLIYG VSNRFSGVPDRF S GS GS GTDF TLKI
SRVEAEDVGVYYCFQATRDPFTFGQGTKLEIKRT
QVTLKE S GPVLVKPTETLTLTC TV S GF SLSSYS VAWIRQP
h 42A5
205 PRT PGKALEWLAE/WRDGDTSYNSRLKSRLTISKDTSKSQVV
- -VH
LTMTNMDPVDTATYYCARGMTLATPFLYVVGQGTLVTV
S S
DVVLTQTPLSLPVTPGEPASISCRSSQSLESRDGNTYLEWY
h-34F8-VL 206 PRT LQKPGQ SPQVLLYG VSNRLSGVPDRF S GS GS GTDFTLKIS
RVEAEDVGVYYCFQATHGPFTFGQGTKLEIKRT
QVQLQESGPGLVKPSETLSLTCTVSGYSISTYHVHWVRQ
h 34F8
207 PRT PPGKGLEWIG/MWRDGDTYYNPKLKSRVTIS VD TSKNQV
- -VH
SLKLSSVTAADTAVYFCARGGTLTTPFTYWGQGTLVTV
S S
QFQLTQPS SVSASVGDRVTITCERSSGD/GDSYVSVVYQQK
h-19F 6-VL 208 PRT PGQPPKNVIYADDQRPSGVPDRFS GSID GS GNS A SLTIS S
LQAEDAADYFCQSYDSNIDFNPVFGGGTKLTVL
EVQLVESGGGLVQPGRSLRLSCTASGFTFSKYAMSVVVR
h 19F6 209 PRT QAPGKGLEWVSA/NYDGSTTYYADSVKGRFTLSRDNSKN
- -VH
TLYLQMNSLRAEDTAVYYCARHPFNNFGIWFA YVVGQG
TLVTVS
Pharmaceutical Compositions
[0053]
The antibodies disclosed herein can be formulated into pharmaceutical
compositions.
The pharmaceutical compositions may further comprise one or more
pharmaceutically acceptable
carriers, excipients, preservatives, or a combination thereof. The
pharmaceutical compositions
can have various formulations, e.g., injectable formulations, lyophilized
formulations, liquid
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formulations, etc. Depending on the formulation and administration route, one
would select
suitable additives, such as adjuvants, carriers, excipients, preservatives.34
[0054]
The pharmaceutical composition can be included in a kit with an instruction
for using
the composition.
Methods of Treatment
[0055]
Provided herein is a method of treating and/or preventing thrombosis in a
subject
suffering from thrombosis and/or at an elevated risk of developing thrombosis.
Also provided is
a method of inhibiting the formation of blood clots in a subject. These
methods entail
administering a therapeutically effective amount of an anti-FXI and/or FXIa
antibody provided
herein to intervene in the intrinsic pathway. In some embodiments, these
methods comprise
administering a pharmaceutical composition comprising an anti-FXI and/or anti-
FXIa antibody as
provided herein to the subject.
[0056]
The methods disclosed herein can be used to prevent and/or treat complications
or
conditions associated with thrombosis in a subject in need thereof. Thrombosis
causes or is
associated with a number of complications or conditions, such as embolic
stroke, venous
thrombosis such as venous thromboembolism (VTE), deep vein thrombosis (DVT),
and
pulmonary embolism (PE), arterial thrombosis such as acute coronary syndrome
(ACS), coronary
artery disease (CAD), and peripheral artery disease (PAD). Other conditions
associated with
thrombosis include, for example, high risk of VTE in surgical patients,
immobilized patients,
patients with cancer, patients with heart failure, pregnant patients, or
patients having other
medical conditions that may cause thrombosis. The methods disclosed herein
relate to a
preventive anticoagulant therapy, that is, thromboprophylaxis.
These methods entail
administering to a subject suffering from a thrombosis-related complication
disclosed above a
therapeutically effective amount of an anti-FXI and/or FXIa antibody as
disclosed herein or a
therapeutically effective amount of a pharmaceutical composition comprising
the anti-FXI and/or
FXIa antibody. The antibody or pharmaceutical composition can be administered
either alone or
in combination with any other therapy for treating or preventing the
thrombosis-related
complications or conditions.
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[0057] Also provided is a method of treating and/or preventing sepsis in a
subject in need
thereof. It was attempted to administer anticoagulants to sepsis patients to
improve mortality or
morbidity. However, the attempt was unsuccessful due to the undesired bleeding
caused by
anticoagulants. The antibodies disclosed herein can be used as a secondary
therapy in
combination with other therapeutic agents for treating sepsis, such as
antibiotics.
[0058] As used herein, the term "subject" refers to mammalian subject,
preferably a human.
A "subject in need thereof' refers to a subject who has been diagnosed with
thrombosis or
complications or conditions associated with thrombosis, or is at an elevated
risk of developing
thrombosis or complications or conditions associated with thrombosis. The
phrases "subject"
and "patient" are used interchangeably herein.
[0059] The terms "treat," "treating," and "treatment" as used herein with
regard to a
condition refers to alleviating the condition partially or entirely,
preventing the condition,
decreasing the likelihood of occurrence or recurrence of the condition,
slowing the progression or
development of the condition, or eliminating, reducing, or slowing the
development of one or
more symptoms associated with the condition. With regard to thrombosis and/or
complications
or conditions associated with thrombosis, "treating" may refer to preventing
or slowing the
existing blood clot from growing larger, and/or preventing or slowing the
formation of blood clot.
In some embodiments, the term "treat," "treating," or "treatment" means that
the subject has a
reduced number or size of blood clots comparing to a subject without being
administered with the
antibodies or functional fragments thereof. In some embodiments, the term
"treat," "treating," or
"treatment" means that one or more symptoms of thrombosis and/or thrombosis-
related
conditions or complications are alleviated in a subject receiving an antibody
or pharmaceutical
composition as disclosed herein comparing to a subject who does not receive
such treatment.
[0060] A "therapeutically effective amount" of an antibody or
pharmaceutical composition as
used herein is an amount of the antibody or pharmaceutical composition that
produces a desired
therapeutic effect in a subject, such as treating and/or preventing
thrombosis. In certain
embodiments, the therapeutically effective amount is an amount of the antibody
or
pharmaceutical composition that yields maximum therapeutic effect. In other
embodiments, the
therapeutically effective amount yields a therapeutic effect that is less than
the maximum
therapeutic effect. For example, a therapeutically effective amount may be an
amount that
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produces a therapeutic effect while avoiding one or more side effects
associated with a dosage
that yields maximum therapeutic effect. A therapeutically effective amount for
a particular
composition will vary based on a variety of factors, including but not limited
to the
characteristics of the therapeutic composition (e.g., activity,
pharmacokinetics,
pharmacodynamics, and bioavailability), the physiological condition of the
subject (e.g., age,
body weight, sex, disease type and stage, medical history, general physical
condition,
responsiveness to a given dosage, and other present medications), the nature
of any
pharmaceutically acceptable carriers, excipients, and preservatives in the
composition, and the
route of administration. One skilled in the clinical and pharmacological arts
will be able to
determine a therapeutically effective amount through routine experimentation,
namely by
monitoring a subject's response to administration of the antibody or the
pharmaceutical
composition and adjusting the dosage accordingly. For additional guidance,
see, e.g., Remington:
The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press,
London, 2012, and
Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th Edition,
McGraw-Hill,
New York, NY, 2011, the entire disclosures of which are incorporated by
reference herein.
[0061] In
some embodiments, a therapeutically effective amount of an antibody disclosed
herein is in the range from about 0.01 mg/kg to about 30 mg/kg, from about 0.1
mg/kg to about
mg/kg, from about 1 mg/kg to about 5 mg/kg.
[0062] It
is within the purview of one of ordinary skill in the art to select a suitable
administration route, such as subcutaneous administration, intravenous
administration,
intramuscular administration, intradermal administration, intrathecal
administration, or
intraperitoneal administration.
For treating a subject in need thereof, the antibody or
pharmaceutical composition can be administered continuously or intermittently,
for an immediate
release, controlled release or sustained release. Additionally, the antibody
or pharmaceutical
composition can be administered three times a day, twice a day, or once a day
for a period of 3
days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, or 4 weeks. The antibody or
pharmaceutical
composition may be administered over a pre-determined time period.
Alternatively, the antibody
or pharmaceutical composition may be administered until a particular
therapeutic benchmark is
reached. In certain embodiments, the methods provided herein include a step of
evaluating one
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or more therapeutic benchmarks to determine whether to continue administration
of the antibody
or pharmaceutical composition.
Method of Producing Antibodies
[0063]
Also provided herein are methods of producing the anti-FXI and/or anti-FXIa
antibodies disclosed herein. In certain embodiments, these methods entail the
steps of cloning a
nucleic acid encoding an anti-FXI and/or anti-FXIa antibody into a vector,
transforming a host
cell with the vector, and culturing the host cell to express the antibody. The
expressed antibody
can be purified from the host cell using any known technique. Various
expression vectors such
as pTT5 vector, and pcDNA3 vector, as well as various host cell lines such as
CHO cells (e.g.
CHO-Kl and ExpiCH0), and HEK193T cells, can be used.
[0064]
Also encompassed by this disclosure are antibodies produced by the method
disclosed
above. The antibodies may have been subjected to one or more post-
translational modifications.
[0065]
The following examples are provided to better illustrate the embodiments and
are not
to be interpreted as limiting the scope of any claimed embodiment. To the
extent that specific
materials are mentioned, it is merely for purposes of illustration and is not
intended to limit the
invention. One skilled in the art may develop equivalent means or reactants
without the exercise
of inventive capacity and without departing from the scope of the invention.
It will be
understood that many variations can be made in the procedures herein described
while still
remaining within the bounds of the present invention. It is the intention of
the inventors that such
variations are included within the scope of the invention.
Examples
Example 1: Materials and methods
[0066]
Materials. Human FXI (Cat No. HFXI 1111), FXIa (Cat No. HFXIa 1111a),
FXIIa (HFXIIa 1212a), and FIX (Cat No. HFIX 1009) were purchased from Enzyme
Research Laboratory (IN, USA).
[0067]
Antibody preparation. Animal immunization and hybridoma screening were
performed at Genscript Inc. (Nanjing, China), and the procedures that were
applied to
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animals in this protocol were approved by the Genscript Institutional Animal
Care and Use
Committee. The experiment was performed in accordance with the approved
guidelines.
Wistar rats were immunized with human FXI, and splenocytes from animals with a
good
immune response were collected for the preparation of hybridomas, which were
subjected to
subcloning by limiting dilution. Finally, several monoclonal hybridoma clones
expressing
the desired anti-FXI antibodies, including 19F6, h-34F8 and 42A5, were
obtained by using
ELISA and functional screening. After determining the amino acid sequences of
their variable
regions, 19F6, h-34F8 and 42A5 were subjected to humanization, resulting in
three humanized
antibodies, h-19F6 , h-34F8 and h-42A5, in an IgG4 form. These three humanized
antibodies were produced in a transient mammalian expression system and
purified by Protein
G chromatography.
[0068] Activated partial thromboplastin time (APTT) and prothrombin time
(PT).
Standard human plasma purchased from Symens Inc. was mixed with equal volume
of various
antibodies at various concentrations from 0 to 400 nIVI for 5 minutes before
being tested on a
CA600 analyzer. In the APTT assay, 50 [IL of the plasma-antibody mixture and
25 [IL of APTT
reagent (SMN 10445709, Symens Inc.) were mixed at 37 C for 4 min. Then 25 [IL
of CaCl2
Solution (25 mM, SMN 10446232, Symens Inc.) was added and time to clot
formation was
determined. In the PT assay, 50 [IL of the plasma-antibody mixture was mixed
with an equal
volume of PT reagent (SMN 10446442, Symens Inc.) at 37 C and time to clot
formation was
determined.
[0069] The effects of the antibodies on APTT and PT in monkey plasma were
also
evaluated using the same methods applied to human plasma. In these assays,
monkey plasma
diluted with an equal volume of phosphate-buffered saline (PBS) was used
instead of the
above mentioned human plasma-antibody mixture.
[0070] FXI activation by FXIIa. Human FXI (500 nM) was pre-incubated at
room
temperature with 11.1.M control IgG4 or h-19F6 or h-34F8 or h-42A5 in PBS for
5 minutes.
At time zero, FXIIa, HK, and kaolin were added so that the final
concentrations were FXI
(250 nM), FXIIa (50 nM), HK(100 nM), and kaolin (0.5 mg/mL). At 0, 30, 60, 120
min
intervals, 50- L samples were collected into dodecyl sulfate sample buffer.
Samples were
size-fractionated on 10% non-reducing gels and transferred to polyvinylidene
fluoride
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membranes. Western blotting was performed to determine the FXI as well as FXIa-
light
chain levels using a mouse anti-human FXI IgG (105, an in-house made antibody
binding
the C-terminal of FXI). The image results were acquired using a ChemiDocMP
Imaging
System with Image Lab Software (Bio-Rad).
[0071] FXIa-mediated FIX activation. Human FIX (200 nM) was incubated with
FXIa (5
nM) in PBS containing 5 mM CaCl2 at room temperature in the presence of 1 [IM
control lgG4,
h-19F6, h-34F8, or h-42A5. At intervals of 0, 15, 30, 45, and 60 min, 50- L
samples were
collected into dodecyl sulfate sample buffer. Samples were size-fractionated
on 10% non-
reducing gels and transferred to polyvinylidene fluoride membranes. Western
blotting was
performed to determine the FIX as well as FIXa levels using goat anti- human
FIX IgG (Affinity
Biologicals). The image results were acquired using a ChemiDocMP Imaging
System with
Image Lab Software (Bio-Rad).
[0072] Surface plasmon resonance (SPR). The interaction of the antibodies
with FXI
was determined by the SPR assay on a Biacore T200 system (Biacore, GE
Healthcare).
Briefly, human IgG capture antibody (Biacore, GE Healthcare) was pre-
immobilized on a
CMS sensor chip (GE Healthcare), and test antibodies were captured by flowing
through the
chip. The final amount of the test antibodies captured was adjusted to an
equal amount of 15
response units (RU) by adjusting the capture time. Then, antigen FXI was
allowed to flow
through the chip for 180 s for association and then for 1200s for
dissociation. FXI was tested
at concentrations of 0.063, 0.313, 0.625, 1.25, 3.125 and 6.25 nM. The data
were collected
and the affinities between the test antibodies and FXI were analyzed using the
Biacore
Evaluation Software.
[0073] To determine the binding sites of test antibodies on FXI, four
mutants of FXI
tagged with 6xHis at the C terminal were first generated by replacing each
apple domain (Al,
A2, A3, and A4) with the corresponding domains from human prekallikrein. Equal
amounts
of each mutant were immobilized on a CMS sensor chip, and test antibodies
(33.3nM) were
allowed to flow through the chip for 180 s for association and then for 1200 s
for dissociation.
The amounts of each antibody captured were recorded in response units (RU)
using the
Biacore Evaluation Software.
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[0074] Epitope binding results of the test antibodies were also analyzed
using the Biacore
T200 system. Briefly, wild-type FXI with 6xHis tag was pre-immobilized on a
CMS sensor
chip (GE Healthcare), and h-19F6, h-34F8, or h-42A5 (5 pg/ml) was successively
injected
into flow cells on the sensor surface at a flow rate of 30 p1/minute to
monitor the change in
response.
[0075] Pharmacodynamics in cynomolgus monkeys. This animal experiment and the
following AV thrombosis experiment were performed at Wincon Inc. (Nanning,
China), and
the procedures applied to animals in this protocol were approved by the Wincon
Institutional
Animal Care and Use Committee. The experiments were performed in accordance
with the
approved guidelines. Animals received an intravenous bolus injection of
different doses
of h-19F6, h -3 4F 8, or h-42A5. Two mL of blood from the superficial veins of
upper limb
was collected into a collection tube containing 3.2% sodium citrate at pre-
dose (time 0) and at
0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h post-dose. Then, tubes were mixed by
gentle inversion ten
times at room temperature. Plasma was collected in labelled tubes and stored
at -20 C
until clotting time analysis. Plasma samples were diluted with an equal volume
of phosphate
buffered saline (pH 7.4) and then subjected to APTT and PT analysis on an
automatic
analyser (CA660, Sysmex Inc.).
[0076] AV shunt thrombosis and bleeding time test. A 30-min post-test
antibody
treatment was administered via intravenous bolus in cynomolgus monkeys. A tail
vein
bleeding time test was then performed, followed by thrombosis induction.
Thrombosis was
induced by connecting a shunt device between the femoral arterial and femoral
venous
cannulas containing a pre-weighed 10-cm-long thread. Blood was allowed to flow
through
the shunt for 10 min. The thrombus formed on the thread was weighed.
Immediately after
the removal of the shunt, blood samples were collected, and the next higher
level of test
antibody was administered. This bleeding/thrombosis process was carried out
four times to
dose the vehicle and three escalating levels (0.1, 0.3, 1 mg/kg) of test
antibody in the same
animal.
[0077] For bleeding time evaluation, a 2-mL syringe was inserted into the
tail vein of the
animals. When the volume of blood in the syringe stopped increasing, the
elapsed time was
recorded manually as the bleeding time.
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[0078] Ferric chloride (FeCl3)-induced thrombosis and bleeding time test.
This
animal experiment was performed at PharmaLegacy Laboratories Inc. (Shanghai,
China), and
the procedures that were applied to animals in this protocol were approved by
the
PharmaLegacy Institutional Animal Care and Use Committee. The experiment was
performed in accordance with the approved guidelines. Cynomolgus monkeys were
pre-
anaesthetized with 1.5 mg/kg of Zoletil, intubated, and ventilated with a
respirator.
Anaesthesia was maintained with isoflurane. Blood pressure, heart rate, and
body
temperature were monitored throughout the entire procedure. The vehicle or 0.3
mg/kg of h-
19F6, h-34F8, or h-42A5 was administered through a limb vein 2 hours before
FeCl3
application. The left femoral artery was exposed and isolated via blunt
dissection. A
Doppler flow probe was set up on the artery, and blood flow was continuously
recorded.
Before applying FeCl3, blood flow was measured for at least 5 minutes. Then,
two pieces of
filter paper pre-soaked with 40% FeCl3 were applied to the adventitial surface
of the vessel
upstream of the probe for 10 minutes. After the filter paper was removed, the
site of
application was washed with saline. Blood flow was continuously measured until
it
decreased to 0. The time to 80% occlusion (blood flow reduced to 20% of the
baseline blood
flow) and the time to 100% occlusion (blood flow reduced to 0) were recorded.
In the same
animals, haemostasis was evaluated using the Surgicutt device and bleeding
time was
manually recorded at pre-dose and 1-hour post-dose. At the end of the study
(approximately
3 hours post-dose), blood samples were collected.
[0079] The binding specificity of test antibodies to human FXI in plasma.
Test
antibodies (h-19F6, h-42A5, and 14E11) were first biotinylated using EZLinkTM
Sulfo-
NHS-LC-Biotinylation Kit (Cat No. 21435, Thermo Fisher Inc.). These antibodies
(25 ng
each) were incubated with 200 !IL of human standard plasma (Siemens Inc.) or
FXI-
deficient plasma (Hyphen Biomed Inc.) for 1 h. Then 50 !IL of Streptavidin-
coated beads
(DynabeadsTM M-280 Streptavidin, Thermo Fisher Inc.) were added to the mixture
to extract
the biotin-containing antigen-antibody complex. After washing with PBS for 3
times, the
antigen-antibody complex was then eluted and subjected to Western blotting
using a mouse
anti-human FXI IgG (105, an in-house made antibody binding the C-terminal of
FXI) as the
primary antibody. The image results were acquired using a ChemiDocMP Imaging
System
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with Image Lab Software (Bio-Rad). In Western blotting, 10 [IL of human
standard plasma
and FXI-deficient plasma served as FXI-positive and FXI-negative controls,
respectively.
[0080] Statistical analysis. Numerical data from multiple experiments are
presented as
means standard error of mean (SEM). One-way ANOVA followed by Dunnett's
multiple
comparisons test was used for the statistical analysis of thrombus weight in
the AV shunt
experiment and both bleeding time tests. The Kruskal-Wallis rank test was
performed for
the statistical analysis of occlusion times in the FeCl3-induced thrombosis
experiment. A
value of P < 0.05 was considered statistically significant.
Example 2: Generation and sequencing of anti-FXI antibodies
[0081] BALB/c mice and Wistar rats were immunized with human FXI, and
splenocytes
from the animals with good immune response were collected for the preparation
of hybridomas,
which were subjected to subcloning by limiting dilution. Twelve monoclonal
hybridoma clones
expressing desired anti-FXI antibodies 3G12, 5B2, 7C9, 7F1, 13F4, 19F6, 21F12,
34F8, 38E4,
42A5, 42F4, and 45H1 were obtained by using capture ELISA and functional
screening.
[0082] To determine the amino acid and nucleotide sequences of the variable
region of the
light (VL) and heavy chain (VH) of these antibodies, cDNAs encoding VL and VH
were cloned
from the corresponding hybridoma cells by standard RT-PCR procedures. The VL
and VH
sequences of exemplary antibodies, including the sequences of CDRs, are shown
in Table 1.
Example 3: Determination of anti-coagulation activity in human plasma using
activated
partial thromboplastin time (APTT) assay and prothrombin time (PT) assay
[0083] APTT assay measures the activity of the intrinsic and common
pathways of
coagulation; whereas PT assay measures the activity of the extrinsic and
common pathways of
coagulation. The antibodies tested in these experiments were 19F6, 34F8, 42A5,
1A6 and 14E11.
Antibodies 1A6 and 14E11 were used as positive controls in this experiment.
The sequences of
the variable regions of the control antibodies were obtained from U.S. Patent
No. 8,388,959 and
U.S. Patent Application Publication No. 2013/0171144 and reformatted to IgG4.
These
antibodies were then expressed using ExpiCHO cell system. The APTT assay and
PT assay were
performed as described above.
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[0084] As shown in Figure 1, all antibodies tested increased APTT in a
concentration-
dependent manner at a relatively low concentration, for example, up to 100 nM
(or up to 200 nM
for 14E11); whereas none of these antibodies had a significant effect on PT
(data not shown).
These results indicate that all of the antibodies tested inhibited the
intrinsic pathway of
coagulation but not the extrinsic pathway.
Example 4: Determination of the anti-coagulation activity in the plasma of non-
human
species using activated partial thromboplastin time (APTT) assay
[0085] Effects of various antibodies, including 19F6, 34F8 and 42A5, on
coagulation were
assessed in the mouse, rat, and monkey plasma using the same method as
described in Example 3.
None of the antibodies tested had any effect on APTT in the mouse and rat
plasma, but all of
them, at a relatively low concentration, concentration-dependently, increased
APTT in the
monkey plasma as shown in Figure 2, indicating that the antibodies tested had
cross-activity with
monkey FXI/FX1a, but not with mouse or rat FXI/FX1a.
Example 5: Humanization of anti-FXI antibodies
[0086] The use of murine monoclonal antibodies directly as therapeutics has
been hindered
by the short half-life and the elicitation of the human anti-murine antibody
responses. One
solution to this problem is to humanize the murine antibodies. Some antibodies
were subjected to
humanization by CDR grafting. Suitable human acceptor frameworks for both VL
and VH of
each murine antibody were identified and varying numbers of back mutations
were introduced to
the selected human frameworks to maintain the structure and/or function of the
resulting antibody.
If the affinity and function of these humanized antibodies were not
substantially inferior to the
corresponding unmodified antibodies, the modified antibodies were considered
successfully
humanized. Three humanized VH and VL sequences of 19F6, 34F8 and 42A5,
described as h-
19F6, h-34F8, and h-42A5, respectively, are shown in Table 2.
Example 6: Determination of the affinity of anti-FXI antibodies to human FXI
[0087] The affinity of anti-FXI/FXIa antibodies to FXI/FXIa were determined
using surface
plasmon resonance (SPR) technology performed on the BIAcore T200 instrument.
The
humanized antibodies were constructed by linking the variable regions of the
antibodies disclosed
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herein to human IgG4 Fc domain and the recombinants were expressed in CHO
cells. These
antibodies were captured onto a Biacore C1\45 sensor chip that was pre-
immobilized with an anti-
human IgG antibody.
[0088] Then different concentrations of the purified antigen FXI or FXIa
(0.005-1 pg/mL)
were allowed to flow through the C1\45 chip for 180 s for association with the
anti-FXI/FXIa
antibody, followed by a time of 1800 s for dissociation. The binding data was
collected and the
affinity between FXI/FXIa and the test antibodies was analyzed using the
Biacore Evaluation
Software provided by GE Healthcare. The SPR sensorgrams of FXI/FXIa binding to
immobilized h-19F6, h-34F8, and h-42A5 are shown in Figure 3. As shown in
Figure 3, the
response (RU) for each antibody became higher with escalating concentrations
of FXI or FXIa.
The dissociation constants (KD) of h-19F6, h-34F8, and h-42A5 to FXI and FXIa
were calculated
and detailed in Table 4. The affinities of each antibody to FXI and FXIa are
considered to be the
same since the difference between them is less than 10 times.
Table 4: KD values of the antibodies to FXI and FXIa
Antibody KD (PM)
FXI FXIa
h-19F6 22.2 25.8
h-34F8 19.5 3.85
h-42A5 35.7 8.14
Example 7: Determination of the binding site of anti-FXI antibodies on FXI
[0089] The binding sites of 19F6 and 42A5 on FXI were determined using the
SPR
technology. Briefly, human IgG capture antibody was pre-immobilized on a
Biacore C1\45 sensor
chip, and recombinant h-19F6 or h-42A5 was captured by flowing through the
chip. An equal
amount (15 relative units) of h-1 9F6 and h-42A5 was captured through
adjustment of the
antibody flowing time. Then wild type FXI or chimeric FXI in which individual
apple domain
was replaced with the corresponding domain from the human prekallikrein
(FXI/PK chimeras)
was allowed to flow through the chip for 180 seconds for association with h-
19F6 or h-42A5,
followed by a time period of 1800 seconds for dissociation. The binding data
was analyzed in a
high performance kinetic mode as only one concentration of FXI, wild-type or
chimeric, was
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tested in the SPR assay. Results showed that both h-19F6 and h-42A5 bound FXI
as well as
FXI/PK chimeras except when the A3 domain of FXI was replaced with the
corresponding PK
domain, indicating that part or the complete epitope of h-19F6 and h-42A5 on
FXI is located in
the A3 domain.
Example 8: Functional neutralization of FXIa by the antibodies
[0090] Human FXIa activity was determined by measuring the cleavage of a
specific,
chromogenic substrate, S-2366 (Diapharma Inc.). For testing the inhibitory
activity of the
antibodies, antibodies h-19F6, h-34F8 and h-42A5 were pre-incubated for 5
minutes at room
temperature with a final concentration of 5 nM of FXIa in PBS (phosphate
buffer saline). Then
an equal volume of 1 mM of S-2366 was added to initiate the FXIa cleavage
reaction and
changes in absorbance at 405 nm was monitored continuously using a M5e plate
reader
(Molecular Devices Inc.). Data were analyzed using the GraphPad Prism software
and are shown
in Figure 4. The calculated apparent Ki for h-1 9F6, h-34F8, and h-42A5 are
0.67, 2.08, and 1.43
nM, respectively. Therefore, all three antibodies tested exhibited satisfying
inhibitory effects on
FXIa at a relatively low concentration.
Example 9: Inhibition of FXIa-mediated FIX activation by the antibodies
[0091] The FXIa-mediated FIX activation was performed as described above.
Anti-FXI
antibodies may modulate the intrinsic pathway by inhibiting FXI activation
and/or by
inhibiting FXIa activity. First the effects of the two antibodies h-19F6 and h-
42A5 on FXIIa-
mediated activation of FXI were tested and it was found that neither h-19F6
nor h-42A5
prevented the conversion of FXI to FXIa mediated by FXIIa (Figures 5C and 5D).
Then, the
effect of these two antibodies on FXIa activity was tested using FIX as the
substrate. As
shown in Figures 5A and 5B, both h-19F 6 and h-42A5 reduced FIX activation in
a
concentration-dependent manner. The inhibitory effect of these two antibodies
on FXIa
activity was further confirmed by using a chromogenic substrate of FXIa, S-
2366. Both
antibodies concentration-dependently inhibited the hydrolysis of S-2366
(Figure 4).
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Example 10: Evaluation of the effects of anti-FXI antibodies on clotting time
in cynomolgus
monkeys
[0092] To find proper animal species for in vivo experiments, the cross-
reactivity of the
antibodies for mouse, rat, and monkey FXI was tested by the APTT assay. The
antibodies
prolonged APTT in monkey plasma but not in mouse or rat plasma (data not
shown). Thus,
monkey models were chosen for evaluating the pharmacodynamic effects of the
three
antibodies on clotting times prior to efficacy studies on thrombosis in vivo.
Cynomolgus
monkeys were intravenously administered with indicated doses of various
antibodies. Blood
from the superficial veins of the upper limb was collected at pre-dose and at
0.5, 1, 3, 6, 12, and
24 hours post-dose, and citrated plasma was prepared for APTT and PT
determination. In the
APTT test, 50 [IL of diluted plasma sample and 25 [IL of APTT reagent (SMN
10445709,
Symens Inc.) were mixed and incubated at 37 C for 4 min. Then 25 [IL of CaCl2
Solution (25
mM, SMN 10446232, Symens Inc.) was added and time to clot formation was
determined. In the
PT test, 50 [IL of diluted plasma sample was mixed with equal volume of PT
reagent (SMN
10446442, Symens Inc.) and incubated at 37 C and time to clot formation was
determined. All
three antibodies tested demonstrated dose-dependently increased APTT as shown
in Figure 6 and
none of them affected PT as shown in Figure 7.
[0093] Both h-19F6 and h-42A5 dose-dependently prolonged APTT (Figures 6B
and 6C).
Notably, h-42A5 increased APTT more strongly than h-19F6 did at the same dose
levels (0.3
and 1 mg/kg), consistent with the antibodies' in vitro effects on human APTT
(Figure 16A).
In addition, neither antibody affected the PT in vivo (Figures 7B and 7C).
Example 11: Evaluation of the effects of anti-FXI antibodies in arteriovenous
(AV) shunt
thrombosis and tail vein bleeding models in cynomolgus monkeys
[0094] Both thrombosis and bleeding time were assessed in the same animal
for multiple
doses of each antibody tested. The antibodies included in this experiment were
h-34F8, h-19F6,
and h42A5. Briefly, bleeding time and thrombosis were sequentially evaluated
at pre-dose and
30 minutes following each administration of the antibody. The
bleeding/thrombosis assessments
were conducted four times: pre-dose and post-dose at three escalating dose
levels (0.1, 0.3 and 1
mg/kg).
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[0095] For AV shunt thrombosis, a shunt device containing a pre-weighed 10-
cm long silk
thread was applied to connecting the femoral arterial and femoral venous
cannulae, and blood
was allowed to flow through the shunt for 10 minutes. Then the thread was
removed from the
shunt and weighed again. Clot weight on the thread was calculated as the
difference of the thread
weight before and after blood flow.
[0096] For bleeding time evaluation, a 2-mL syringe was inserted into the
tail vein of the
animals. When the volume of blood in the syringe stopped increasing, the
elapsed time was
recorded manually as the bleeding time.
[0097] All antibodies dose-dependently reduced the thrombus weight as shown
in Figure 8
and none of them prolonged the tail vein bleeding time as shown in Figure 9.
Effects of h-1 9F 6
and h-42A5 on thrombosis and haemostasis were evaluated in monkey models of AV
shunt
thrombosis and tail vein bleeding. Intravenous injection of h-19F6 resulted in
a dose-
dependent reduction of clot weight, and a significant reduction was observed
at 1 mg/kg
dose (Figure 8B). Regarding h-42A5-treated animals, clot weight was
significantly reduced
at all test dose levels in a dose-dependent manner (Figure 8C). No significant
change in
bleeding time was noted following treatment with h-19F6 or h-42A5 (Figures 9B
and 9C).
Example 12: Evaluation of the effects of anti-FXI antibodies on ferric
chloride¨induced
artery thrombosis and template bleeding time in cynomolgus monkeys
[0098] Cynomolgus monkeys were pre-anesthetized with 1.5 mg/kg of Zoletil,
intubated, and
ventilated with a respirator. Anesthesia was maintained with isoflurane. The
blood pressure,
heart rate, and body temperature were monitored throughout the entire
procedure. The antibodies
tested, including h-34F8, h-19F6, and h-42A5, or the vehicle control were
administered through
limb vein by injection 2 hours before FeCl3 application. The left femoral
artery was exposed and
isolated via blunt dissection. A Doppler flow probe was set up on the artery
and the blood flow
was continuously recorded. Before applying FeCl3, the blood flow was measured
for at least 5
minutes. Then two pieces of filter paper pre-soaked with FeCl3 were applied to
the adventitial
surface of the vessel upstream from the probe for 10 minutes. After the filter
paper was removed,
the site of application was washed with saline. Blood flow was continuously
measured until it
decreased to 0. The time to 80% occlusion (blood flow reduced to 20% of the
baseline blood
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flow) and the time to 100% occlusion (blood flow reduced to 0) were recorded.
In the same
animal, template bleeding time was assessed at pre-dose and 1 hour post-dose.
[0099] The effects of all three antibodies on FeCl3-induced arterial
thrombosis were
investigated. Four groups of monkeys were treated with the vehicle control, h-
34F8, h-19F6, or
h-42A5 for 2 hours, respectively, and FeCl3 was applied on the left femoral
artery of each animal
to induce thrombosis. Downstream blood flow velocity was monitored. The time
to 80% and to
100% thrombotic occlusion in the vehicle control group was 14.66 1.30 min
and 18.50 1.76
min, respectively. Pretreatment with 0.3 mg/kg of h-34F8 or h-42A5
significantly delayed the
time to 80% occlusion to 59.53 16.95 min and 40.80 7.94 min, and the time
to 100%
occlusion to 70.40 20.76 min and 50.61 9.48 min, respectively, as shown in
Figure 10.
Prolongation of the time to 80% occlusion (26.43 5.72 min) and to 100%
occlusion (32.78
5.09 min) was also observed in monkeys treated with h-19F6, although there was
no statistically
significant difference when compared to the vehicle control group as shown in
Figure 10.
[00100] The effects of the antibodies on haemostasis was assessed in terms of
template
bleeding time. No significant difference was noted between pre-dose and 1 hour
post-dose for
each test article (Figure 11A, 11B, and 11C). The bleeding time change
following h-34F8, h-
19F6, and h-42A5 treatment was not different from that following the vehicle
control treatment
(Figure 11D).
[00101] The effects of h-19F6 and h-42A5 antibodies on haemostasis were
assessed by a
skin laceration-caused bleeding test in the same animals with FeCl3-induced
arterial
thrombosis (n=5 per group). The bleeding times were recorded at pre-dose and 1-
hour post-
dose. No significant difference in bleeding time was observed between the pre-
dose and 1-
hour post-dose for either antibody or among the three groups 1-hour post-dose
(Figure 18).
[00102] The effects of the antibodies on ex vivo clotting times of monkey
plasma were also
evaluated. As expected, treatments with 0.3 mg/kg of h-3 4F8, h-1 9F6, and h-
42A5 significantly
prolonged APTT by 3.29 0.20, 1.67 0.09, and 2.87 0.10 fold,
respectively, while no
increase in APTT was observed upon vehicle control treatment, as shown in
Figure 12A. In
addition, treatments with h-34F8, h-19F6, or h-42A5 had no effect on PT as
shown in Figure 12B.
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[00103] Thus, it was unexpectedly discovered that the antibodies disclosed
herein did not
have any adverse effects of prolonged bleeding while effectively inhibiting
the intrinsic pathway
of coagulation.
Example 13: Evaluation of the effects of modified anti-FXI antibodies on
clotting time in
cynomolgus monkeys for an extended period of time
[00104] Two additional CMC optimized, humanized anti-FXI antibodies, shown
in Figures
14 and 15 as "modified h-19F6" and "modified h-42A5," were evaluated for their
effects on
clotting time in cynomolgus monkeys for an extended period of time, e.g., for
up to 14 days, by
APTT and PT assays as described in Example 10. The heavy chain and light chain
sequences of
these two antibodies are shown in Table 3. Cynomolgus monkeys were
intravenously
administered with 0.6 mg/kg or 2.0 mg/kg of the tested antibodies. Blood from
the superficial
veins of the upper limb was collected at pre-dose and at 0.5 hour, 2 hours, 6
hours, 12 hours, 24
hours, 48 hours, 96 hours, 168 hours, 240 hours, 336 hours post-dose. Both
modified antibodies
tested demonstrated dose-dependently increased APTT as shown in Figure 14 and
none of them
affected PT as shown in Figure 15. Both modified antibodies demonstrated
efficacy for an
extended period, up to 7 days, up to 10 days, or up to 14 days.
[00105] Thus, it was unexpectedly discovered that the modified antibodies
disclosed herein
did not show any adverse effects of prolonged bleeding while effectively
inhibiting the intrinsic
pathway of coagulation for an extended period of time, up to 14 days.
Example 14: Effects on clotting times of standard human plasma
[00106] Antibodies h-19F6 and h-42A5 were added to normal human plasma, after
which
the APTT (Figure 16A) and PT (Figure 16B) were determined (N = 3). Both h-19F6
and h-42A5
antibodies prolonged the activated partial thromboplastin time (APTT) of
standard human
plasma in a concentration-dependent manner (Figure 16A). The maximum levels of
inhibition of FXI activity in the plasma for h-19F6 and h-42A5 were
approximately 97% and
99.5%, respectively, based on the correlation curve between plasma F XI level
and APTT
established (data not shown). Neither antibody affected the PT of human plasma
(Figure
16B).
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Example 15: Binding properties of h-19F6 and h-42A5 to FXI
[00107] The binding specificity of h-19F6 and h-42A5 to FXI were first
verified as they
reacted with FXI in standard human plasma and no reaction was detected in
human FXI-
deficient plasma (Figure 17). Biotinylated test antibodies were incubated with
human
normal plasma or FXI-deficient plasma. The FXI-antibody complex in the plasma
was
eluted and subject to Western blotting using a mouse anti-human FXI IgG as the
primary
antibody. In Western blotting, 10 [IL of human standard plasma or FXI-
deficient plasma
were served as FXI-positive and FXI-negative controls. A previously reported
anti-FXI
antibody, 14E1117, showed the same binding profile as the two antibodies did
(Figure 17).
[00108] The affinities of h-19F 6 and h-42A5 to FXI were determined using
surface plasmon
resonance (SPR) technology. The test antibodies were captured on a sensor
chip, and then
indicated concentrations of FXI were allowed to flow through the chip.
Sensorgrams for h-
19F6 (Figure 19A) and h-42A5 (Figure 19B) were obtained. The dissociation
constants for
h-19F6 and h-42A5 were 22 and 36 pM, respectively (Figures 19A and 19B).
[00109] The binding sites of these two antibodies on FXI were then determined.
FXI is a
homodimer consisting of 4 tandem apple domains (A1-4) and a catalytic domain.
Four
mutants of FXI were generated by replacing each apple domain with
corresponding domains
from human prekallikrein and tested the binding properties of h-19F6 or h-42A5
to the 4
mutants of FXI using SPR. Equal amounts of the 4 mutant FXIs in which the Al,
A2, A3, or
A4 domain was replaced with the corresponding domain from prekallikrein were
immobilized on a sensor chip, and test antibodies (5 p.g/mL) were allowed to
flow through
the chip for association. The amounts of each antibody captured were recorded.
The
experiment was performed twice, and a representative result is depicted.
Unexpectedly, both
antibodies predominantly bound to the A3 domain of FXI, as replacement of the
A3 domain
resulted in much less binding of either antibody compared with the replacement
of the other
3 apple domains (Figure 19C). Another antibody, 01A6, a reported anti-FXI
antibody used
as a positive control, also specifically bound to the A3 domain of FXI,
consistent with a
previous study.21 However, it was hypothesized that h-19F6 and h-42A5 bind
different sites
of FXI because they have comparable affinities to FXI but quite different
inhibitory
potencies with respect to FXI activity, as indicated in Figure 16. This
hypothesis was tested
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by epitope binding using a Biacore T200 system. Indeed, the binding of h-19F6
to FXI did
not prevent the following binding of h-42A5 to FXI, indicating that the two
antibodies bind
different sites in the A3 domain of FXI (Figure 19D). Then the flowing order
of these two
antibodies were changed and it was found that binding of h-42A5 to FXI did not
prevent the
following binding of h-19F6 to FXI (data not shown).
Example 16: Binding properties of h-19F6 and h-42A5 to FXIa
[00110] The antibodies bound to FXIa with the good affinities with which they
bound to
FXI (Figures 20A and 20B). The affinities of h-19F6 and h-42A5 to FXI were
determined
using surface plasmon resonance (SPR) technology. The dissociation constants
for h-19F6
and h-42A5 were 26 and 81 pM, respectively (Figures 20A and 20B). The test
antibodies
were captured on a sensor chip, and then indicated concentrations of FXIa were
allowed to
flow through the chip. Sensorgrams for h-19F6 (Figure 20A) and h-42A5 (Figure
20B) were
obtained.
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