U.S. patent application number 15/117027 was filed with the patent office on 2017-09-07 for dual targeting of tafi and pai-1.
The applicant listed for this patent is Centre Hospitalier Universitaire de Caen, INSERM (Institut National de la Sante et de la Recherche Medicale), KATHOLIEKE UNIVERSITEIT LEUVEN, Universite de Caen Basse Normandie. Invention is credited to Simon DE MEYER, Paul DECLERCK, Nick GEUKENS, Ann GILS, Marina RUBIO, Denis VIVIEN, Tine WYSEURE.
Application Number | 20170253664 15/117027 |
Document ID | / |
Family ID | 53777354 |
Filed Date | 2017-09-07 |
United States Patent
Application |
20170253664 |
Kind Code |
A2 |
DECLERCK; Paul ; et
al. |
September 7, 2017 |
DUAL TARGETING OF TAFI AND PAI-1
Abstract
Disclosed herein is a bispecific inhibitor for use in treating
thrombotic disorders, such as acute thrombotic disorders like
stroke and thromboembolism. The bispecific inhibitor is based on
monoclonal antibodies targeting TAFI and PAI-1, and shows efficacy
in the presence or the absence of plasminogen activators such as
tissue-type plasminogen activator (tPA).
Inventors: |
DECLERCK; Paul; (Leuven,
BE) ; DE MEYER; Simon; (Leuven, BE) ; GEUKENS;
Nick; (Leuven, BE) ; GILS; Ann; (Leuven,
BE) ; RUBIO; Marina; (Caen, FR) ; VIVIEN;
Denis; (Caen, FR) ; WYSEURE; Tine; (Leuven,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KATHOLIEKE UNIVERSITEIT LEUVEN
INSERM (Institut National de la Sante et de la Recherche
Medicale)
Universite de Caen Basse Normandie
Centre Hospitalier Universitaire de Caen |
Leuven
Paris
Caen
Caen |
|
BE
FR
FR
FR |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160347859 A1 |
December 1, 2016 |
|
|
Family ID: |
53777354 |
Appl. No.: |
15/117027 |
Filed: |
February 9, 2015 |
PCT Filed: |
February 9, 2015 |
PCT NO: |
PCT/EP2015/052624 PCKC 00 |
371 Date: |
August 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61937323 |
Feb 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/38 20130101;
C07K 2317/622 20130101; C07K 2317/565 20130101; A61P 7/02 20180101;
A61P 9/10 20180101; C07K 2317/76 20130101; A61K 2039/505 20130101;
G01N 2800/22 20130101; C07K 2317/31 20130101; C07K 2317/56
20130101; C07K 2317/24 20130101; C07K 16/468 20130101; C07K
2317/626 20130101 |
International
Class: |
C07K 16/38 20060101
C07K016/38 |
Claims
1-19. (canceled)
20. A method of treating or preventing an acute thrombotic disorder
in a patient, comprising the step of administering a bispecific
antibody against TAFI and PAI-1, wherein said antibody comprises a.
a first targeting domain that specifically binds to Thrombin
Activatable Fibrinolysis Inhibitor (TAFI) and comprises
complementary determining regions (CDRs) represented by the amino
acid sequences SEQ ID NO:1 of CDR1H, SEQ ID NO:2 of CDR2H, SEQ ID
NO:3 of CDR3H, SEQ ID NO:4 of CDR1L, SEQ ID NO:5 of CDR2L, and SEQ
ID NO:6 of CDR3L, and b. a second targeting domain that
specifically binds to Plasminogen Activator Inhibitor-1 (PAI-1) and
comprises complementary determining regions (CDRs) represented by
SEQ ID NO:7 of CDR1H, SEQ ID NO:8 of CDR2H, SEQ ID NO:9 of CDR3H,
SEQ ID NO:10 of CDR1L, SEQ ID NO:11 of CDR2L and SEQ ID NO:12 of
CDR3L.
21. The method according to claim 20, wherein a. said first
targeting domain that binds to Thrombin Activatable Fibrinolysis
Inhibitor (TAFI) comprises a VH region represented by an amino acid
sequence that is at least 80% identical to SEQ ID NO:13 and a VL
region represented by an amino acid sequence that is at least 80%
identical to SEQ ID NO:14 and comprises complementary determining
regions (CDRs) represented by the amino acid sequences SEQ ID NO:1
of CDR1H, SEQ ID NO:2 of CDR2H, SEQ ID NO:3 of CDR3H, SEQ ID NO:4
of CDR1L, SEQ ID NO:5 of CDR2L, and SEQ ID NO:6 of CDR3L; and b.
said second targeting domain that binds to Plasminogen Activator
Inhibitor-1 (PAI-1) and comprises a VH region represented by an
amino acid sequence that is at least 80% identical to SEQ ID NO:15
and a VL region represented by an amino acid sequence that is at
least 80% identical to SEQ ID NO:16, and comprises complementary
determining regions (CDRs) represented by SEQ ID NO:7 of CDR1H, SEQ
ID NO:8 of CDR2H, SEQ ID NO:9 of CDR3H, SEQ ID NO:10 of CDR1L, SEQ
ID NO:11 of CDR2L and SEQ ID NO:12 of CDR3L.
22. The method according to claim 20, wherein said first targeting
domain that binds to Thrombin Activatable Fibrinolysis Inhibitor
(TAFI) comprises a VH region represented by an amino acid sequence
that is at least 90% identical to SEQ ID NO:13 and comprises a VL
region represented by an amino acid sequence that is at least 90%
identical to SEQ ID NO:14, and wherein said second targeting domain
that binds to Plasminogen Activator Inhibitor-1 (PAI-1) comprises a
VH region represented by an amino acid sequence that is at least
90% identical to SEQ ID NO:15 and comprises a VL region represented
by an amino acid sequence that is at least 90% identical to SEQ ID
NO:16.
23. The method according to claim 20, wherein said first targeting
domain that binds to Thrombin Activatable Fibrinolysis Inhibitor
(TAFI) comprises a VH region represented by an amino acid sequence
that is at least 95% identical to SEQ ID NO:13 and comprises a VL
region represented by an amino acid sequence that is at least 95%
identical to SEQ ID NO:14, and wherein said second targeting domain
that binds to Plasminogen Activator Inhibitor-1 (PAI-1) comprises a
VH region represented by an amino acid sequence that is at least
95% identical to SEQ ID NO:15 and comprises a VL region represented
by an amino acid sequence that is at least 95% identical to SEQ ID
NO:16.
24. The method according to claim 20, wherein said first targeting
domain that binds to Thrombin Activatable Fibrinolysis Inhibitor
(TAFI) comprises a VH region represented by an amino acid sequence
that is at least 98% identical to SEQ ID NO:13 and comprises a VL
region represented by an amino acid sequence that is at least 98%
identical to SEQ ID NO:14, and wherein said second targeting domain
that binds to Plasminogen Activator Inhibitor-1 (PAI-1) comprises a
VH region represented by an amino acid sequence that is at least
98% identical to SEQ ID NO:15 and comprises a VL region represented
by an amino acid sequence that is at least 98% identical to SEQ ID
NO:16.
25. The method according to claim 20, wherein said bispecific
antibody is humanized.
26. The method according to claim 20, for treating brain lesions in
an acute thrombotic disorder.
27. The method according to claim 20, wherein the acute thrombotic
disorder is selected from the group consisting of, acute peripheral
arterial occlusion, middle cerebral artery occlusion (MCAO), and
thromboembolism such as deep vein thromboembolism and lung
embolism.
28. The method according to claim 20, in a combination treatment
with tPA.
29. The method according to claim 20, wherein said treatment is
performed without administration of tPA, prior, together of after
the administration of the bispecific antibody.
30. The method according to claim 20, wherein said acute thrombotic
disorder is characterized by presence of a platelet-rich blood
clot.
Description
FIELD OF INVENTION
[0001] The present invention relates generally to treatment and
prevention of thrombotic disorders such as stroke and
thromboembolism by dual inhibition of plasminogen activator
inhibitor 1 (PAI-1) and Thrombin-Activatable Fibrinolysis Inhibitor
(TAFI). The dual inhibition may be mediated by a bispecific
antibody derivative that binds to both targets.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] This application includes an electronically submitted
sequence listing in .pdf format. The .pdf file contains a sequence
listing entitled "19893-18-Sequence_Listing.pdf" created on Aug. 5,
2016 and is 43 bytes in size. The sequence listing contained in
this .pdf file is part of the specification and is hereby
incorporated by reference herein in its entirety.
BACKGROUND
[0003] Following an acute cardiovascular accident, the only
treatment currently available to recanalize an occluded blood
vessel is systemic delivery of a high dose of plasminogen
activators. While effective when administered soon after the event,
plasminogen activators also cause debilitating side effects such as
intracranial haemorrhage and neurotoxicity. In addition, successful
restoration of blood flow is not guaranteed because of low
recanalization and high reocclusion rates, even when high doses of
plasminogen activators are administered [Saver J L. et al. (2011) J
Thromb Haemost. 9 Suppl 1,333-343]. Accordingly, there remains a
need in the art for effective treatments of occluded blood vessels,
for example by promoting fibrinolysis or thrombolysis.
[0004] One of the causes for thrombolytic failure is the presence
of circulating inhibitors of fibrinolysis, such as
Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) and plasminogen
activator inhibitor 1 (PAI-1) [Fernandez-Cadenas I et al. (2007) J
Thromb Haemost. 5,1862-1868]. Both molecules slow down the tissue
type-plasminogen activator (tPA)-mediated formation of plasmin, the
key enzyme in fibrinolysis, although through distinct mechanisms
(as reviewed in Rijken D C & Lijnen H R (2009) J Thromb
Haemost. 7, 4-13). TAFI, a 56 kDa proenzyme with a plasma level of
4-15 .mu.g/ml, can be activated into TAFIa by thrombin, alone or in
complex with thrombomodulin, or plasmin. Through its
carboxypeptidase activity, TAFIa is able to cleave off C-terminal
Lys residues exposed on partially degraded fibrin, which serve as a
co-factor function in the tPA-mediated activation of plasminogen
into plasmin. PAI-1 (45 kDa glycoprotein with a plasma level of
5-50 ng/ml and a concentration within platelets of 200 ng/ml) is
the main inhibitor of tPA and belongs to the serine protease
inhibitors (serpin) superfamily. The active form of PAI-1 can
irreversibly neutralize the activity of tPA by forming a 1:1
stoichiometric covalent complex, accompanied by deformation of
catalytic triad of the serine protease.
[0005] Given their complementary roles in inhibiting fibrinolysis,
one approach to promoting fibrinolysis is dual inhibition of TAFI
and PAI-1. Simultaneous targeting of TAFI and PAI-1 has been
attempted in several studies. However, the results did not
consistently indicate that dual inhibition of TAFI and PAI-1
improved thrombolysis as compared to single inhibition. In one
study, complementary roles of TAFI and PAI-1, as well as a third
molecule .alpha..sub.2-AP, were characterized in tPA induced
thrombolysis assays in the presence or absence of inhibitors of
TAFI, PAI-1, and/or .alpha..sub.2-AP [Mutch N J. et al. (2007) J
Thromb Haemost. 5, 812-817]. Depending on the type of thrombus, the
assays indicated either a role for all three molecules or a
substantial contribution of .alpha..sub.2-AP and TAFI, with a minor
contribution from PAI-1. Similarly, single and double knockout
studies in mice suggested that thrombolytic effects in certain
assays were due to inhibition of TAFI rather than PAI-1
[Vercauteren E et al. (2012) J Thromb Haemost. 10, 2555-2562].
[0006] Notably, a dual targeting strategy based on bispecific
antibody derivatives (diabodies) has shown promise. The diabody
T12D11.times.33H1F7, based on monoclonal antibodies which bind TAFI
and PAI-1, was shown to have a stimulating effect on fibrinolysis
which exceeded the effect observed when its component monoclonal
antibodies (MA) were tested separately. In addition, new monoclonal
antibodies against TAFI and PAI-1 exhibit unique features.
MA-RT36A3F5 and MA-TCK26D6 both inhibit mouse and rat TAFI, with
each MA acting through distinct mechanisms: the former destabilizes
TAFIa, whereas the latter impairs the plasmin-mediated activation
of TAFI and also interferes with the interaction of TAFIa on fibrin
[Hillmayer K et al. J (2008) Thromb Haemost. 6, 1892-1899;
Vercauteren E et al. (2011) Blood 117, 4615-4622; Semeraro F, et
al. (2013) J Thromb Haemost. 11, 2137-2147]. MA-33H1F7 and MA-MP2D2
inhibit mouse and rat PAI-1, by converting the active form into a
substrate form of PAI-1 which is cleaved by tPA [Debrock S. &
Declerck P J. (1997) Biochim Biophys Acta. 1337, 257-266; Van De
Craen B. et al. (2011) Thromb Res. 128, 68-76]. In vivo studies
have shown a beneficial effect of the above mentioned antibodies on
the rate of survival and paralysis in mice after thromboembolic
challenge [Vercauteren (2011) cited above, Van De Craen cited
above]. Recently, the MA antibodies MA-33H1F7 and MA-TCK26D6 which
specifically recognize the corresponding human antigens were
adapted to make the bispecific antibody derivative
Db-TCK26D6.times.33H1F7, and a strong profibrinolytic effect of the
diabody was demonstrated in vitro [Wyseure T et al. (2013) J Thromb
Haemost. 11, 2069-2071]. However, no dual targeting studies to date
have conclusively demonstrated a role for inhibitors or diabodies
in treating specific thrombotic disorders in vivo. In addition, no
studies have provided evidence for viable treatments for thrombotic
disorders based on inhibitors of fibrinolysis or thrombolysis as
alternatives to plasminogen activators.
SUMMARY OF INVENTION
[0007] Described herein is the diabody Db-TCK26D6.times.33H1F7 for
use in treating thrombotic disorders, such as stroke and
thromboembolism. Db-TCK26D6.times.33H1F7 may be administered either
before or after the onset of the thrombotic disorder, and moreover,
may be administered in the presence or the absence of plasminogen
activators such as tPA.
[0008] The present disclosure relates to a bispecific antibody
derivative for use in treating an acute thrombotic disorder in a
patient, comprising a first targeting domain that binds to Thrombin
Activatable Fibrinolysis Inhibitor (TAFI) and comprises
complementary determining regions (CDRs) represented by amino acid
sequences that are at least 80% identical to each of CDR1H of SEQ
ID NO:1, CDR2H of SEQ ID NO:2, CDR3H of SEQ ID NO:3, CDR1L of SEQ
ID NO:4, CDR2L of SEQ ID NO:5, and CDR3L of SEQ ID NO:6; and a
second targeting domain that binds to Plasminogen Activator
Inhibitor-1 (PAI-1) and comprises complementary determining regions
(CDRs) represented by amino acid sequences that are at least 80%
identical to each of CDR1H of SEQ ID NO:7, CDR2H of SEQ ID NO:8,
CDR3H of SEQ ID NO:9, CDR1L of SEQ ID NO:10, CDR2L of SEQ ID NO:11,
and CDR3L of SEQ ID NO:12, wherein the bispecific antibody
derivative is administered after onset of the acute thrombotic
disorder.
[0009] In some embodiments, the amino acid sequences in the
bispecific antibody derivatives are at least 80%, 85%, 90%, 95%,
99% or 100% identical to the amino acid sequences disclosed in SEQ
ID NO:1-18. In some embodiments, the bispecific antibody derivative
is for use in an acute thrombotic disorder that is at least one of
acute ischemic stroke (AIS), middle cerebral artery occlusion
(MCAo), thromboembolism, deep vein thrombosis, myocardial
infarction (MI), pulmonary embolism, peripheral arterial disease,
thrombosis of liver and/or kidneys, or catheter blockage. For
example, the acute thrombotic disorder may be AIS. The acute
thrombotic disorder may be MCAo.
[0010] In certain embodiments, the bispecific antibody derivative
is administered between 0-15 hours after onset of symptoms of the
acute thrombotic disorder. In some embodiments, the bispecific
antibody derivative is administered up to 3 hours after onset of
symptoms of the acute thrombotic disorder. In some embodiments, the
bispecific antibody derivative is administered up to 4.5 hours
after onset of symptoms of the acute thrombotic disorder. In some
embodiments, the bispecific antibody derivative is administered up
to 12 hours after onset of symptoms of the acute thrombotic
disorder.
[0011] The bispecific antibody derivative may be administered
without tPA. In some embodiments, the bispecific antibody
derivative is administered without tPA during a time period of up
to 90 minutes after the onset of the acute thrombotic disorder, for
example, 0-90 minutes after onset.
[0012] In some embodiments, the bispecific antibody derivative is
administered with tPA. For example, the bispecific antibody
derivative may be administered 1 hour after administration of
tPA.
[0013] In some embodiments, bispecific antibody derivative is for
use in an acute thrombotic disorder that is characterized by
presence of a fibrin-rich blood clot. The acute thrombotic disorder
may be characterized by presence of a platelet-rich blood clot.
[0014] In certain embodiments, the bispecific antibody derivative
is humanized.
[0015] A further aspect of the present disclosure relates to a
bispecific antibody derivative for use in treating an acute
thrombotic disorder in a patient, comprising a first targeting
domain that binds to Thrombin Activatable Fibrinolysis Inhibitor
(TAFI) and comprises a VH region represented by an amino acid
sequence that is at least 80% identical to SEQ ID NO:13 and a VL
region represented by an amino acid sequence that is at least 80%
identical to SEQ ID NO:14; and a second targeting domain that binds
to Plasminogen Activator Inhibitor-1 (PAI-1) and comprises a VH
region represented by an amino acid sequence that is at least 80%
identical to SEQ ID NO:15 and a VL region represented by an amino
acid sequence that is at least 80% identical to SEQ ID NO:16,
wherein the bispecific antibody derivative is administered after
onset of the acute thrombotic disorder. In some embodiments, a
bispecific antibody derivative for use in treating an acute
thrombotic disorder in a patient, comprising a first domain
represented by an amino acid sequence that is at least 80%
identical to SEQ ID NO:17; and a second domain represented by an
amino acid sequence that is at least 80% identical to SEQ ID NO:18,
wherein the bispecific antibody derivative is administered after
onset of the acute thrombotic disorder.
[0016] Yet another aspect of the present disclosure relates to a
bispecific antibody derivative for use in treating an acute
thrombotic disorder in a patient, comprising a first targeting
domain that binds to Thrombin Activatable Fibrinolysis Inhibitor
(TAFI) and comprises complementary determining regions (CDRs)
represented by amino acid sequences that are at least 80% identical
to each of CDR1H of SEQ ID NO:1, CDR2H of SEQ ID NO:2, CDR3H of SEQ
ID NO:3, CDR1L of SEQ ID NO:4, CDR2L of SEQ ID NO:5, and CDR3L of
SEQ ID NO:6; and a second targeting domain that binds to
Plasminogen Activator Inhibitor-1 (PAI-1) and comprises
complementary determining regions (CDRs) represented by amino acid
sequences that are at least 80% identical to each of CDR1H of SEQ
ID NO:7, CDR2H of SEQ ID NO:8, CDR3H of SEQ ID NO:9, CDR1L of SEQ
ID NO:10, CDR2L of SEQ ID NO:11, and CDR3L of SEQ ID NO:12, wherein
the bispecific antibody derivative is administered after onset of
the acute thrombotic disorder and is administered without tPA. In
some embodiments, the bispecific antibody derivative is
administered without tPA, and within a time period that is no more
than 90 minutes from the onset of the acute thrombotic
disorder.
[0017] An aspect of the present invention relates to bispecific
antibodies use in treating or preventing an acute thrombotic
disorder in a patient. Such antibodies comprise a first targeting
domain that specifically binds to Thrombin Activatable Fibrinolysis
Inhibitor (TAFI) and comprises complementary determining regions
(CDRs) represented by the amino acid sequences SEQ ID NO:1 of
CDR1H, SEQ ID NO:2 of CDR2H, SEQ ID NO:3 of CDR3H, SEQ ID NO:4 of
CDR1L , SEQ ID NO:5 of CDR2L , and SEQ ID NO:6 of CDR3L; and a
second targeting domain that specifically binds to Plasminogen
Activator Inhibitor-1 (PAI-1) and comprises complementary
determining regions (CDRs) represented by SEQ ID NO:7 of CDR1H, SEQ
ID NO:8 of CDR2H, SEQ ID NO:9 of CDR3H, SEQ ID NO:10 of CDR1L, SEQ
ID NO:11 of CDR2L and SEQ ID NO:12 of CDR3L.
[0018] Embodiments hereof include bispecific antibodies wherein the
first targeting domain that binds to Thrombin Activatable
Fibrinolysis Inhibitor (TAFI) comprises a VH region represented by
an amino acid sequence that is at least 80% identical to SEQ ID
NO:13 and a VL region represented by an amino acid sequence that is
at least 80% identical to SEQ ID NO:14; and the second targeting
domain that binds to Plasminogen Activator Inhibitor-1 (PAI-1) and
comprises a VH region represented by an amino acid sequence that is
at least 80% identical to SEQ ID NO:15 and a VL region represented
by an amino acid sequence that is at least 80% identical to SEQ ID
NO:16.
[0019] Embodiments hereof are bispecific antibodies that are
humanized.
[0020] In specific embodiments these bispecific antibodies are for
use in treating or preventing brain lesions resulting from an acute
thrombotic disorder
[0021] In specific embodiments the acute thrombotic disorder is
selected from the group consisting of, acute peripheral arterial
occlusion, middle cerebral artery occlusion (MCAo), and
thromboembolism such as deep vein thromboembolism and lung
embolism.
[0022] In certain embodiments the bispecific antibodies are for use
in treating or preventing an acute thrombotic disorder in a patient
in a combination treatment with tPA.
[0023] In other embodiments the bispecific antibodies are for use
in treating or preventing an acute thrombotic disorder in a
patient, where the treatment is performed without administration of
tPA, prior, together of after the administration of the bispecific
antibody.
[0024] The above mentioned acute thrombotic disorder is in specific
embodiment characterized by the presence of a platelet-rich blood
clot.
[0025] Another aspect of the present invention relates to methods
for treating or preventing an acute thrombotic disorder in a
patient, comprising the step of administering a bispecific antibody
against TAFI and PAI. Herein the antibody comprises a first
targeting domain that specifically binds to Thrombin Activatable
Fibrinolysis Inhibitor (TAFI) and comprises complementary
determining regions (CDRs) represented by the amino acid sequences
SEQ ID NO:1 of CDR1H, SEQ ID NO:2 of CDR2H, SEQ ID NO:3 of CDR3H,
SEQ ID NO:4 of CDR1L , SEQ ID NO:5 of CDR2L , and SEQ ID NO:6 of
CDR3L; and a second targeting domain that specifically binds to
Plasminogen Activator Inhibitor-1 (PAI-1) and comprises
complementary determining regions (CDRs) represented by SEQ ID NO:7
of CDR1H, SEQ ID NO:8 of CDR2H, SEQ ID NO:9 of CDR3H, SEQ ID NO:10
of CDR1L, SEQ ID NO:11 of CDR2L and SEQ ID NO:12 of CDR3L.
[0026] The methods of the invention provide several advantages
compared to existing therapies.
[0027] The methods of the present invention wherein a diabody
against TAFI and PAI-1 is used have, compared to tPA, less risks of
causing intracranial haemorrhage and neurotoxicity. The diabodies
of the present invention are of use in reducing lesion size in
patients suffering from a brain lesion. Brain lesions may be caused
by thrombotic disorders, such as stroke, acute ischemic stroke
(AIS), and/or middle cerebral artery occlusion (MCAo).
[0028] Compared to tPA which has short activity upon administration
(about 15 minutes), diabodies can bind to their targets for a much
longer time period.
[0029] High doses of tPA can result in unwanted enzymatic activity
of plasmin. The use of a high dose of diabody is less critical.
Antibody which does not bind PAI-1 or TAFI has no side effects.
[0030] Compared to tPA, the diabodies show a reduction in bleeding
time. Accordingly the diabodies of the present invention have the
advantageous property of reducing the risk of unwanted bleeding,
such as intracranial haemorrhage.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1A-1B shows the expression levels of CDR-grafted scFv.
A. Modeled structure of CDR of MA-RT36A3F5 (circles) in the
framework of scFv-4D5. B. Immunoblot showing periplasmic extracts
containing CDR-grafted scFv-RT36A3F5-4D5 (lane 1), scFv-T12D11 as
control (lane 2), scFv-RT36A3F5-T12D11 (lane 3) and
scFv-RT36A3F5-4D5DM (lane 4), detected via anti-His-tag polyclonal
antibody.
[0032] FIG. 2 shows a schematic representation of bispecific
inhibitors: Db-RT36A3F5.times.33H1F7 (Db),
Db-RT36A3F5-4D5.times.33H1F7 (CDR-grafted Db),
scDb-33H1F7.times.RT36A3F5 (scDb) and
scDb-33H1F7.times.RT36A3F5-4D5 (CDR-grafted scDb).
[0033] PH: variable region heavy chain anti-PAI-1 antibody; PL:
variable region light chain anti-PAI-1 antibody; TH: variable
region heavy chain anti-TAFI antibody; TL: variable region light
chain anti-TAFI antibody; TH': humanised variable region heavy
chain anti-TAFI antibody; TL': humanised variable region light
chain anti-TAFI antibody.
[0034] FIG. 3A-3B shows the plasma stability and profibrinolytic
effect during in vitro clot lysis. A. Graph representing stability,
determined by an ELISA-based assay to measure residual binding
capacity towards TAFI and PAI-1 simultaneously.
(sc)Db-variants(Db-RT36A3F5.times.33H1F7 (Db),
Db-RT36A3F5-4D5.times.33H1F7 (CDR-grafted Db),
scDb-33H1F7.times.RT36A3F5 (scDb) and
scDb-33H1F7.times.RT36A3F5-4D5 (CDR-grafted scDb)) at 10 .mu.g/ml
were incubated in citrated rat plasma at 37.degree. C. The control
Db was Db-T12D11.times.33H1F7] At time points zero, 1 hour and 3
hours, an aliquot was analyzed and bound protein was relatively
expressed towards that of time point zero (residual binding in %,
mean.+-.SEM, n=3-9). B. Graph representing stability (% residual
binding after 3 hours at 37.degree. C. in plasma, mean.+-.SEM,
n=3-9) vs. profibrinolytic properties of (sc)Db-variants at an
8-fold molar excess over TAFI during clot lysis in rat plasma
(expressed as relative lysis (mean.+-.SEM, n=3) to that of
MA-RT36A3F5 at a 4-fold molar excess over TAFI).
[0035] FIG. 4A-4B shows the profibrinolytic effect of MA (single or
combined addition of MA-TCK26D6 and MA-33H1F7) and
Db-TCK26D6.times.33H1F7 during thromboelastometric measurements
using human blood (A) and blood from endotoxemic mice (B). Graph
representing (A) degree of lysis (.DELTA. L45, %; mean.+-.SEM;
n=6-12) and (B) relative .DELTA. AUC (mean.+-.SEM, n=3-6) in the
presence of MA-33H1F7, MA-TCK26D6, the combined addition of MA or
diabody. Statistical significance is indicated by asterisks
(*p<0.05; **p<0.01; ***p<0.001).
[0036] FIG. 5 shows an in vivo evaluation of MA in a
thromboembolism model induced by systemic administration of
thromboplastin. Graph representing fibrin contents in lungs
injected with saline, MA-TCK26D6 at 5 mg/kg or MA-33H1F7 at 1 mg/kg
(mean.+-.SEM, n=5-7). Baseline levels were obtained by isolating
lungs from mice without thrombotic challenge (mean.+-.SEM, n=5).
Statistical significance is indicated by asterisks (*p<0.05;
**p<0.01; ***p<0.001).
[0037] FIG. 6A-6B shows an in vivo evaluation of MA and Db in a
thromboembolism model using endotoxemic mice. Graph representing
fibrin contents in lungs from endotoxemic mice injected with (A.)
vehicle, MA-33H1F7 at 10 mg/kg, MA-TCK26D6 at 5 mg/kg, MA-TCK26D6
at 5 mg/kg+MA-33H1F7 at 10 mg/kg or (B.) vehicle, MA-33H1F7 at 1
mg/kg, MA-TCK26D6 at 1 mg/kg, MA-TCK26D6 at 1 mg/kg+MA-33H1F7 at 1
mg/kg, Db-TCK26D6.times.33H1F7 at 0.8 mg/kg (mean.+-.SEM, n=5-10).
Baseline levels were obtained by isolating lungs from healthy mice
without thrombotic challenge (mean.+-.SEM, n=5). Statistical
significance is indicated relative to vehicle (*p<0.05;
**p<0.01; ***p<0.001).
[0038] FIG. 7A-7G shows in vivo evaluation of MA in a mouse model
of transient mechanical MCAo. FIGS. 7A and 7D show lesion size
(mm.sup.3), FIGS. 7B and 7E show the Bederson score (0-5), FIGS. 7C
and 7F show the Grip test score (0-5), measured 24 hours post
occlusion in mice treated with vehicle (PBS), negative control IgG,
MA-TCK26D6 at 6 mg/kg and MA-TCK26D6 at 25 mg/kg (FIG. 7A-7C,
mean.+-.SEM, n=8-12) and vehicle (PBS), negative control IgG and
MA-33H1F7 at 6 mg/kg (FIG. 7D-7F, mean.+-.SEM, n=14-16). FIG. 7G
shows fibrinogen contents in ipsilateral side of brain (fold
increase vs. contralateral), measured at 24 hours post occlusion in
mice treated with vehicle (PBS), negative control IgG, MA-33H1F7 at
6 mg/kg and MA-TCK26D6 at 25 mg/kg (mean.+-.SEM, n=4-5).
Statistical significance is indicated as follows: *p<0.05;
**p<0.01; ***p<0.001. (the control MA is MA-T30E5)
[0039] FIG. 8A-8D shows the in vivo evaluation of MA and Db in a
mouse model of transient mechanical MCAo. FIG. 8A shows lesion size
(mm.sup.3), FIG. 8B shows the Bederson score (0-5), FIG. 8C shows
the Grip test score (0-5) measured at 24 hours post occlusion in
mice treated with vehicle (PBS), negative control IgG, MA-33H1F7 at
1 mg/kg, MA-TCK26D6 at 1 mg/kg, MA-TCK26D6 at 1 mg/kg+MA-33H1F7 at
1 mg/kg or Db at 0.8 mg/kg (mean.+-.SEM, n=10-12). FIG. 8D shows
fibrinogen contents in ipsilateral side of brain (fold increase vs.
contralateral), measured at 24 hours post occlusion in mice treated
with vehicle (PBS), negative control IgG, MA-TCK26D6 at 1 mg/kg
+MA-33H1F7 at 1 mg/kg or Db at 0.8 mg/kg (mean.+-.SEM, n=3-4).
Statistical significance is indicated as follows: *p<0.05;
**p<0.01; ***p<0.001. (the control MA is MA-NB27B3)
[0040] FIG. 9A-9F shows in vivo evaluation of diabody as single
treatment or as adjuvans to thrombolytic treatment in a mouse model
of thrombin-induced MCAo. Graphs representing following parameters
measured 24 hours post clot onset in mice treated with PBS, tPA (10
mg/kg), diabody (Db-TCK26D6.times.33H1F7) at 0.8 mg/kg) and
combination therapy (diabody 0.8 mg/kg+tPA 10 mg/kg): (A) lesion
size (mm3) (B) angiographic score (0-2) and (C) % reduction in
cerebral blood flow (CBF) mean.+-.SEM, n=6-8). Statistical
significance is indicated relative to PBS (*p<0.05; **p<0.01;
***p<0.001).
[0041] Additional time points after thrombin-induced occlusion are
presented in FIGS. 9D, 9E, and 9F. Lesion volume (mm3) at 24 h post
occlusion (upper panel) and representative T2-weighted images 24 h
post occlusion (lower panel)of mice treated with vehicle, tPA (10
mg/kg), Db (0.8 mg/kg) or a combination of Db (0.8 mg/kg) and tPA
(10 mg/kg) 20 min post occlusion (FIG. 9D, n=6-8), 90 min post
occlusion (FIG. 9E, n=9-10) and 240 min post occlusion (FIG. 9F,
n=7-9). Dotted lines delineate stroke lesions. Data are represented
as mean.+-.SEM. *, p<0.05; **, p<0.01; ns=not significant.
tPA indicates recombinant tissue-type plasminogen activator; Db,
diabody.
[0042] FIG. 10A-10D shows in vivo evaluation of diabody in a mouse
model of FeCl.sub.3-induced MCAo. Graphs representing (A)
difference in cerebral blood flow (CBF) at 1 hour post occlusion
(B) lesion size (mm3) (C) angiographic score (0-2) and (D) %
reduction in CBF at 24 hours post occlusion, in mice treated with
PBS, tPA at 10 mg/kg and diabody (Db-TCK26D6.times.33H1F7) at 1.6
mg/kg and at 3.6 mg/kg (mean.+-.SEM, n=8-15). Statistical
significance is indicated relative to PBS (*p<0.05; **p<0.01;
***p<0.001).
[0043] FIG. 11A-11C shows In vitro and in vivo expression of scDbs
against TAFI and PAI-1. Graphs comparing the following properties
of a series of scDbs and their variants (A) in vitro production
expressed as secreted protein in conditioned medium (B) in vitro
stability in plasma at 37.degree. C. up to 72 hours, expressed as
residual binding (%) and (C) in vivo expression after intramuscular
DNA injection and electroporation.
[0044] FIG. 12A-12B shows tail bleeding time and accumulative
bleeding up to 60 min.in mice treated with vehicle (PBS); tPA at 1
and 10 mg/kg; diabody at 0.8 mg/kg and 3.6 mg/kg; and Db (0.8
mg/kg)+tPA (10 mg/kg). FIGS. 12A and 12B show the effect of tPA
and/or diabody on bleeding time and haemoglobin levels.
[0045] FIG. 13 shows systemic pharmacokinetics of the diabody after
intravenous injection. Graph shows plasma levels (.mu.g/ml,
mean.+-.SEM) plotted against time (min) after IV injection of
diabody at 0.8 mg/kg in 6 mice (red arrow indicates circulating
half-life=121 min) FIG. 13A shows the time (in minutes) until
initial cessation of tail bleeding as monitored in mice, and (B)
accumulative bleeding (haemoglobin loss) up to 60 min, measured as
haemoglobin (g/dL) (median, n=9-16 mice/group; *, p<0.05;
**p<0.01; ***p<0.005). tPA indicates recombinant tissue-type
plasminogen activator; Db, diabody.
[0046] FIG. 14A-14B shows the evaluation of the effect of the
diabody (Db) on cortical neuronal death with or without
NMDA-induced excitotoxicity. In FIG. 14A, cortical neurons were
exposed to NMDA (as a full kill condition (FK); 500 .mu.mol/L) or
diabody (0.5 -50 .mu.g/ml); In FIG. 14B cortical neurons were
exposed to NMDA (500 .mu.mol/L (full kill, FK) or 12.5 .mu.mol/L),
Db (5 .mu.g/ml) or rtPA (20 .mu.g/ml), either alone or in
combination, during 24 hours before measurement of neuronal death
(N=2 independent cultures, n=2-4, *p<0.05; ns=not significant).
tPA indicates recombinant tissue-type plasminogen activator; Db,
diabody; NMDA, N-methyl-D-aspartate.
DETAILED DESCRIPTION
[0047] The present disclosure relates to the use of bispecific
antibody derivatives in treating thrombotic disorders. The
bispecific antibody derivatives target TAFI and PAI-1 and inhibit
both proteins in a dual targeting strategy. In some embodiments,
the bispecific antibody derivative is a diabody known as
Db-TCK26D6.times.33H1F7, and may be used in treating acute
thrombotic disorders. In some embodiments, the bispecific antibody
derivative is administered after onset of the acute thrombotic
disorder. In certain embodiments, the bispecific antibody
derivative is administered to patients at risk for developing
thrombotic disorders, either acute or chronic thrombotic
disorders.
[0048] Exemplary sequences of bispecific antibody derivatives or
portions thereof are described herein (SEQ ID NOS: 1-18). In
addition, bispecific antibody derivatives of the present disclosure
may be identical, substantially identical, homologous, or similar
to the exemplary sequences described herein.
[0049] "Sequence identity" refers to two amino acid sequences or
subsequences that are identical, or that have a specified
percentage of amino acid residues that are the same (e.g., 60% or
65% identity, preferably, 70%-95% identity, more preferably,
>95% identity), when compared and aligned for maximum
correspondence over a window of comparison, or over a designated
region, as measured using a sequence comparison algorithm as known
in the art, or by manual alignment and visual inspection. In
certain embodiments, the described identity exists over a region
that is at least about 5 to 10 amino acids in length.
[0050] Specific "designated regions" in the context of the present
invention are the CDR regions or the present invention. These CDR
regions are typically conserved (100% sequence identity compared to
the reference sequence), although one or more substations may be
allowable in one or more CDRs as long as the functional properties
of the reference antibody are maintained. With CDR regions ranging
from 5 to almost 20 amino acids, typical embodiments of a modified
CDR of an antibody sequences have a sequence identity in the CDR
region which is at least 75, 80, 85, 90, 92, 94, 95% to the
reference CDR sequence.
[0051] Outside the CDR regions the sequence of a variable heavy or
light chain may be less restricted while still maintaining the
function of the antibody. Thus a variable chain may be at least 75,
80, 85, 90, 92, 95, 97, 98 or 99% identical to a reference sequence
of a variable chain while one, two, or all three CDR sequences have
one amino acid difference with the corresponding reference CDR
sequence or wherein all CDR regions are identical with those of the
reference sequence.
[0052] A difference at a certain position can be a change into any
of the other 19 amino acids or can be a so-called "conservative
substitution"
[0053] It is a well-established principle of protein chemistry that
such "conservative amino acid substitutions," can frequently be
made in a protein without altering either the conformation or the
function of the protein. Such changes include substituting any of
isoleucine (I), valine (V), and leucine (L) for any other of these
hydrophobic amino acids; aspartic acid (D) for glutamic acid (E)
and vice versa; glutamine (Q) for asparagine (N) and vice versa;
and serine (S) for threonine (T) and vice versa. Substituting any
of tryptophan (W), tyrosine (Y), and phenylalanine (F) for any
other of these aromatic amino acids and vice versa. Other
substitutions can also be considered conservative, depending on the
environment of the particular amino acid and its role in the
three-dimensional structure of the protein. For example, glycine
(G) and alanine (A) can frequently be interchangeable, as can
alanine and valine (V). Methionine (M), which is relatively
hydrophobic, can frequently be interchanged with leucine and
isoleucine, and sometimes with valine. Lysine (K) and arginine (R)
are frequently interchangeable in locations in which the
significant feature of the amino acid residue is its charge and the
differing pK's of these two amino acid residues are not
significant. "Bispecific antibody" refers to an antibody based
construct that can simultaneously bind to two different antigens.
In the context of the present invention this means specific binding
to TAFI and specific binding to PAI-1.
[0054] "Diabody" refers to a specific type of bispecific antibody
which comprise a heavy chain variable domain (VH) of one antibody
connected to a light-chain variable domain (VL) of another antibody
on the same polypeptide chain (VH-VL). By using a linker that is
too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites. In a
diabody antigen-binding sites point in opposite directions.
[0055] Typically these are complexes of ScFv constructs. Different
configurations are possible. A possible configuration is a complex
of two polypeptides, namely:
[0056] a fusion protein of the VH region of an anti-TAFI antibody
and the VL region of an anti PIA-1 antibody, with
[0057] a fusion protein of the VL region of an anti-TAFI antibody
and the VH region of an anti-PIA-1 antibody. Other examples are a
single peptide chain with two VH and two VL regions, yielding
tandem scFv's. Or scFv's with linker peptides that are too short
for the two variable regions to fold together (about five amino
acids), forcing scFvs to dimerize.
[0058] "Targeting domain" refers to the part of the bispecific
antibody that is required to obtain specific antigen binding
(antigen binding domain) with one of the antigens.
[0059] "Fibrinolysis" refers to the degradation of fibrin within a
blood clot.
[0060] "Thrombolysis" refers to the degradation of a blood clot by
inter alia, breakdown of fibrin threads and other structural
elements which form a clot.
[0061] TAFI (Thrombin-Activatable Fibrinolysis Inhibitor) is also
known as Carboxypeptidase B2 (CPB2), Carboxypeptidase U (CPU) or
plasma carboxypeptidase B (pCPB). TAFI is an enzyme that reduces
fibrinolysis by removing fibrin C-terminal residues that are
important for the binding and activation of plasminogen.
[0062] PAI-1 (Plasminogen Activator Inhibitor-1 (PAI-1), is also
known as endothelial plasminogen activator inhibitor or serpin E1.
PAI- is a serine protease inhibitor that tissue plasminogen
activator (tPA) and urokinase (uPA).
[0063] Thrombus refers to a clot in the cardiovascular system
formed during life from blood constituents. Clots may be occlusive
or attached are attached to vessel or heart wall without
obstructions. Exemplary types of thrombi are fibrin clots formed by
deposits of fibrin and white or pale clots mainly composed of
platelets.
[0064] "tPA" (tissue plasminogen activator) refers to the wild type
protein, but also covers modified versions known as reteplase,
tenecteplase
[0065] "Thrombosis" is a condition characterized by the formation
of a blood clot inside a blood vessel, and is thought to result
from an abnormality in one or more of hypercoagulability,
endothelial injury/dysfunction, and hemodynamic changes of stasis
and turbulence (together known as Virchow's triad). Thrombosis can
lead to vessel blockage at the site of clot formation, or to vessel
blockage at a distance from the site of origin (i.e., embolism). In
both cases, obstruction of the vessel disrupts the supply of oxygen
to the tissues supplied by the vessel, resulting in hypoxia,
anoxia, and infarction. Accordingly, many pathological conditions
arise from thrombosis, ranging from deep vein thrombosis to
pulmonary embolism to arterial thrombosis which cause heart attacks
and strokes, and more.
[0066] "Thrombotic disorders" as used herein includes but is not
limited to deep vein thrombosis (DVT), pulmonary embolism (PE),
coronary artery disease (CAD) and acute coronary syndrome (ACS),
central retinal artery occlusion (CRAO), age related macular
degeneration (AMD) and thrombotic neurological disorders, including
stroke, acute ischemic stroke (AIS), middle cerebral artery
occlusion (MCAo), acute peripheral arterial occlusion (APAO) and
more.
[0067] The disorders may also be thrombotic neurological disorders
comprising diseases, disorders or conditions which directly or
indirectly affects the normal functioning or anatomy of a subject's
nervous system, including but not limited to, cerebrovascular
insufficiency, cerebral ischemia or cerebral infarction such as
stroke, retinal ischemia (diabetic or otherwise), glaucoma, retinal
degeneration, multiple sclerosis, ischemic optic neuropathy,
reperfusion following acute cerebral ischemia, perinatal
hypoxic-ischemic injury, or intracranial haemorrhage of any type
(including, but not limited to, epidural, subdural, subarachnoid or
intracerebral haemorrhage).
[0068] In certain embodiments, the thrombotic disorder is
hereditary in origin. In certain embodiments, the thrombotic
disorder is acquired. The thrombotic disorder may be acute, chronic
and/or recurring. In certain embodiments, the thrombotic disorder
is acute, and is at least one of acute ischemic stroke (AIS),
middle cerebral artery occlusion (MCAo), thromboembolism, deep vein
thrombosis, myocardial infarction (MI), pulmonary embolism,
peripheral arterial disease, thrombosis of liver and/or kidneys, or
catheter blockage. The thrombotic disorder may be an occlusive
syndrome in the cerebral vascular system, for example, causing
cerebral infarcts due to stroke or ischemic stroke. In some
embodiments, the acute thrombotic disorder is AIS. In certain
embodiments, the acute thrombotic disorder is MCAo.
Bispecific Antibody Derivatives for Use in Treating Thrombotic
Disorders
[0069] Bispecific antibody derivatives represent the smallest
format currently available to achieve bispecificity when starting
from two IgG's to allow efficient penetration into the blood clot.
The dual specificity confers inhibition of TAFI and PAI-1 at the
same time, same localization, and same concentration, which leads
to a similar pharmacokinetic profile and biodistribution. In some
embodiments, the bispecific antibody derivatives for use in
treating thrombotic disorders are based on monoclonal antibodies
(MAs). For example, exemplary MAs which target TAFI are MA-RT36A3F5
and MA-TCK26D6 [6, 7] [Hillmayer et al. cited above; Vercauteren
(2011) cited above], while exemplary MAs which targets PAI-1 are
MA-33H1F7 and MA-MP2D2 [De Brock cited above, Van De Craen cited
above]. One exemplary bispecific antibody derivative is
DbTCK26D6.times.33H1F7 [Wyseure T et al. (2013) J Thromb Haemost.
11, 2069-2071]. In certain embodiments, the efficacy of bispecific
antibody derivatives surpasses that of either MA administered
alone.
[0070] In some embodiments, the bispecific antibody derivative
comprises a first targeting domain that binds to Thrombin
Activatable Fibrinolysis Inhibitor (TAFI) and comprises
complementary determining regions (CDRs) represented by amino acid
sequences that are at least 80% identical to each of CDR1H of SEQ
ID NO:1, CDR2H of SEQ ID NO:2, CDR3H of SEQ ID NO:3, CDR1L of SEQ
ID NO:4, CDR2L of SEQ ID NO:5, and CDR3L of SEQ ID NO:6; and a
second targeting domain that binds to Plasminogen Activator
Inhibitor-1 (PAI-1) and comprises complementary determining regions
(CDRs) represented by amino acid sequences that are at least 80%
identical to each of CDR1H of SEQ ID NO:7, CDR2H of SEQ ID NO:8,
CDR3H of SEQ ID NO:9, CDR1L of SEQ ID NO:10, CDR2L of SEQ ID NO:11,
and CDR3L of SEQ ID NO:12.
[0071] For example, the bispecific antibody derivative may comprise
a first targeting domain that binds to Thrombin Activatable
Fibrinolysis Inhibitor (TAFI) and comprises a VH region represented
by an amino acid sequence that is at least 80% identical to SEQ ID
NO:13 and a VL region represented by amino acid sequence that is at
least 80% identical to each of SEQ ID NO:14; and a second targeting
domain that binds to Plasminogen Activator Inhibitor-1 (PAI-1) and
comprises a VH region represented by an amino acid sequence that is
at least 80% identical to SEQ ID NO:15 and a VL region represented
by amino acid sequence that is at least 80% identical to SEQ ID
NO:16.
[0072] In certain embodiments, a bispecific antibody derivative
comprises a first domain that comprises an amino acid sequence that
is at least 80% identical to SEQ ID NO:17; and a second domain that
comprises an amino acid sequence that is at least 80% identical to
SEQ ID NO:18.
[0073] SEQ ID NO:17 comprises at the N terminal side the sequence
with SEQ ID NO:13 and at the C terminal side the sequence of SEQ ID
NO 16.
[0074] SEQ ID NO:18 comprises at the N terminal side the sequence
with SEQ ID NO:15 and at the C terminal side the sequence of SEQ ID
NO 14.
[0075] In some embodiments, the amino acid sequences in the
bispecific antibody derivatives are at least 80%, 85%, 90%, 95%,
99% or 100% identical to the amino acid sequences disclosed in SEQ
ID NO:1-18. For example, in SEQ ID NO:2, the second amino residue
(marked "X") may be either Val or Ile (and correspondingly, in SEQ
ID NO:13, the fifty first amino acid residue (marked "X") may be
either Val or Ile. In certain embodiments, the amino acid sequences
in the bispecific antibody derivatives have variations in amino
acid residues which do not significantly affect the binding
properties of the bispecific antibody derivatives to their targets.
In some embodiments, variations in amino acid residues may affect
the stability of the bispecific antibody derivative.
[0076] In some embodiments, the bispecific antibody derivative, for
example, as disclosed herein is humanized.
[0077] The bispecific antibodies disclosed herein may not be
neurotoxic in contrast to tPA, and may be used for reducing lesion
size in patients suffering from a brain lesion. Brain lesions may
be caused by thrombotic disorders, such as stroke, acute ischemic
stroke (AIS), and/or middle cerebral artery occlusion (MCAo).
Thrombo-inflammation in patients suffering from a thrombotic
disorder may be reduced by the bispecific antibodies.
[0078] A dangerous side effect of thrombolytic treatment is
increased bleeding risk, for example from intracranial haemorrhage.
The bispecific antibodies described herein may be used to reduce or
minimize the bleeding and/or bleeding risk, as they did not prolong
bleeding in an animal model for bleeding risk, in contrast to tPA.
In addition, the relatively short half-life of the bispecific
antibodies may minimize side effects such as intracranial
haemorrhage in patients treated with the bispecific antibodies.
[0079] A further consequence of thrombotic disorders such as
stroke, acute ischemic stroke (AIS), and/or middle cerebral artery
occlusion (MCAo) are neurological impairments. In some embodiments,
the bispecific antibodies disclosed herein may be used for treating
neurological impairments, such as motor, sensory, and/or cognitive
impairments. Impairments and/or limitations in limb flexion,
lateral push, grip, and more may be treated with the bispecific
antibodies disclosed herein.
Administration
[0080] Timing of thrombolytic treatment is critical. In the case of
acute thrombotic disorders such as acute myocardial infarction,
acute ischemic stroke, or acute massive pulmonary embolism, tPA is
typically administered to break down clots within 15 hours of the
stroke, for example, as soon as possible after onset of stroke
symptoms, at 0-6 hours, or at 4.5 hours. Similarly, in some
embodiments, the bispecific antibody derivative as disclosed herein
is administered between 0-15 hours after onset of symptoms of the
thrombotic disorder. The thrombotic disorder may be an acute
thrombotic disorder, such as a stroke, for example, AIS or MCAo. In
certain embodiments, the bispecific antibody is administered at 0.5
hours, 1 hour, 1.5 hours, 3 hours, 4 hours, 4.5 hours, or more
hours after onset of symptoms of the thrombotic disorder, for
example, at 4.5 hours after onset of symptoms. In some embodiments,
the bispecific antibody derivative is administered 12 hours after
onset of symptoms of the thrombotic disorder. The bispecific
antibody derivative may be administered intravenously, or directly
into the blood clot.
[0081] To date, plasminogen activators such as tPA are the only
thrombolytic agents approved by the US FDA, and plasminogen
activators such as tPA remain the primary first-line treatment for
acute thrombotic disorders. However, some patients do not respond
to tPA treatment and further interventions are needed. Accordingly,
in some embodiments, the bispecific antibody derivative is
administered together with a plasminogen activator, for example,
tPA. For example, the bispecific antibody derivative may be
administered simultaneously with tPA, as a combination treatment.
The bispecific antibody derivative may also be administered after
tPA is administered first. In some embodiments, the bispecific
antibody derivative is administered 1 hour after administration of
tPA. If a patient does not respond to tPA within 1 hour, the
bispecific antibody derivative may be administered as a further
intervention.
[0082] A surprising and unexpected result of the present disclosure
is that bispecific antibody derivatives were equally effective in
the presence or in the absence of tPA when administered up to 90
minutes post-occlusion. Additionally, bispecific antibody
derivatives had a superior thrombolytic effect than tPA. Thus, the
bispecific antibody derivatives disclosed herein may be used as
thrombolytic agents in place of a plasminogen activator such as
tPA. One aspect of the present disclosure relates to the use of the
bispecific antibody derivatives disclosed herein for use in
treating an acute thrombotic disorder, wherein the bispecific
antibody derivative is administered without tPA. In certain
embodiments, the acute thrombotic disorder is characterized by the
presence of a fibrin-rich blood clot. In some embodiments, the
acute thrombotic disorder is characterized by the presence of a
platelet-rich blood clot. In some embodiments, the bispecific
antibody derivative is administered without tPA during a time
period of up to 90 minutes after the onset of the acute thrombotic
disorder, for example, 0-90 minutes after onset. Thus, the
bispecific antibody derivative may be administered at 0, 10, 15,
20, 30, 40, 45, 50, 60, 70, 80, or 90 minutes after onset of the
acute thrombotic disorder.
[0083] In some embodiments, when treatment for an acute thrombotic
disorder is administered at least 90 minutes of onset of the acute
thrombotic disorder, the bispecific antibody derivative is
administered together with tPA. The combination of bispecific
antibody derivative and tPA may be administered at 90 minutes after
onset, or at 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5
hours, 5 hours, 5.5 hours, 6 hours, or more after onset. In certain
embodiments, administration of the diabody does not lead to
deleterious neurotoxic effects. When administered in combination
with tPA, the bispecific antibody derivative may potentiate the
thrombolytic effect of tPA without potentiating the adverse side
effects observed with tPA alone. The bispecific antibody
derivatives as disclosed herein may also be combined with
antiplatelet treatments such as aspirin, clopidogrel, and
dipyridamole; anticoagulant treatments such as heparin, warfarin,
and dabigatran; and/or surgical interventions such as
revascularization, carotid endartectomy, carotid angioplasty,
intra-arterial thrombolysis, and mechanical embolus removal in
cerebral ischemia (MERCI).
[0084] The diabodies of the present invention can be delivered as
one bolus or can be administered over a longer period of time.
[0085] The total amount of diabody, administered to a patient can
range from 0,1, 0,5, 1 or kg body weight up to 2 or 5 mg/kg body
weigth.
[0086] Since the administration of an excess of antibody has no
detrimental side effects, a total dosis of 10, 20, 40, 80 or 100 mg
of diabody may be administered regardless from the body weight of
the patient.
Prevention
[0087] Thrombotic disorders are common, particularly in elderly
populations and/or in patients who have been previously affected by
a thrombotic disorder. Thrombotic disorders may be either acute or
chronic. Additional risk factors for stroke such as AIS and/or MCAo
include advanced age, alcohol use, atherosclerosis, atrial
fibrillation, use of birth control pills, diabetes, poor diet,
family history of stroke, fibromuscular dysplasia, high blood
pressure, high cholesterol, hypercoagulability (either hereditary
or acquired), inflammation, low birth weight, migraine, obesity,
patent foramen ovale, physical inactivity, postmenopausal hormone
therapy, previous stroke, certain races/ethnicities, sickle cell
disease, sleep apnoea, transient ischemic attack, tobacco use, and
more. Risk factors for venous thrombosis, such as deep vein
thrombosis (DVT) which is a frequent cause of pulmonary embolism,
include but are not limited to advanced age, major surgery,
orthopaedic surgery, cancer, immobilization, pregnancy,
antiphospholipid syndrome, trauma, minor leg injury, previous
venous thrombosis, use of oral contraceptives, hormonal replacement
therapy, central venous catheters, inflammatory diseases or
autoimmune disease, nephrotic syndrome, obesity, infection, HIV,
polycythaemia vera, and chemotherapy, as well as hereditary risk
factors including but not limited to antithrombin deficiency,
protein C deficiency, protein S deficiency, Factor V Leiden,
Prothrombin G20210A, dysfibrogenemia, and non-O blood type.
Additional risk factors include but are not limited to low levels
of protein S, activated protein C resistance, high Factor VIII
levels, hyperhomocysteinemia, and/or high levels of fibrinogen,
Factor IX, and/or Factor XI.
[0088] A further aspect of the present disclosure relates to a
bispecific antibody for use in patients at risk for developing an
acute or chronic thrombotic disorder, comprising a first targeting
domain that binds to Thrombin-Activatable Fibrinolysis Inhibitor
(TAFI) and comprises complementary determining regions (CDRs)
represented by amino acid sequences that are at least 80% identical
to each of CDR1H of SEQ ID NO:1, CDR2H of SEQ ID NO:2, CDR3H of SEQ
ID NO:3, CDR1L of SEQ ID NO:4, CDR2L of SEQ ID NO:5, and CDR3L of
SEQ ID NO:6; and a second targeting domain that binds to
Plasminogen Activator Inhibitor-1 (PAI-1) and comprises
complementary determining regions (CDRs) represented by amino acid
sequences that are at least 80% identical to each of CDR1H of SEQ
ID NO:7, CDR2H of SEQ ID NO:8, CDR3H of SEQ ID NO:9, CDR1L of SEQ
ID NO:10, CDR2L of SEQ ID NO:11, and CDR3L of SEQ ID NO:12, wherein
the bispecific antibody derivative is administered before onset of
the acute or chronic thrombotic disorder. In certain embodiments,
the acute or chronic thrombotic disorder is thromboembolism.
[0089] For example, the bispecific antibody derivative may comprise
a first targeting domain that binds to Thrombin-Activatable
Fibrinolysis Inhibitor (TAFI) and comprises a VH region represented
by an amino acid sequence that is at least 80% identical to SEQ ID
NO:13 and a VL region represented by an amino acid sequence that is
at least 80% identical to SEQ ID NO:14; and a second targeting
domain that binds to Plasminogen Activator Inhibitor-1 (PAI-1) and
comprises a VH region represented by an amino acid sequence that is
at least 80% identical to SEQ ID NO:15 and a VL region represented
by an amino acid sequence that is at least 80% identical to SEQ ID
NO:16.
[0090] In certain embodiments, a bispecific antibody derivative
comprises a first domain that comprises an amino acid sequence that
is at least 80% identical to SEQ ID NO:17; and a second domain that
comprises an amino acid sequence that is at least 80% identical to
SEQ ID NO:18.
[0091] In some embodiments, the amino acid sequences in the
bispecific antibody derivatives are at least 80%, 85%, 90%, 95%,
99% or 100% identical to the amino acid sequences disclosed in SEQ
ID NO:1-18. For example, in SEQ ID NO:2, the second amino residue
(marked "X") may be either Val or Ile (and correspondingly, in SEQ
ID NO:13, the fifty first amino acid residue (marked "X") may be
either Val or Ile.
INCORPORATION BY REFERENCE
[0092] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
EQUIVALENTS
[0093] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
EXAMPLES
[0094] Having provided a general disclosure, the following examples
help to illustrate the general disclosure. These specific examples
are included merely to illustrate certain aspects and embodiments
of the disclosure, and they are not intended to be limiting in any
respect. Certain general principles described in the examples,
however, may be generally applicable to other aspects or
embodiments of the disclosure.
Example 1
Improving Expression and Efficacy of an Unstable Bispecific
Inhibitor (Db-RT36A3F5.times.33H1F7) Against TAFI and PAI-1 Through
Antibody Engineering
A. CDR-Grafting to Engineer Stable Variable Domains
[0095] In a previous study, scFv-RT36A3F5 was generated, but could
not be produced by bacteria and corresponding
Db-RT36A3F5.times.33H1F7 was found to be unstable resulting in a
diminished effect on clot lysis. Thus, the variable domains of
MA-RT36A3F5 were optimized by complementarity determining region
(CDR)-grafting onto the stable scaffolds of scFv-4D5 (FIG. 1A)
[Jung S. & Pluckthun A. (1997) Protein Eng. 10, 959-966. This
article discloses the 4D5 humanised antibody used in the CDR
grafting]. Two approaches were performed: (i) structure
alignment-based strategy of scFv-RT36A3F5 and of scFv-4D5,
generating scFv-RT36A3F5-4D5DM (in collaboration with prof Marc
Demaeyer) and (ii) evidence-based strategy, generating
scFv-RT36A3F5-4D5 [Ewert S. et al. (2004) Methods. 34, 184-199].
The latter approach was also performed on the stable scaffolds of
scFv-T12D11 (an anti_TAFI antibody disclosed in de Develter et al.
2008 J Thromb Haemost. 6, 1884-1889, generating
scFv-RT36A3F5-T12D11. Western blot analysis revealed that solely
scFv-RT36A3F5-4D5 was properly expressed and secreted (FIG. 1B,
lane 1) and therefore, these CDR-grafted variable domains were used
in corresponding (sc)Db constructs.
B. Production of Bispecific Antibody-Based Inhibitors from
MA-RT36A3F5 and MA-33H1F7
[0096] Four bispecific inhibitors were formed out of MA-RT36A3F5
and MA-33H1F7: Db-RT36A3F5.times.33H1F7 (Db),
Db-RT36A3F5-4D5.times.33H1F7 (CDR-grafted Db),
scDb-33H1F7.times.RT36A3F5 (scDb with an additional flexible linker
between the variable domains of MA-RT36A3F5) and
scDb-33H1F7.times.RT36A3F5-4D5 (CDR-grafted scDb) (FIG. 2). As a
result of CDR-grafting, bacterial and eukaryotic expression of Db
and scDb, respectively, were elevated (for CDR-grafted Db
approximately 1.5 mg/L culture corresponding to a two-fold increase
vs. Db and for CDR-grafted scDb 11.+-.1 mg/L culture medium
corresponding to a ten-fold increase vs. scDb).
C. Inhibitory Properties of Bispecific Inhibitors
[0097] Inhibitory properties of the parental antibodies were
preserved in Db, CDR-grafted Db and CDR-grafted scDb as confirmed
by functional assays Inhibitory properties of scDb could not be
evaluated due to insufficient production.
D. Stability and Profibrinolytic Properties of Diabodies in
Citrated Rat Plasma
[0098] Out of all constructs, only CDR-grafted scDb exhibited a
similar stability as the control diabody, Db-T12D11.times.33H1F7
(88.+-.13% residual binding activity after three hours at
37.degree. C.; FIG. 3A). With a relative profibrinolytic effect of
0.81.+-.0.23, CDR-grafted scDb was also the most potent construct
compared to MA-RT36A3F5 (FIG. 3B). The contribution of the effect
of the PAI-1 inhibiting moiety could not be evaluated in the
plasma-based assay system due to the low baseline plasma levels of
PAI-1.
[0099] In conclusion, our efforts to increase the plasma stability
of an unstable bispecific antibody-based inhibitor against rat TAFI
and PAI-1 resulted in a CDR-grafted scDb, exhibiting a seven-fold
increased stability and profibrinolytic effect. This antibody
derivative cross-reacts with mouse TAFI and mouse PAI-1, allowing
further in vivo evaluation in mice and rats.
Example 2
In Vitro Evaluation of the Profibrinolytic Properties of a Novel
Bispecific Inhibitor Against TAFI and PAI-1
A. Generation of Db-TCK26D6.times.33H1F7
[0100] Based on the successful generation of stable scFvs with
preserved inhibitory capacity of the respective parental antibodies
(MA-TCK26D6 and MA-33H1F7), Db-TCK26D6.times.33H1F7 was generated.
This diabody contains two polypeptide chains as depicted in FIG. 2
left bottom. The first one is a fusion protein of the VH chain of
the anti TAFI antibody and the VL chain of the anti PAI-1 antibody.
The second one is a fusion protein of the VL chain of the anti TAFI
antibody and the VH chain of the anti PAI-1 antibody.
[0101] The production level of Db-TCK26D6.times.33H1F7 was
approximately 2 mg/L culture.
B. Characterization of the Inhibitory Effect Towards TAFI and
PAI-1
[0102] Inhibitory properties of the parental antibodies against
human and mouse TAFI and PAI-1 were preserved in
Db-TCK26D6.times.33H1F7 as confirmed by functional assays.
Moreover, Db-TCK26D6.times.33H1F7 remained stable after incubation
in human, mouse and rat plasma after 8 hours at 37.degree. C.
C. Effect of Db-TCK26D6.times.33H1F7 During Thromboelastometric
Analysis in Whole Blood
[0103] To evaluate the profibrinolytic effect due to TAFI and PAI-1
inhibition, Db-TCK26D6.times.33H1F7 was incubated in human whole
blood from four individuals and its effect was analysed by
thromboelastometry [Wyseure T. et al. (2013) J. Thromb. Haemost.
11, 2069-2071]. The combined addition of both MAs as well as the
addition of diabody facilitated fibrinolysis to a very significant
degree (p<0.001), whereas the addition of a single MA caused
only a modest effect (FIG. 4A).
[0104] The effect of Db-TCK26D6.times.33H1F7 was also evaluated in
whole blood from mice. Since PAI-1 levels are extremely low in mice
(serum levels, mean.+-.SD, n=4, 3.0.+-.0.3 ng/ml for mice vs.
267.+-.114 ng/ml for humans), thromboelastometric analysis in blood
from mice is insensitive to PAI-1. To increase PAI-1 levels in
mouse blood, we induced experimental endotoxemia through
intraperitoneal injection of LPS (0.5 mg/kg) prior to collection of
blood for thromboelastometric analysis. The combined addition of
both MAs as well as the addition of diabody facilitated
fibrinolysis to a significant degree (p<0.05), whereas the
addition of a single MA caused no significant effect (FIG. 4B).
[0105] Thus, Db-TCK26D6.times.33H1F7 exhibits strong
profibrinolytic properties in vitro.
Example 3
In Vivo Evaluation of the Profibrinolytic Properties of a
Bispecific Inhibitor Against TAFI and PAI-1
1. Complementary Effect of Dual TAFI/PAI-1 Inhibition After
Systemic Thrombotic Challenge
[0106] Mice, pre-treated with a dose of MA-TCK26D6 or MA-33H1F7
targeting all circulating antigen, were subjected to
thromboembolism by systemic administration of thromboplastin.
Fibrin deposition in lungs was only decreased to baseline levels
upon administration of a TAFI inhibitor (FIG. 5). Since PAI-1
levels are extremely low in mice, no effect of PAI-1 inhibition was
detected.
[0107] To evaluate simultaneous inhibition of TAFI and PAI-1 in
this model, endotoxemia was induced to upregulate PAI-1 levels in
plasma. Fibrin deposition in the lungs was reduced through TAFI
inhibition with MA-TCK26D6 (5 mg/kg) or through PAI-1 inhibition
with MA-33H1F7 (10 mg/kg). However, this reduction did not reach a
maximal degree. After administering a mixture of MA-TCK26D6 (5
mg/kg) and MA-33H1F7 (10 mg/kg), fibrin levels in lungs returned to
baseline (FIG. 6A). This maximal effect disappeared when lowering
the dosages of MA to 1 mg/kg (FIG. 6B). Upon treatment with diabody
(Db-TCK26D6.times.33H1F7 at 0.8 mg/kg, i.e. a dose which targets
the same amount of TAFI and PAI-1 as achieved with the combined MA
each at 1 mg/kg), a maximal effect of fibrin clearance from lungs
was obtained.
[0108] As demonstrated, simultaneous inhibition of TAFI and PAI-1
results in an additive effect on fibrin removal in a
thromboembolism model which is most effective through
Db-TCK26D6.times.33H1F7.
2. Effect of Db-TCK26D6.times.33H1F7 in Mouse Models for Acute
Ischemic Stroke
[0109] i. Monofilament-Mediated MCAo
[0110] Transient occlusion was accomplished by advancing a
monofilament into the MCA. This model was used to assess the effect
of TAFI and/or PAI-1 inhibition on cerebral ischemia/reperfusion
injury. This model typically yields large lesion volumes in
untreated mice which have measurable neurological/motor defects.
Paramount in preclinical evaluation of stroke is to assess
neurological parameters in addition to lesion size. Interestingly,
in this model, tPA has a well-described deleterious effect through
aggravating neuronal damage after focal cerebral ischemia [Wang Y F
et al. (1998) Nat Med. 4, 228-231]. Treatment with either
MA-TCK26D6 at 25 mg/kg or MA-33H1F7 at 6 mg/kg caused reduced brain
lesions (FIG. 7A, 7D) and concomitant neurological and motor
recovery 24 hours post occlusion (FIG. 7B-C, 7E-F). In addition,
the brains of treated mice contained less fibrin(ogen) in the
ipsilateral side compared to those of control mice (FIG. 7G). The
control IgG in control IgG was MA-T30E5 in FIG. 7 and MA-NB27B3 in
FIG. 8.Treatment with either one of the parental antibody at 1
mg/kg did not alter lesion sizes
[0111] or neurological/motor scores 24 hours post occlusion (FIG.
8A-C). However, the combined administration of the antibodies
substantially reduced brain lesions 1.9-fold (FIG. 8A). Moreover,
the diabody at a corresponding dose caused a similar reduction in
lesion size (2.3-fold) however concomitantly improved neurological
and motor scores (FIG. 8A-C). Lesion sizes were 76.+-.11 mm.sup.3
with vehicle, 81.+-.11 mm.sup.3 with control IgG, 43.+-.8 mm.sup.3
with combination of MA and 35.+-.8 mm.sup.3 with diabody. In
addition, western blot analysis revealed that the combination of
parental antibodies or diabody effectively reduced massive fibrin
deposition induced by reperfusion injury by at least 2-fold (FIG.
8D; p<0.05; n=3-4 mice/group).
ii. Thrombin-Mediated MCAo
[0112] A model of thromboembolic stroke by thrombin injection was
used in which clots are rich in fibrin and thus susceptible to be
thrombolysed by tPA. The efficacy of the diabody was compared to
that of tPA, the current thrombolytic agent. In order to mimic the
clinical procedure of thrombolysis, the administration of tPA was
performed by an initial bolus of 10% volume followed by 90%
infusion during 40 min because of the short half-life of
circulating tPA (5 min) [Chandler W L et al. (1997) Circulation.
96, 761-768]. 24 h post occlusion, complete recanalization of the
arterial lumen occurred in all groups including the vehicle group
(median angiographic score=2, FIG. 9B). At the same time point,
Speckle contrast imaging showed a 40% reduction in tissue perfusion
in the MCA territory distal to the occlusion in the vehicle group
(FIG. 9C). Interestingly, brain perfusion was virtually restored by
the combination of diabody and tPA (FIG. 9C; p<0.05 vs. vehicle;
n=6-8 mice/group), while diabody or tPA separately did not
significantly increase perfusion. Lesion volume was reduced by
administration of tPA, however this reduction was not statistically
significant (37.+-.13 mm.sup.3 vs. 26.+-.12 mm.sup.3; FIGS. 9A;
p=0.203; n=6-8 mice/group). In contrast, early diabody
administration (0.8 mg/kg) at 20 minutes post-occlusion, regardless
of the co-administration of tPA, substantially reduced lesion
volume at 24 h (15.+-.4 mm.sup.3 for diabody and 15.+-.8 mm.sup.3
for diabody+tPA; p<0.01 and p<0.05 vs. vehicle respectively;
n=6-8 mice/group FIG. 9A). [FIGS. 9A and 9D show the same
conditions]. Treatments were also delayed to a clinically more
relevant time point, e.g. 90 min post occlusion (intermediate time
point) [Hacke W. et al. (2004) Lancet 363, 768-774], complete
recanalization was also observed at 24 h post occlusion in all
treatment groups (median angiographic score=2). Intermediately
delayed administration of diabody nor infusion of tPA had any
beneficial effect on the lesion volume (25.+-.3 mm.sup.3 (vehicle)
vs. 24.+-.3 mm.sup.3 (tPA) vs. 21.+-.4 mm.sup.3 (Db); FIG. 9E;
n=9-10 mice/group). However, at the same treatment time point
diabody administration prior to tPA infusion resulted in a
significantly reduced lesion volume (15.+-.2 mm.sup.3 (Db+tPA);
p<0.05 vs. vehicle; n=10 mice/group; FIG. 9E).
[0113] None of the treatments had an effect on lesion sizes in this
model when administered at 240 min post occlusion (late time point,
FIG. 9F).
[0114] At 90 min post stroke onset and onwards, tPA treatment does
not always result in a beneficial outcome, presumably because of
the increased stability of the clot (i.e. clot retraction)
resulting in thrombolytic resistance, the neurotoxic effect of tPA
to the progressively damaged brain and/or the increased risk for
haemorrhagic transformation. In the present example, neither tPA
treatment nor diabody treatment at 90 min post occlusion reduced
the lesion volumes. However, the combined treatment of the diabody
and tPA resulted in a significantly decreased lesion volume,
underscoring the potential clinical benefit of adding the diabody
to current thrombolytic treatment. At a later treatment time point
of 4 h post occlusion, a tendency towards increased lesion volumes
after tPA treatment, alone or with diabody, was observed (FIG. 9F).
In correspondence to the in vitro neurotoxicity data (FIG. 14), the
diabody also had no deleterious effect in vivo.
iii. FeCl.sub.3-Mediated MCAo
[0115] Platelet-rich clots are more resistant to treatment with tPA
[Kim E Y et al. (2006) Neurology 67, 1846-1848]. Therefore, a
FeCl.sub.3-induced MCAo model was used in which clots are rich in
platelets and thus mimic this clinically relevant issue. As
expected, tPA was not effective in (i) increasing CBF 1 h post
occlusion (laser Doppler tracings; FIG. 10A), (ii) ameliorating the
angiographic score 24 h post occlusion (FIG. 10C), (iii) reducing
lesion volume 24 h post occlusion (FIG. 10C) or (iv) increasing
brain reperfusion 24 h post occlusion (Speckle contrast imaging,
FIG. 10D). The diabody administered at 1.6 mg/kg significantly
increased CBF 1 h (FIG. 10A; p<0.05 vs. vehicle; n=8-15
mice/group) and the angiographic score 24 h post occlusion (FIG.
10C; p<0.05 vs. vehicle; n=8-15 mice/group), however no
amelioration of brain perfusion or lesion volume was observed 24 h
post occlusion (FIG. 10B, D). In contrast, at 3.6 mg/kg the diabody
significantly increased CBF at 1 h post occlusion (FIG. 10A;
p<0.05 vs. vehicle; n-8-15 mice/group) which resulted in a
significantly increased angiographic score (FIG. 10C; p<0.05;
n=8-15 mice/group), reduced lesion volume (FIG. 10B; p<0.05 vs.
vehicle; n=8-15 mice/group), and increased brain perfusion (FIG.
10D; p<0.05 vs. vehicle; n=8-14 mice/group) at 24 h.
[0116] In conclusion, the strong fibrinolytic enhancer designated
as Db-TCK26D6.times.33H1F7 showed a robust in vivo performance in a
set of mouse models of stroke.
Example 4
In Vivo Expression of Bispecific Inhibitors Against TAFI and
PAI-1
[0117] scDbs were expressed in vivo as they can be efficiently
produced in eukaryotic cells. The following constructs were
generated against TAFI and PAI-1 and cloned into pcDNA3.1.:
scDb-33H1F7.times.RT36A3F5, scDb-TCK26D6.times.33H1F7 and
scDb-TCK26D6.times.MP2D2. In vitro expression levels in HEK293T
cells ranged from 0.6-1.1 .mu.g/ml (FIG. 11A).
scDb-33H1F7.times.RT36A3F5 was further optimized by CDR-grafting
into scDb-33H1F7.times.RT36A3F5-4D5. which resulted in a ten-fold
higher expression and seven-fold increased plasma stability (after
three hours of incubation at 37.degree. C.). In mice, peak plasma
levels after gene transfer were 584.+-.79 ng/ml (n=6) and 188.+-.19
ng/ml (n=4) for scDb-33H1F7.times.RT36A3F5-4D5 at day 3 and
scDb-TCK26D6.times.33H1F7 at day 6, respectively, however no
expression of scDb-TCK26D6.times.MP2D2 could be detected (FIG.
11C). As the obtained plasma levels were too low for
pharmacological evaluation, pharmacokinetics of the scDbs were
altered to prolong the circulating half-life. To this end, an
affinity-engineered albumin binding domain [Jonsson A et al. (2008)
Protein Eng Des Sel. 21, 515-527] was fused to the C-terminus of
scDb-TCK26D6.times.33H1F7 and scDb-TCK26D6.times.MP2D2, which were
the only constructs that remained stable during incubation in
plasma at 37.degree. C. up to three days (FIG. 11B). The albumin
binding constructs were designated as
scDb-TCK26D6.times.33H1F7.times.ABDH and
scDb-TCK26D6.times.MP2D2.times.ABDH. Unfortunately, a two- to
four-fold reduction in expression was observed in vitro for these
constructs (FIG. 11A). However, in vivo expression levels were
two-fold increased at day 9 (287.+-.28 ng/ml, n=5) for the albumin
binding variant of scDb-TCK26D6.times.33H1F7, whereas expression of
the albumin binding variant of scDb-TCK26D6.times.MP2D2 was not
detectable (FIG. 11C).
Example 6
Assessment of Bleeding and Pharmacokinetics
[0118] Additional tail bleeding experiments were performed to
compare the effects of an IV injection of tPA at two different
doses: the dose equivalent to that used in clinical practice for
humans (1 mg/kg) and to that typically used in mice (10 mg/kg).
Diabody (Db-TCK26D6.times.33H1F7) was injected at 0.8 mg/kg and 3.6
mg/kg. IV administration of diabody up to 3.6 mg/kg did not alter
tail bleeding time or accumulative haemoglobin loss after 60 min
tail incubation, whereas both doses of tPA prolonged bleeding time
and increased haemoglobin loss (FIG. 12A and FIG. 12B; n=9-16
mice/group). Co-administration of diabody (0.8 mg/kg) and tPA (10
mg/kg), the treatment regimen tested in the thrombin-mediated MCAo
model, did not further increase the tail bleeding time nor
haemoglobin loss compared to tPA administration alone.
[0119] Alternatively, no cerebral haemorrhages were observed in
either mechanical or thrombotic MCAo stroke models after any
treatment.
[0120] The circulating half-life of diabody after IV administration
in mice was 121 min (FIG. 13) which allows bolus injection as acute
treatment.
MATERIALS & METHODS
[0121] Production of Diabodies (Db) and Single-Chain Diabodies
(scDb)
[0122] Antibody derivatives were produced by cloning the variable
domains (V.sub.H, V.sub.L) from a hybridoma cell line producing
monoclonal antibody. DNA fragments containing Db or scDb were
designed for bacterial production (via periplasmic secretion) and
eukaryotic production (via extracellular secretion), respectively.
The DNA fragments were synthetically produced and were further
cloned into pSKID2 for production of Db in E. coli RV308 and into
pcDNA3.1. for production of scDb using HEK293T cells. The
His.sub.6-tagged antibody derivatives were purified on a
Ni.sup.++-column and prior to in vivo evaluation endotoxins were
removed by anion-exchange chromatography.
Quantification of (sc)Db
[0123] (sc)Dbs were quantified by an ELISA based on the
simultaneous binding towards PAI-1 and TAFI. Briefly, a microtiter
plate coated with mouse PAI-1 was used to bind (sc)Db and detection
was performed via subsequent incubation with mouse TAFI, followed
by addition of MA-TCK32G12-HRP against TAFI, which was subsequently
developed using o-phenylenediamine as chromogenic substrate.
TAFI Neutralization Assay
[0124] The ability of antibody derivatives to inhibit TAFI was
quantified by using a chromogenic assay to measure residual TAFIa
activity and was compared to the inhibitory properties of the
parental monoclonal antibody (MA). TAFI was incubated with MA or
sc(Db), at concentrations ranging from 0.06- to 8-fold molar ratio
over TAFI, before or after activation by thrombin/thrombomodulin or
plasmin, depending on the working mechanism of the MA. Residual
TAFIa activity was determined using Hippuryl-Arg as a substrate,
followed by a colorimetric reaction.
PAI-1 Neutralization Assay
[0125] The ability of antibody derivatives to inhibit active PAI-1
was quantified by using a plasminogen-coupled chromogenic method
and was compared to the inhibitory properties of the parental MA.
PAI-1 was pre-incubated (2 hours, room temperature) with MA or
(sc)Db at concentrations ranging from 0.06- to 32-fold molar ratio
over PAI-1. After a consecutive incubation with tPA (15' 37.degree.
C.), plasminogen was added and the extent of conversion to plasmin,
was quantified by a chromogenic substrate.
In Vitro Plasma Clot Lysis Assay
[0126] Pooled rat citrated plasma was pre-incubated (10',
37.degree. C.) with MA or sc(Db), followed by the addition of
CaCl.sub.2 and t-PA. Clot lysis was then monitored over time
through measurement of the turbidity (OD.sub.405 nm) by a
microtiter plate reader. The degree of fibrinolysis was expressed
as the area under the curve over a time frame of 180 min. The
retrieved data were normalized to the value obtained in the
presence of MA (at a concentration corresponding to the equivalent
number of binding sites to the respective antigens as that of
sc(Db)).
Rotational Thromboelastometry
[0127] Citrated whole blood from four healthy donors or from mice
(healthy or endotoxemic by intraperitoneal injection of LPS (0.5
mg/kg) six hours prior to the start of the experiment) was
pre-incubated with MA or Db (at concentrations yielding an
equivalent number of binding sites to the respective antigens).
Clotting and subsequent fibrinolysis was initiated by
thromboplastin, CaCl.sub.2 and tPA. For human blood, fibrinolysis
was determined by the decrease in amplitude at 45 minutes after
initial clotting relative to the maximal amplitude (L
(%)=[(A.sub.max-A.sub.45)/A.sub.max]*100). In each run, baseline
lysis (L.sub.wo) was determined using the same blood sample without
MA or Db (L.sub.wo never exceeded 12%). Specific inhibitor-enhanced
lysis was then determined as .DELTA.L (%)=L.sub.inhibitor-L.sub.wo.
For mouse blood, specific inhibitor-enhanced fibrinolysis was
determined as the difference in area under the curve (AUC from
clotting time to clotting time+120 minutes) between saline
(AUC.sub.wo) and treated condition (AUC.sub.inhibitor) relative to
the AUC.sub.wo (relative
.DELTA.AUC=[(AUC.sub.wo-AUC.sub.inhibitor)/AUC.sub.wo]*100.
In Vivo Models
Thromboembolism Model
[0128] MA, diabody or saline (0.9% NaCl) was injected intravenously
(IV) in overnight fasted non-anesthetized SWISS mice (healthy or
endotoxemic by intraperitoneal injection of LPS (0.5 mg/kg) three
hours prior to the start of the experiment). Five minutes later,
thromboembolism was induced by IV injection of thromboplastin. Mice
were anaesthetized by pentobarbital (60 mg/kg intraperitoneally)
and 15 minutes post thrombotic challenge lungs were perfused with
10 IU/ml heparin. Then the lungs were isolated and homogenized.
Washed homogenate of (left) lung was incubated with 2 .mu.M
microplasmin in order to convert fibrin into solubilized fibrin
degradation products for subsequent quantification of fibrin
degradation products using a cross-reacting ELISA towards mouse
fibrinogen. Fibrin content in lungs was expressed as fibrinogen
equivalents (.mu.g/ml).
Thrombin- and FeCl.sub.3-Mediated MCAo Model
[0129] Anesthetized SWISS mice (by inhalation of 2%
isoflurane/oxygen mixture) were placed on a stereotaxic device to
expose the right middle cerebral artery (MCA) by craniectomy. In
situ occlusion, as confirmed by Laser Doppler flowmetry, was
performed by micro-injection of murine alpha-thrombin into the MCA
[Orset C et al. (2007) Stroke. 38, 2771-2778] or by application of
a filter paper saturated with 20% FeCl.sub.3 [Karatas H et al.
(2011) J Cereb Blood Flow Metab. 231, 1452-1460]. Db or PBS
(vehicle) was injected IV 15 minutes post clot onset. Five minutes
later, tPA (10 mg/kg) or saline was administered via a tail vein
catheter (10% as bolus and 90% infused over 40 minutes). 24 hours
after initial occlusion, cerebral blood flow was mapped on the
ipsi- and contralateral side by exposing the skull to a Speckle
contrast imager (Moor FLPI-2, Moor instruments).Reduction of blood
flow was expressed relative to the contralateral side. Brain lesion
volume was determined by T2-weighted MRI, angiographic score in the
MCA (0=occlusion, 1=partial recanalization and 2=complete
recanalization) was determined by MR angiography and T2*-weighted
MRI was used to exclude the occurrence of haemorrhages.
[0130] To assess different treatment time points in the
thrombin-mediated model, diabody (Db) or vehicle (PBS) was injected
IV via a tail vein catheter at certain time points post occlusion:
an early (15 min), intermediate (90 min) or late (240 min) time
point. Five min after diabody or vehicle administration, tPA (10
mg/kg) or saline was administered IV (10% as bolus and 90% infused
over 40 min) Brain lesion volume was determined by T2-weighted MRI
and angiographic score in the MCA (0=occlusion, 1=partial
recanalization and 2=complete recanalization) was determined by MR
angiography and T2*-weighted MRI was used to exclude the occurrence
of haemorrhages.
Monofilament-Mediated MCAo Model
[0131] After a midline skin incision in the neck of anesthetized
C57BL/6 mice (by inhalation of 2% isoflurane/oxygen mixture), the
proximal common carotid artery and the external carotid artery were
ligated. The origin of the right MCA was occluded by inserting a
standardized silicon rubber-coated 6.0 nylon monofilament via the
right internal carotid artery. After 60 minutes of in situ
occlusion, the intraluminal monofilament was withdrawn and 5
minutes after reperfusion, MA or PBS was injected IV. 24 hours
after initial occlusion, mice were subjected to functional tests:
the modified Bederson test and the grip test to assess neurological
and motoric function, respectively. Mice were then euthanized and
brains were harvested to determine lesion volumes (by
2,3,5-triphenyl-tetrazolium chloride staining).
Neurological Tests
[0132] 24 hours post occlusion (MCAo model), mice were subjected to
the modified Bederson test and the grip test to assess global
neurological function and motoric function, respectively. This
modified Bederson test uses the following scoring system: 0, no
deficit; 1, forelimb flexion; 2, decreased resistance to lateral
push; 3, unidirectional circling; 4, longitudinal spinning; 5, no
movement.
[0133] The grip test was performed in which a mouse was placed on a
wooden bar (3 mm diameter, 40 cm long) attached to 2 vertical
supports 40 cm above a flat surface. When placing the mouse on the
bar midway between the supports, the experiment was rated according
to the following system: 0, falls off; 1, hangs onto bar by 2
forepaws; 2, same as for 1, but attempts to climb onto bar; 3,
hangs onto bar by 2 forepaws plus 1 or both hind paws; 4, hangs
onto bar by all 4 paws plus tail wrapped around bar; 5, escape
(mouse able to reach one of the supports). Assessment was performed
blinded.
Lesion Quantification
[0134] 24 hours post occlusion (MCAo model), mice were euthanized.
Brains were quickly harvested and cut into 2-mm-thick coronal
sections using a mouse brain slice matrix. The presence of cerebral
haemorrhages was assessed visually. The slices were stained with 2%
2,3,5-triphenyl-tetrazolium chloride (Sigma-Aldrich, St. Louis,
Mo.) in PBS to distinguish healthy tissue from unstained
infarctions. Stained slices were photographed with a digital Nikon
D70 camera, and infarct areas (white) were measured blindly using
Image J software (National Institutes of Health, Bethesda,
Md.).
Protein Extraction and Western Blot Analysis
[0135] Ischemic tissue including the cortex and basal ganglia was
dissected from formalin-fixed TTC-stained brain slices and
homogenized in RIPA buffer (25 mmol/L Tris pH 7.4, 150 mmol/L NaCl,
1% NP40) containing 0.1% SDS and 0.25% protease inhibitor cocktail
(Roche) as previously described with slight modifications. 39
Samples were homogenized using a CLI12 mixer followed by incubation
at 4.degree. C. for 20 min and subsequent sonication on ice. Then
tissue lysates were centrifuged at 15,000.times.g for 20 min at
4.degree. C. and supernatants were subjected to Western blot
analysis as follows. 30 .mu.g of total protein was loaded,
electrophoresed on a SDS-polyacrylamide gel and transferred to a
nitrocellulose membrane. After blocking for 1 h with blocking
buffer (5% nonfat dry milk, 50 mmol/L Tris-HCl pH 7.5, 150 mmol/L
NaCl, 0.05% Tween-20) membranes were incubated with either
anti-Fibrinogen polyclonal antibody (AP00766PU-N, Acris; diluted
1:500) or anti-Actin MA (MAB1501, Millipore; diluted 1:500) at
4.degree. C. overnight or for 1 hour, respectively. Then membranes
were washed followed by incubation with HRP-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch; diluted 1:14000)
(fibrinogen) or goat anti-mouse IgG (Dako; diluted 1:2000) (actin)
for 60 min at room temperature. Blots were developed using
SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific) and signal was detected with the LAS-4000 mini imager
(GE Healthcare).
Tail Bleeding Assay
[0136] Mouse tail vein bleeding times were determined with a
tail-clipping assay, as described previously. Mice were
administered with PBS, diabody, tPA as a single administration or
diabody 5 min prior to tPA as a co-administration via proximal tail
vein injection. Five min post injection, a distal 3 mm segment of
the tail was clipped and the amputated tail was immersed
immediately in 0.9% isotonic saline at 37.degree. C. Bleeding time
was monitored until initial cessation of bleeding (i.e. no
rebleeding within 30 s). Experiments were conducted blinded to
treatments. Accumulative haemoglobin loss was determined over a
period of 60 min after tail-clipping. Subsequent to centrifugation
(10 min at 2000.times.g), blood cells were resuspended in 1 mL
isotonic saline, and the haemoglobin content was measured on a
Cell-Dyn 3500R counter (Abbott, Diegem, Belgium).
Determination of Circulating Half-Life
[0137] Db-TCK26D6.times.33H1F7 (0.8 mg/kg) was administered IV via
tail vein injection in mice (n=6). Prior to the experiment, blood
was withdrawn on 0.38% trisodium citrate (=pre-sample). Post
injection, blood was withdrawn on 0.38% trisodium citrate at
several time points: 5 min, 45 min, 3 hours, 6 hours and 24 hours.
Diabody concentrations in corresponding plasma samples were
determined by an ELISA based on the simultaneous binding of the
diabody towards PAI-1 and TAFI. Wells of polystyrene microtiter
plates were incubated with 200 .mu.l recombinant mouse PAI-1 in PBS
(pH 7.4; 4 .mu.g/ml) for 72 hours at 4.degree. C., emptied and
treated for two hours with PBS supplemented with 1% (m/v) bovine
serum albumin. After washing, serial two-fold dilutions (180 .mu.l)
of plasma samples were added to the wells and incubated overnight
at 4.degree. C. Then, the wells were washed and incubated with 170
.mu.l mouse TAFI (0.1 .mu..mu.g/ml) for 2 hours at room
temperature. Subsequently, plates were washed and 160 .mu.l
HRP-conjugated MA-TCK32G12 (directed against TAFI) was added to the
wells followed by incubation for 2 hours at room temperature. All
washing steps were performed with PBS containing Tween 80 (0.002%)
and dilutions were made in PBS containing Tween 80 (0.002%) and
bovine serum albumin (0.1% m/v). The ELISA was developed using 150
.mu.l of 0.1 mol/L citrate-0.2 mol/L sodium phosphate buffer, pH
5.0, containing 300 .mu.g/mL o-phenylenediamine and 0.01% hydrogen
peroxide. After 30 min at room temperature the peroxidase reaction
was stopped with 50 .mu.l 4 mol/L H2S04. The absorbance was
measured at 492 nm. Db-TCK26D6.times.33H1F7 was used as
calibrator.
Gene Transfer by In Vivo Electroporation
[0138] Anesthetized SWISS mice (by inhalation of 2%
isoflurane/oxygen mixture) were pre-bled. Both quadriceps muscles
received an injection of hyaluronidase three hours prior to
injection of plasmid DNA (pcDNA3.1. containing scDb), followed by
electroporation. Mice were bled via retro-orbital puncture to
prepare citrated plasma in order to determine expression levels
(cfr. 3.2) up to 15 days post DNA injection.
Neurotoxicity
[0139] Neuronal cultures were prepared from Swiss mouse embryos
(embryonic day 14). Cortices were dissected and dissociated in
DMEM, and plated on 24-well plates coated with poly-D-lysine (0.1
mg/ml) and laminin (0.02 mg/ml). Cells were cultured in DMEM
supplemented with 5% foetal bovine serum, 5% horse serum (both from
Invitrogen, Cergy Pontoise, France) and 2 mM glutamine. Cultures
were maintained at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere. To inhibit glial proliferation, cytosine
.beta.-D-arabinoside (10 .mu.M) was added after 3 days in vitro
(DIV) to the cortical cultures.
[0140] Excitotoxicity was induced at 12-13 DIV by exposure to NMDA
(10 .mu.M) in serum-free DMEM supplemented with 10 .mu.M of glycine
for 24 hours. NMDA was applied alone or together with rtPA (20
.mu.g/ml) and/or diabody (5 .mu.g/ml). As a control, the diabody
was added to the neuronal culture at 12-13 DIV at several
concentrations (0.5 .mu.g/ml-50 .mu.g/ml) in the absence of NMDA.
After 24 hours, neuronal death was quantified by measurement of the
activity of lactate dehydrogenase (LDH) released from damaged cells
into the bathing medium (Roche Diagnostics, Mannheim, Germany). The
LDH level corresponding to the maximal neuronal death (full kill,
FK) was determined in sister cultures exposed to 500 .mu.M NMDA.
Background LDH levels were determined in sister cultures subjected
to control washes. Experimental values were measured after
subtracting LDH.sub.min and then normalized to
LDH.sub.max-LDH.sub.min to express the results in percentage of
neuronal death.
Statistical Analysis
[0141] All quantitative data are presented as mean and standard
error of mean (SEM). Circulating half-life of the diabody was
retrieved after nonlinear fitting of plasma levels plotted against
time (Graphpad Prism Version 5, GraphPad Software, Inc., San Diego,
Calif., USA). Statistical analysis was performed with GraphPad
Prism Version 5 (GraphPad Software). Curves from thromboelastometry
(retrieved from the Export tool) were integrated with GraphPad
Prism 5. A chi-square test was performed to compare angiographic
scores from different treatment groups. Outliers were excluded by
performing the Grubb's test. Prior to statistical analysis, a
D'Agostino and Pearson normality test was used to check data
distribution. One-way ANOVA with Bonferroni's multiple comparison
test was used for statistical comparison of lesion volumes and
speckle contrast imaging data after FeCl.sub.3-induced MCAo and an
unpaired students t-test was used for statistical comparison of
lesion volumes after mechanical tMCAo. Kruskal-Wallis ANOVA with
Dunn's multiple comparison test was used for statistical comparison
of: (i)) thromboelastometric parameters for lysis, .DELTA. L and
relative .DELTA. AUC, (ii) lung fibrinogen equivalents in the
venous thromboembolism model; (iii) lesion volumes and speckle
contrast imaging data in the IIa-induced MCAo model; (iv) laser
Doppler data in the FeCl.sub.3-induced MCAo model and (v) tail
bleeding times and haemoglobin contents. A Mann-Whitney test was
performed for statistical analysis of neurological/motor data,
fibrinogen levels after mechanical tMCAo and in vitro neurotoxicity
data. P-values less than 0.05 were considered significant.
Sequences Disclosed in the Application
[0142] Underlined text: CDR sequences
[0143] N terminal Met Ala residues in SEQ ID 17 and 18 are from the
PelB signal peptide
[0144] Bold text: synthetic linkers and tags
TABLE-US-00001 SEQ ID NO Description Sequence SEQ ID NO: 1 VH DNNMD
TCK26D6 CDR1 SEQ ID NO: 2 VH SXYSNNGGTIYNQKFKG (where X may be V or
I) TCK26D6 CDR2 SEQ ID NO: 3 VH EMSDGPYWFFDV TCK26D6 CDR3 SEQ ID
NO: 4 VL RASENIFRNLV TCK26D6 CDR1 SEQ ID NO: 5 VL SATNLVD TCK26D6
CDR2 SEQ ID NO: 6 VL QHFWGTPRT TCK26D6 CDR3 SEQ ID NO: 7 VH 33H1F7
DTYIH CDR1 SEQ ID NO: 8 VH 33H1F7 RIDPANGNTKYDSKFQD CDR2 SEQ ID NO:
9 VH 33H1F7 GDYDYVYFDY CDR3 SEQ ID NO: 10 VL 33H1F7 RASQDISNFLD
CDR1 SEQ ID NO: 11 VL 33H1F7 YTSRLHS CDR2 SEQ ID NO: 12 VL 33H1F7
QQGNTFPPT CDR3 SEQ ID NO: 13 VH QVQLQQSGPELVKPGASVKISCKASGYTFTDNNM
TCK26D6 DWAKQSHGKSLEWIGSXYSNNGGTIYNQKFKGK
ATLNVDTSSSTAYMELRSLTSEDTAVYYCAREMS DGPYWFFDVWGTGTTVTVSG (where X
may be V or I) SEQ ID NO: 14 VL DIQMTQSPASLSVSVGETVTITCRASENIFRNLVW
TCK26D6 YQQKQGKSPQLLVYSATNLVDGVPSRFSGSGSGT
QYSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTK LEIKR SEQ ID NO: 15 VH 33H1F7
QVQLQQSGAEVVKPGASVKLACTASGFNIKDTYI
HWVKQGPEQGLEWIGRIDPANGNTKYDSKFQDK
ATITADTSSNTAYLHLSSLTSEDTAVYYCVRGDY DYVYFDYWGQGTTVTVSS SEQ ID NO:16
VL 33H1F7 DIQMTQSPSSLSASLGDRVTISCRASQDISNFLDW
YQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTD
YSLTISKLEQEDIATYFCQQGNTFPPTFGGGTKLEI KR SEQ ID Polypeptide 1
MAQVQLQQSGPELVKPGASVKISCKASGYTFTDN NO: 17
NMDWAKQSHGKSLEWIGSIYSNNGGTIYNQKFK
GKATLNVDTSSSTAYMELRSLTSEDTAVYYCARE
MSDGPYWFFDVWGTGTTVTVSGAKTTPKLGGDI
QMTQSPSSLSASLGDRVTISCRASQDISNFLDWYQ
QKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYS
LTISKLEQEDIATYFCQQGNTFPPTFGGGTKLEIKR ADAAAAGSEQKLISEEDLNSHHHHHH SEQ
ID Polypeptide 2 MAQVQLQQSGAEVVKPGASVKLACTASGFNIKD NO: 18
TYIHWVKQGPEQGLEWIGRIDPANGNTKYDSKFQ
DKATITADTSSNTAYLHLSSLTSEDTAVYYCVRG DYDYVYFDYWGQGTTVTVSSAKTTPKLGGDIQ
MTQSPASLSVSVGETVTITCRASENIFRNLVWYQQ
KQGKSPQLLVYSATNLVDGVPSRFSGSGSGTQYS
LKINSLQSEDFGSYYCQHFWGTPRTFGGGTKLEIK RADTAPTGSEQKLISEEDLNSHHHHHH
Sequence CWU 1
1
1815PRTMus musculus 1Asp Asn Asn Met Asp 1 5 217PRTMus
musculusPRT(2)..(2)Xaa may be Val or Ile 2Ser Xaa Tyr Ser Asn Asn
Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 312PRTMus
musculus 3Glu Met Ser Asp Gly Pro Tyr Trp Phe Phe Asp Val 1 5 10
411PRTMus musculus 4Arg Ala Ser Glu Asn Ile Phe Arg Asn Leu Val 1 5
10 57PRTMus musculus 5Ser Ala Thr Asn Leu Val Asp 1 5 69PRTMus
musculus 6Gln His Phe Trp Gly Thr Pro Arg Thr 1 5 75PRTMus musculus
7Asp Thr Tyr Ile His 1 5 817PRTMus musculus 8Arg Ile Asp Pro Ala
Asn Gly Asn Thr Lys Tyr Asp Ser Lys Phe Gln 1 5 10 15 Asp 910PRTMus
musculus 9Gly Asp Tyr Asp Tyr Val Tyr Phe Asp Tyr 1 5 10 1011PRTMus
musculus 10Arg Ala Ser Gln Asp Ile Ser Asn Phe Leu Asp 1 5 10
117PRTMus musculus 11Tyr Thr Ser Arg Leu His Ser 1 5 129PRTMus
musculus 12Gln Gln Gly Asn Thr Phe Pro Pro Thr 1 5 13121PRTMus
musculusPEPTIDE(51)..(51)Xaa may be Val or Ile 13Gln Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30
Asn Met Asp Trp Ala Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35
40 45 Gly Ser Xaa Tyr Ser Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Asn Val Asp Thr Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Met Ser Asp Gly Pro Tyr
Trp Phe Phe Asp Val Trp Gly 100 105 110 Thr Gly Thr Thr Val Thr Val
Ser Gly 115 120 14108PRTMus musculus 14Asp Ile Gln Met Thr Gln Ser
Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile
Thr Cys Arg Ala Ser Glu Asn Ile Phe Arg Asn 20 25 30 Leu Val Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr
Ser Ala Thr Asn Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr
Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 15119PRTMus musculus 15Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ala Cys Thr
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val
Lys Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile
Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Ser Lys Phe 50 55 60 Gln
Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70
75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Val Arg Gly Asp Tyr Asp Tyr Val Tyr Phe Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 16108PRTMus
musculus 16Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp
Ile Ser Asn Phe 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Asp Gly
Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile Ser Lys Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala
Thr Tyr Phe Cys Gln Gln Gly Asn Thr Phe Pro Pro 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 17266PRTMus
musculus 17Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr 20 25 30 Asp Asn Asn Met Asp Trp Ala Lys Gln Ser
His Gly Lys Ser Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Ser Asn
Asn Gly Gly Thr Ile Tyr Asn Gln 50 55 60 Lys Phe Lys Gly Lys Ala
Thr Leu Asn Val Asp Thr Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Glu
Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Ala Arg Glu Met Ser Asp Gly Pro Tyr Trp Phe Phe Asp Val 100 105 110
Trp Gly Thr Gly Thr Thr Val Thr Val Ser Gly Ala Lys Thr Thr Pro 115
120 125 Lys Leu Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser 130 135 140 Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln Asp 145 150 155 160 Ile Ser Asn Phe Leu Asp Trp Tyr Gln Gln
Lys Pro Asp Gly Thr Val 165 170 175 Lys Leu Leu Ile Tyr Tyr Thr Ser
Arg Leu His Ser Gly Val Pro Ser 180 185 190 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser 195 200 205 Lys Leu Glu Gln
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn 210 215 220 Thr Phe
Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235
240 Ala Asp Ala Ala Ala Ala Gly Ser Glu Gln Lys Leu Ile Ser Glu Glu
245 250 255 Asp Leu Asn Ser His His His His His His 260 265
18264PRTMus musculus 18Met Ala Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Val Val Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ala Cys Thr
Ala Ser Gly Phe Asn Ile Lys 20 25 30 Asp Thr Tyr Ile His Trp Val
Lys Gln Gly Pro Glu Gln Gly Leu Glu 35 40 45 Trp Ile Gly Arg Ile
Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Ser 50 55 60 Lys Phe Gln
Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr 65 70 75 80 Ala
Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Val Arg Gly Asp Tyr Asp Tyr Val Tyr Phe Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro
Lys Leu 115 120 125 Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser 130 135 140 Val Gly Glu Thr Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Phe 145 150 155 160 Arg Asn Leu Val Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu 165 170 175 Leu Val Tyr Ser Ala
Thr Asn Leu Val Asp Gly Val Pro Ser Arg Phe 180 185 190 Ser Gly Ser
Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu 195 200 205 Gln
Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr 210 215
220 Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp
225 230 235 240 Thr Ala Pro Thr Gly Ser Glu Gln Lys Leu Ile Ser Glu
Glu Asp Leu 245 250 255 Asn Ser His His His His His His 260
* * * * *