Note: Descriptions are shown in the official language in which they were submitted.
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"Treatment of coagulopathy with hyperfibrinolysis"
The invention relates to the field of coagulopathy with hyperfibrinolysis.
More
particularly, this invention relates to the treatment of haemophila diseases
such as
haemophilia A or haemophilia B. The present invention claims priority of the
PCT
application PCT/EP2009/004218 which is hereby fully incorporated in terms of
disclosure.
Haemophilia is a group of hereditary genetic disorders that impair the body's
ability to
control blood clotting or coagulation, which is used to stop bleeding when a
blood
vessel is broken. Haemophilia A, the most common form, results from a mutation
in the
gene for Factor VIII; haemophilia B, also known as Christmas disease, results
from a
mutation in the gene for Factor IX. Haemophilia B, like haemophilia A, is X-
linked and
accounts for approximately 12% of haemophilia cases. The symptoms are
identical to
those of haemophilia A: excessive bleeding upon injury; and spontaneous
bleeding,
especially into weight-bearing joints, soft tissues, and mucous membranes.
Repeated
bleeding into joints results in haemarthrosis, causing painful crippling
arthropathy that
often necessitates joint replacement. Haematomas in soft tissues can result in
pseudo
tumors composed of necrotic coagulated blood; they can obstruct, compress, or
rupture into adjacent organs and can lead to infection. Once formed the
haematomas
are difficult to treat, even with surgery. Recovery of nerves after
compression is poor,
resulting in palsy. Those bleeding episodes that involve the gastrointestinal
tract,
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central nervous system, or airway/retroperitoneal space can lead to death if
not
detected. Intracranial bleeding is a major cause of death in haemophiliacs.
There are estimated to be 100,000 cases of congenital haemophilia in the
United
States. Of these, approximately 20,000 are cases of haemophilia B, the blood
of such
patients being either totally devoid of Factor IX or seriously deficient in
plasma Factor
IX component. The disease therefore exists in varying degrees of severity,
requiring
therapy anywhere from every week up to once or twice a year. The completely
deficient
cases require replacement therapy once every week; the partially deficient
cases
require therapy only when bleeding episodes occur, which may be as seldom as
once
a year. The bleeding episodes in congenital, partially deficient cases are
generally
caused by a temporarily acquired susceptibility rather than by injury alone.
Intravenous
injection of a sufficiently large amount of fresh plasma, or an equivalent
amount of
fresh blood temporarily corrects the defect of a deficient subject. The
beneficial effect
often lasts for two or three weeks, although the coagulation defect as
measured by in
vitro tests on the patient's blood appears improved for only two or three
days.
Such therapy with fresh plasma or fresh blood is effective but it has several
serious
drawbacks: (1) it requires ready availability of a large amount of fresh
plasma; (2)
requires hospitalization for the administration of the plasma; (3) a great
many of the
patients become sensitized to repeated blood or plasma infusions and
ultimately
encounter fatal transfusion reactions; (4) at best plasma can only partially
alleviate the
deficiency; and (5) prolonged treatment or surgery is not possible because the
large
amounts of blood or plasma which are required will cause acute and fatal
edema.
An improved therapy includes intravenous replacement therapy with Factor VIII
or
Factor IX concentrates. However, also this therapy suffers from several
disadvantages:
(1) when treating major bleeding episodes tissue damage remains even after
prompt
detection and treatment; (2) a great many of the patients become refractory to
the
coagulation factors and develop inhibitory antibodies against the coagulation
factors
(so called haemophilia with inhibitors); (3) despite the improved virus
inactivation
methods there is still an increased risk of contamination with fatal viruses
such as HIV
and hepatitis C (it is estimated that more than 50% of the haemophilia
population, over
10,000 people, contracted HIV from the tainted blood supply in the USA); (4),
the
isolated and especially the recombinant clotting factors are very expensive
and not
generally available in the developing world.
A treatment or prevention of bleeding beyond a replacement therapy is a
challenge
because bleeding in haemophilia is a complex pathophysiological process that
may be
attributable to triple defects: (1) a reduced thrombin generation via the
extrinsic
pathway at low tissue factor concentration, (2) a reduced secondary burst of
thrombin
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generation via the intrinsic pathway, and (3) a defective downregulation of
the
fibrinolytic system by the intrinsic pathway.
The fact that a reduced thrombin generation results in a reduced clotting
propensity
and therefore an increased risk of bleeding is generally accepted. However,
work in the
past decade indicates that also a defective downregulation of the fibrinolysis
may play
a role in haemophilia. As a result haemophila can be also classified as a
coagulopathy
with hyperfibrinolysis.
A recent publication supports this assumption by showing in vitro that when a
clot is
formed in Factor VIII depleted plasma (FVIII-DP) and supplemented with tissue
plasminogen activator tPA, fibrinolysis is not adequately downregulated and as
a result
the clot lyses prematurely (Broze and Higuchi, Blood 1996, 88: 3815-3823;
Mosnier et
al.; Thromb. Haemost. 2001, 86: 1035-1039). Furthermore, it could be shown
that this
"premature lysis" is due to reduced or absent activation of thrombin-
activatable
fibrinolysis inhibitor (TAFI) (Broze and Higuchi, 1996) and that in FVIII-DP,
an activated
TAFI containing mixture increases clot lysis time. It was concluded that
stabilized TAFI
can be used for the treatment of haemophilia (WO02/099098).
TAFI plays a crucial role in the downregulation of fibrinolysis, which is
required for
formation of stable clots. TAFI also known as plasma procarboxypeptidase B2 or
procarboxypeptidase U is a plasma zymogen that, when exposed to the thrombin-
thrombomodulin complex, is converted by proteolysis at Arg92 to a basic
carboxypeptidase (TAFIa or activated TAFI) that inhibits fibrinolysis. It
potently
attenuates fibrinolysis by removing the C-terminal lysine and arginine
residues from
fibrin which are important for the binding and activation of plasminogen.
As discussed above thrombomodulin (TM) in complex with thrombin is responsible
for
the TAFI activation. Thrombomodulin is a membrane protein that acts as a
thrombin
receptor on the endothelial cells lining the blood vessels. Thrombin is a
central enzyme
in the coagulation cascade, which converts fibrinogen to fibrin, the matrix
clots are
made of. Initially, a local injury leads to the generation of small amounts of
thrombin
from its inactive precursor prothrombin. Thrombin, in turn, activates
platelets and,
second, certain coagulation factors including factors V and VIII. The latter
action gives
rise to the so-called thrombin burst, a massive activation of additional
prothrombin
molecules, which finally results in the formation of a stable clot.
When bound to thrombomodulin, however, the activity of thrombin is changed in
direc-
tion: A major feature of the thrombin-thrombomodulin complex is its ability to
activate
protein C, which then downregulates the coagulation cascade by proteolytically
inacti-
vating the essential cofactors Factor Va and Factor Villa (Esmon et al., Ann.
N. Y.
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Acad. Sci. (1991), 614:30-43), thus affording anticoagulant activity. The
thrombin-
thrombomodulin complex is also able to activate the thrombin-activatable
fibrinolysis
inhibitor (TAFI), which then antagonizes fibrinolysis (see above).
Mature human TM is composed of a single polypeptide chain of 559 residues and
consists of five domains: an aminoterminal "lectin-like" domain, an "6 EGF-
like repeat
domain" comprising six epidermal growth factor (EGF)-like repeats, an 0-
glycosylation
domain, the transmembrane domain and a cytoplasmic domain with following
localisation (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3):
Approx. amino acid position Domain
-18 - -1 Signal sequence
1 - 226 N-terminal domain (lectin-like)
227 - 462 6 EGF-like repeat domains
307 - 345 EGF-like repeat domain 3
333 - 344 c-loop of EGF-like repeat domain 3
347 - 386 EGF-like repeat domain 4
387 - 422 EGF-like repeat domain 5
423 - 462 EGF-like repeat domain 6
463 - 497 0-linked GI cos lation
498 - 521 Transmenbrane domain
522 - 557 Cytoplasmic domain
Table 3: domain structure of thrombomodulin (numbered according to SEQ ID
NO:1).
Various structure-function studies using proteolytic fragments of rabbit TM or
deletion
mutants of recombinant human TM have localized its activity to the last three
EGF-like
repeats. The smallest mutant capable of efficiently promoting TAFI activation
contained
residues including the c-loop of epidermal growth factor-3 (EGF3) through
EGF6. This
mutant is 13 residues longer than the smallest mutant that activates C; the
latter
consisted of residues from the interdomain loop connecting EGF3 and EGF4
through
EGF6.
As discussed above the replacement therapy for treating coagulation disorders
such as
haemophilia does not meet the medical needs. Importantly, no drug besides the
coagulation factors used for the replacement therapy is available which can
prevent or
treat haemophilia patients.
Thus, despite the long-standing need for the development of therapies to
prevent or
treat coagulopathy with hyperfibrinolysis, in particular haemophilia, progress
has been
slow, and therapeutics that are safe and effective are still missing.
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Thus, it is the objective of the present invention to provide novel means for
the
treatment of coagulopathy with hyperfibrinolysis.
This objective is solved by providing a medicament for the treatment of
coagulopathy
with hyperfibrinolysis in a mammal, in particular in humans, comprising a
thrombomodulin analogue exhibiting at therapeutically effective dosages an
antifibrinolytic effect. Particularly suitable pharmaceutically active
proteins and peptides
are also provided which can be used according to the invention.
This novel approach is based on the surprising findings that a thrombomodulin
can be
modified in a way that it exhibits an antifibrinolytic activity that prevail
its profibrinolytic
activity even at high plasma concentrations, in particular at concentrations
of more than
15 nM, in particular more than 20, 30, 40 or 50 nM (at least up to 100 nM).
Hence
these TM analogues exhibit an antifibrinolytic effect, and are thus suitable
for the use
according to the invention. In most favourable embodiments the TM analogues
can
have a prevailing antifibrinolytic activity in concentration even up to 200 nM
or more,
even more preferred up to 300 nM or 500 nM.
This antifibrinolytic effect was shown in plasma from haemophilia patients
(which is
depleted for Factor VIII; FVIII-DP) and in whole blood or plasma of dogs with
haemophilia ATherewith it was demonstrated that such a thrombomodulin analogue
can be used as a therapeutic.
So far the therapeutic use of thrombomodulin for the treatment of haemophilia
was not
regarded as a real option because it was known from rabbit lung thrombomodulin
(rITM) that it always has both anti- and profibrinolytic activities even at
rather low
concentrations (see Mosnier and Bouma; Arterioscler. Thromb. Vasc. Biol. 2006;
26:
2445 - 2453; especially Figure 5). At plasma concentrations of less than 15 nM
rITM
increased clot lysis time whereas at plasma concentrations greater than 15 nM
a
marked decrease in lysis time was demonstrated (Mosnier et al., 2001, Mosnier
and
Bouma, 2006) with a profibrinolytic effect as the final result. This
profibrinolytic effect at
higher concentrations prohibits any therapeutical use in haemophilia since a
potential
overdosing or individual variabilites in susceptibility would fatally
aggravate, prolong or
even cause bleeding events.
According to the invention various options exist which lead to TM analogues
that
exhibit an antifibrinolytic effect and thus are suitable for the treatment
according to the
invention.
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In one embodiment thrombomodulin analogues can be used with reduced binding
affinity to thrombin. Consequently they can prolong the clot lysis in normal
plasma and
FVIII-DP, e.g. up to 100 nM (Figure 4) or up to 500 nM (Figure 10)
The importance of these findings is that these thrombomodulin analogues
exhibit an
antifibrinolytic effect without a deleterious profibrinolytic effect even at
high
concentrations. This concentration exceeds by far the therapeutically
effective
dosages. Therefore the TM analogues enable the treatment of coagulopathy with
hyperfibrinolysis.
Without bound to this theory the inventors have shown that this therapeutic
potential of
the TM analogues can be explained by the fact that they show a markedly
reduced
affinity towards thrombin. This was shown by Bajzar et al. (J. Biol. Chem
1996; 271:
16603-16608) who found a KD value of 23 nM in contrast to the KD value of 0.2
nM
observed for the binding between thrombin and rabbit lung thrombomodulin
(Esmon et
al., Ann. NY. Acad. Sci. 1986, 485: 215-220).
Hence, according to one embodiment of the invention thrombomodulin analogues
can
be used for the treatment of coagulopathy with hyperfibrinolysis which have a
reduced
binding affinity towards thrombin compared to the rabbit lung thrombomodulin.
In particular, a thrombomodulin analogue can be used which exhibits a KD for
thrombin
binding of more than 0.2 nM, preferably more than 1 nM, 2 nM, 4 nM, 5 nM, 7.5
nM,
nM, 12.5 nM, 15nM, 17.5 nM, 20 nM, 22.5nM, or 25 nM, and more preferably a KD
value in a range between 10 and 30 nM or 10 and 100 nM or more. In specific
embodiments TM analogues are used with a KD for thrombin binding of about 50,
60 or
70 nM.
In a further embodiment of the invention, the reduced profibrinolytic activity
of a
thrombomodulin analogue can be due to a reduced ability to activate protein C
(so
called "cofactor activity"). Since the protein C activation results in an
upregulation of
fibrinolysis (Mosnier et al., 2001) a reduced cofactor activity will prolong
the clot lysis
time. The person skilled in art knows several strategies to reduce the
cofactor activity
of thrombomodulin, such as e.g. changes in the glycosylation, secondary or
tertiary
structure of the protein or preferably changes in the primary structure e.g.
by mutation
of one or more amino acids.
In a yet another embodiment TM analogues can be used which have a reduced
cofactor activity compared to the thrombomodulin analogue TMEM388L, where TME
denotes to an analogue consisting of the six EGF domains only.
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According to the invention a thrombomodulin analogue can also be used which
has an
increased ability to activate TAFI (so called "TAFI activation activity")
since TAFI
activation results in a downregulation of fibrinolysis (Mosnier and Bouma,
2006). For
the person skilled in art there are several strategies to increase the TAFI
activation
activity by thrombomodulin such as changes in the glycosylation, secondary or
tertiary
structure of the protein or preferably changes in the primary structure e.g.
by mutation
of one or more amino acids.
Particularly, this invention also provides for a thrombomodulin analogue which
has a
significantly increased ratio of TAFI activation activity to cofactor activity
compared to
the thrombomodulin analogue TMEM388L.
Notably, according to the invention the TM analogue used for the treatment of
coagulopathy has one or more of the above described features, namely:
(i) a binding affinity towards thrombin that is decreased compared to the
rabbit lung thrombomodulin, and/or a binding affinity towards thrombin
with a kD value of more than 0.2 nM;
(ii) a reduced cofactor activity compared to cofactor activity of the TM
analogue TMEM388L, or
(iii) an increased ratio of TAFI activation activity to cofactor activity as
compared to the TM analogue TMEM388L.
In an embodiment of the invention, thrombomodulin can be used to treat human
patients with any coagulopathy that occurs with a prominently or even slightly
reduced
fibrinolysis compared to normal subjects. In particular the following diseases
can be
treated with the thrombomodulin analogue: haemophilia A, haemophilia B,
haemophilia
C, von Willebrandt disease (vWD),. acquired von Willebrandt disease, Factor X
deficiency, parahaemophilia, hereditary disorders of the clotting factors I,
II, V, or VII,
haemorrhagic disorder due to circulating anticoagulants (including
autoantibodies
against coagulation factors such as Factor VIII) or acquired coagulation
deficiency.
It will be understood that the therapeutic success that can be maintained or
achieved
by the treatment of the invention depends on the nature and the degree of the
disease
in any particular patient.
Specific embodiments of the invention relate to the prophylactic treatment of
coagulopathy to prevent bleeding or to the acute treatment when bleeding
occurs ("on
demand"). The bleeding events to be treated with the thrombomodulin analogue
can
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occur in every organ or tissue in the organism, most importantly in the
central nervous
system e.g. as intracranial bleeding, in the joints, the muscles, the
gastrointestinal tract,
the respiratory tract, the retroperitoneal space or soft tissues.
For the preventive treatment the TM analogue can be given to the patient at
regular
intervals over an extended period. However, also multiple dosing for a rather
restricted
time period ("subchronic treatment") is possible.
In one embodiment of the invention the thrombomodulin analogue is given in
advance
of a higher bleeding risk, e.g. a surgery or a tooth extraction.
In a further embodiment of the invention the thrombomodulin analogue is
administered
to patients that are refractory to standard therapy such as the transfusion of
blood or
plasma or the replacement therapy using coagulation factors.
According to the invention the TM analogue can be administered in multiple
doses
preferably once daily but also bidaily, or every third, fourth, fifth, sixth
or seven days
over a total time period of less than one week to four weeks, more preferably
as
chronic administration. Thus, according to the invention a pharmaceutical
composition
is provided, which is suitable for allowing a multiple administration of the
thrombomodulin analogue.
The TM analogue is given preferably non-orally as a parenteral application
e.g. by
intravenous or subcutaneous application. An intravenous or subcutaneous bolus
application is possible. Thus, according to the invention a pharmaceutical
composition
is provided, which is suitable for a parenteral administration of
thrombomodulin.
In one embodiment of the invention the thrombomodulin analogue is a soluble TM
analogue, in particular a TM analogue where the cytoplasmic domain is deleted
and
the transmembrane domain is completely or partially deleted.
In a preferred embodiment of the invention the thrombomodulin analogue
comprises at
least one structural domain selected from the group containing EGF3, EGF4,
EGF5, or
EGF6, preferably the EGF domains EGF1 to EGF6, more preferably the EGF domains
EGF3 to EGF6 and most preferably the EGF domains EGF4 to EGF6 and particularly
the fragment including the c-loop of epidermal growth factor-3 (EGF3) through
EGF6.
Various forms of soluble thrombomodulin are known to the skilled person, e.g.
the so
called ART-123 developed by Asahi Corporation (Tokyo, Japan) or the
recombinant
soluble human thrombomodulin Solulin, currently under development by PAION
.Deutschland GmbH, Aachen (Germany). The recombinant soluble thrombomodulin,
i.e.
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a soluble thrombomodulin without a modification of the amino acid sequence, is
subject
of the Asahi patent EPO 312 598 B1.
Solulin is a soluble, as well as protease and oxidation-resistant analogue of
human
thrombomodulin and thus exhibits a long life in vivo. Solulin's main feature
lies in its
broad mechanism of action since it not exclusively inhibits thrombin. It also
activates
TAFI and the natural protein C / protein S pathway. As a result of its reduced
thrombin
binding Solulin inhibits fibrinolysis even up to high concentrations. Solulin
and its
modifications are particular embodiments of the invention.
Solulin is inter alia subject of the European patent EP 0 641 215 131, EP 0
544 826 B1
as well as EP 0 527 821 131. Solulin contains modifications compared to the
sequence
of native human thrombomodulin (SEQ. ID NO. 1) at the following positions: G -
3V,
Removal of amino acids 1-3, M388L, R456G, H457Q, S474A and termination at
P490.
This numbering system is in accordance with the native thrombomodulin of SEQ.
ID
NO. 1 and SEQ ID NO:3. The sequence of Solulin as one preferred embodiment of
the
invention is shown in SEQ ID NO: 2.
However, notably, according to the invention also thrombomodulin analogues can
be
used, which comprise only one or more of the above mentioned properties, or of
the
properties outlined in the above mentioned European patent EP 0 544 826 B1,
EP 0 641 215 131 and EP 0 527 821 B1.
Furthermore, according to the invention thrombomodulin analogues can be used,
which
comprise only one or more of the above mentioned properties, or of the
properties
outlined in the publication by Wnag et al., 2000, J. Biol CHem. 275: 22942-
22947.
Particularly preferred thrombomodulin analogues applicable according to the
invention
are those that have one or more of the following characteristics:
(i) they exhibit oxidation resistance,
(ii) they exhibit protease resistance,
(iii) they have homogeneous N- or C-termini,
(iv) they have been post-translationally modified, e.g., by glycosylation of
at least
some of the glycosylation sites of native thrombomodulin (SEQ ID NO: 1),
(v) they have linear double-reciprocal thrombin binding properties,
(vi) they are soluble in aqueous solution in relatively low amounts of
detergents
and typically lack a transmembrane sequence,
(vii) they are lacking a glycosaminoglycan chain.
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The manufacture of these analogues used in this invention is disclosed in the
above
mentioned European patents relating to Solulin.
In one embodiment of the invention only the six EGF domains of Solulin can be
used,
in particular a Solulin fragment consisting of the EGF4 to EGF6 domain.
In an embodiment a thrombomodulin analogue with reduced cofactor activity as
known
from the W093/25675 Al can be used. A series of thrombomodulin analogues is
described herein having about 50% or less of the cofactor activity of the
control human
soluble thrombomodulin (TMEM388L).
More particularly said thrombomodulin analogues upon binding to thrombin,
exhibit a
modified cofactor activity as compared to binding with TMEM388L of less than
or equal
to 50%, said analogue having amino acid substitutions at one or more positions
corresponding to the amino acid position as given in SEQ ID NO:1 or SEQ ID
NO:3:
ab) 355Asn;
ae) 359GIn;
af) 363Leu;
ai) 368 Tyr;
aj) 371Val;
ak) 374Glu;
al) 376Phe;
am) 384 His;
an) 3a5Arg;
ba) 387Gln;
bb) 389Phe;
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
bf) 403Thr;
bg) 408Glu;
bh) 41Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp;
bm) 417Asp;
bn) 4201le;
ca) 423Asp;
cb) 4241le;
cc) 425Asp;
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cd) 426Glu;
ce) 428GIu;
cf) 429Asp;
cg) 432Phe;
ch) 434Ser;
ci) 436Val;
cj) 438His;
ck) 439Asp;
cl) 440Leu;
cm) 443Thr;
cn) 444Phe;
co) 445GIu;
cp) 456Arg;
cq) 4581le; or
cr) "Asp
Most preferred are TM analogues with only one of the above listed
substitutions. In one
embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2) with
one or
more, preferably one, of the above mutations. Accordingly the invention also
relates to
proteins according to SEQ ID NO:2 with at least one, in a specific embodiment
with
exactly one, of the above mutations. . In one embodiment one amin o acid at
the given
position is deleted instead of substituted. In a further embodiment the
invention
encompasses a Solulin fragment, in particular a Solulin fragment consisting of
the
EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above
mutations.
The Solulin or the Solulin fragment can contain at least one, or exactly one
(e.g. a
mutation in the position 376), of the above mutations in the amino acid
positions 371 to
389. If the Solulin or the Solulin fragments contains a mutation in position
376 (e.g.
F376A) a second mutation selected from the above mutations is possible.
For convenience the designation to the left, e.g. aa) are identical for each
modified site.
The first letter represents the EGF domain, where a is EGF4; b is EGF5 and c
is EGF6.
The second letter represents the relative position of the modification with
regard to
other residues in the listing. Also provided herein are nucleic acids encoding
the TM
analogues described above.
The following analogues constitute a preferred subset of the above given
analogues
wherein the analogues have 25% or less of the cofactor activity of the
control,
TMEM388L. These analogues have one or more amino acid substitutions,
preferably
only one (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3):
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ae) 359GIn;
aj) 371Vai;
ak) 374Glu;
al) 376Phe;
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
bg) 408Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bI) 416Asp;
bm) 417Asp;
bo) 423Asp;
bp) 4241le;
bq) 425Asp;
cd) 426Glu;
ce) 429Asp;
ck) 439Asp;
cn) 444Phe; or
cr) "Asp.
In one embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2)
with
one or more, preferably one, of the above mutations. Accordingly the invention
also
relates to proteins according to SEQ ID NO:2 with at least one, in a specific
embodiment with exactly one, of the above mutations. In one embodiment one
amin o
acid at the given position is deleted instead of substituted. In a further
embodiment the
invention encompasses a Solulin fragment, in particular a Solulin fragment
consisting
of the EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above
mutations. The Solulin or the Solulin fragment can contain at least one, or
exactly one
(e.g. a mutation in the position 376), of the above mutations in the amino
acid positions
371 to 389. If the Solulin or the Solulin fragments contains a mutation in
position 376
(e.g. F376A) a second mutation selected from the above mutations is possible.
The modifications set forth above with regard to protease activity, aliphatic
substitutions, oxidation resistance and uniform termini are also applicable
for the above
analogues having less than 50% of the cofactor activity of the control.
Preferred are those listed above having less than 30% of the activity of the
control.
These analogues are represented by mutations in domain 4. These analogues have
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one or more amino acid substitutions, preferably only one (amino acid position
as given
in SEQ ID NO:1 or SEQ ID NO:3):
ae) 359GIn;
aj) 371Val; or
al) 376Phe.
In one embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2)
with
one or more, preferably one, of the above mutations. Accordingly the invention
also
relates to proteins according to SEQ ID NO:2 with at least one, in a specific
embodiment with exactly one, of the above mutations. . In one embodiment one
amin o
acid at the given position is deleted instead of substituted. In a further
embodiment the
invention encompasses a Solulin fragment, in particular a Solulin fragment
consisting
of the EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above
mutations. The Solulin or the Solulin fragment can contain at least one, or
exactly one
(e.g. a mutation in the position 376), of the above mutations in the amino
acid positions
371 to 389. If the Solulin or the Solulin fragments contains a mutation in
position 376
(e.g. F376A) a second mutation selected from the above mutations is possible.
There are also described herein analogues having an essentially unmodified KD
value
compared to TMEM388L. EGF5 and EGF6 are known to play an important role in
high
affinity binding to thrombin, whereas EGF4 with a less critical role in
binding is critical
for conferring cofactor activity to the TM/thrombin complex. For this reason
those
analogues having modifications in the EGF repeats 5 and 6 can have almost the
same
cofactor activity but a reduced KD compared to TMEM388L, e.g. (S406A).
Analogues
having modifications in the EGF repeats 5 and 6 which resulted in reduced
cofactor
activity are listed below. These analogues have one or more amino acid
substitutions,
preferably only one (amino acid position as given in SEQ ID NO:1 or SEQ ID
NO:3):
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
bf) 403Thr;
bg) 408Glu;
bi) 413Tyr;
bj) 41411e;
bk) 415Leu;
bl) 416Asp;
bm) 417Asp;
ca) 423Asp;
cb) 4241le;
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cc) 425Asp;
cd) 426Glu;
cf) 429Asp;
ck) 439Asp;
cn) 444Phe; or
cr) 461Asp
The above analogues may also grouped by their respective domains (i.e., EGF4,
EGF5
or EFG6) as well as by their respective relative activity. For example the TM
analogues
with a EGF4 sequence modification having approximately 50% of the control
cofactor
activity are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3):
bb) 355Asn;
ae) 359GIn;
af) 363Leu;
ai) 368 Tyr;
aj) 371Val;
ak) 374GIu;
al) 376Phe;
am) 384 His; or
an) 385Arg.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions.
Those in EGF4 having less than 25% of the cofactor activity of the control are
(amino
acid position as given in SEQ ID NO:1 or SEQ ID NO:3):
ae) 359GIn;
aj) 371Val; or
al) 376Phe.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions.
In EGF5, the following modifications resulted in analogues having at least a
50%
reduction in cofactor activity (amino acid position as given in SEQ ID NO:1 or
SEQ ID
NO:3):
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
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bf) 403Thr;
bg) 408Glu;
bh) 41Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp;
bm) 417Asp; or
bn) 4201le.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. Among these analogues are those where the
analogues
have an essentially unmodified kCat/Km compared to TMEM388L.
In EGF5, the analogues can be further subgrouped according to those
modifications
resulted in analogues having at least a 75% reduction in cofactor activity
(amino acid
position as given in SEQ ID NO:1 or SEQ ID NO:3):
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
bg) 408Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp; or
bm) 417Asp.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. Among these analogues are those with
essentially
unmodified kCat/Km compared to TMEM388L. Nucleic acids encoding the above
analogues are also provided.
With regard to EGF6 the groups are provided below. Those having a cofactor
activity of
less than 50% of the control are (amino acid position as given in SEQ ID NO:1
or SEQ
ID NO:3):
ca) 423Asp;
cb) 4241le;
CC) 425Asp;
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cd) 426GIu;
ce) 426GIu;
cf) 429Asp;
cg) 432Phe;
ch) 434Ser;
ci) 436Val;
cj) 438His;
ck) 439Asp;
cl) 44OLeu;
cm) 4`13Thr;
cn) "4Phe;
co) 5Glu;
cp) 456Arg;
cq) 4581 le; or
cr) 46'Asp.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions.
Those having a cofactor activity of less than 25% of the control are (amino
acid position
as given in SEQ ID NO:1 or SEQ ID NO:3):
ca) 423Asp;
cb) 4241le;
CC) 425Asp;
cd) 426GIu;
cf) 429Asp;
ck) . 439Asp;
cn) 444Phe; or
cr) 46'Asp.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. The preferred analogues are those set forth
above with
additional modifications for solubility, protease resistance, oxidation
resistance as well
as uniform terminal ends. The nucleic acids encoding these analogues are also
a part
of the claimed invention. As with the other groups, these analogues include
those
wherein said analogue has an essentially unmodified kCat/Km compared to
TMEM388L.
The analogues can be further subgrouped according to those possessing a
modified
amino acid at a certain position, wherein said analogue has essentially
equivalent KD
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for thrombin compared to an analogue having at said position the native
residue,
wherein said position corresponds to (amino acid position as given in SEQ ID
NO:1 or
SEQ ID NO:3):
bb) 355Asn; or
ae) 359GIn.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. These analogues may have a modified kCat/Km of
less
than 30% of the control.
The following sites embrace described analogues having a modified KD or
kCat/Km
compared to an analogue having at said position the native residue, wherein
said
position corresponds to (amino acid position as given in SEQ ID NO:1 or SEQ ID
NO:3):
af) 363Leu;
aj) 371 Val;
ak) 374Glu;
al) 376Phe;
am) 384 His;
an) 385Arg;
bc) 398Asp;
bd) 400Asp; or
be) 402Asn.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. These further include those analogues having
both a
modified KD and kCat/Km, especially those having been modified by at least
20%.
The following sites describe analogues having a lower cofactor activity and a
KD or
kCat/Km that is essentially equivalent when compared to an analogue having at
said
position the native residue, wherein said position corresponds to (amino acid
position
as given in SEQ ID NO:1 or SEQ ID NO:3):
bg) 408Glu;
bh) 41Glu;
bi) 413Tyr;
bj) 414lie;
bk) 415Leu;
bl) 416Asp;
bm) 417Asp;
bn) 4201le;
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ca) 423Asp;
cb) 4241le;
cc) 425Asp;
cd) 426GIu;
ce) 428Glu;
cf) 429Asp;
cg) 432Phe;
ch) 434Ser;
ci) 436Val;
cj) 438His;
ck) 439Asp;
cl) 4`'OLeu;
cm) 443Thr;
cn) "Phe;
Co) 445GIu;
cp) 456Arg;
cq) 4-9811e; or
cr) 'Asp.
Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions.
The following positions describe a subgrouping of those modifications which
resulted in
at least a 75% reduction in cofactor activity yet essentially little change in
kcat/Km
(amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3):
bg) 4o8Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp;
bm) 417 Asp;
ca) 423Asp;
cb) 4241le;
cc) a25Asp;
cd) 426GIu;
cf) 429Asp;
ck) 439Asp;
cn) 4"Phe; or
cr) 46'Asp.
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Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only
one of
the above listed substitutions. A further subgrouping can be made of the above
modifications wherein the KD for thrombin is modified by at least 30%.
This invention further provides for methods. More specifically there is
described herein
a method useful for screening for analogues of thrombomodulin which exhibit a
modified Kd for thrombin binding, comprising the steps of:
a) making an amino acid substitution at a position (amino acid
position as given in SEQ ID NO:1 or SEQ ID NO:3):
bg) 408Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 41'Asp;
bm) 417Asp;
bn) 4201le;
ca) 423Asp;
cb) 4241le;
cc) 425Asp;
cd) 426GIu;
ce) 428GIu;
cf) 429Asp;
cg) 432Phe;
ch) 434Ser;
ci) 436Val;
cj) 438His;
ck) 439Asp;
cl) 44OLeu;
cm) 443Thr;
cn) 444Phe;
CO) 445Glu;
cp) 456Arg;
cq) 45611e;
cr) 461Asp; and
b) comparing the KD for thrombin to a control molecule.
As used within these methods TM analogues, e.g. Solulin or Solulin fragments,
with
only one amino acid substitutions are preferred. Various embodiments of this
invention
include those wherein said KD is modified by at least 33%, or where said
modification is
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an amino acid substitution, or wherein said control molecule is TMEM388L. A
preferred
grouping of modifications for use in the method are (amino acid position as
given in
SEQ ID NO:1 or SEQ ID NO:3):
bg) 408Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp;
bm) 417 Asp;
ca) 423Asp;
cb) 4241le;
cc) 425Asp;
cd) 426Glu;
cf) 429Asp;
ck) 439Asp;
cn) 444Phe; or
cr) 46'Asp.
As used within these methods TM analogues, e.g. Solulin or Solulin fragments,
with
only one amino acid substitutions are preferred.
Ananother method is described herein which is useful for screening for
analogues of
thrombomodulin which possess a modified cofactor activity upon binding to
thrombin,
comprising the steps of:
a) making an amino acid substitution at a position (amino acid
position as given in SEQ ID NO:1 or SEQ ID NO:3.):
bb) 355Asn;
ae) 359GIn; and
b) comparing the rate of cofactor activity upon binding to thrombin
with the rate of a control molecule.
As used within these methods TM analogues, e.g. Solulin or Solulin fragments,
with
only one amino acid substitutions are preferred.
In a preferred embodiment of the invention the thrombomodulin analogue has a
modification of the phenylalanine residue at position 376 (Phe376X; SEQ ID
NO:1 or
SEQ ID NO:3). This residue can be chemically or biochemically modified or
deleted by
methods that are well known for the person skilled in art. The phenylalanine
residue is
preferably substituted with an aliphatic amino acid, more preferably with
glycine,
alanine, valine, leucine, or isoleucine and most preferably substituted with
alanine. It
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was demonstrated that a substitution of Phe376 by alanine ("F376A")
substantially
decreased the cofactor activity of the thrombomodulin analogue while
preserving the
TAFI activation activity (see Figure 7). As a result the F376A-TM analogue has
an
increased ratio of TAFI activation activity versus cofactor activity. In one
embodiment of
the invention Solulin contains the Phe376X, in particular the F376A
substitution.
In a further embodiment of the invention the thrombomodulin analogue has a
modification of the glutamine residue at position 387 (SEQ ID NO:1 or SEQ ID
NO:3).
The glutamine residue is preferably substituted with the following amino
acids, ordered
in decreasing cofactor activity of the resulting mutant GIn387X-TM analogue
(see
Figure 8A): Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr,
Leu, Asn, Phe,
Asp, Cys. In one embodiment of the invention Solulin contains this GIn387X
substitution; in a further embodiment this substitution within Solulin
contains the
GIn387X substitution together with the above F376X substitution.
In another embodiment of the invention the thrombomodulin analogue has a
modification of the methionine residue at position 388 (SEQ ID NO:1 or SEQ ID
NO:3).
The methionine residue is preferably substituted with the following amino
acids,
ordered in decreasing cofactor activity of the resulting mutant Met388X-TM
analogue
(see Figure 8B): GIn, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp,
Asn, Lys, Gly,
Glu, Asp, Cys. In one embodiment Solulin contain this substitution together
with one or
both of the above Phe376X and GIn387X substitutions.
In a further embodiment of the invention the thrombomodulin analogue has a
modification of the phenylalanine residue at position 389 (SEQ ID NO:1 or SEQ
ID
NO:3). The phenylalanine residue is preferably substituted with the following
amino
acids, ordered in decreasing cofactor activity of the resulting mutant Phe389X-
TM
analogue (see Figure 8C): Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, Ile,
Asn, Ser, Arg,
Lys, Met, Tyr, Gly, Cys, Pro. In one embodiment Solulin can contain this
substitution,
which can be further combined with one or more, preferably all, of the above
Phe376X,
GIn387X or Met388X substitutions.
In another embodiment of the invention the interdomain loop of the TM, e.g.
Solulin,
consisting of the three amino acids Gln387, Met388 and Phe389 is partially or
completely
deleted or inserted by one or more amino acids, preferably by an alanine
residue (see
Figure 8D).
For the TM analogues with modifications at positions Phe376, Gln387, Met388 or
Phe389,
the TM analogue can be a full length or a soluble TM analogue, comprising the
EGF
domains EGF1 to EGF6, preferably comprising the EGF domains EGF3 to EGF6. In a
preferred embodiment these analogues contain the substitutions that are given
in the
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TM analogue Solulin. In a more preferred embodiment these Solulin-derived TM
analogues consist only of EGF1 to EGF6, in particular of the EGF domains EGF3
to
EGF6 or from the c-loop of EGF3 to EGF6 (these three fragments are denominated
as
Solulin fragments).
In an embodiment of the invention the thrombomodulin analogue is used in its
oxidised
form. Several techniques are known to the skilled person for a controlled
oxidation of
proteins. The TM analogue is preferably oxidised using chloramine T, hydrogen
peroxide or sodium periodate.
The invention further pertains to a method that is useful for screening TM
analogues to
be used for the treatment of coagulopathy with hyperfibrinolysis. This method
comprises a first step of modifying the amino acid sequence of thrombomodulin
by
insertion, deletion or substitution of one or more amino acids, preferably in
the EGF
domains EGF1 to EGF6, more preferably in the EGF domains EGF3 to EGF6, and
most preferably between the amino acid positions Asp319 and Asp461. For the
person
skilled in the art several techniques are known to modify protein sequences
e.g. by
site-directed mutagenesis or random mutagenesis with subsequent selection.
In a second step the modified TM analogue is compared with a control protein
for one
or more of the following characteristics selected from the group consisting
of: binding
affinity to thrombin (KD value), cofactor activity, TAFI activation activity
or TAFIa
potential, ratio of TAFI activation activity and cofactor activity, effect of
protein
oxidation, effect on clot lysis in time in an in vitro assay, or the effect in
a coagulation-
associated animal model.
As a control protein, a thrombomodulin protein or analogue is used, preferably
a rabbit
lung thrombomodulin or a human TM analogue comprising the six EGF domains. The
TM analogue can have the native amino acid sequence or alternatively can
possess
one or more modifications such as the M388L substitution.
The invention further relates to a method of treating coagulopathy with
hyperfibrinolysis, comprising the administration of a therapeutically
effective amount of
a thrombomodulin analogue exhibiting an antifibrinolytic effect.
Particularly these methods of treatment comprise TM analogues exhibiting one
or
more of the following features in comparison with a control protein: a
decreased
binding affinity towards thrombin, a binding affinity towards thrombin with a
ko value of
more than 0.2 nM, a significantly reduced cofactor activity, or an increased
ratio of
TAFI activation activity to cofactor activity. As a control protein, a
thrombomodulin
protein or analogue is used, preferably a rabbit lung thrombomodulin or a
human TM
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analogue comprising the six EGF domains. The TM analogue can have the native
amino acid sequence or alternatively can possess one or more modifications
such as
the M388L substitution.
In a further embodiment the invention relates to a thrombomodulin analogue
with
reduced cofactor activity upon binding to thrombin as compared to TMEM338L
with
a.) an amino acid sequence according to SEQ ID NO:2 or
b) an amino acid sequence according to SEQ ID NO:4 or
c) with an amino acid sequence which has at least 90%, more preferred at least
95%,
most preferred at least 98% identity to the amino acid sequences according to
SEQ
ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
d) a thrombomodulin fragment consisting essentially of the 6 EGF-like repeat
domains of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (amino acid position 227 to
462 as numbered in SEQ ID NO:1), the EGF-like repeat domain 3 to the EGF-like
repeat domain 6 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (amino acid
position
307 to 462 as numbered in SEQ ID NO:1) or from the c-loop of the EGF-like
repeat
domain 3 to the EGF-like repeat domain 6 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID
NO:4 (amino acid position 333 to 462 as numbered in SEQ ID NO:1),
whereas the phenylalanine in position 376 (as numbered according to SEQ ID
NO:1)
is deleted or substituted by glycine, alanine, leucine, isoleucine or valine.
In yet another embodiment of the invention the thrombomodulin further
comprises a
deletion or substitution of the glutamine residue at position 387 (as numbered
in SEQ
ID NO:1), whereas the substitution preferably is substituted with Met, Thr,
Ala, Glu,
His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys.
In a further embodiment the invention relates to a thrombomodulin which
further
comprises a deletion or substitution of the methionine residue at position 388
(as
numbered in SEQ ID NO:1), whereas the methionine residue preferably is
substituted
with Gin, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys,
Gly, Glu, Asp,
Cys.
In a further embodiment the thrombomodulin further comprises a deletion or
substitution of the phenylalanine residue at position 389 (as numbered in SEQ
ID
NO:1), whereas the phenylalanine preferably is substituted with Val, Glu, Thr,
Ala,
His, Trp, Asp, Gin, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro.
In another embodiment the thrombomodulin comprises a combination of a first
and a
second amino acid modification as depicted in table 4.
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In yet another embodiment the thrombomodulin comprises a combination of a
first, a
second and a third amino acid modification as depicted in table 5.
The invention relates further to a thrombomodulin analogue and its medical use
for the
treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and terminating at P490,
whereby this thrombomodulin analogue further comprises a sequence modification
of
one or more of the subsequent amino acid positions (the numbering is related
to the
amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1):
a) 359GIn;
b) 398Asp;
c) 40OAsp;
d) 402Asn;
e) 408GIu;
f) 413Tyr;
g) 4141le;
h) 415Leu,
i) 417Asp;
j) 439Asn.
In a further preferred aspect of the invention the amino acids as listed from
a) to j) are
substituted with alanine.
The invention relates further to a thrombomodulin analogue and its medical use
for the
treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) S474A, and terminating at P490,
whereby this thrombomodulin analogue further comprises a sequence modification
of
one or more of the subsequent amino acid positions (the numbering is related
to the
amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1):
a) -188Met, with a modification other than a change into Leu
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b) 416Asp;
c) a23Asp;
d) a25Asp;
e) 426Glu;
f) 429Asp;
g) 4`10Leu;
h) 4G1Asp.
In one aspect of the invention the amino acids as listed from a) to h) are
substituted
with alanine.
The invention relates further to a thrombomodulin analogue and its medical use
for the
treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) S474A, and terminating at P490,
whereby this thrombomodulin analogue comprises an oxidation of the 388 Met
residue
(numbering is related to the amino acid sequence of mature thrombomodulin
depicted
in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue and its medical
use for
the treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and terminating at P490,
whereby this analogue comprises a mutation of the amino acid 387GIn, which is
substituted by an amino acid selected from the group consisting of Lys, Gly,
Ile, Trp,
Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering is
related to the
amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue and its medical
use for
the treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO: 1) and comprises one or more of the subsequent modifications:
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a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) S474A, and terminating at P490,
whereby this analogue comprises a mutation of the amino acid 388 Met, which is
substituted by an amino acid selected from the group consisting of Ile, Phe,
His, Arg,
Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which is
deleted
(numbering is related to the amino acid sequence of mature thrombomodulin
depicted
in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue and its medical
use for
the treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and terminating at P490,
whereby this analogue comprises a mutation of the amino acid 389Phe, which is
substituted by an amino acid selected from the group consisting of Ser, Arg,
Lys, Met,
Tyr, Gly, Cys or Pro, or which is deleted (numbering is related to the amino
acid
sequence of mature thrombomodulin depicted in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue and its medical
use for
the treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and terminating at P490,
whereby this analogue comprises an insertion of a hydrophobic amino acid,
preferably
Ala between one of the following pairs of amino acids: 386 Cys and 387 Gin,
387 Gin and
388Leu, 388Leu and 389Phe, 389Phe and 390Cys (numbering is related to the
amino acid
sequence of mature thrombomodulin depicted in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue and its medical
use for
the treatment of coagulopathy with hyperfibrinolysis that has an amino acid
sequence
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corresponding to the amino acid sequence of mature thrombomodulin (depicted in
SEQ
ID NO:1) and comprises one or more of the subsequent sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and terminating at P490,
whereby this analogue comprises a mutation of the amino acid 376Phe, which
preferably
is a Phe376A1a mutation (numbering is related to the amino acid sequence of
mature
thrombomodulin depicted in SEQ ID No: 1).
In a more preferred aspect of the invention relates to Solulin as depicted in
SEQ ID
NO:2, which comprises sequence modifications of one or more, preferably
exactly
one, of the subsequent amino acid positions (the numbering is related to the
amino
acid sequence of mature thrombomodulin depicted in SEQ ID No: 1):
a) 369GIn;
b) 398Asp;
c) 400Asp;
d) 402Asn;
e) 408GIu;
f) 4' 3Tyr;
g) 41411e;
h) 4'5Leu,
i) 417Asp;
j) 43Asn.
The above amino acids as listed from a) to j) are preferably substituted by
alanine.
In one embodiment Solulin as depicted in SEQ ID NO:2 comprises modifications
in one
or more, preferably one, of the subsequent amino acid positions (the numbering
is
related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1):
a) 388 Met, with a modification other than a change into Leu
b) 416Asp;
c) 423Asp;
d) 425Asp;
e) 426GIu;
f) 429Asp;
g) 440Leu;
h) 46'Asp.
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On one aspect of the invention the amino acids as listed from a) to h) are
substituted
by alanine.
The Solulin as used in the invention can comprise a mutation of the amino acid
387 Gin,
which is substituted by an amino acid selected from the group consisting of
Lys, Gly,
Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering
is related
to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1).
In a further aspect of the invention Solulin comprises a mutation of the amino
acid
388Met, which is substituted by an amino acid selected from the group
consisting of Ile,
Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys,
or which is
deleted (numbering is related to the amino acid sequence of mature
thrombomodulin
depicted in SEQ ID No: 1).
Furthermore, the invention relates to a Solulinwhich comprises a mutation of
the amino
acid 389Phe, which can be substituted by an amino acid selected from the group
consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted
(numbering is
related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1).
In addition, in one embodiment of the invention Solulin comprises an insertion
of a
hydrophobic amino acid, preferably Ala between one of the following pairs of
amino
acids: 386Cys and 387 Gin, 387 Gin and 388Leu, 388Leu and 389Phe, 389Phe and
390Cys
(numbering is related to the amino acid sequence of mature thrombomodulin
depicted
in SEQ ID No: 1)..
Solulin can be modified in order to comprise an oxidised 388 Met residue
(numbering is
related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1). Furthermore, it can comprise a mutation of the amino acid 376Phe, which
preferably
is a Phe376AIa mutation (numbering is related to the amino acid sequence of
mature
thrombomodulin depicted in SEQ ID No: 1).
In a preferred aspect of the invention relates to a thrombomodulin analogue
that
essentially consists of the six EGF domains as given by residues 227 to 462 of
SEQ ID
NO:1, or residues 224 to 459 of SEQ ID:NO 2, which can comprise the sequence
modifications of one or more, preferably one, of the subsequent amino acid
positions
(the numbering is related to the amino acid sequence of mature thrombomodulin
depicted in SEQ ID No: 1):
a) 359GIn;
b) 398Asp;
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c) 400Asp;
d) 402Asn;
e) 408Glu;
f) 41 3Tyr;
g) 4141le;
h) 4'5Leu,
i) 417 Asp;
j) 439Asn.
In one aspect of the invention the amino acids as listed from a) to j) are
substituted by
alanine.
The TM analogue of the invention can also essentially consist of the six EGF
domains
as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ
ID:NO
2, whereas each of these fragments can comprise the sequence modifications in
one
or more, preferably one, of the subsequent amino acid positions (the numbering
is
related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1):
a) 388 Met, with a modification other than a change into Leu
b) 416Asp;
c) 42sAsp;
d) 42sAsp;
e) 426Glu;
f) 429Asp;
g) 44 Leu;
h) 4s'Asp.
The amino acids as listed from a) to h) are preferably substituted by alanine.
The invention further relates to TM analogues that essentially consists of the
six EGF
domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459
of
SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the
amino
acid 387Gln, which can be substituted by an amino acid selected from the group
consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or
which is deleted
(numbering is related to the amino acid sequence of mature thrombomodulin
depicted
in SEQ ID No: 1).
The invention also relates to TM analogues that essentially consist of the six
EGF
domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459
of
SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the
amino
acid 388Met, which can be substituted by an amino acid selected from the group
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consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly,
Glu, Asp or
Cys, or which is deleted (numbering is related to the amino acid sequence of
mature
thrombomodulin depicted in SEQ ID No: 1).
Furthermore, the invention relates to TM analogues that essentially consist of
the six
EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to
459
of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the
amino acid 389Phe, which can be substituted by an amino acid selected from the
group
consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted
(numbering is
related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1).
In addition, the invention relates to TM analogues that essentially consist of
the six
EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to
459
of SEQ ID:NO 2, whereas each of these fragments can comprise an insertion of a
hydrophobic amino acid, preferably Ala between one of the following pairs of
amino
acids: 386Cys and 387Gln, 387GIn and 388 Met, 388 Met and 369Phe, 369Phe and
390Cys
(numbering is related to the amino acid sequence of mature thrombomodulin
depicted
in SEQ ID No: 1).
Furthermore the invention relates to TM analogues that essentially consist of
the six
EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to
459
of SEQ ID:NO 2, whereas each of these fragments can comprise an oxidised 388
Met
residue (numbering is related to the amino acid sequence of mature
thrombomodulin
depicted in SEQ ID No: 1).
Furthermore the invention relates to TM analogues that essentially consist of
the six
EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to
459
of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the
amino acid 376Phe, which preferably is a Phe376Ala mutation (numbering is
related to
the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1).
In one embodiment of the invention a TM analogue is claimed that essentially
consists
of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or
residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can
comprise
modifications of one or more, preferably one, of the subsequent amino acid
positions
(the numbering is related to the amino acid sequence of mature thrombomodulin
depicted in SEQ ID No: 1):
a) 359GIn;
b) 398Asp;
c) 400Asp;
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d) 402Asn;
e) 408GIu;
f) 4' 3Tyr;
g) 4141 le;
h)415 Leu,
i) 417 Asp;
j) 439Asn.
The amino acids as listed from a) to j) can be substituted by alanine.
In one embodiment of the invention a TM analogue is claimed that essentially
consists
of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or
residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can
comprise
modifications of one or more, preferably one, of the subsequent amino acid
positions
(the numbering is related to the amino acid sequence of mature thrombomodulin
depicted in SEQ ID No: 1):
a) 388 Met, with a modification other than a change into Leu
b) 416 Asp;
c) 423Asp;
d) 425Asp;
e) 426Glu;
f) 429Asp;
g) 44 Leu;
h) 48'Asp.
The amino acids as listed from a) to h) can be substituted by alanine.
The invention further relates to a thrombomodulin analogue that essentially
consists of
the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or
residues
308 to 459 of SEQ ID:NO 2, whereas each of those fragments can comprise a
mutation
of the amino acid 387GIn, which can be substituted by an amino acid selected
from the
group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro,
or which is
deleted (numbering is related to the amino acid sequence of mature
thrombomodulin
depicted in SEQ ID No: 1).
The invention also relates to a thrombomodulin analogue that essentially
consists of
the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or
residues
308 to 459 of SEQ ID:NO 2, whereas each of the fragments can comprise a
mutation
of the amino acid 388Met, which can be substituted by an amino acid selected
from the
group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn,
Lys, Gly, Glu,
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Asp or Cys, or which is deleted (numbering is related to the amino acid
sequence of
mature thrombomodulin depicted in SEQ ID No: 1).
Furthermore, the invention relates to a thrombomodulin analogue that
essentially
consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID
NO:1,
or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can
comprise a mutation of the amino acid 389Phe, which can be substituted by an
amino
acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys
or Pro, or
which is deleted (numbering is related to the amino acid sequence of mature
thrombomodulin depicted in SEQ ID No: 1).
In addition, the invention relates to a thrombomodulin analogue that
essentially
consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID
NO:1,
or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can
comprise an insertion of a hydrophobic amino acid, preferably Ala between one
of the
following pairs of amino acids: 386Cys and 387Gln, 387Gln and 388Met, 388Met
and 389Phe,
389Phe and 390Cys (numbering is related to the amino acid sequence of mature
thrombomodulin depicted in SEQ ID No: 1).
In a further aspect of the invention a thrombomodulin analogue is claimed that
essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462
of SEQ
ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of the fragments
can
comprise an oxidised 388 Met residue (numbering is related to the amino acid
sequence
of mature thrombomodulin depicted in SEQ ID No: 1).
In a further aspect of the invention relates to a thrombomodulin analogue that
essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462
of SEQ
ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these
fragments
can comprise a mutation of the amino acid 376Phe, which preferably is a
Phe376Ala
mutation (numbering is related to the amino acid sequence of mature
thrombomodulin
depicted in SEQ ID No: 1).
In a preferred aspect of the invention a thrombomodulin analogue is claimed
that
essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as
given by
residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2,
whereas
each of these fragments comprise modifications of one or more, preferably one,
of the
subsequent amino acid positions (the numbering is related to the amino acid
sequence
of mature thrombomodulin depicted in SEQ ID No: 1):
a) 359GIn;
b) 398Asp;
c) 400Asp;
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d) 402Asn;
e) 408Glu;
f) 4' 3Tyr;
g) 4141 le;
h) 4'5Leu,
i) 417 Asp;
j) 439Asn.
The amino acids as listed from a) to j) can be substituted by alanine.
In one aspect of the invention a thrombomodulin analogue is claimed that
essentially
consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by
residues
333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each
of
these fragments comprise modifications of one or more, preferably one, of the
subsequent amino acid positions (the numbering is related to the amino acid
sequence
of mature thrombomodulin depicted in SEQ ID No: 1):
a) 388 Met, with a modification other than a change into Leu
b) 416 Asp;
c) 423Asp;
d) 425Asp;
e) 426Glu;
f) 429Asp;
g) 44 Leu;
h) 46'Asp.
The amino acids as listed from a) to h) are preferably substituted by alanine.
The invention further relates to a thrombomodulin analogue that essentially
consists of
the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to
462
of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these
fragments can comprise a mutation of the amino acid 387G1n, which can be
substituted
by an amino acid selected from the group consisting of Lys, Gly, Ile, Trp,
Tyr, Leu, Asn,
Phe, Asp, Cys, or Pro, or which can be deleted (numbering is related to the
amino acid
sequence of mature thrombomodulin depicted in SEQ ID No: 1).
The invention also relates to a thrombomodulin analogue that essentially
consists of
the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to
462
of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these
fragments can comprise a mutation of the amino acid 388 Met, which can be
substituted
by an amino acid selected from the group consisting of Ile, Phe, His, Arg,
Pro, Val, Thr,
Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which can be deleted
(numbering is
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related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID
No:
1).
Furthermore, the invention relates to a thrombomodulin analogue that
essentially
consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by
residues
333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each
of
these fragments can comprise a.mutation of the amino acid 389Phe, which can be
substituted by an amino acid selected from the group consisting of Ser, Arg,
Lys, Met,
Tyr, Gly, Cys or Pro, or which can be deleted (numbering is related to the
amino acid
sequence of mature thrombomodulin depicted in SEQ ID No: 1).
In addition the invention relates to a thrombomodulin analogue that
essentially consists
of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333
to
462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereby this
thrombomodulin analogue comprises an insertion of a hydrophobic amino acid,
preferably Ala between one of the following pairs of amino acids: 386CyS and
387Gln,
387GIn and 388Met, 368Met and 369Phe, 389Phe and 390Cys (numbering is related
to the
amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1).
In a preferred aspect of the invention a thrombomodulin analogue is claimed
that
essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as
given by
residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2,
whereas
each of these fragments can comprise an oxidized 388 Met residue (numbering is
related
to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1).
The invention relates preferably to a thrombomodulin analogue that essentially
consists
of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333
to
462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of
these
fragments can comprise a mutation of the amino acid 376Phe, which preferably
is a
Phe376AIa mutation (numbering is related to the amino acid sequence of mature
thrombomodulin depicted in SEQ ID No: 1).
According to the invention the TM analogues according to SEQ ID NO:2 can
contain
two sequence modifications (a "first amino acid modification" and a "second
amino acid
modification"), which are depicted in table 4.
In yet another embodiment of the invention the TM analogues according to SEQ
ID NO
:4 can contain two sequence modifications (a "first amino acid modification"
and a
"second amino acid modification"), which are depicted in table 4.
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In yet another embodiment of the invention the TM analogues according to SEQ
ID NO
3 can contain two sequence modifications (a "first* amino acid modification"
and a
"second amino acid modification"), which are depicted in table 4.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 2 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
2
(amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2
(amino
acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
4.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 3 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
3
(amino acid position 307 to 462 as numbered in SEQ ID NO:1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3
(amino
acid position 333 to 462 as numbered in SEQ ID NO:1) whereas each.of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
4.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 4 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
4
(amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4
(amino
acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
4.
No. First amino acid modification Second amino acid modification
1 Phe 376AIa GIn387L s
2 Phe 376AIa GIn387GIy
3 Phe 376AIa Gln387IIe
4 Phe 376AIa Gln387Trp
Phe 376AIa Gln387Tyr
6 Phe 376AIa Gln387Leu
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7 Phe 376AIa GIn387Asn
8 Phe 376AIa Gln387Phe
9 Phe 376AIa Gln387Asp
Phe 376AIa Gln387Cys
11 Phe 376AIa Gln387Pro
12 Phe 376AIa Met38811e
13 Phe 376AIa Met388Phe
14 Phe 376AIa Met388His
Phe 376AIa Met388Arg
16 Phe 376AIa Met388Pro
17 Phe 376AIa Met388Val
18 Phe 376AIa Met388Thr
19 Phe 376AIa Met388Ser
Phe 376AIa Met388AIa
21 Phe 376AIa Met388Trp
22 Phe 376AIa Met388Asn
23 Phe 376AIa Met388Lys
24 Phe 376AIa Met388G1
Phe 376AIa Met388GIu
26 Phe 376AIa Met388Asp
27 Phe 376AIa Met388Cys
28 Phe 376AIa Phe389Ser
29 Phe 376AIa Phe389Arg
Phe 376AIa Phe389Lys
31 Phe 376AIa Phe389Met
32 Phe 376AIa Phe389Tyr
33 Phe 376AIa Phe389G1
34 Phe 376AIa Phe389Cys
Phe 376AIa Phe389Pro
36 Phe 376AIa Deletion GIn387
37 Phe 376AIa Deletion Met388
38 Phe 376AIa Deletion Phe389
39 Phe 376AIa Ala insert 386/387
Phe 376AIa Ala insert 387/388
41 Phe 376AIa Ala insert 388/389
42 Phe 376AIa Ala insert 389/390
43 Phe 376Val Gln387Lys
44 Phe 376Val Gln387G1
Phe 376Val Gln38711e
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46 Phe 376Val Gln387Trp
47 Phe 376Val Gln387Tyr
48 Phe 376Val Gln387Leu
49 Phe 376Val Gln387Asn
50 Phe 376Val Gln387Phe
51 Phe 376Val Gln387Asp
52 Phe 376Val Gln387Cys
53 Phe 376Val Gln387Pro
54 Phe 376Val Met38811e
55 Phe 376Val Met388Phe
56 Phe 376Val Met388His
57 Phe 376Val Met388Arg
58 Phe 376Val Met388Pro
59 Phe 376Val Met388Val
60 Phe 376Val Met388Thr
61 Phe 376Val Met388Ser
62 Phe 376Val Met388A1a
63 Phe 376Val Met388Trp
64 Phe 376Val Met388Asn
65 Phe 376Val Met388Lys
66 Phe 376Val Met388G1
67 Phe 376Val Met388G1u
68 Phe 376Val Met388Asp
69 Phe 376Val Met388Cys
70 Phe 376Val Phe389Ser
71 Phe 376Val Phe389Arg
72 Phe 376Val Phe389Lys
73 Phe 376Val Phe389Met
74 Phe 376Val Phe389Tyr
75 Phe 376Val Phe389GIy
76 Phe 376Val Phe389Cys
77 Phe 376Val Phe389Pro
78 Phe 376Val Deletion Gln387
79 Phe 376Val Deletion Met388
80 Phe 376Val Deletion Phe389
81 Phe 376Val Ala insert 386/387
82 Phe 376Val Ala insert 387/388
83 Phe 376Val Ala insert 388/389
84 Phe 376Val Ala insert 389/390
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85 Phe 376 Ile GIn387L s
86 Phe 376 Ile Gln387GIy
87 Phe 376 Ile GIn3871Ie
88 Phe 376 Ile GIn387Trp
89 Phe 376 Ile GIn387T r
90 Phe 376 Ile GIn387Leu
91 Phe 376 Ile GIn387Asn
92 Phe 376 Ile GIn387Phe
93 Phe 376 Ile GIn387Asp
94 Phe 376 Ile GIn387Cys
95 Phe 376 Ile GIn387Pro
96 Phe 376 Ile Met38811e
97 Phe 376 Ile Met388Phe
98 Phe 376 Ile Met388His
99 Phe 376 Ile Met388Arg
100 Phe 376 Ile Met388Pro
101 Phe 376 Ile Met388VaI
102 Phe 376 Ile Met388Thr
103 Phe 376 Ile Met388Ser
104 Phe 376 Ile Met388AIa
105 Phe 376 Ile Met388Trp
106 Phe 376 Ile Met388Asn
107 Phe 376 Ile Met388Lys
108 Phe 376 Ile Met388G1
109 Phe 376 Ile Met388GIu
110 Phe 376 Ile Met388Asp
111 Phe 376 Ile Met388Cys
112 Phe 376 Ile Phe389Ser
113 Phe 376 Ile Phe389Arg
114 Phe 376 Ile Phe389Lys
115 Phe 376 Ile Phe389Met
116 Phe 376 Ile Phe389Tyr
117 Phe 376 Ile Phe389GIy
118 Phe 376 Ile Phe389Cys
119 Phe 376 Ile Phe389Pro
120 Phe 376 Ile Deletion GIn387
121 Phe 376 Ile Deletion Met388
122 Phe 376 Ile Deletion Phe389
123 Phe 376 Ile Ala insert 386/387
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124 Phe 376 Ile Ala insert 387/388
125 Phe 376 Ile Ala insert 388/389
126 Phe 376 Ile Ala insert 389/390
127 Phe 376 Leu Gln387Lys
128 Phe 376 Leu Gln387G1
129 Phe 376 Leu GIn387lle
130 Phe 376 Leu Gln387Trp
131 Phe 376 Leu GIn387T r
132 Phe 376 Leu Gln387Leu
133 Phe 376 Leu Gln387Asn
134 Phe 376 Leu GIn387Phe
135 Phe 376 Leu GIn387Asp
136 Phe 376 Leu Gln387Cys
137 Phe 376 Leu GIn387Pro
138 Phe 376 Leu Met3881le
139 Phe 376 Leu Met388Phe
140 Phe 376 Leu Met388His
141 Phe 376 Leu Met388Arg
142 Phe 376 Leu Met388Pro
143 Phe 376 Leu Met388Val
144 Phe 376 Leu Met388Thr
145 Phe 376 Leu Met388Ser
146 Phe 376 Leu Met388A1a
147 Phe 376 Leu Met388Trp
148 Phe 376 Leu Met388Asn
149 Phe 376 Leu Met388Lys
150 Phe 376 Leu Met388GIy
151 Phe 376 Leu Met388GIu
152 Phe 376 Leu Met388Asp
153 Phe 376 Leu Met388Cys
154 Phe 376 Leu Phe389Ser
155 Phe 376 Leu Phe389Arg
156 Phe 376 Leu Phe389Lys
157 Phe 376 Leu Phe389Met
158 Phe 376 Leu Phe389Tyr
159 Phe 376 Leu Phe389GIy
160 Phe 376 Leu Phe389Cys
161 Phe 376 Leu Phe389Pro
162 Phe 376 Leu Deletion Gln387
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163 Phe 376 Leu Deletion Met388
164 Phe 376 Leu Deletion Phe389
165 Phe 376 Leu Ala insert 386/387
166 Phe 376 Leu Ala insert 387/388
167 Phe 376 Leu Ala insert 388/389
168 Phe 376 Leu Ala insert 389/390
169 Phe 376AIa As 416Ala
170 Phe 376AIa As 423Ala
171 Phe 376AIa As 425 Ala
172 Phe 376AIa Glu426Ala
173 Phe 376AIa As 429Ala
174 Phe 376AIa Leu440Ala
175 Phe 376AIa As 461 Ala
176 Phe 376AIa Gln359Ala
177 Phe 376AIa As 398AIa
178 Phe 376AIa Asp400Ala
179 Phe 376AIa Asn402AIa
180 Phe 376AIa GIu408Ala
181 Phe 376AIa T r413Ala
182 Phe 376AIa IIe414Ala
183 Phe 376AIa Leu415AIa
184 Phe 376AIa As 417Ala
185 Phe 376AIa Asn439Ala
Table 4: First and second amino acid modifications
According to the invention the TM analogues according to SEQ ID NO 2 can each
contain the modifications (a "first, second and third amino acid
modification") as
depicted in table 5. Table 5 depicts an alanine substitution for Phe376
("First amino
acid modification"). In certain embodiments of the invention the "first amino
acid
modification" is constituted either by glycine, valine, leucine or isoleucine.
These
modifications are each combined with the "second amino acid modification" and
"third
amino acid modification" as given in table 5. These further embodiments of the
invention are collectively summarised as "modifications as depicted in table
5.
In yet another embodiment of the invention the TM analogues according to SEQ
ID NO
4 can contain two sequence modifications (a "first amino acid modification"
and a
"second amino acid modification"), which are depicted in table 5.
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In yet another embodiment of the invention the TM analogues according to SEQ
ID NO
3 can contain two sequence modifications (a "first amino acid modification"
and a
"second amino acid modification"), which are depicted in table 5.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 2 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
2
(amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2
(amino
acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
5.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 3 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
3
(amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3
(amino
acid position 333 to 462 as numbered in SEQ ID NO1) whereas each of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
5.
In yet another thrombomodulin fragment consisting essentially of the 6 EGF-
like repeat
domains of SEQ ID NO 4 (amino acid position 227 to 462 as numbered in SEQ ID
NO
1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO
4
(amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop
of
the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4
(amino
acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF
domain containing fragments can contain two sequence modifications (a "first
amino
acid modification" and a "second amino acid modification"), which are depicted
in table
5.
No. 1st as modification 2nd as modification 3 rd as modification
1 Phe 376AIa As 416AIa GIn359AIa
2 Phe 376AIa As 416Ala Asp398AIa
3 Phe 376AIa As 416AIa As 400Ala
4 Phe 376AIa As 416AIa Asn402AIa
Phe 376AIa As 416AIa GIu408AIa
6 Phe 376AIa As 416AIa T r413AIa
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7 Phe 376AIa As 416Ala Ile414Ala
8 Phe 376AIa Asp4l6AIa Leu4l5AIa
9 Phe 376AIa Asp4l6AIa As 417Ala
Phe 376AIa Asp4l6AIa Asn439AIa
11 Phe 376AIa Asp4l6AIa GIn387C s
12 Phe 376AIa As 416Ala Gln387Pro
13 Phe 376AIa As 416AIa Oxidated Met388
14 Phe 376AIa As 416AIa Met388Asp
Phe 376AIa As 416AIa Met388Cys
16 Phe 376AIa As 416AIa Phe389Cys
17 Phe 376AIa As 416Ala Phe389Pro
18 Phe 376AIa Asp4l6AIa Deletion GIn387
19 Phe 376AIa Asp4l6Ala Deletion Met388
Phe 376AIa As 416AIa Deletion Phe389
21 Phe 376AIa As 416Ala Ala insert 386/387
22 Phe 376AIa As 416Ala Ala insert 387/388
23 Phe 376AIa Asp4l6AIa Ala insert 388/389
24 Phe 376AIa As 416Ala Ala insert 389/390
Phe 376AIa As 423AIa Gln359Ala
26 Phe 376AIa As 423AIa Asp398AIa
27 Phe 376AIa Asp423AIa Asp400Ala
28 Phe 376AIa As 423AIa Asn402AIa
29 Phe 376AIa As 423AIa Glu408AIa
Phe 376AIa As 423AIa T r413AIa
31 Phe 376AIa As 423AIa Ile414AIa
32 Phe 376AIa As 423AIa Leu415Ala
33 Phe 376AIa As 423AIa As 417Ala
34 Phe 376AIa As 423AIa Asn439AIa
Phe 376AIa As 423AIa Gln387Cys
36 Phe 376AIa As 423AIa Gln387Pro
37 Phe 376AIa As 423AIa Oxidated Met388
38 Phe 376AIa As 423AIa Met388Asp
39 Phe 376AIa As 423AIa Met388Cys
Phe 376AIa As 423AIa Phe389Cys
41 Phe 376AIa As 423AIa Phe389Pro
42 Phe 376AIa As 423AIa Deletion GIn387
43 Phe 376AIa As 423AIa Deletion Met388
44 Phe 376AIa As 423AIa Deletion Phe389
Phe 376AIa As 423AIa Ala insert 386/387
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46 Phe 376AIa As 423AIa Ala insert 387/388
47 Phe 376AIa As 423AIa Ala insert 388/389
48 Phe 376AIa As 423AIa Ala insert 389/390
49 Phe 376AIa As 425AIa Gln359Ala
50 Phe 376AIa Asp425AIa As 398Ala
51 Phe 376AIa As 425AIa Asp400Ala
52 Phe 376AIa As 425AIa Asn402AIa
53 Phe 376AIa As 425AIa Glu408AIa
54 Phe 376AIa As 425AIa T r413Ala
55 Phe 376AIa As 425AIa Ile414Ala
56 Phe 376AIa As 425AIa Leu415Ala
57 Phe 376AIa As 425AIa As 417Ala
58 Phe 376AIa As 425AIa Asn439Ala
59 Phe 376AIa As 425AIa Gln387Cys
60 Phe 376AIa Asp425AIa Gln387Pro
61 Phe 376AIa As 425AIa Oxidated Met388
62 Phe 376AIa As 425AIa Met388Asp
63 Phe 376AIa As 425AIa Met388Cys
64 Phe 376AIa As 425AIa Phe389Cys
65 Phe 376AIa As 425AIa Phe389Pro
66 Phe 376AIa As 425AIa Deletion Gln387
67 Phe 376AIa As 425AIa Deletion Met388
68 Phe 376AIa As 425AIa Deletion Phe389
69 Phe 376AIa As 425AIa Ala insert 386/387
70 Phe 376AIa As 425AIa Ala insert 387/388
71 Phe 376AIa As 425AIa Ala insert 388/389
72 Phe 376AIa As 425AIa Ala insert 389/390
73 Phe 376AIa GIu426AIa Gln359Ala
74 Phe 376AIa GIu426AIa As 398AIa
75 Phe 376AIa GIu426AIa Asp400Ala
76 Phe 376AIa GIu426AIa Asn402Ala
77 Phe 376AIa GIu426AIa Glu408AIa
78 Phe 376AIa GIu426AIa T r413Ala
79 Phe 376AIa GIu426AIa Ile414Ala
80 Phe 376AIa GIu426AIa Leu415Ala
81 Phe 376AIa GIu426AIa As 417Ala
82 Phe 376AIa GIu426AIa Asn439AIa
83 Phe 376AIa GIu426AIa Gln387Cys
84 Phe 376AIa GIu426AIa Gln387Pro
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85 Phe 376AIa GIu426Ala Oxidated Met388
86 Phe 376AIa GIu426Ala Met388Asp
87 Phe 376AIa GIu426Ala Met388Cys
88 Phe 376AIa GIu426Ala Phe389Cys
89 Phe 376AIa GIu426Ala Phe389Pro
90 Phe 376AIa GIu426Ala Deletion GIn387
91 Phe 376AIa GIu426Ala Deletion Met388
92 Phe 376AIa GIu426Ala Deletion Phe389
93 Phe 376AIa GIu426Ala Ala insert 386/387
94 Phe 376AIa GIu426Ala Ala insert 387/388
95 Phe 376AIa GIu426Ala Ala insert 388/389
96 Phe 376AIa GIu426Ala Ala insert 389/390
97 Phe 376AIa As 429AIa GIn359Ala
98 Phe 376AIa As 429AIa As 398Ala
99 Phe 376AIa As 429AIa As 400Ala
100 Phe 376AIa As 429AIa Asn402AIa
101 Phe 376AIa As 429AIa GIu408Ala
102 Phe 376AIa As 429AIa T r413AIa
103 Phe 376AIa As 429AIa IIe414Ala
104 Phe 376AIa As 429AIa Leu415Ala
105 Phe 376AIa As 429AIa As 417Ala
106 Phe 376AIa As 429AIa Asn439Ala
107 Phe 376AIa As 429AIa GIn387C s
108 Phe 376AIa As 429AIa GIn387Pro
109 Phe 376AIa As 429AIa Oxidated Met388
110 Phe 376AIa As 429AIa Met388Asp
111 Phe 376AIa As 429AIa Met388Cys
112 Phe 376AIa As 429AIa Phe389Cys
113 Phe 376AIa As 429AIa Phe389Pro
114 Phe 376AIa As 429AIa Deletion GIn387
115 Phe 376AIa As 429AIa Deletion Met388
116 Phe 376AIa As 429AIa Deletion Phe389
117 Phe 376AIa As 429AIa Ala insert 386/387
118 Phe 376AIa As 429AIa Ala insert 387/388
119 Phe 376AIa As 429AIa Ala insert 388/389
120 Phe 376AIa As 429AIa Ala insert 389/390
121 Phe 376AIa Leu440Ala GIn359Ala
122 Phe 376AIa Leu440Ala As 398Ala
123 Phe 376AIa Leu440Ala As 400Ala
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124 Phe 376AIa Leu440Ala Asn402AIa
125 Phe 376AIa Leu440Ala Glu408AIa
126 Phe 376AIa Leu440Ala T r413Ala
127 Phe 376AIa Leu440Ala Ile414Ala
128 Phe 376AIa Leu440Ala Leu4l5AIa
129 Phe 376AIa Leu440Ala As 417Ala
130 Phe 376AIa Leu440Ala Asn439Ala
131 Phe 376AIa Leu440Ala Gln387Cys
132 Phe 376AIa Leu440Ala Gln387Pro
133 Phe 376AIa Leu440Ala Oxidated Met388
134 Phe 376AIa Leu440Ala Met388Asp
135 Phe 376AIa Leu440Ala Met388Cys
136 Phe 376AIa Leu440Ala Phe389Cys
137 Phe 376AIa Leu440Ala Phe389Pro
138 Phe 376AIa Leu440Ala Deletion Gln387
139 Phe 376AIa Leu440Ala Deletion Met388
140 Phe 376AIa Leu440Ala Deletion Phe389
141 Phe 376AIa Leu440Ala Ala insert 386/387
142 Phe 376Ala Leu440Ala Ala insert 387/388
143 Phe 376AIa Leu440Ala Ala insert 388/389
144 Phe 376AIa Leu440Ala Ala insert 389/390
145 Phe 376AIa As 461Ala Gln359Ala
146 Phe 376AIa As 461 Ala Asp398AIa
147 Phe 376AIa As 461 Ala Asp400Ala
148 Phe 376AIa As 461 Ala Asn402AIa
149 Phe 376AIa As 461 Ala Glu408AIa
150 Phe 376AIa As 461 Ala T r413Ala
151 Phe 376AIa As 461 Ala l le414Ala
152 Phe 376AIa As 461 Ala Leu415Ala
153 Phe 376AIa As 461 Ala As 417AIa
154 Phe 376AIa As 461 Ala Asn439AIa
155 Phe 376AIa As 461Ala Gln387Cys
156 Phe 376AIa As 461 Ala Gln387Pro
157 Phe 376AIa As 461 Ala Oxidated Met388
158 Phe 376AIa As 461 Ala Met388Asp
159 Phe 376AIa As 461 Ala Met388Cys
160 Phe 376AIa As 461 Ala Phe389Cys
161 Phe 376AIa As 461Ala Phe389Pro
162 Phe 376AIa As 461 Ala Deletion GIn387
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163 Phe 376AIa As 461 Ala Deletion Met388
164 Phe 376AIa As 461 Ala Deletion Phe389
165 Phe 376AIa As 461 Ala Ala insert 386/387
166 Phe 376AIa As 461Ala Ala insert 387/388
167 Phe 376AIa As 461 Ala Ala insert 388/389
168 Phe 376AIa As 461 Ala Ala insert 389/390
Table 5: First, second and third modifications
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The following tables gives an overview on the reduced cofactor activities that
results
from specific amino acid modifications of thrombomodulin analogues.
as modification Cofactor activity Normalized Source
against
F376A - 5% M388L Wang 2000, Fig. 4B
-65% TAFI act.)
M388L -180% M388L W093125675, Fig. 2B
-90% TaFI act.) wild type
D398A -20% M388L W093125675, Fig. 2B
D400A -2% M388L W093125675, Fig. 2B
N402A -10% M388L W093125675, Fig. 2B
E408A -24% M388L W093/25675, Fig. 2B
Y413A -2% M388L W093/25675, Fig. 2B
1414A -5% M388L W093125675, Fig. 2B
L415A -5% M388L W093/25675, Fig. 2B
D416A -4% M388L W093125675, Fig. 2B
D417A -20% M388L W093/25675, Fig. 2B
D423A -12% M388L W093/25675, Fig. 2C
1424A -2% M388L W093/25675, Fig. 2C
E426A -2% M388L W093/25675, Fig. 2C
N429A -10% M388L W093/25675, Fig. 2C
N439A -2% M388L W093125675, Fig. 2C
L440A -25% M388L W093/25675, Fig. 2C
D461A -9% M388L W093125675, Fig. 2C
Gln387Lys -30% wild type Clarke 1993, Figure 2
Gln387G1 -30% wild type Clarke 1993, Figure 2
Gln38711e -30% wild type Clarke 1993, Figure 2
Gln387Trp -28% wild type Clarke 1993, Figure 2
Gln387Tyr -30% wild type Clarke 1993, Figure 2
Gln387Leu -25% wild type Clarke 1993, Figure 2
Gln387Asn -25% wild type Clarke 1993, Figure 2
GIn387Phe -20% wild type Clarke 1993, Figure 2
Gln387Asp -18% wild type Clarke 1993, Figure 2
Gln387Cys 2% wild type Clarke 1993, Figure 2
Gln387Pro 0% wild type Clarke 1993, Figure 2
Met38811e 48% wild type Clarke 1993, Figure 2
Met388Phe 40% wild type Clarke 1993, Figure 2
Met388His 40% wild type Clarke 1993, Figure 2
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Met388Ar 35% wild a Clarke 1993, Figure 2
Met388Pro 35% wild a Clarke 1993, Figure 2
Met388Val 30% wild a Clarke 1993, Figure 2
Met388Thr 22% wild type Clarke 1993, Figure 2
Met388Ser 22% wild type Clarke 1993, Figure 2
Met388Ala 22% wild type Clarke 1993, Figure 2
Met388Trp 20% wild type Clarke 1993, Figure 2
Met388Asn 20% wild type Clarke 1993, Figure 2
Met388Lys 15% wild type Clarke 1993, Figure 2
Met388GIy 15% wild type Clarke 1993, Figure 2
Met388Glu 15% wild type Clarke 1993, Figure 2
Met388Asp 8% wild type Clarke 1993, Figure 2
Met388Cys 5% wild type Clarke 1993, Figure 2
Phe389Ser 5% wild type Clarke 1993, Figure 2
Phe389Arg 45% wild tvpe Clarke 1993, Figure 2
Phe389Lys 45% wild type Clarke 1993, Figure 2
Phe389Met 45% wild type Clarke 1993, Figure 2
Phe389Tyr 45% wild type Clarke 1993, Figure 2
Phe389G1 25% wild type Clarke 1993, Figure 2
Phe389Cys 15% wild type Clarke 1993, Figure 2
Phe389Pro 8% wild type Clarke 1993, Figure 2
Deletion GIn387 2% wild t e Clarke 1993, Figure 3
Deletion Met388 4% wild type Clarke 1993, Figure 3
Deletion Phe389 5% wild type Clarke 1993, Figure 3
Ala insert 386/387 5% wild type Clarke 1993, Figure 3
Ala insert 387/388 3% wild type Clarke 1993, Figure 3
Ala insert 388/389 10% wild type Clarke 1993, Figure 3
Ala insert 389/390 5% wild type Clarke 1993, Figure 3
Table 6: First and second amino acid modifications
Therapeutic treatment according to the invention
In one aspect of the invention the thrombomodulin analogues as disclosed in
here are
used for treatment of coagulopathy with hyperfibrinolysis in patients, who
possess anti-
factor VIII antibodies. These antibodies can inhibit factor VIII activity. In
the typical
case, they arise as alloantibodies during replacement therapy of haemophilia A
patients. They can be responsible for the failure of FVIII replacement therapy
in
haemophilia A patients. Thus, according to one aspect of the invention the
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thrombomodulin analogues disclosed herein can be used as rescue medication for
haemophilia patients who are non-responders for factor VIII.
According to a further aspect of the invention, patients, who are deficient in
factor VIII,
are treated with a combination of factor VIII and the TM analogues of the
invention.
Factor VIII and the TM analogue can be administered either concomitantly or
sequentially. The patients are treated preferably with recombinant factor VIII
or a
recombinant B-domain-deleted factor VIII molecule, more preferably Octocog-
alfa or
moroctocog-alfa. In one embodiment isolated human factor VIII can be used,
e.g.
Aafact .
According to the invention also haemophilia patients can be treated who had
been
treated with factor VIII in the past or are currently under factor VIII
treatment.
Accordingly these patients have a pharmaceutically effective factor VIII
level.
In a further aspect of the invention, the claimed thrombomodulin analogues can
be
used to screen haemophilia patients for the presence of factor VIII antibodies
since the
presence of factor VIII antibodies causes characteristic changes in the
thromboelastogram (see Figure 11A vs. Figure 12A).
Embodiments of the invention
In one aspect of the invention the use of a thrombomodulin analogue for the
manufacture of a medicament for the treatment of coagulopathy with
hyperfibrinolysis
is claimed, whereas said TM analogue is characterized by exhibiting at
therapeutically
effective dosages an antifibrinolytic effect.
In a further aspect of the invention the use of a thrombomodulin analogue for
the
manufacture of a medicament for the treatment of coagulopathy with
hyperfibrinolysis
is claimed, whereas the thrombomodulin analogue exhibits one or more of the
following
features:
(i) a binding affinity towards thrombin that is decreased compared to
the rabbit lung thrombomodulin, and/or a binding affinity towards
thrombin with a kp value of more than 0.2 nM;
and/or
(ii) a reduced cofactor activity compared to cofactor activity of the TM
analogue TMEM388L,
(iii) an increased ratio of TAFI activation activity to cofactor activity as
compared to the TM analogue TMEM388L.
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In a further aspect of the invention the use of the above described
thrombomodulin
analogues is claimed, whereas the coagulopathy with hyperfibrinolysis is
selected from
the group of diseases as follows: haemophilia A, haemophilia B, haemophilia C,
von
Willebrandt disease (vWD), acquired von Willebrandt disease, Factor X
deficiency,
parahemophilia, hereditary disorders of the clotting factors I, II, V, or VII,
haemorrhagic
disorder due to circulating anticoagulants or acquired coagulation deficiency.
In a further aspect of the invention said thrombomodulin analogues can be used
to
treat one or more of the bleeding events selected from the group consisting
of:
intracranial or other CNS haemorrhage, bleeding in joints, microcapillaries,
muscles,
the gastrointestinal tract, the respiratory tract, the retroperitoneal space
or soft tissues
The intracranial bleeding event treated with the thrombomodulin analogue of
the
invention can be an intra-axial, an extra-axial or a subarachnoid haemorrhage
(SAH) or
an epidural or subdural haematoma. Preferably a patient with SAH is treated,
more
preferably a aneurismal bleeding after SAH is treated.
In a preferred aspect of the invention the thrombomodulin analogue according
to the
invention is used to treat hyperfibrinolysis after physical trauma, preferably
a CNS
trauma. A physical trauma as defined herein refers to a body wound or shock
produced
by sudden physical injury, as from violence or accident. The physical trauma
encompasses polytrauma, head injury, chest trauma, abdominal trauma, extremity
trauma, facial trauma, genitourinary system trauma, pelvic trauma and soft
tissue
injury.
In a further aspect of the invention the thrombomodulin analogue used for the
treatment of coagulopathy with hyperfibrinolysis, is given in combination with
a further
fibrinolysis inhibitor. In particular a substance which corrects the normal
adhesion of
platelets can be used such as Etamsylate. Preferably an inhibitor of
proteolytic
enzymes can be used, which more preferably is an inhibitor of plasmin such as
aprotinin. In even more preferred aspect of the invention the further
antifibrinolytic drug
blocks the lysine-binding site of plasmin, such as epsilon-aminocaproic acid
or
tranexamic acid.
In a preferred aspect of the invention the patients which are treated with the
thrombomodulin analogues have anti- factor VIII antibodies.
In another aspect of the invention the patients which are treated with the
thrombomodulin analogues are further treated with factor VIII, preferably
recombinant
factor VIII or a recombinant B-domain-deleted factor VIII molecule, more
preferably
Octocog-alfa or moroctocog-alfa.
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In a preferred aspect of the invention the thrombomodulin analogues according
SEQ ID
NOs: 5 to 11 are given in the above described doses or dose ranges.
In a further aspect of the invention the thrombomodulin analogue used for the
treatment of coagulopathy with hyperfibrinolysis, is given in combination with
factor VIII,
preferably recombinant factor VIII a recombinant B-domain-deleted factor VIII
molecule,
more preferably octocog-alfa or moroctocog-alfa.
In one aspect of the invention the thrombomodulin analogue is administered at
the time
of a bleeding episode of the coagulopathy with hyperfibrinolysis or in advance
of an
increased bleeding risk, e.g. a surgery or a tooth extraction.
In a further aspect of the invention the thrombomodulin analogue is
administered to
patients that are refractory to blood/plasma transfusion or coagulation factor
replacement therapy.
In one aspect of the invention the thrombomodulin analogue is administered in
multiple
doses, preferably once daily, bidaily, or every third, fourth, fifth, sixth or
seven days
over a total time period of less than one week to four weeks, more preferably
as
chronic administration.
In a preferred aspect of the invention the thrombomodulin analogues according
SEQ ID
NOs: 5 to 11 are administered according to the administration schemes
described
above.
In a preferred aspect of the invention the thrombomodulin analogue is given as
parenteral application, preferable as intravenous or subcutaneous application.
In one aspect of the invention the thrombomodulin analogue is given in an
amount to
yield a plasma concentration in the subject to be treated of less than 5 nM/L,
preferably
of less than 3nM/L and more preferably of less than 1.5 nM/L.
In a further aspect of the invention the thrombomodulin analogue is titrated
so that the
plasma concentration is between 0.1 nM/L and 5nM/L, preferably between 0.1
nM/L
and 3 nM/L.
In a preferred aspect of the invention the thrombomodulin analogues according
SEQ ID
NOs: 5 to 11 are used to yield the above disclosed plasma concentrations.
In another aspect of the invention the thrombomodulin analogue is given to the
subject
to be treated in a dose between 0.1 pg/kg and 140 pg/kg, preferably in a dose
between
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0.5 pg/kg and 40 pg/kg, more preferably in a dose between 0.5 pg/kg and 4
pg/kg and
specifically in a dose between 0.75 and pg/kg (kg refers to kg bodyweight of
the
subject to be treated).
In a further aspect of the invention the thrombomodulin analogue is given in a
dose of,
0.75, 1.5, 2.5 or 4.0 pg/kg which equals to a body weight adjusted dose of
0.6, 1, 3, or
4.0 mg/patient.
In a further preferred aspect of the invention the thrombomodulin analogues
according
SEQ ID NOs: 5 to 11 are given in the above described doses or dose ranges.
In a more preferred aspect of the invention the thrombomodulin analogue is a
soluble
TM analogue.
In an even more preferred aspect of the invention the thrombomodulin analogue
is a
human soluble TM analogue.
In one aspect of the invention said thrombomodulin analogue comprises at least
one
structural domain selected from the group containing EGF3, EGF4, EGF5, EGF6,
preferably comprising the fragment EGF3-EGF6 and more preferably comprising
the
EGF domains 1-6.
In a further aspect of the invention said thrombomodulin analogue consists of
EGF
domains EGF1 to EGF6, and more preferably consists of the EGF domains EGF3 to
EGF6.
In one aspect of the invention the thrombomodulin analogue has an amino acid
sequence corresponding to the amino acid sequence of mature thrombomodulin
(depicted in SEQ ID NO:1 or SEQ ID NO:3) and comprises one or more of the
following
modifications:
a) removal of amino acids 1-3
b) M388L
c) R456G
d) H457Q
e) S474A, and terminating at P490.
In a further aspect of the invention the thrombomodulin analogue has an amino
acid
sequence which comprises a sequence of at least 85%, or at least 90% or 95%
sequence identity with SEQ ID NO: 2.
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In a preferred aspect of the invention the thrombomodulin analogue has an
amino acid
modification at one or more positions corresponding to natural sequence at
(according
to SEQ ID NO: 1 or SEQ ID NO:3):
ab) 355Asn;
ae) 359GIn;
af) 36'Gln;
ag) 363Leu;
ah) 364Asn;
ai) 368Tyr;
aj) 371Val;
ak) 374GIu;
al) 376Phe;
am) 384 His;
an) 385Arg;
ba) 387GIn;
bb) 389Phe;
bc) 398Asp;
bd) 400Asp;
be) 402Asn;
bf) 403Thr;
bg) 408GIu;
bh) 411Glu;
bi) 413Tyr;
bj) 4141le;
bk) 415Leu;
bl) 416Asp;
bm) 417Asp;
bn) 4201le;
bo) 423Asp;
bp) 4241le;
bq) 425Asp;
br) 426GIu;
ca) 428Glu;
cb) 429Asp;
cc) 432Phe;
cd) 434Ser;
ce) 436 Val;
cf) 438His;
cg) 439Asp;
ch) 4`10Leu;
ci) 443Thr;
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cj) 44Phe;
ck) 'Glu;
co 456Arg;
cm) 458 lie; or
cn) 46,Asp
In a further aspect of the invention the thrombomodulin analogue has a
modification of
the phenylalanine at position 376 according to SEQ ID NO:1 or SEQ ID NO:3,
preferably substituted with an aliphatic amino acid, more preferably
substituted with
glycine, alanine, valine, leucine, or isoleucine and most preferably
substituted with
alanine.
In a further aspect of the invention the thrombomodulin analogue has a
modification of
one or more of the following amino acids according SEQ ID NO:1 or SEQ ID NO:3:
a) 387Gln;
b) 36Met;
b) 39Phe,
whereby the amino acids are deleted, inserted by one or more additional
amino acids or preferably substituted.
In a further aspect of the invention the thrombomodulin analogue is used in
its oxidised
form, preferably oxidised with chloramine T, hydrogen peroxide or sodium
periodate.
In a further aspect of the invention a thrombomodulin analogue is us used,
whereas
one or more of methionine residues within the TM analogue are oxidised,
preferably
the methionine residue at position 388 (according SEQ ID NO:1 or SEQ ID NO:3).
In another aspect of the invention a method for screening for analogues of
thrombomodulin suitable for the treatment of coagulopathy with
hyperfibrinolysis is
claimed, whereas the thrombomodulin exhibits one or more of the following
features:
(i) a reduced binding affinity towards thrombin,
(ii) a reduced cofactor activity,
(iii) an increased TAFI activation activity,
comprising the steps of:
a) making one or more amino acid substitution of the thrombomodulin
sequence (SEQ ID NO:1 or SEQ ID NO:3), preferably of the amino
acid positions listed in claim 15;
b) comparing the modified analogue with a control molecule, preferable
a rabbit lung TM or a soluble human TM analogue with regard to
one or more of the following characteristics:
ba) binding affinity to thrombin (KD value);
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bb) cofactor activity;
bc) TAFI activation activity or TAFIa potential;
bd) ratio of TAFI activation activity and cofactor activity;
be) effect of protein oxidation;
bf) effect on clot lysis in time in an in vitro assay; or
bg) effect in a coagulation-associated animal model.
In another aspect of the invention a method of treating coagulopathy with
hyperfibrinolysis is claimed, comprising administering a therapeutically
effective
amount of a thrombomodulin analogue according to any of the claims 1 to 20.
In another aspect of the invention a thrombomodulin analogue according to SEQ
ID
NO:2 is claimed which has a modification of the phenylalanine at position 376
(numbering according to SEQ ID NO:1), preferably substituted with an aliphatic
amino
acid, more preferably substituted with glycine, alanine, valine, leucine, or
isoleucine
and most preferably substituted with alanine.
In a preferred aspect of the invention a thrombomodulin analogue is us used as
given
in Figure 19 and depicted by the amino acid sequences SEQ ID NO:5 to SEQ ID
NO:11.
These preferred TM analogues are:
1. A TM fragment extending from as 4 to as 490 having the following amino acid
exchanges: Phe376Ala, Met388Leu, Arg456GIy, His457GIn and Ser474AIa (equals
SEQ ID NO: 5).
2. A TM fragment extending from as 227 to as 462 (= EGF1-6) having the
following
amino acid exchanges: Phe376Ala, Met388Leu, Arg456GIy and His457GIn (equals
SEQ ID NO: 6).
3. A TM fragment extending from as 333 to as 462 having the following amino
acid
exchanges: Phe376Ala, Met388Leu, Arg456GIy and His457GIn (equals SEQ ID NO:
7).
4. A TM fragment extending from as 227 to as 462 having the following amino
acid
exchanges: Phe376Ala and Met388Leu (equals SEQ ID NO: 8).
5. A TM fragment extending from as 333 to as 462 having the following amino
acid
exchanges: Met388Ala, Arg456GIy and His457GIn (equals SEQ ID NO: 9).
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6. A TM fragment extending from as 333 to as 462 having the following amino
acid
exchanges: Met388Leu, Arg456Gly, His457GIn and Glu461AIa (equals SEQ ID NO:
10).
7. A TM fragment extending from as 333 to as 462 having the following amino
acid
exchanges: Phe376Ala, Met388Ala, Arg456Gly and His457GIn (equals SEQ ID NO:
11).
Definitions
As used in the context of the present invention the term "antifibrinolytic
effect" shall
refer to the ability of a thrombomodulin analogue to prolong the clot lysis
time (as
described in Example I) compared to identical assay conditions without
addition of the
thrombomodulin analogue. The antifibrinolytic effect is due to a prevalence of
the
antifibrinolytic activity of the TM analogue compared to its profibrinolytic
activity.
As used herein the term "profibrinolytic effect" shall refer to the ability of
a
thrombomodulin analogue to significantly reduce the clot lysis time in an in
vitro assay
(as described in Example I) compared to identical assay conditions without
addition of
the thrombomodulin analogue.
The terms "significantly reduce" and "significantly prolong" as used herein
refers to a
prolongation or reduction of the clot lysis time that is significantly
different from the
basis value at the p= 0.1 level and/or refers to a prolongation or reduction
that exceeds
10%, preferably 20%, more preferably 30% and most preferably 40%, 50%, 60%,
70%,
80% 100%, 150% Or 200%.
As used in the context of the present invention the words "treat," "treating"
or
"treatment" refer to using the TM analogues of the present invention or any
composition
comprising them to either prophylactically prevent a bleeding event, or to
mitigate,
ameliorate or stop a bleeding event. They encompass either curing or healing
as well
as mitigation, remission or prevention, unless otherwise explicitly mentioned.
Also, as
used herein, the word "patient" refers to a mammal, including a human.
As used herein the term "coagulopathy with hyperfibrinolysis" shall refer to a
coagulopathy as a disease affecting the coagulability of the blood, whereby a
markedly
increased fibrinolysis causes, aggravates or prolongs bleeding events.
As used in the context of the present invention the term "thrombomodulin
analogue"
refers to both protein and peptides having the same characteristic biological
activity as
membrane-bound or soluble thrombomodulin. Biological activity is the ability
to act as a
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receptor for thrombin and increase the activation of TAFI, or other biological
activity
associated with native thrombomodulin.
The term "binding affinity" used herein refers to the strength of the affinity
between the
thrombomodulin analogue and thrombin and is described by the dissociation
constant
K0. The KD value for the binding affinity between thrombin and thrombomodulin
may be
determined by equilibrium methods, (e.g. enzyme-linked immunoabsorbent assay
(ELISA) or radioimmunoassay (RIA)) or kinetics (e.g. BIACORETM analysis), for
example. The binding affinity is preferably analysed using a kinetics assay as
described in Example II of the present invention.
"KD" refers to the relative binding affinity between the TM analogue and
thrombin. High
KD values represent low binding affinity. The precise assays and means for
determining
KD are provided in example II.
The term "cofactor activity" as used herein refers to the ability of the
thrombomodulin
analogues to complex with thrombin and potentiate the ability of thrombin to
activate
protein C. The assay procedures used to measure cofactor activity are given in
Example III of the present invention.
The terms "TAFI activation activity" as used herein refers to the ability of
the
thrombomodulin analogues to complex with thrombin and potentiate the ability
of
thrombin to activate TAFI. The assay procedures used to measure TAFI activity
is
given in Example IV of the present invention.
"Km" refers to the Michaelis constant and is derived in the standard way by
measuring
the rates of catalysis measured at different substrate concentrations. It is
equal to the
substrate concentration at which the reaction rate is half of its maximal
value. The "Km"
for the TM analogues of the present invention is determined by keeping
thrombin
concentrations at a constant level (e.g. 1 nM) and using saturation levels of
TM (e.g.
100 nM or greater) depending on the KD. Reactions are carried out using
increasing
concentrations of protein C (e.g., 1-60 NM). Km and kcat are then determined
using
Lineweaver-Burke plotting or nonlinear regression analysis.
"TME" refers to an analogue of TM consisting of the six EFG repeats (amino
acids 227
to 462 according to SEQ ID NO:1 or SEQ ID NO:3).
"TMEM388L" refers to an analogue of TM consisting of the six EFG repeats (aa
227 to
462) with a substitution of the native methionine at position 388 (based on
SEQ ID
NO:3) by an leucine residue.
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The term "therapeutically effective amount" is defined as the amount of active
ingredient that will reduce the symptoms associated with coagulopathy with
hyperfibrinolysis, such as bleeding events. "Therapeutically effective" also
refers to any
improvement in disorder severity, frequency or duration of incidence compared
to no
treatment.
The term õsequence modification" as used in the context of the present
invention
relates to the modification of a primary amino acid sequence, in particular by
amino
acid substitution, deletion or insertion. Where not otherwise explicitly
defined this term
means the substitution of one amino acid by another amino acid, which
substantially
differs from the first amino acid in terms of its polarity, hydrophilic or
hydrophobic
property, acidic or basic property, size or aromaticity, respectively.
Given the common classes of amino acids, namely classes of acidic, basic,
polar,
nonpolar, negatively charged, positively charged, aromatic and aliphatic amino
acids,
the concept of sequence modifications as of the invention preferably requires
a
substitution of one amino acid with another amino acid of a different class of
amino
acids. In a preferred aspect an amino acid is selected as a substituent which
has an
"opposite characteristic" of the amino acid to be substituted. The subsequent
amino
acid (aa) substitutions are particularly suggested: acidic as vs. basic aa,
polar as vs.
nonpolar aa, negatively charged as vs. positively charged aa, aromatic as vs.
aliphatic
aa.
A particular embodiment of the sequence modification of the present invention
is the
substitution of an amino acid by an aliphatic amino acid such as glycin,
alanine, valine,
leucine and isoleucine, most preferred is the substitution with alanine.
The sequence modification as of the invention can also include a substitution
with a
non-natural amino acid. A "non-natural amino acid" refers to an amino acid
that is not
one of the 20 common amino acids or pyrrolysine or selenocysteine. The term
"non-
natural amino acid" includes, but is not limited to, amino acids that occur
naturally by
modification of a naturally encoded amino acid (including but not limited to,
the 20
common amino acids or pyrrolysine and selenocysteine) but are not themselves
incorporated into a growing polypeptide chain by the translation complex.
Examples of
naturally-occurring amino acids that are not naturally-encoded include, but
are not
limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine,
and 0-
phosphotyrosine, ornithine, taurine, The sequence modification includes also
an
attachment of other groups to the amino acid. These groups include acetate,
phosphate, various lipids and carbohydrates, which preferably changes the
chemical
nature of the amino acid. The sequence modification further includes oxidation
or
reduction of the respective amino acid, preferably of a Met or Cys residue.
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I. EXAMPLE
Clot lysis assay in human plasma
Using a model of in vitro clot lysis the ability of soluble thrombomodulin
(Solulin) to
decrease or increase the clot lysis time in mixtures of normal plasma and
Factor VIII
deficient plasma was tested.
1. Test system
Within the plasma compositions the clotting was initiated in vitro by mixing
thrombin
(Factor Ila), calcium chloride and phosphatidylcholine/phosphatidylserine
(PCPS)
vesicles. Time course of coagulation and fibrinolysis were determined with a
turbidity
assay, and the "TAFIa potential" using a functional assay.
2. Experimental procedures
Materials. Thrombin and fibrinogen were prepared as described in Walker et al.
(J.Biol.
Chem. 1999; 274: 5201-5212) with one exception: for the fibrinogen
preparation, the
solution was made to 1.2% PEG-8000 instead of 2% PEG-8000 by the addition of
40%
(w/v) PEG-8000 in water, subsequent to 1 -alanine precipitation. This change
in
protocol allowed for a greater yield of fibrinogen. QSY-FDPs (fibrin
degradation
products that are covalently attached to the quencher, QSY9 C5-maleimide) and
TAFIa
standards used in the TAFIa assay were prepared as described (Kim et al.,
2008; Anal.
Biochem 372: 32-40; Neill et al., 2004; Anal. Biochem. 330: 332-341) and
recombinant
human Pg (S741 C) and the fluorescein derivative (5IAF-Pg) were prepared as
described by Horrevoets et al. (J. Biol. Chem 1997; 272: 2176-2182). S525C-
prothrombin was purified and fluorescently labelled with 5-
iodoamidofluorescein (5IAF)
as previously described by Brufatto et al. (J. Biol. Chem. 2001; 276: 17663-
17671).
QSY9 C5-maleimide and 5-iodoamidofluorescein were purchased from Invitrogen
Canada Inc. (Burlington, ON, Canada). Plasmin was purchased from Haematologic
Technologies Inc. (Essex Junction, VT, USA) and recombinant human soluble
thrombomodulin (Solulin; sTM) was provided from Paion Deutschland GmbH
(Aachen,
Germany). Normal human pooled plasma (NP) was obtained from healthy donors at
the blood bank in the Kingston General Hospital (KGH) in Kingston, Ontario,
Canada,
and FVIII-deficient plasma (FVIII-DP) was purchased from Affinity Biologicals,
Inc.
(Hamilton, ON, Canada). TAFI-deficient plasma (TDP) was prepared by affinity
chromatography of normal plasma on a column of immobilized anti-human TAFI
monoclonal antibody, as described by Schneider et al., (J. Biol. Chem. 2002;
277:
1021-1030). The plasmin inhibitor D-Val-Phe-Lys chioromethyl ketone (VFKck),
the
thrombin inhibitor D-Phe-Pro-Arg chioromethyl ketone (PPAck) and potato tuber
carboxypeptidase inhibitor (PTCI) were purchased from Calbiochem (San Diego,
CA,
USA). Tissue-type plasminogen activator (Activase; tPA) was purchased from the
pharmacy at KGH (Kingston, ON, Canada). All other reagents were of analytical
quality.
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3. Methods.
Clot lysis assays and the preparation of samples to determine the extent of
TAFI
activation.
FVIII-DP was mixed with NP so that the final percentage of NP was 0, 1, 6, 10,
50 or
100% (0-100% NP). Before mixing, each plasma was diluted to an optical density
of 32
and added to an equal volume of a solution containing 1.5 nM tPA, 40 pM PCPS
and
20 mM CaC12 in the presence or absence of 20 nM thrombin (final
concentrations: 0.75
nM tPA, 20 pM PCPS, 10 mM CaC12, 10 nM thrombin) and the samples were divided
into multiple Eppendorf tubes and placed in a 37 C water bath. Clotting and
lysis were
stopped in these tubes at various time points by the addition of 10 pM PPAck
and 10
pM VFKck to selectively inhibit thrombin and plasmin, respectively. The
samples were
mixed vigorously, then centrifuged for 30 s at 16 000 g (room temperature) and
immediately placed on ice to prevent thermal inactivation of TAFIa. The
supernatant of
each sample was serially diluted by 5-fold with TAFI-deficient plasma and
TAFIa was
measured using a functional assay described by Kim et al. (Anal. Biochem 2008;
372:
32-40). Identical experiments were conducted in a covered, 96-well plate and
the
turbidity was monitored at 400 nm over time using a SpectraMax Plus
spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) to determine the
timing
of coagulation and fibrinolysis. Similar experiments were conducted in the
presence or
absence of soluble thrombomodulin (0-100 nM) at 4 tPA concentrations (0.25,
0.75,
1.5 and 3 nM) to determine the effect of sTM on TAFI activation and lysis
times. These
experiments were also conducted in the presence of 5 pM PTCI to show the TAFIa
dependent prolongation of lysis in normal and FVIII-deficient plasma.
Determination of the time course of prothrombin activation in normal and FVIII-
deficient plasma.
Normal and FVIII-deficient plasmas (0-100% NP) were supplemented with the
prothrombin derivative (5IAF-ll; 300 nM final) as well as 20 pM PCPS and 10 mM
CaCl2 in the presence of 10 nM thrombin to initiate clotting. These
experiments were
conducted in an opaque, plastic-covered 96-well plate. A SpectraMax GeminiXS
(Molecular Devices, Sunnyvale, CA, USA) was used to monitor fluorescence
intensities
over time at 37 C with excitation and emission wavelengths of 495 nm and 535
nm,
respectively, employing a 530-nm emission cut-off filter. Fluorescence was
normalized
(0-1) to reflect the baseline and maximal fluorescence, which correlates with
full
prothrombin activation.
Determination of the TAFIa potential.
The area under the TAFIa plots was chosen as a parameter to quantify the
effect of
TAFIa over the course of the experiments. This parameter was designated the
"TAFIa
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potential" by analogy with the "thrombin potential" defined by Hemker et al.
(Thromb.
Haemost. 1993; 85: 5-11). TAFIa potential, like thrombin potential, is
proportional to the
amount of substrate cleaved and is explained mathematically, as follows:
dS k.t [TAFIa] [S], (I)
T t
Km
where dS/dt is the rate of substrate consumption and S is the substrate.
If S is constant (i.e. limited consumption of S),
dS = -S /Tent [TAFIa] (2)
dt K.
dS = -S K [TAFIa] di (3)
For some interval 0 to t,
S(r) t
dS = -S K t` / [TAFIa] di (4)
1/ m
s(0) 0
Realizing that the integral on the right in equation (4) is the area under the
TAFIa plot,
AS(i) = -S Km (area under curve) (5)
AS(t) = -S---t (TAFIa Potential) (6)
4. Results
Clot lysis time is increased by addition of normal plasma to FVIII-deficient
plasma.
Clotting was initiated with 10 nM Factor lla, 10 mM CaCl2 and 20 pM PCPS
vesicles to
create a model where the clot structure is insensitive to the FVIII
concentration.
Because the clot structure is similar regardless of the FVIII concentration,
the effect of
FVIII on tPA-dependent (0.75 nM) clot lysis can be determined. Using this
lysis model,
lysis times increased as the percentage of normal plasma increased. Figure 1
shows
the clot lysis profiles for FVIII-DP with 0 - 100% added normal plasma and the
lysis
times are summarized in Fig. 1 (inset). In FVIII-DP the lysis time is 37 min
and can be
increased by approximately 50% by the addition of normal plasma.
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10% normal plasma is sufficient to restore clot lysis in FVIII-DP.
At 10% normal plasma the shortened lysis time associated with FVIII-DP has
been
corrected to that observed in normal plasma (see Fig. 1, inset).
50% of the TAFIa potential is sufficient to restore clot lysis in FVIII-DP.
TAFI activation was measured in normal, FVIII-deficient and mixed plasmas to
quantify
the effect of FVIII on the time course of activation. A functional assay was
used to
measure TAFIa over the time course of clotting and lysis and the results are
presented
in Figure 2. When thrombin, calcium ions and PCPS were used to initiate
clotting in
FVIII-DP, approximately 30 pM TAFIa was measured after 5 min. As the
percentage of
normal plasma increased so too did the peak concentration of TAFIa. Although
the
lysis time was corrected by supplementing FVIII-DP with 10% normal plasma,
this was
not sufficient to fully correct TAFI activation. By calculating the area under
the TAFIa
time course plots (Fig. 2A) it was determined that approximately the same
TAFIa
potential (Fig. 2B) was achieved over the first 50 min in normal plasma and
50%
normal plasma (16 800 pM mins and 14 100 pM mins, respectively) but FVIII-DP
plasma mixed with 10% normal plasma had a TAFIa potential of only 50% of the
TAFIa
potential in normal plasma.
There is a strong correlation between lysis time and TAFIa potential.
In order to quantify the relationship between lysis time and TAFI activation
over the
range 0-100% FVIII, log lysis time vs. log TAFIa potential was plotted (Fig.
2B, inset).
As expected, the data show a strong positive correlation between lysis time
and TAFIa
potential in plasma containing 0-100% FVIII. The TAFI activation profile in
Fig. 2A can
be rationalized by analyzing prothrombin activation in plasma (Fig. 3) because
thrombin is the activator of TAFI. The general trend is that as the percentage
of normal
plasma increased, the rate of prothrombin activation also increased (which can
be
determined by examining the slope of the curve in Fig. 3). An exception occurs
with
normal plasma. In normal plasma the rate of prothrombin activation is lower
than in
FVIII-DP mixed with 50% normal plasma. While the rate is slower in normal
plasma,
prothrombin activation persists for about twice as long as in FVIII-DP mixed
with 50%
normal plasma. In every experiment, the timing of prothrombin activation
corresponds
well with TAFI activation. Normal plasma was also clotted using calcium ion
and PCPS,
without added thrombin. Calcium-induced coagulation does not occur
immediately; it
takes approximately 15 min for the clot to form in normal plasma. At this
time,
prothrombin activation enters the propagation phase and as a result, TAFI is
activated.
The extent and timing of TAFI activation with respect to clot formation is the
same
whether clotting is initiated in the presence or absence of added thrombin,
which
suggests that TAFI activation is a result of thrombin generated in situ and
not of
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thrombin added to induce clotting. In the presence of thrombin there was a
TAFIa
potential of 16,800 pM min compared with 14,150 pM min in the absence of
thrombin.
Soluble thrombomodulin prolongs clot lysis in normal and FVIII-deficient
plasma.
In normal plasma, peak TAFIa levels and TAFIa potential increased from 600 pM
and
16 800 pM min, respectively, in the absence of sTM to approximately 6000 pM
and
150,000 pM min, respectively, in the presence of 10 nM sTM. This increase in
TAFI
activation resulted in a 70% increase in the lysis time. The effect of 10 nM
sTM on the
relative prolongation of lysis in FVIII-DP was similar to normal plasma in
that lysis was
prolonged by 65% when FVIII-DP was clotted and lysed in the presence of sTM.
In the
presence of 10 nM sTM, 750 pM TAFIa was present at peak TAFIa concentration
compared with 30 pM in the absence of sTM. In the time from clot initiation to
the clot
lysis time the TAFIa potential was measured to be 12 800 pM min in the
presence of 10
nM sTM compared with 600 pM min in the absence of sTM.
The increase of clot lysis time in normal and FVIII-deficient plasma depends
on
tPA and sTM concentrations.
The effect of TAFI activation on lysis time was analyzed over a range of tPA
and sTM
concentrations to determine if the lysis defect in FVIII-DP could be corrected
by
stimulating TAFI activation. The lysis times summarized in Fig. 4 are relative
to lysis
times from similar experiments containing PTCI, which is an inhibitor of
TAFIa. In the
presence of PTCI, there is no functional TAFIa so the relative lysis times
presented in
Fig. 4 are representative of TAFIa-dependent prolongation of lysis. At the
lowest
concentration of tPA (0.25 nM), the maximal TAFIa-dependent prolongation of
lysis (2-
fold) was observed when 1 nM sTM was added to normal plasma. Supplementing
FVIII-DP with sTM caused a dose-dependent prolongation of the lysis time (Fig.
4).
When 100 nM sTM was added to FVIII-DP the lysis time was fully corrected to
that
seen in normal plasma. As the tPA concentration increased, a higher
concentration of
sTM was required to get maximal TAFIa-dependent prolongation of lysis. For
example,
when 1.5 nM tPA (Fig. 4) is present, 25 nM sTM is required to maximize the
TAFIa
dependent prolongation of lysis in normal plasma and 100 nM sTM is required in
FVIII-
DP. Also, as tPA is increased in these clot lysis experiments TAFIa appears to
have a
much greater effect on lysis time (up to 5.2-fold at 1.5 nM tPA compared with
2.3-fold
at 0.25 nM tPA). It appears that as the tPA concentration is increased, the
concentration of sTM required to get any TAFIa-dependent prolongation of lysis
also
increases. At 0.25 nM tPA, no sTM was required to get prolongation of lysis in
normal
plasma whereas 25 nM sTM was required to get prolongation of lysis when 3 nM
tPA
(Fig. 4) was added to normal plasma. In order to show how the actual lysis
times are
affected by tPA and sTM the lysis times in TAFIa inhibited normal and FVIII-
deficient
plasma are presented in Table 1.
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Thrombomodulin very substantially promotes TAFI activation and prolongs lysis
in both normal and FVIII-deficient plasma.
In normal plasma TAFI activation is shown to be significantly increased in the
presence
of 10 nM sTM (=; 6000 pM TAFIa at its peak level) compared to the absence of
sTM
(o; 600 pM TAFIa; see Figure 5 A). The accompanying clot-lysis profile reveals
that the
addition of lOnM sTM resulted in a 70% increase in the lysis time. In FVIII-DP
supplemented with 1OnM sTM TAFIa was measured to be 750 pM at its peak
compared to 30 pM in the absence of sTM (see Figure 5 B). The increase in TAFI
activation resulted in a 60% prolongation of lysis compared to FVIII-DP
lacking sTM.
ll. EXAMPLE
Analysis of binding affinity between thrombin and thrombomodulin
Using a fluorescent kinetics assay the affinity expressed as a KD value was
determined
for the binding between thrombin and the thrombomodulin analogue.
1. Test system
The affinity for the binding between thrombin and the thrombomodulin analogue
was
determined using a fluorescent kinetics assay and expressed as a KD value.
2. Experimental procedures
Materials.
The human thrombin was isolated from plasma as described by Bajzar et al. (J.
Biol.
Chem. 1995; 270: 14477-14484). Recombinant soluble thrombomodulin (Solulin)
was
obtained from PAION Deutschland GmbH (Aachen, Germany). All other reagents
were
obtained from Sigma in analytical quality.
Methods.
Measurement of the Binding of Thrombin to Thrombomodulin and TAFI
The binding of thrombin to thrombomodulin was measured as an equilibrium
binding
assay. A solution containing thrombin (20 nM), thrombomodulin (1.54 NM), and
DAPA
(20 nM, dansylarginine N-3-(ethyl-1,5-pentanediyl)amide, a fluorescent,
reversible
thrombin inhibitor) in 0.02 M Tris-HCI, 0.15 M NaCl, 5.0 mM CaCl2, 0.01% Tween
80, pH 7.4, was added in small successive aliquots to an otherwise identical
solution
that lacked thrombomodulin. The additions were performed in a cuvette fitted
with a
magnetic stirrer in the sample compartment of a Perkin-Elmer model LS50B
spectrofluorimeter. Intensity values were continuously recorded with
excitation and
emission wavelengths of 280 and 545 nm, respectively. A 430-nm cut-off filter
was
used in the emission beam. The data were analyzed as follows. The intensity of
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fluorescence, /, was assumed to be the sum of intensities from thrombin-DAPA
(T=D)
and thrombin-thrombomodulin-DAPA (T=TM=D). That is, 1= i,=[T=D] + i2-[T-TM-D],
where
i, and i2 are the coefficients of fluorescence for T-D and T=TM-D (since
excitation was at
280 nm, the emission from free DAPA was negligible). Because TM does not
appreciably alter the Km for either protein C activation or TAFI activation
(see Bajzar et
al., 1996; J. Biol. Chem. 271: 16603-16608), it can be assumed that it does
not alter
the affinity of the thrombin-DAPA interaction.
Thus [T-D] = ([T] + [T=D])/(1 + KpAPA/[DAPA])
and [T-TM-D] = ([T-TM] + [T-TM-D])/(1 + KDAPA/[DAPA]),
where KpAPA is the dissociation constant for the thrombin-DAPA interaction.
Therefore,
I = ii-([T] + [T=D])/(1 + KpAPA/[DAPA]) + i2([T=TM] + [T=TM-D])/(1 +
KDAPA/[DAPA]).
If f and b are defined as the fractions of thrombin, respectively, free and
bound to
thrombomodulin, and [T]o is the total concentration of thrombin, then
f = ([T] + [T-D])/[Tlo, b = ([T=TM]+[T=TM-D])/[T]o and f + b = 1. The
fluorescence intensity
then is given by / = i,-f[T]o/(1 + KDApA/[DAPA]) + i2-b[7]o/(1 +
KDApA/[DAPA]). If Io is
defined as the initial intensity when no thrombomodulin has been added, then f
= 1 and
/o = I1[T]o/(1 + KpAPA/[DAPA]). Similarly, if /max is defined as the intensity
upon saturation
of thrombin with thrombomodulin, then b = 1 and /max = 12mo/(1 +
KDApA/[DAPA]). Thus,
I =10=f + lma=b. Substituting 1 - b for f then gives: I = /o + (/max - 10)-b
or A/ = Almax-b.
Normalizing to the initial intensity gives (Al/lo) = (AImax/lo)-b. If DAPA
binds T and T=TM
with equal affinity, then TM binds T and T=D with equal affinity.
Therefore, with KTM defined as the dissociation constant for the thrombin-
thrombomodulin interaction, [T][TM] = KTM[T-TM]; [T=D][TM] = KTM[T-TM-D]; and
([T] + [T-D])-[TM] = KTM([T-TM] + [T-TM=D]). The last expression is identical
to
f-[TM] = KTM=b. Since f = 1 - b and [TM] = [TM]o - b-[T]o, where [TM]o is the
total
thrombomodulin concentration, the following equation is obtained: (1 -
b)([TM]o -
b-[T]o) = KTM-b. This is a quadratic equation in b, which when solved and
substituted in
the expression above for (Al/Io) gives the equation: (Al/lo) = (A
/max//o)=0.5-(KTM + [Tlo + [TM]o - ((KTM + [T]o + [TM]o)2 - 4=mo'[TM]o)1/2).
This latter
equation expresses the relationship between fluorescence intensity values, the
nominal
concentrations of thrombomodulin and thrombin, the dissociation constant for
the
thrombin-thrombomodulin interaction, and the fluorescence intensity increment
that
signals the interaction of thrombomodulin with thrombin-DAPA. Intensity data
were fit to
the above equation by nonlinear regression analysis, with [TM]o as the
independent
variable and KTM and A/max as best-fit parameters.
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3. Results
Thrombin binds to soluble thrombomodulin with an affinity of KD= 23 14nM.
The binding of thrombin to soluble thrombomodulin was measured by perturbation
of
the fluorescence of DAPA. As depicted in Figure 6, the titration curve showed
a
increase of the relative fluorescence for the concentration range of soluble
thrombomodulin between 0 and 75nM. The data analysis revealed that thrombin
binding to soluble thrombomodulin was characterized by KD= 23 14nM.
Ill. EXAMPLE
Analysis of cofactor activity for mutated thrombomodulin analogues
Using a fluorescent kinetics assay the affinity expressed as a KD value was
determined
for the binding between thrombin and the thrombomodulin analogue.
1. Experimental procedures
Materials and Methods.
The ability of TM mutants to act as cofactor for thrombin-mediated activation
of protein
C was assayed directly in the shockates. Recombinant human protein C was from
Dr.
John McPherson, Genzyme Corp., Framingham, MA., and was purified as described
(BioTechnology 1990; 8: 655-661). Twenty five pl of each shockate was mixed
with
equal volumes of recombinant human protein C (final concentration of 0.3 NM)
and
human alpha thrombin (Sigma Chemicals, St. Louis, MO) at a final concentration
of
1 nM in a microtiter plate. All reagents used were diluted in 20 mM Tris,
pH7.4/100 mM
NaCI/3.75 mM CaCl2/0.1 % NaN3 (wN) containing 5 mg/ml bovine serum albumin.
Mixtures were incubated for 1 hr at 37 C and the reaction was terminated by
addition of
25 pl of hirudin at 800 units/ml (Sigma Chemicals, St. Louis, MO). The amount
of
activated protein C was determined by addition of 100 pl of chromogenic
substrate D-
valyl-L-leucyl-L-arginine-p-nitroanilide (S-2266) (1mM). The change is
measured by the
absorbance at 405 nm with time using a plate reader. Data is recorded as
milliOD
unit/min and determined for each sample by measuring the absorbance every 10
seconds for 15 minutes using a Molecular Devices plate reader. All assays
contained
triplicate shockate samples each of DH5 alpha cells transfected with either
pSELECT-1
vector (no TM), pTHR211 (wild type) or pMJM57 (pTHR211 with methionine at 388
altered to leucine), as internal controls. Cofactor activities of TM mutants
were
expressed as mean of that obtained for pMJM57.
Statistical Analysis.
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Each mutant was assayed for activity at least twice (three times for those
mutants for
which only two positive clones were isolated), and all the data were included
in the
determination of the significance of difference using Student t-Test.
Coefficient of
variation between plates was 16.7% (n=18).
Western blot analysis
E. coli shockates were run in 10% Tris-tricine SDS PAGE under reduced
conditions
according to the manufacturer's specifications (Novex Inc., San Diego, CA).
Reduced
and alkylated samples were prepared by boiling shockates in sample buffer
(62.5 mM
Tris, pH6.8, 2% SDS, 10% glycerol, 0.0025% bromophenol blue) containing 10 mM
dithiothreitol for 10 minutes, followed by incubation with 50 mM
iodoacetamide.
Proteins were transferred to nitrocellulose filter in transfer buffer (192 mM
glycine, 25
mM Tris, pH8.3, 20% methanol) at 4 C. The nitrocellulose filter was blocked
with a
blocking buffer (1% bovine serum albumin in 10 mM Tris, pH7.5, 0.9% NaCl,
0.05%
NaN3), and then incubated with mouse polyclonal antiserum (raised against
reduced
and alkylated EGF domain of human thrombomodulin) in the blocking buffer.
After
washing with a washing buffer (10 mM Tris, pH7.5, 0.9% NaCl, 0.05% NaN3, 0.05%
Tween 20), the filter was incubated with biotinylated goat anti-mouse IgG
antibodies in
the blocking buffer containing 0.05% Tween 20. Proteins were detected using
the
Vectastain ABC solution (Vector Laboratories, Burlingame, CA) and ECL
detection
system (Amersham Corporation, Arlington Heights, IL) according to the
manufacturer's
specifications.
IV. EXAMPLE
Analysis of thrombomodulin analogues for TAFI and protein C activation
Using a fluorescent kinetics assay the affinity expressed as a KD value was
determined
for the binding between thrombin and the thrombomodulin analogue.
1. Experimental procedures
Proteins and Reagents.
Truncated forms of thrombomodulin comprising Solulin (residues 4-490), THE
(residues
227-462), TMEc-loop 3-6 (residues 333-462), and TMEi4-6 (residues 345-362)
were
prepared as described by Parkinson et al. (Biochem. Biophys. Res. Commun.
1992;
185: 567 - 576). Sf9 cells were transfected with the TM constructs, and the
proteins
were isolated from the media by a combination of chromatography procedures
utilizing
anion exchange, gel filtration, and thrombin affinity. Purity, assessed by SDS-
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polyacrylamide gel electrophoresis and silver staining, was 95% or greater.
Human
plasma TAFI was isolated as described by Bajzar et al. (J. Biol. Chem. 1995;
270:
14477 - 14484). Human protein C and thrombin were prepared as described by
Bajzar
and Nesheim (J. Biol. Chem. 1993; 268: 8608 - 8616). The thrombin inhibitor
dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was synthesized as
described
by Nesheim et al. (Biochemistry 1979; 18: 996 - 1003). Point mutants resulting
from
alanine scanning were generated from the TMEM388L construct. Proteins were
expressed in Escherichia coli. The procedures and preparation of periplasmic
extracts
have been described by Nagashima et al., (J. Biol. Chem. 1993; 268: 8608 -
8616).
HEPES, the basic carboxypeptidase substrate hippuryl-arginine, cyanuric
chloride, and
1,4-dioxane were obtained from Sigma. All other reagents were of analytical
quality.
Measurement of the rates of Protein C and TAFI activation with point mutants
of
thrombomodulin analogues.
For the activation of TAFI, a 20-pl aliquot of each periplasmic extract was
preincubated
with thrombin (13 nM final) in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl2
for
min at room temperature. The mixtures were then incubated with purified
recombinant TAFI (18 nM final) and a substrate, hippuryl-arginine (1.0 mM
final), in a
total volume of 60 pl for 60 min. The amount of activated TAFI was quantitated
by
measuring the hydrolysis of hippuryl-arginine to hippuric acid, followed by
conversion of
hippuric acid to a chromogen with 80 pl of phosphate buffer (0.2 M, pH 8.3)
and 60 pl of
3% cyanuric acid in dioxane (w/v). After thorough mixing, absorbance of the
clear
supernatant was measured at 382 nm. The amount of thrombin-dependent
activation of
TAFI was calculated by subtracting the background absorbance produced in the
absence of thrombin for each mutant. Activation of protein C by TMEM388L-
alanine
mutants was assayed as follows.
All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI,
pH7.4,
100 mM NaCl, 2.5 mM CaCl2, 0.5 % BSA). Samples and TM standards (0-1 nM) were
incubated for 60 min in 60 pl total volume at 37 C in a 96-well plate with 0.5
pM protein
C and 1 nM thrombin to generate APC before being quenched with 20 pl of
hirudin
(0.16 U/pl, 570 nM). The amount of APC formed was determined by monitoring the
hydrolysis of S-2266 (100pi of 1 mM) at 1-min intervals at 405 nm using a
plate reader
(Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of
activated protein C / min (37 C).
All assays contained extracts of DH5a cells transfected with either pSelect-1
vector (no
TME), wild-type TME(M388), or TME(M388L) as internal controls. Cofactor
activities of
TME(M388L) alanine mutants were expressed as percentages of the activity of
TME(M388L). Each TM mutant was assayed for both protein C and TAFI activation
in
duplicate using three independent preparations of extracts.
2. Results
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The results obtained with the TM mutants (Figure 7) indicate that five out of
eigth
mutants have a substantially reduced cofactor activation. From these five
mutants four
mutants show also a concomitant reduced activation activity of TAFI. Only the
mutation
at F376A resulted in a profound loss in protein C activation, but only in a
modest
reduction in TAFI activation. Intriguingly, the difference in importance of
Phe376 for TAFI
and protein C activation suggests the requirements for thrombomodulin
structure are
more constrained when protein C is the substrate of the thrombin-
thrombomodulin
complex.
V. EXAMPLE
Analysis of Met-specific TM mutants for protein C activation with regard to
oxidation.
Using specific methionine mutants of thrombomodulin analogues the role of
these
residues for cofactor activation also with respect to protein oxidation was
analysed
using a protein C activation assay.
1. Experimental procedures
Proteins and Reagents.
Human recombinant protein C was from Genzyme Corp. (Boston, MA). Bovine
thrombin was from Miles Laboratories Inc. (Dallas, TX). D-Val-Leu-L-Arg-p-
nitroanilide
was prepared as described by Glaser et al. (Prep. Biochem. 11975; 5: 333 -
348).
Human alpha-thrombin (4,000 NIH U/mg), bovine serum albumin (fraction V) and
chloramine T were from Sigma Chemical Co. (St. Louis, MO).
Expression of TME (Sf9).
All procedures were performed at 4 C. The DNA sequence encoding the six EGF-
like
repeats of TM (amino acids 227 - 462) was linked to the signal sequence of the
insect
protease, hypodermin A, and the hybrid gene placed under control of the
polyhedron
gene promoter in the baculovirus shuttle vector pTMHY101. Recombinant virus
was
generated using standard techniques. Mutant analogues described were prepared
by
use of a mutator site-specific mutagenesis kit (Stratagene, Inc., La Jolla,
CA) and virus
was prepared for expression in the baculovirus system by the same methods.
Purification and oxidation with Chloramine T
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Growth media containing secreted mutants of THE (Sf9) was clarified by
centrifugation,
lyophilized and redisolved in 1:10 volume of 0.2% NEM-Ac, pH 7, and 0.008%
Tween
80. Aliquots were treated with either 5 pl of H2O or 5 pl of 100 mM chloramine
T;
incubated for 20 min at room temperature; oxidant removed by dilution;
desalted on
NAP-5 columns (20 mM Tris-HCI, 0.1 M NaCl, 2.5 mM CaCl2, 5 mg/ml BSA, pH 7.4;
Pharmacia Inc.); and assayed for activation of protein C as follows.
Measurement of TM cofactor activity (APC assay)
All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI,
pH7.4,
100 mM NaCl, 2.5 mM CaCI2, 0.5 % BSA). Samples and TM standards (0-1 nM) were
incubated for 60 min in 60 pl total volume at 37 C in a 96-well plate with 0.5
pM protein
C and 1 nM thrombin to generate APC before being quenched with 20 pl of
hirudin
(0.16 UIpl, 570 nM). The amount of APC formed was determines by monitoring the
hydrolysis of S-2266 (100 pl of 1 mM) at 1-min intervals at 405 nm using a
plate reader
(Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of
activated protein C/min (37 C).
2. Results
Reduced cofactor activity of TM by oxidation of Met388.
Mutant and wild-type THE (Sf9) were expressed in insect cells, treated with
chloramines T, assayed for cofactor activity and the results compared (Table
2). When
TME is treated with an oxidant such as chloramine T it looses approx. 85% of
its
cofactor activity (see Table 2). Site-specific mutations of Met291 and Met388
demonstrate
that inactivation of THE (Sf9) is due to oxidation of a single methionine.
Derivatives
that retain Met388 were inactivated by chloramine T to a similar extent (>80%)
whereas
the Met388Leu mutant was resistant. Mutants in which Met291 is replaced were
active
but were not resistant to oxidative inactivation.
VI. EXAMPLE
Analysis of TM analogues with mutations of the interdomain loop between EGF4
and EGF5 (GIn387, Met388, Phe389)
Using specific mutants of thrombomodulin analogues the role of these residues
and
their oxidation was analysed using a protein C activation assay.
1. Experimental procedures
Plasmid constructions. A thrombomodulin fragment consisting of only the EGF-
like
domains (TME) was expressed in E.coli as follows, DNA fragment coding for TME
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(residues 227-462) of full length TM was obtained by polymerase chain reaction
from
human genomic DNA using primers 5'-
CCGGGATCCTCAACAGTCGGTGCCAATGTGGCG-3' and 5'-
CCGGGATCCTGCAGCGTGGAGAACGGCGGCTGC-3'. This fragment was placed
under the control of a (3-lactamase promoter and signal sequence in pKT279. An
EcoRV-Bglll fragment of the resultant plasmid and a Scal-Sacl fragment of
pGEM3zf-
containing the f1 origin of replication was then inserted into the pSelect-1
vector at the
EcoRV-BamHl and Scal-Sacl site, respectively, to construct E.coli expression
plasmid
pTHR21 1. Plasmids coding for TM mutants at position 387, 388, or 389 were
constructed using a site-directed mutagenesis procedure described in the
altered sites
in vitor mutagenesis kits with a single stranded pTHR211 DNA template. Each
primer
of the site-specific mutation was confirmed by restriction analysis.
To measure cofactor activity of the mutants, the individual E.coli cultures
expressing
mutant proteins were centrifuged, washed, and the cell pellets incubated (10
min, 4 C)
in 20% sucrose, 300mM Tris-HCI, pH 8.0, 1 mM EDTA, 0.5mM MgCI2. Shockates were
prepared by centrifugation of cell pellets treated with 0.5 mM MgCI2 (10 min,
4 C) and
assayed in the APC assay. The data are an average of the results from each of
three
independent clones.
Measurement of TM cofactor activity (APC assay)
All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI,
pH7.4,
100 mM NaCl, 2.5 mM CaCl2, 0.5 % BSA). Samples and TM standards (0-1 nM) were
incubated for 60 min in 60 pl total volume at 37 C in a 96-well plate with 0.5
pM protein
C and 1 nM thrombin to generate APC before being quenched with 20 pl of
hirudin
(0.16U/pi, 570 nM). The amount of APC formed was determined by monitoring the
hydrolysis of S-2266 (100 pl of 1 mM) at 1-min intervals at 405 nm using a
plate reader
(Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of
activated protein C/min (37 C).
2. Results
Reduced cofactor activity of TM by mutation of the interloop domain.
Using the site-directed mutagenesis, TM mutants that have either an altered
amino
acid, a deletion or an insertion at positions 387, 388, or 389 were expressed
(Figure 8).
The cofactor activity of the TM mutants are an average obtained from three
independent clones and are expressed as a percentage of the activity found for
TME(Sf9)WT. Gel scans on the Western blots were performed using a polyclonal
antibody against TM for all new mutants at position 388 and for selected
mutants at
position 387. These scans gave approximately equivalent amounts of TM,
indicating
that expression differences cannot account for the observed activity
differences. In
addition, in an independent substitution at position 387 (Figure 8A), 388
(Figure 8B),
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389 (Figure 8C), or insertions and deletions anywhere in the inter-domain loop
(Figure
8D) result in analogues which generally are poorer cofactors in the APC assay
then
wildtype TME. Analogues where Gln387 is replaced by Thr, Met or Ala retain
>70%
cofactor activity, but substitution with Glu reduces this to 58% of control,
and all other
amino acids result in >50% loss. Only the substitution of Met388 with Leu
results in a
substantially higher cofactor activity (1.8-fold) than wildtype. All other
substitutions of
Met388 except Gln and Tyr resulted in >50% loss of cofactor activity. TM
cofactor
activity is less sensitive to amino acid replacement of Phe389 and nine of the
point
mutants at this position retain >70% of the activity found in the control. Pro
or Cys
substitution at any positions reduced the activity to >10% except for
Met388Pro which
retained 30% activity. Varying the length of the interdomain loop between EGF4
and
EGF5 by either deleting individual amino acids or inserting an Ala into each
of the four
possible positions resulted in mutants with less than 10% of the activity of
wild type
TME.
VII. EXAMPLE
Analysis of fibrinolysis in canine haemophilic plasma
Using models of in vitro clot lysis and thrombelastography the ability of
soluble
thrombomodulin (Solulin) to decrease or increase the clot lysis time was
tested in
whole blood or plasma of dogs with haemophilia A.
Materials and methods
Clot Lysis assay
The clot lysis assay was performed as described in Example I of the present
invention.
Thromboelastrography
Thrombelastography was performed as described in Prasad et al., 2008; Blood
111:
672-679. Canine haemophilic whole blood ( Factor VIII neutralizing
antibodies)
(320pL) was added to channels of a Haemoscope TEG 5000 (Haemonetics Corp.
Braintree, MA) containing a 40 pL solution of 90 nM thrombin, 9 nM tPA and 0 -
390
nM sTM. After mixing thoroughly, the pin was seated and coagulation and
fibrinolysis
was monitored continuously. The Haemoscope TEG 5000 allows for measurement of
the clot time, clot kinetics, clot strength and clot stability (fibrinolysis)
by measuring the
torque on a wire which is connected to the clot through the pin. As a clot
forms, the
torque on the pin increases and is represented by an increase in the amplitude
(output). Similarly, during fibrinolysis, degradation of the clot results in a
decrease of
the torque and a decrease in the amplitude.
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The inhibitory antibodies (inhibitors) developed by some of the dogs at the
Queen's
University haemophilic dog colony have been described previously by Giles et
al.,
1984; Blood 63:451-456 and by Tinlin et al., 1993; Thromb Haemost 69: 21-24.
The haemophilic dog plasma with inhibitors was drawn from a dog with an
inhibitor titre
of >150 Bethesda Units (>5 B.U. is considered untreatable with Factor VIII
replacement
therapy).
Results
Solulin prolongs clot lysis even at a concentration of 500nM.
In canine hemophilic plasma Solulin dose-dependently increased the clot lysis
time
with an approx. 2-fold prolongation in the presence of 25 nM Solulin, reaching
a
plateau of approx. 9-fold prolongation at 200 - 500 nM Solulin, which
represents the
highest dose tested (see Figure 9).
Conclusion: The pronounced antifibrinolytic effect of Solulin in hemophilic
plasma
even at very high concentrations strongly argues for an efficacious and safe
use of
thrombomodulin analogues in Haemophilia.
Solulin prolongs clot lysis in whole blood from a canine with haemophilia A.
In the thromboelastogram the maximum amplitude of the torsion wire is a
measure of
the maximum clot strength. In these experiments (see Figure 11A) sequential
clot
formation and lysis was induced by transfering canine hemophilic whole blood
into a
cuvette containing 90 nM thrombin and 9 nM rt-PA. Solulin was present at
concentrations of 0 - 390 nM. An amplitude of approx. 85 mm was observed in
the
absence of Solulin. Solulin at concentrations of 100 and 250 nM completely
blocked
clot lysis: Amplitudes were larger and remained unchanged for up to 80 to 90
minutes
(end of experiment). A further increase of Solulin concentration to 390 nM was
still
associated with a larger amplitude and a slower clot lysis compared with
control
experiments (no Solulin) (Figure 11A).
In this experiment the area under the clot-lysis-curve (AUCL) can be used for
quantification of the clot firmness, whereby Solulin at 100 and 250 nM
increased this
parameter by greater than 5-fold (Figure 11 B). Prolongation of clot-"lysis
time in the
same experiments is plotted in Fig. 11 C. Lysis time was increased from 20 min
in the
absence of Solulin to 3 hours at 100 and 250 nM Solulin.
Conclusion: The antifibrinolytic effect of Solulin shown by
thrombelastrography is in
line with the results of the above clot lysis assay. Furthermore Solulin
appears to
increase clot firmness.
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Solulin prolongs clot lysis in whole blood from a canine with haemophilia A in
the presence of anti-Factor VIII antibodies.
These thrombelastography experiments on blood known to contain a high titre of
anti-
Factor VIII antibodies (> 150 Bethesda Units) were performed as described
above:
Sequential clot formation and lysis was induced by transfering canine
haemophilic
whole blood into a cuvette containing 90 nM thrombin and 9 nM rt-PA. Solulin
was
present at concentrations of 0 - 3510 nM. Without Solulin, an amplitude of
approx. 20
mm was observed (see Figure 12A). Solulin dose dependently increased the
amplitudes to approx. 60 mm at concentrations of 100 and 250 nM, reaching an
amplitude of approx. 90 mm at 390 nM.
Furthermore Solulin dose-dependently prolonged the clot-lysis time from
approx. 18
min in the control to approx. 46 min at 390 nM Solulin (see Fig. 12A).
Increased clot
firmness is also indicated by a more than 10fold enhancement of the Area Under
the
Elasticity Curve (0 nM Solulin compared to 390 nM; see Fig. 12B).
Conclusion: The virtually complete inhibition of Factor VIII activity by the
anti-Factor
VIII antibodies reduced but failed to suppress the ability of Solulin to
prolong clot lysis.
Viewed from a different angle, this experiment reveals that the presence of
residual
Factor VIII activity in haemophilic whole blood (absence of antibodies, see
Fig. 1 1A) is
sufficient to augment the efficacy of Solulin to prolong clot-lysis.
This supports the use of the claimed thrombomodulin analogues in haemophilic
patients treated with Factor VIII. Furthermore also patients, who suffer from
haemophilia and still possess functional Factor VIII concentrations could be
treated
with the claimed thrombomodulin analogues. Finally also patients, who are
lacking
Factor VIII or show high titres of anti-Factor VIII antibodies are amenable
for a
treatment with the claimed thrombomodulin analogues.
VIII. EXAMPLE
Ratio of TAFI activation vs. protein C activation for different TM analogues
Several TM analogue as depicted in Figure 19 and given in SEQ ID NO: 5-11 were
generated. The solulin analog with a Phe 376Ala exchange was tested in vitro
for
protein C and TAFI activation in comparison with solulin.
Materials and methods
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Measurement of TM cofactor activity (APC assay)
. A DNA fragment encoding Solulin with the selected mutation was synthesised
and
cloned into a pGAEx vector (a standard HEK293-derived vector). The pGAEx
vector
includes a standard promoter-, leader- and excretion sequence for HEK293 cells
and a
His-6-Tag at the C-terminus of the protein. HEK293 cells were transfected with
the
pGAEx-Solulin vector (transient transfection) and cultured in suspension in a
1 liter
scale. Six days after transfection the protein was harvested by a Ni-HiTrap
column
(linear gradient from 20-500mM Imidazole in PBS, 500 mM NaCl). Target for
purification was the His-6-Tag. After dialysis (16 h against PBS) the proteins
were
analysed. The protein was quantified by Coomassie blue staining of an SDS-
PAGE.
Thereafter, protein identity was verified by Western Blot (labelled anti-His-6-
Antibodies).
Measurement of TM cofactor (APC assay) and TAFI activity
The TM analogues were tested in an assay buffer containing 0 to 2 pM TAFI or
protein
C, respectively, 0.5 nM thrombin, 5 mM calcium chloride and 25nM solulin or
the solulin
Phe376A1a analogue, respectively.
The reactions were quenched with D-phenylalanyl-L-prolyl-L-arginine
chloromethyl
ketone (PPACK) for TAFI experiments or hirudin for protein C experiments.
TAFIa and activated protein C activity was determined using N-(4-
methoxyphenylazoformyl)-Arg-OH (AAFR) and pyroGlu-Pro-Arg-pNA-HCI (S-2366),
respectively. The amount of TAFIa or aPC generated in 10 minutes was
determined by
comparing the rate of substrate hydrolysis to a set of standards. Using the
standards,
we determined the kinetics of TAFI and protein C activation by Ila-sTM.
Results
The exchange of Phe376 against Ala leads to an increased ratio of TAFI vs.
protein C
activation rates, the increase being more pronounced at lower substrate
concentrations
While Michaelis-Menten constants could be determined for the activation of
TAFI (Fig.
15, upper panel), no such constants could be derived for protein C activation
(Fig. 15,
lower panel).
The ratio of activation rates of TAFI and protein C (TAFI/protein C) by
solulin and
Phe376A1a at various substrate concentrations is illustrated in Fig. 16. The
high
TAFI/PC ratio of Solulin could be further improved by the amino acid exchange
from
Phe376 to Ala.
Conclusion:
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The increase in the TAFI/protein C ratio, which can be used as a measure of
TAFI
preference, occurred at relevant substrate (TAFI or protein C) concentrations,
being
most pronounced at the lowest concentration tested (250 nm), which is close to
the
physiological concentrations of both TAFI (- 75 - 220 nM) and protein C (- 70
nM) and
corroborates the contention that both solulin and its variant are suitable for
preferential
TAFI activation. On the other hand, especially the protein C activation by
Phe376AIa
proved to be so slow that it must be considered unlikely that it could enhance
protein C
activation to any appreciable extent at physiological protein C concentrations
(- 70
nM). Thus, the relevant effect of the additional mutation in Phe376AIa can be
seen in
sizable reductions of both the TAFI and protein C activating capabilities,
which still
allow TAFI activation, but practically preclude protein C activation under
physiological
conditions.
Furthermore these results confirm the general importance of single amino acid
mutations and in particular of Phe376AIa since the beneficial effect of this
mutation is
not only observed for a Solulin fragment extending from EGF1 to EGF6 but also
for the
full length soluble thrombomodulin including the N-terminal lectin-like
domain, the six
EGF-domains and the O-linked glycosylation domain.
IX. EXAMPLE
In vivo analysis of fibrinolysis in dog model of haemophilia
Objective of the study
Restitution of factor VIII (fVIIl) is the mainstay of treatment of patients
with haemophilia
A. While providing effective treatment in the majority of cases, a sizable
number of
patients develop neutralizing anti-fVIIl antibodies, a problem which cannot be
overcome
so far with the available adjunctive therapeutic options. Other downsides of
fVIll
therapy include complicated dose determination and the need for intravenous
administration and regular monitoring.
There has been increasing evidence that, besides the dominating failure to
produce
fVlll in sufficient quantities, also an unrestrained activity of tissue
plasminogen activator
(tPA) contributes to the condition. Normally, the growing and maturing clot is
protected
from tPA by activation of the Thrombin-Activatable Fibrinolysis Inhibitor
(TAFI). This is
achieved by a complex formed by thrombin, generated in the process of vascular
lesion
closure, and thrombomodulin, a protein constitutive to the endothelial cell
membrane.
The thrombin/thrombomodulin complex converts a plasma zymogen, TAFI, to its
active
form (TAFIa), which acts by removing carboxyl-terminal lysines and arginines
from clot
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fibrin to impair the ability tPA to bind and, in this conformation, activate
plasminogen.
This control of tPA efficacy is faulty in Haemophilia A, due to the
insufficient thrombin
generation and probably also a decreased availablity of the zymogen, TAFI.
Thus, one
of the hallmarks of Hemophilia A is a premature clot lysis by tPA.
In-vitro work has shown that Solulin is able to correct premature clot lysis
in plasma
from haemophiliacs, partially or entirely, dependent on the concentration of
tPA. It is
the aim of the present study to test whether the in vitro efficacy can be
confirmed in
vivo, using the well-established model of haemophilic dogs. Pilot experiments
have
ascertained that haemophilic dog plasma, like human plasma, allows Solulin to
correct
premature clot lysis, justifying the use of this animal model for this
purpose.
Study substance
Identification: Solulin
Physical state: clear colorless solution, pH 7.0
Dissolution buffer : sodium phosphate, 13.4; potassium chloride, 2.7; sodium
chloride, 137 (all in mmol/I); mannitol, 5%
Content of vial: 3 mg Solulin in 1 ml (3 mg/ml)
Vial handling: Use ethanol to wipe the vial septum, then insert a sterile
needle through
the septum to draw the required volume. Store vials with residual volume in
the
refrigerator.
Storage conditions and time: To be stored in the refrigerator at 2-6 C.
Residual
volumes can be kept in the refrigerator for 28 days. Within this period, they
can be used
for further experiments, but it is recommended to use separate vials for each
dog.
Preparation of dosing formulations
The appropriate volume is taken from the original Solulin vial(s) and
complemented
with 0.9 % NaCl to achieve a dosing volume of 3 ml.
The final dose formulations will be prepared freshly for each experimental
day.
Animals: Species and maintenance
The study was conducted in dogs with haemophilia A (haemophilia A dogs)
characterized as "cured" FVIII gene therapy dogs. After canine FVIII gene
therapy,
these dogs had undetectable levels of plasma FVIII (< 1%) but showed
shortening of
their whole blood clot times and did not experience further spontaneous
bleeding
episodes for at least.3 years after treatment (normal frequency approximately
5/yr).
These dogs may be regarded as showing partial phenotypic correction after gene
transfer. They best recapitulate the clinical picture documented in
approximately 10%
of severe haemophilia A patients (plasma FVIII < 1%) who rarely bleed. Such a
profile
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also mirrors the outcome of a low dose FVIII prophylaxis protocol. The age and
body
weight of the animals were approximately 3 years and 10-15 kgs, respectively.
Except
for the haemophilia trait, only healthy animals were included into the study.
Experimental procedures
Pilot study (Pharmacokinetics and in vitro efficacy)
As a first step, the pharmacokinetics of Solulin was explored in haemophilic
dogs
together with pharmacodynamic effects on coagulation/fibrinolysis. Based on in
vitro
efficacy data available on dog plasma and pharmacokinetic modelling, Solulin
was
administered at a dose of 0.5 mg/kg in a volume of 3 mL by intravenous bolus
injection.
According to the results obtained in a first dog, a second dog was treated the
same
way or at an adapted dose of Solulin. Thereafter, a decision was made on
initiation of
the main study or further exploration of pharmacokinetics.
Preparation of blood samples
Two different sets of blood samples were taken from contralateral legs (see
Table
below).
Full blood is harvested on citrate buffer (final volume: 1 part of 3.13 %
sodium citrate
mixed with 9 parts of blood) and processed immediately (see preparation
below).
Unless assayed in close temporal relationship as indicated for the individual
splits, they
are adequately labeled and stored at -80 C until further use.
Samples for
Time relative to
dosing (min) TEG, whole blood clotting, Thrombin Generation
PK,TAFIa, PT/aPTT Potential
Sample 1
Sample 2
Blood volume; Blood volume;
drawn and splitted for whole drawn from contralateral
blood assays and plasma leg and processed
generation separately
Predose 14 ml 1 ml*
0: Dosing
30 14 ml 1 ml*
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60 14 ml
120 14 ml 1 ml*
480 14 ml
* preceded by'1 ml, which is discarded
Sample 1
The first milliliter to be discarded was taken at the times indicated in the
table above,
citrated, and split in five parts.
14 mis of citrated whole blood were portioned for split I (thrombelastography
x 5 mis
whole blood) and split 2 (whole blood clotting; 2 mis whole blood).
The remaining volume was centrifuged at 1500 g for 15 minutes. The resulting
supernatant was split into three portions (split 3, Solulin concentration, 0.5
ml plasma;
split 4, activated TAFI, 0.5 ml plasma; split 5, PT/aPTT, 0.5 ml plasma and
stored at
-80 C until further use.
Sample 2:
Sample 2 (2 ml; the first ml. to be discarded) was taken from the leg
contralateral to the
sample 1 side and citrated. Sampling times are indicated in the table above.
This sample is for determination of thrombin generation potential and needs
special
precautions. It is important to avoid contact activation, which means that the
sample
should be drawn with the widest needle possible (preferably not from a
butterfly
needle).
The required volume of plasma is 0.5 ml. There is a detailed description of
plasma
preparation for thrombin generation potential:
1st centrifugation step:
o Put in the following centrifugation parameters:
- Break power: 9
- Rotor velocity 3790 at r=156 mm
- duration: 5 minutes.
o Centrifuge the sample for 5 minutes at 2500 g
(for the Hettich Rotina 35 R this is reached at the setting 3790 rpm at r=156
mm)
o Use a plastic disposable Pasteur pipette to transfer the plasma supernatant
to a
labelled plastic centrifuge tube. Be careful not to suck up any cells from the
buffycoat, leave 0.5 cm plasma on top of the buffy coat.
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2nd centrifugation step:
o Apply the following centrifugation parameters:
- Temperature: 18 C
- Centrifugation velocity: 11000 RPM at r=156 mm
- Duration: 10 minutes
o Centrifuge the plasma for 10 minutes at 10000 g.
(for the Universal 30 RF this is reached at the setting 11000 rpm at r=156 mm)
o Pour or pipette in one movement the content of the centrifuge tube into a
labelled
tube (to avoid mixing of sediment with supernatant).
o When applicable, combine the content of several identical plasmas into one
large
plastic tube.
Pharmacokinetic and pharmacodynamic assays
Thrombelasto_graphy. Thrombelastography was performed in citrated whole blood.
340
pl of whole blood are added to channels of a Haemoscope TEG 5000 (Haemonetics
Corp. Braintree, MA) containing a 20 pL solution of 1/15000 dilution (18X) of
Innovin,
270mM CaCIZ and 18nM tPA. After mixing thoroughly, the pin was seated and
coagulation and fibrinolysis were monitored continuously. The Haemoscope TEG
5000 allows for measurement of the clot time, clot kinetics, clot strength and
clot
stability (fibrinolysis) by measuring the torque on a wire which is connected
to the clot
through the pin.
Whole Blood Clotting Time: Whole blood clotting time was determined by
incubating
citrated whole blood at 37 C in a glass test tube with continuous monitoring
for clot
formation.
Pharmacokinetics. Plasma concentrations were determined using an ELISA
validated
for dog plasma. This study will be performed under the responsibility of the
sponsor.
Results will be integrated in the final report.
Activated TAFI. TAFIa was determined using the methods of Kim et al. (Anal
Biochem.
(2008), 372(1):32-40). Briefly, fibrin degradation products containing a
covalently
attached quencher moiety (QSY-FDP) bind to fluorescently labelled plasminogen
(F-
Pg). When F-Pg is bound to QSY-FDP, the fluorescence is quenched. When TAFIa
is
added, F-Pg binding sites are removed and F-Pg is released from QSY-FDP
resulting
in an increase in fluorescence. The rate of this fluorescence increase is
proportional to
the TAFIa concentration. Using plasma spiked with known concentrations of
TAFIa a
standard curve was developed and used to determine the TAFIa concentration in
plasma samples.
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Coagulation assays. Diluted PT (dPT) and aPTT assays have been performed. The
dilute PT assay involve a 1/5 dilution of test platelet poor plasma. TriniCLOT
PT HTF
thromboplastin reagent was added to the plasma and then following a 180 sec
incubation, the sample is re-calcified. The aPTT was performed with TriniCLOT
Automated APTT Reagent. Again, a 180 sec incubation is performed and then the
sample is re-calcified. All studies were performed on a Coag-A-Mate MAX
(bioMerieux)
automated coagulometer.
Endogenous thrombin potential. Endogenous thrombin potential was determined
using
the Calibrated Automated Thrombogram (CAT) assay in a 96-well plate
fluorimeter.
The CAT assay is a proprietary method of CoagScope B.V. (Maastricht, The
Netherlands).
Briefly, the CAT assay measures the concentration of thrombin in clotting
plasma over
time. Citrated plasma is triggered with PPP reagent that contains Tissue
Factor and
phospholipids. The plasma is recalcified with another reagent containing
Calcium
Chloride and a fluorogenic Substrate (FluCa). This Substrate is converted by
thrombin
into a fluorophore, the fluorescence being followed in a fluorometer.
Simultaneously
another sample of the same plasma is measured in the presence of Thrombin
Calibrator (a calibrated amount of thrombin activity). Comparing the results
of these
parallel measurements allows the calculation of thrombin concentration over
time.
Main study (Cuticle bleeding assay)
Animal number, study drug and dose level
Three haemophilic dogs were studied to test the effect of Solulin on blood
coagulation
and fibrinolyis. Solulin was administered by intravenous bolus injection at a
dose
selected according to the pilot study described under 8.1.
Anesthesia and supportive medications
Premedication: Hydrocortisone 100 mgs and Benadryl 50 mgs IV
Maintenance: 1-2% Isoflurane
Pain management post-surgery: Buprenorphine
Time course and procedures
The course of the experiment is visualized in the table below. 15 min after
anaesthesia,
a baseline cuticle test is performed, followed by a 15 min observation period.
Thereafter, nominally 30 min post-anaesthesia, the dogs receive an intravenous
bolus
injection of Solulin, based on the actual body weight. After a 30 min exposure
to
Solulin, a second cuticle assay is performed on a contralateral paw. With the
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subsequent observation period of 15 min, the total time of anaesthesia is
approximately
75 min.
For the cuticle bleeding test, the dogs are placed in the lateral recumbency
position,
and all hair around the nail bed is carefully removed by clipping around
the.base of the
claw to be used for the cuticle bleeding time assay. Silicone grease is
applied to the
claw to prevent blood tracking back underneath the nail. The apex of the
cuticle is
visualized, and the nail severed proximal to the dorsal nail groove using a
spring-
loaded sliding blade guillotine clipper. The animal's paw is subsequently
positioned
over the edge of the operating table and blood from the severed cuticle
allowed to fall
freely. The number of blood drops in each of the subsequent 15 minutes is
recorded
and converted to a cumulative score for the 15 minutes. After 15 minutes of
observation, if the cuticle is still bleeding, the site of injury is
cauterized by topical
application of silver nitrate.
All assays are performed by the same experienced veterinary technologist.
Results and Discussion
Dogs with severe haemophilia A were studied in vivo. Led by the solulin
concentrations
of several hundred nM found effective in the in vitro experiments presented in
example
VII, a first dog was injected with a dose of 500 ug/kg to see whether the
improvement
in the thrombelastogram observed after in vitro addition of solulin could be
confirmed in
vivo. Contrary to expectations, this dose was not only ineffectve, it even
severely
inhibited clot formation and strength (Fig. 17). This effect was manifest 30
min post-
administration and sustained at 2 hours.
At the end of the day, the decrease in clot formation was still evident, but
the inhibition
seemed to slowly subside. As the 24 hour test was technically flawed, the
experiment
could be considered a failure. Even so, it was decided to draw a final sample
at 48
hours to see whether the animal had recovered. This seemed to be the case,
but, to
some surprise, there seemed to be even a trend to a slight improvement over
the
baseline thrombelastogram. Encouraged by this notion, a further sample was
investigated 72 hours post solulin and, much unexpected, a clear improvement
in clot
formation and strength was recognized. This led to the conclusion that much
lower
doses than anticipated from the in vitro studies were effective and even
required, as
larger doses establish higher plasma concentrations, which then would
deteriorate clot
properties, a hypothesis that was tested in a further severely haemophilic dog
(Fig 18).
The second dog received a much lower dose of solulin (10 ug/kg) than the first
one.
This experiment confirmed the hypothesis: A higher plasma concentration was
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ineffective at improving clot properties, whereas concentrations as low as 0.2
nM were
able to improve it.
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Figure legends
Fig. 1: Clot-lysis profiles and lysis times of factor VIII deficient plasma
(FVIII-DP),
normal plasma (NP) and FVIII-DP mixed with NP. Clot lysis profiles are shown
for 0 (-
), 1 (====), 6 (---), 10 (-==-==-), 50 (---) and 100% (-=-=-) NP. From the
clot-lysis
profiles, the lysis time was determined by taking the time at which the clot
has been
degraded to one half of its highest optical density. In the inset, the lysis
times are
summarized, with the general trend being an increase in lysis time as the
percentage of
NP (and consequently amount of FVIII) is increased. The effect of adding NP on
lysis
time reaches a plateau at 10% NP.
Fig. 2: Thrombin activatable fibrinolysis inhibitor (TAFI) activation in
plasma containing
various percentages of FVIII: (A) When FVIII-deficient plasma (FVIII-DP) is
mixed with
normal plasma (NP) TAFI activation is enhanced. In FVIII-DP only 30 pM TAFIa
was
measured at its peak (=) compared with -600 pM TAFia in 50% NP (0) and 100%NP
(A). These experiments were conducted in triplicate and the data represent the
mean
SE. The TAFIa potential (B), defined here as the area under the time course of
activation plot (A) from the time of clot initiation to the last time point,
increases as the
percentage of NP increases to a plateau at 50% NP. The TAFIa potentials of 50%
NP
and 100% NP are similar (14,100 pM min and 16,800 pM min, respectively)
despite the
shape of their respective TAFI activation plots being quite different. The
relationship
between lysis time (Fig. 1, inset) and TAFia potential, as it relates to FVIII
levels, is
presented (Fig. 2B, inset) using a plot of log lysis time vs. log TAFia
potential. As
expected, the data show a strong positive correlation between lysis time and
TAFIa
potential in plasma containing 0 - 100% FVIII.
Fig. 3: Prothrombin activation in plasma containing various percentages of
FVIII. The
time course of prothrombin activation is shown for FVIIIDP mixed with 0 (=), 1
(^), 6
(A), 10 (0), 50 (0) and 100% NP (Li). Generally, the rate of prothrombin
activation
increases as the percentage of NP increases. At 50% NP prothrombin activation
occurs at a high rate (as determined by examining the slope of each plot) and
appears
to be over within 15 min, whereas 100% NP has a slower rate of prothrombin
activation
over a longer time period.
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Fig. 4 A-D: The effect of sTM on thrombin activatable fibrinolysis inhibitor
(TAFI)
activation in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP)
at various
concentrations of both sTM (0 - 100 nM) and tPA (0.25nM in Figure 4A, 0.75 nM
in
Figure 4B, 1.5 nM in Figure 4C, and 3 nM in Figure 4D). The TAFIa-dependent
defect
in prolonging lysis in FVIII-DP is corrected by the addition of 100 nM sTM to
plasma
containing 0.25 nM tPA. As the concentration of tPA is increased only partial
correction
of the lysis defect is observed in FVIII-DP in the presence of 100 nM sTM. In
these
experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create
a
condition in which there is no functional TAFIa. Therefore, any increase in
lysis, as
presented by the ratio lysis time/lysis time + PTCI is TAFIa dependent.
Fig. 5: TAFI activation and clot lysis profiles in normal plasma (NP) (A) and
FVIII-
deficient plasma (FVIII-DP) (B) in the presence of 10 nM thrombomodulin (=) or
without
thrombomodulin (o). The accompanying clot-lysis profile is shown (-) and the
clot lysis
profile for no sTM is shown as a reference (---).These experiments were
conducted in
triplicate and the data represents the meant SE.
Fig. 6: Thrombin binding to thrombomodulin. Binding of thrombin. to
thrombomodulin was determined by titrating 1.5 ml of a solution composed of
thrombin
(20 nM) and DAPA (20 nM) in 20 mM Tris-HCI, 150 mM NaCl, 5.0 mM Ca2+, 0.01%
Tween 80 with 1.54 pM thrombomodulin in an identical solution. Fluorescence
intensity
was measured (A,ex = 280 nm, Xem = 545 nm).
Fig. 7: Relative cofactor activities of point mutants in TAFI and protein C
activation. Alanine-scanning mutagenesis was used to prepare point mutations
in
soluble thrombomodulin. Rates of protein C and TAFI activation (relative to
the rate of
activation with mutant TMEM388L) are shown for TAFI (solid bars) and protein C
(hatched bars).
Fig. 8: Mutations of the interdomain loop between EGF4 and EGF5. Three
independent plasmids were constructed in E.coli for each mutant. Shockates
were
prepared, assayed for cofactor activity by the APC assay, and samples were
analysed
on Western blots (not shown). Activity values are the average from three
separate
clones. Panel A, substitution mutants at Gln387; panel B, substitution at
Met386; panel
C, substitution mutants at Phe389; panel D, deletions and alanine insertions
in the
interdomain loop. The activity measured for shockates from E.coli transfected
with the
control plasmid, pSelect, lacking the TM insert is shown. See Clarke et al.
(J. Biol.
Chem. 1993; 268:6309-6315) for additional details.
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Fig. 9: Schematic diagram of the pro- and antifibrinolytic effects of
thrombomodulin (modified after Mosnier and Bouma, Arterioscler. Thomb. Vasc.
Biol.
2006; 26: 2445 - 2453). The increase in clot lysis time at low TM
concentrations is
attributable to stimulation of TAFI activation and illustrates the
antifibrinolytic activity of
TM. At higher concentrations of the rabbit lung TM the clot lysis time
decrease because
of the activation of protein C and inhibition of TAFI activation; illustrating
the
profibrinolytic activity of rabbit lung TM (solid line). Note that above 15 nM
the
profibrinolytic activity of rabbit lung TM exceeds the antifibrinolytic
activity resulting in
an overall profibrinolytic effect. In contrast the soluble TM analogue shows
only an
antifibrinolytic effect (dashed line).
Fig. 10: Relationship between TAFIa-dependent Fold propagation of clot lysis
and added Solulin. The dose-dependent increase in clot lysis time is
attributable to
stimulation of TAFI activation and illustrates the antifibrinolytic activity
of TM.
Fig. 11A: Solulin-dependent change in clot appearance in whole blood from a
canine with haemophilia A (as measured by thrombelastrography)
Fig. 11B: Solulin-dependent increase of clot firmness in whole blood from a
canine with haemophilia A. The area under the clot-lysis curve was measured by
thrombelastrography as a function of Solulin (sTM) concentration.
Fig. 11C: Solulin-dependent dependent prolongation of lysis in whole blood
from
a canine with haemophilia A. The clot-lysis time as measured by
thrombelastography
as a function of Solulin (sTM) concentration.
Fig. 12A: Solulin-dependent prolongation of lysis in whole blood from a canine
with hemophilia A and inhibitory anti-factor VIII antibodies (as measured by
thrombelastrography)
Fig. 12B: Solulin-dependent increase of clot firmness in whole blood from a
canine with haemophilia A in the presence of inhibitory anti-factor VIII
antibodies. The area under the clot-lysis curve was measured by
thrombelastrography
as a function of Solulin (sTM) concentration.
Fig. 13: Figure showing Table 1: Summary of the data used to construct Fig. 4,
including the absolute lysis time in the presence of PTCI to enable
determination of the
lysis time under each condition. In all cases, the lysis time is expressed
relative to that
obtained in the presence of the TAFIa inhibitor, PTCI. TAFI, thrombin
activatable
fibrinolysis inhibitor; PTCI, potato tuber carboxypeptidase inhibitor.
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Fig. 14: Figure showing Table 2: Chloramine T oxidation of site-specific
mutant
analogues of TME (Sf9). The results after chloramine T treatment were
expressed as
a percentage of the activity after control treatment. *Average of duplicate
determinations and deviation from the mean.
Fig. 15: Activation rates of TAFI (upper panel) and protein C (lower panel) by
solulin
and the solulin mutant, Phe376AIa (both present at 25 nM) at rising substrate
concentrations.
Fig. 16: Ratio of TAFI activation vs. protein C activation for Solulin (black
bar)
compared to a Solulin analogue with Phe376AIa mutation ((light grey bar) as
determined at TM concentrations of 0.25, 0.5, 0.75, 1, 1.5 and 2 NM. The most
right
bars indicate the ratios combined from easurements at all concentrations.
Fig. 17: Thrombelastography of blood samples from a dog with severe
haemophilia A.
Samples were taken at different times after intravenous injection of solulin
at a dose of
500 pg/kg.
Fig. 18: Bell-shaped concentration dependence of improvement of clot formation
and
stability in the blood from a severely haemophilic dog. Solulin was injected
at a dose of
pg/kg and plasma samples were taken at the indicated times for determination
of
the thrombelastogram. At each thrombelastogram, time of sampling and plasma
concentration of solulin are indicated.
Fig. 19: Overview on TAFI-specific thrombomodulin analogues
Table legends
Table 1: Summary of the data used to construct Fig. 4, including the absolute
lysis
time in the presence of PTCI to enable determination of the lysis time under
each
condition. In all cases, the lysis time is expressed relative to that obtained
in the
presence of the TAFIa inhibitor, PTCI. TAFI, thrombin activatable fibrinolysis
inhibitor;
PTCI, potato tuber carboxypeptidase inhibitor.
Table 2: Chloramine T oxidation of site-specific mutant analogues of TME (Sf9)
The results after chloramine T treatment were expressed as a percentage of the
activity after control treatment. *Average of duplicate determinations and
deviation from
the mean.
