U.S. patent application number 15/146313 was filed with the patent office on 2016-11-03 for compounds suitable for treatment of haemophilia.
The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Gert Bolt, Jesper Haaning, Jens Jacob Hansen, Ole Hvilsted Olsen, Ditte Maria Karpf, Marianne Kjalke, Maj Petersen, Frederik Rode, Kirstine Roepstorff, Lars Thim.
Application Number | 20160318991 15/146313 |
Document ID | / |
Family ID | 48573612 |
Filed Date | 2016-11-03 |
United States Patent
Application |
20160318991 |
Kind Code |
A1 |
Bolt; Gert ; et al. |
November 3, 2016 |
Compounds Suitable for Treatment of Haemophilia
Abstract
The present invention relates to VWF compounds as well as
compositions suitable for treatment of blood clotting diseases.
Inventors: |
Bolt; Gert; (Vaerloese,
DK) ; Karpf; Ditte Maria; (Veksoe Sjaelland, DK)
; Rode; Frederik; (Hedehusene, DK) ; Haaning;
Jesper; (Birkeroed, DK) ; Roepstorff; Kirstine;
(Ballerup, DK) ; Thim; Lars; (Gentofte, DK)
; Petersen; Maj; (Vaerloese, DK) ; Kjalke;
Marianne; (Frederikssund, DK) ; Hvilsted Olsen;
Ole; (Broenshoej, DK) ; Hansen; Jens Jacob;
(Jyllinge, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
48573612 |
Appl. No.: |
15/146313 |
Filed: |
May 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14396907 |
Oct 24, 2014 |
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PCT/EP2013/055106 |
Mar 13, 2013 |
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15146313 |
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61641434 |
May 2, 2012 |
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61752612 |
Jan 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0019 20130101;
C07K 14/755 20130101; A61P 7/04 20180101; A61K 38/37 20130101 |
International
Class: |
C07K 14/755 20060101
C07K014/755; A61K 9/00 20060101 A61K009/00; A61K 38/37 20060101
A61K038/37 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 2012 |
EP |
12165301.8 |
Jan 9, 2013 |
EP |
13150576.0 |
Claims
1. A VWF fragment comprising up to 1200 amino acids, wherein said
VWF fragment comprises the TIL' domain.
2. A VWF fragment according to claim 1, wherein said fragment
comprises the TIL' and the E' domains.
3. A VWF fragment according to claim 1, wherein said VWF fragment
comprises one or two amino acid substitution(s) of the 1099 and/or
1142 cysteine(s).
4. A VWF fragment according to claim 1, wherein less than 5% of
said VWF fragment are in the form of oligomers and/or
multimers.
5. A VWF fragment according to claim 1, wherein said VWF fragment
is part of a dimer.
6. A VWF fragment according to claim 1, wherein said VWF fragment
is a monomer.
7. A VWF fragment according to claim 1, wherein said fragment
comprises an amino acid sequence selected from the list consisting
of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO:
21.
8. A VWF fragment according to claim 1, wherein said fragment
comprises SEQ ID NO: 9, wherein the 1099 cysteine residue is
substituted with another amino acid.
9. A VWF fragment according to claim 8, wherein the 1099 cysteine
residue is substituted with serine.
10. A VWF fragment according to claim 1, wherein said fragment
comprises an amino acid sequence selected from the list consisting
of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, wherein the
1099 and the 1142 cysteine residues are substituted with another
amino acid.
11. A VWF fragment according to claim 10, wherein the 1099 and the
1142 cysteine residues are substituted with serine.
12. A pharmaceutical composition comprising: (i) a VWF fragment
according to claim 1; and (ii) a FVIII molecule.
13. A pharmaceutical composition according to claim 12, wherein
said FVIII molecule comprises a truncated B domain at a size of
5-700 amino acids.
14. A pharmaceutical composition according to claim 12, wherein
FVIII is a B domain truncated variant, wherein the amino acid
sequence of said truncated B domain is derived from the wt FVIII B
domain amino acid sequence as set forth in SEQ ID NO: 1.
15. A pharmaceutical composition according to claim 14, wherein
said B domain comprises an O-glycan linked to the Ser 750 amino
acid residue as set forth in SEQ ID NO: 1.
16. A pharmaceutical composition according to claim 12, wherein
said FVIII molecule is conjugated with at least one half-life
extending moiety.
17. A pharmaceutical composition according to claim 12, wherein at
least one half life extending moiety is covalently attached to an
O-glycan present in the FVIII B domain.
18. A pharmaceutical composition according to claim 12, wherein the
bioavailability of said FVIII molecule is at least 5% following
subcutaneous administration.
19. A pharmaceutical composition according to claim 12, wherein the
molar ratio between FVIII and VWF is 1:1.
20. A pharmaceutical formulation according to claim 12, wherein the
concentration of FVIII is at least 500 IU/ml.
21. A pharmaceutical formulation according to claim 12, wherein the
amount of FVIII bound to VWF fragment is at least 70% of the total
amount of FVIII in said formulation.
22. A pharmaceutical composition according to claim 12 for use in
treating haemophilia, wherein said pharmaceutical composition is
for subcutaneous administration.
23. A pharmaceutical composition wherein said composition comprises
a VWF fragment, wherein the amino acid sequence of said VWF
fragment is selected from the list consisting of: SEQ ID NO: 4, SEQ
ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,
SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
24. A pharmaceutical composition according to claim 23 for use in
treatment of von willebrand disease by intravenous or subcutaneous
administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/396,907, filed Oct. 24, 2014 (Pending), which is a 35 U.S.C.
.sctn.371 National Stage application of International Application
PCT/EP2013/055106 (WO 2013/083858), filed Mar. 13, 2013, which
claimed priority of European Patent Application 12165301.8, filed
Apr. 24, 2012 and European Patent Application 13150576.0, filed
Jan. 9, 2013; this application claims priority under 35 U.S.C.
.sctn.119 of U.S. Provisional Application 61/641,434; filed May 2,
2012 and U.S. Provisional Application 61/752,612; filed Jan. 15,
2013; the contents of which are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 12, 2013 (modified on May 2, 2016), is named
8490US02_Sequences_ ST25.txt and is 144,232 bytes in size.
TECHNICAL FIELD
[0003] The present invention relates to treatment and/or
prophylaxis of haemophilia.
BACKGROUND
[0004] Protein replacement therapy by intravenous administration of
coagulation factors is currently used for treating patients
suffering from haemophilia. For patient convenience and compliance,
extravascular (e.g. subcutaneous (s.c.) or intradermal)
administration would be preferable to the existing intravenous
(i.v.) injections. There are furthermore potential safety
advantages associated with extravascular administration, since many
patients could avoid intravenous port surgery as well as the risk
of infection and clots associated with insertion of such
catheters.
[0005] S.c. administration of FVIII in FVIII deficient mice is
disclosed in Shi et al, Haemophilia, 2012, DOI:
10.1111/j.1365-2516.2011.02735.x. The bioavailability of FVIII is
herein reported to be low (about 1%).
[0006] S.c. administration of FVIII and VWF is furthermore
disclosed in WO08151817 but no dose response relationship between
the FVIII dose and the achieved circulating FVIII concentration is
disclosed. In WO815817, the (Unit) ratio of VWF over FVIII was
larger than 5:1, corresponding to a 150-250 fold molar excess of
the concentration of VWF protein as compared to that of FVIII. From
a practical and economical pint of view, this type of ratios are,
however, not desirable. In WO08151817, it is furthermore shown that
the immunogenicity in mice of s.c. administered FVIII is
significantly reduced when FVIII is co-formulated with VWF.
[0007] In WO10062768, it is disclosed that PEGylation of FVIII can
improve the bioavailability of FVIII in connection with
subcutaneous injection into mice, whereas co-formulation with VWF
does not improve the bioavailability of FVIII.
[0008] There is a need in the art for compounds and/or
pharmaceutical compositions suitable for extravascular
administration in treatment and/or prophylaxis of patients
suffering from blood clotting diseases such as haemophilia A with
or without inhibitors, and/or von Willebrand disease, as such
administration forms would alleviate the burden of i.v. treatment
both related to the infusion as such and also the risk of
infections due to implanted portable catheters. Such compounds and
compositions are preferably safe (i.e. have a low risk of
immunogenicity) and/or have a high bioavailability and/or are
preferably easy to handle in connection with production and
formulation processes.
SUMMARY
[0009] The present invention relates to a recombinant VWF fragment
comprising 1200 amino acids or less, such as e.g. the TIL' domain
or the TIL'/E' domain (Zhou et al. Blood 2012; 120(2): 449-458).
The present invention furthermore relates to a pharmaceutical
composition comprising: (i) a VWF fragment according to the
invention and (ii) FVIII molecule (full-length/truncated B
domain/conjugated). The present invention furthermore relates to
use thereof for treatment of haemophilia, e.g. by extravascular
administration. Such compounds and compositions will preferably
result in a relatively high FVIII bioavailability and/or a
relatively low risk of FVIII immunogenicity in connection with
extravascular co-administration of FVIII.
DESCRIPTION
[0010] In one aspect of the invention, VWF fragments according to
the invention co-administered with FVIII molecules having a
prolonged in vivo circulatory half-life have a surprisingly high
bioavailability in connection with extravascular (e.g. s.c.)
administration thereof.
[0011] The inventors of the present invention have furthermore made
the surprising observation that bioavailability of FVIII molecules
may be significantly improved upon extravascular co-administration
with similar molar amounts of VWF fragments according to the
invention. Alternatively, high bioavailability may be achieved
through extravascular co-administration of a pool of FVIII
molecules, wherein the majority of said FVIII molecules are bound
to VWF fragments according to the invention. Interestingly, full
length VWF does not have a positive impact on the bioavailability
of FVIII. Preferably, VWF should be in the form of a VWF fragment
that comprise the TIL' or the TIL'/E' domains. Compounds and
compositions according to the present invention are thus useful for
treatment and prophylaxis of haemophilia patients (in particular
haemophilia A patients) with and without inhibitors, as well as for
immune tolerance induction (ITI) of haemophilia patients with
inhibitors.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1: FVIII activity in plasma after subcutaneous
administration of 10000 U/kg "N8-GP" with or without
co-administration of 7.7 times the molar dose of VWF TIL'/E'/D3/A1
relatively to N8-GP. Data are mean and standard deviation of
measurements from n=2 FVIII KO mice per time point. "N8-GP" is a
glyco-PEGylated FVIII molecule produced as described in Examples
1+2 in WO2009108806.
[0013] FIG. 2: FVIII antigen in plasma after subcutaneous
administration of 10000 U/kg N8-GP with or without
co-administration of 7.7 times the molar dose of VWF TIL'/E'/D3/A1
relatively to N8-GP. Data are mean and standard deviation of
measurements from n=2 FVIII KO mice per time point
[0014] FIG. 3: FVIII activity in plasma after subcutaneous
administration of 2500 U/kg N8-GP with or without co-administration
of 7.7 times the molar dose of VWF TIL'/E'/D3/A1 relatively to
N8-GP. Data are mean and standard deviation of measurements from
n=2 FVIII KO mice per time point
[0015] FIG. 4: FVIII antigen in plasma after subcutaneous
administration of 2500 U/kg N8-GP with or without co-administration
of 7.7 times the molar dose of VWF TIL'/E'/D3/A1 relatively to
N8-GP. Data are mean and standard deviation of measurements from
n=2 FVIII KO mice per time point.
[0016] FIG. 5: FVIII activity in plasma after subcutaneous
administration of 5000 or 20000 IU/kg wt FVIII (N8, turoctocog
alfa) with or without co-administration of 7.7 times the molar dose
of VWF TIL'/E'/D3/A1 relatively to FVIII, respectively. Data are
mean and standard deviation of measurements from n=2 FVIII KO mice
per time point. "N8"/"turocotog alfa" is a B domain truncated FVIII
molecule produced as described in Example 1 in WO2009108806.
[0017] FIG. 6: FVIII antigen in plasma after subcutaneous
administration of 5000 or 20000 IU/kg wt FVIII (N8, turoctocog
alfa) with or without co-administration of 7.7 times the molar dose
of VWF TIL'/E'/D3/A1 relatively to FVIII. Data are mean and
standard deviation of measurements from n=2 FVIII KO mice per time
point.
[0018] FIG. 7: FVIII antigen in plasma after subcutaneous
administration of 5000 IU/kg FVIII (N8, turoctocog alfa) with or
without co-administration of 7.7 times the molar dose of VWF
TIL'/E'/D3/A1 relatively to FVIII. Data are mean and standard
deviation of measurements from n=2 FVIII KO mice per time
point.
[0019] FIG. 8: FVIII activity in plasma after subcutaneous
administration of 5000 IU/kg FVIII (N8, turoctocog alfa) with or
without co-administration of 7.7 times the molar dose of VWF
TIL'/E'/D3/A1 relatively to FVIII. Data are mean and standard
deviation of measurements from n=2 FVIII KO mice per time
point.
[0020] FIG. 9: VWF variant (764-865 SEQ ID NO:5) binding to FVIII
(N8, turoctocog alfa) at 20.degree. C. The upper panel shows raw
data of heat released upon each titration. Lower panel shows
binding isotherm obtained from integrating raw data. Data analysis
shows that VWF variant (SEQ ID NO:5) binds to FVIII in an
exothermic reaction with a stoichiometry of 1.14, .DELTA.H of -5.82
kcal/mole, .DELTA.S of 9.8 cal/mol/deg and a K.sub.d of 0.33 .mu.M.
"F8/N8/turoctocog alfa" is a B domain truncated FVIII molecule
produced as disclosed in Example 1 in WO2009108806.
[0021] FIG. 10: s.c. administrated N8-GP is haemostatic effective
in vivo. The left panel shows blood loss in FVIIIKO mice treated
s.c. with N8-GP or vehicle 24 hr before tail transection, or i.v. 5
min before tail transection. N8-GP'' is a glyco-PEGylated FVIII
molecule produced as described in Examples 1+2 in WO2009108806. The
right panel shows clot times in whole blood from the mice ex vivo
using ROTEM.
[0022] FIG. 11: SEC-UV (280 nm) chromatograms for FVIII,
TIL'/E'/D3/A1 III, and a mixture of FVIII and TIL'/E'/D3/A1 III in
155 mM NaCl, 10 mM Calciumacetat, 10% Isopropanol at 25.degree.
C.
[0023] FIG. 12: SEC-UV (280 nm) chromatograms for FVIII, TIL'/E'/D3
II, and a mixture of FVIII and TIL'/E'/D3 II in 155 mM NaCl, 10 mM
Calciumacetat, 10% Isopropanol at 25.degree. C.
DEFINITIONS
[0024] The term "treatment", as used herein, refers to the medical
therapy of any human or other vertebrate subject in need thereof.
Said subject is expected to have undergone physical examination by
a medical practitioner, or a veterinary medical practitioner, who
has given a tentative or definitive diagnosis which would indicate
that the use of said specific treatment is beneficial to treating a
disease in said human or other vertebrate. The timing and purpose
of said treatment may vary from one individual to another,
according to the subject's health. Thus, said treatment may be
prophylactic, palliative, symptomatic and/or curative.
[0025] Mode of administration: Compounds and pharmaceutical
compositions according to the invention may be administered
parenterally, such as e.g. intravenously or extravascularly (such
as e.g. intradermally, intramuscularly, subcutaneously, etc).
Compounds and pharmaceutical compositions according to the
invention may be administered prophylactically and/or
therapeutically and/or on demand. According to the present
invention, several advantages are associated with extravascular
administration of compounds/pharmaceutical compositions according
to the present invention. Extravascular administration is easier,
simpler, and associated with less pain, inconvenience, and
complications (and thus potentially resulting in better compliance)
which is of potential benefit to all patients but of particular
benefit for children and small infants.
[0026] Combination treatments/co-administration: Combined
administration of two or more active compounds (e.g. FVIII and
VWF/VWF fragments according to the invention having the ability to
bind to FVIII) may be achieved in a number of different ways. In
one embodiment, the two active compounds may be administered
together in a single composition. In another embodiment, the two
active compounds may be administered in separate compositions as
part of a combined therapy. For example, the first compound may be
administered before, after, or concurrently with the second
compound. In case FVIII and VWF fragment are administered
extravascularly (e.g. subcutaneously) as two separate
pharmaceutical compositions, they are preferably administered in
close proximity in order to benefit from the improved
bioavailability that can be obtained when administering these two
types of compounds together (i.e. the injection sites should be
separated by no more than 5 cm, preferably no more than 4 cm,
preferably no more than 3 cm, preferably no more than 2 cm, and
most preferably no more than 1 cm). The two compounds should
preferably also be injected within about an hour, preferably within
about 30 minutes, preferably within about 15 minutes, and most
preferably within about 5 minutes.
[0027] Factor VIII: Factor VIII (FVIII) is a large, complex
glycoprotein that is primarily produced by hepatocytes. Human FVIII
comprises2351 amino acids, including a signal peptide, and contains
several distinct domains as defined by homology. There are three
A-domains, a unique B-domain, and two C-domains. The domain order
can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. The chains are
connected by bivalent metal ion-bindings. The A1-A2-B chain is
termed the heavy chain (HC) while the A3-C1-C2 is termed the light
chain (LC). Small acidic regions C-terminal of the A1 (the a1
region) and A2 (the a2 region) and N-terminal of the A3 domain (the
a3 region) play important roles in its interaction with other
coagulation proteins, including thrombin and von Willebrand factor
(VWF), the carrier protein for FVIII.
[0028] Endogenous FVIII molecules circulate in vivo as a pool of
molecules with B domains of various sizes, the shortest having
C-terminal at position 740, i.e. at the C-terminal of A2-a2, and
thus contains no B domain. These FVIII molecules with B-domains of
different length all have full procoagulant activity. Upon
activation with thrombin, FVIII is cleaved C-terminal of A1-a1 at
position 372, C-terminal of A2-a2 at position 740, and between a3
and A3 at position 1689, the latter cleavage releasing the a3
region with concomitant loss of affinity for VWF. The activated
FVIII molecule is termed FVIIIa. The activation allows interaction
of FVIIIa with phospholipid surfaces like activated platelets and
activated factor IX (FIXa), i.e. the tenase complex is formed,
allowing efficient activation of factor X (FX).
[0029] The terms "Factor VIII(a)" and "FVIII(a)" include both FVIII
and FVIIIa. Similarly, the term "Factor VIII" and "FVIII" may
include both FVIII and FVIIIa. "Factor VIII" or "FVIII" as used
herein refers to a human plasma glycoprotein that is a member of
the intrinsic coagulation pathway and is essential to blood
coagulation. "Wldtype(wt)/native FVIII" is the human FVIII molecule
derived from the full length sequence as shown in SEQ ID NO: 1
(amino acid 1-2332). "FVIII(a)" includes natural allelic variants
of FVIII(a) that may exist and occur from one individual to
another. FVIII(a) may be plasma-derived or recombinantly produced,
using well known methods of production and purification. The degree
and location of glycosylation, tyrosine sulfation and other
post-translation modifications may vary, depending on the chosen
host cell and its growth conditions.
[0030] Pharmaceutical compositions according to the present
invention may comprise native or B domain-truncated FVIII molecules
wherein the remaining domains correspond closely to the sequences
as set forth in amino acid numbers 1-740 and 1649-2332 of SEQ ID
NO: 3. In such molecules, as well as in FVIII comprising the
full-length B domain amino acid sequence, mutations may be
introduced. Amino acid modifications, such as substitutions,
insertions, and deletions, may be introduced into the molecule in
order to modify the binding capacity of FVIII with various other
components such as low-density lipoprotein receptor-related protein
(LRP) and related receptors, various other receptors, other
coagulation factors, cell surfaces, introduction and/or abolishment
of glycosylation sites, etc. Other mutations that do not abolish
FVIII activity may also be accommodated in the FVIII molecules
herein.
[0031] FVIII molecules herein
(molecules/variants/derivatives/analogues/conjugates) are capable
of functioning in the coagulation cascade in a manner that is
functionally similar, or equivalent, to wt/endogenous FVIII,
inducing the formation of FXa via interaction with FIXa on an
activated platelet and supporting the formation of a blood clot.
FVIII activity can be assessed in vitro using techniques well known
in the art. Clot analyses, FX activation assays (often termed
chromogenic assays), thrombin generation assays and whole blood
thrombo-elastography are examples of such in vitro techniques.
FVIII molecules according to the present invention have FVIII
activity that is at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%,
100% or even more than 100% of that of native human FVIII.
[0032] Endogenous full length FVIII is synthesized as a
single-chain precursor molecule. Prior to secretion, the precursor
is cleaved into the heavy chain and the light chain. Recombinant B
domain-deleted or truncated FVIII can be produced by means of two
different strategies. Either the heavy chain without the B-domain
and the light chain are synthesized individually as two different
polypeptide chains (two-chain strategy) or the B domain-deleted or
truncated FVIII is synthesized as a single precursor polypeptide
chain (single-chain strategy) that is cleaved into the heavy and
light chains in the same way as the full-length FVIII
precursor.
[0033] In a B domain-deleted or truncated FVIII precursor
polypeptide, produced by the single-chain strategy, the heavy and
light chain moieties are often separated by a linker. To minimize
the risk of introducing immunogenic epitopes in the B
domain-deleted FVIII, the sequence of the linker is preferably
derived from the FVIII B-domain. In the B domain of full length
FVIII, amino acid 1644-1648 constitutes this recognition site. The
thrombin cleavage site leading to removal of the linker on
activation of B domain-deleted FVIII is located in the heavy chain.
Thus, the size and amino acid sequence of the linker is unlikely to
influence its removal from the remaining FVIII molecule by thrombin
activation. Deletion/truncation of the B domain is an advantage for
production of FVIII. Nevertheless, parts of the B domain can be
included in the linker without reducing the productivity. The
negative effect of the B domain on productivity has not been
attributed to any specific size or sequence of the B domain.
TABLE-US-00001 SEQ ID NO: 1: wt human FVIII (Ser750 residue shown
in bold and underline)
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL
FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA
VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASD
PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA
VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR
KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL
MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL
TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL
APDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILG
PLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD
FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP
LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAG
VQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLS
VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNR
GMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS
TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP
HGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFT
PESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDN
TSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLES
GLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKT
NKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRM
LMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKML
FLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKV
VVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEK
KETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQD
FRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPN
TSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPS
TLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIR
PIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTL
EMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHI
YQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVA
TESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILS
LNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREI
TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI
AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG
ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA
EPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG
LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR
APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN
ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVEC
LIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKL
ARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQ
FIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR
LHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF
ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKS
LLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPP
LLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY
The B domain in FVIII spans amino acids 741-1648 of SEQ ID NO: 1.
The B domain is cleaved at several different sites, generating
large heterogeneity in circulating plasma FVIII molecules. The
exact function of the heavily glycosylated B domain is unknown.
What is known is that the B domain is dispensable for FVIII
activity in the coagulation cascade. Recombinant FVIII is thus
frequently produced in the form of B domain-deleted/truncated
variants. In a preferred embodiment, the FVIII molecule is produced
by an expression vector encoding a FVIII molecule comprising a 21
amino acid residue L (linker) sequence with the following sequence:
SEQ ID NO:2: SFSQNSRHPSQNPPVLKRHQR (the O-glycan is attached to the
underlined S). Alternative preferred B domain linker sequences may
lack one or more of the amino acid residues set forth in SEQ ID
NO:2, e.g. the C-terminal R in SEQ ID NO:2. Preferred FVIII
molecules are B domain deleted/truncated variants comprising an
O-glycan attached to the Ser 750 residue shown in SEQ ID
NO:1--optionally being conjugated to a polymeric (half life
extending) moiety via this O-glycan.
[0034] The inventors of the present invention have made the
surprising observation that B domain deleted FVIII molecules
according to the invention having a B domain of a size from about
100 to about 400 amino acids ((preferably 150-650, more preferably
150-600, more preferably 150-550, more preferably 150-500, more
preferably 150-450, more preferably 150-400, more preferably
150-350, more preferably 200-700, more preferably 200-600, more
preferably 200-500, more preferably 200-400, more preferably
200-300, and most preferably about 200 to 250) have a surprisingly
high bioavailability in connection with extravascular (e.g. s.c.)
administration compared to e.g. FVIII molecules having the entire B
domain intact as well FVIII molecules having no or only a few amino
acids (e.g. 15-30 amino acids) intact. Such molecules may or may
not comprise the Ser750 residue according to SEQ ID NO:1. A simple
and safe way of producing FVIII having improved bioavailability
upon subcutaneous/intradermal administration is thus provided. It
is plausible that the in vivo circulatory half-life of FVIII having
B domains of 100 to about 400 amino acids may be prolonged by
conjugating/fusing such variants with a half-life extending moiety.
An example of a FVIII molecule comprising a 226 amino acid B domain
is shown in SEQ ID NO:3:
TABLE-US-00002 SEQ ID NO: 3: (226 amino acid B domain variant):
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL
FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA
VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASD
PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA
VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR
KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL
MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL
TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL
APDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILG
PLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD
FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP
LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAG
VQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLS
VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNR
GMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS
TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP
HGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFT
PESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDN
TSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLES
GLMNSQESSWGKNVSHHHHHHSQNPPVLKRHQREITRTTLQSDQEEIDYD
DTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSP
HVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA
EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY
FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP
AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN
YRFHAINGYIMDTLPGLVMAQDQRIRVWLLSMGSNENIHSIHFSGHVFTV
RKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFL
VYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK
EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY
RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME
LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGR
SNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSS
QDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH
QIALRMEVLGCEAQDLY
[0035] Von Willebrand Factor (VWF) is a blood glycoprotein involved
in hemostasis. It is deficient or defective in von Willebrand
disease which is the most common hereditary bleeding disorder. VWF
is a large multimeric glycoprotein present in blood plasma and
produced constitutively in endothelium, megakaryocytes, and
subendothelial connective tissue. The basic VWF monomer is a 2050
amino acid protein. Each monomer contains a number of specific
domains with a specific function, including the TIL' or TIL'/E'
domain (Zhou et al. Blood 2012; 120(2): 449-458) which binds to
FVIII. FVIII is bound to VWF while inactive in circulation and is
released from VWF by the action of thrombin. FVIII(a) not bound to
VWF is rapidly cleared and/or degraded. It is shown herein, that
full-length VWF does not have the ability to significantly increase
bioavailability of extra-vascularly co-administered FVIII despite
of its inherent FVIII protective effects.
[0036] The full length VWF molecule is thus a very complex protein.
The prepro VWF consists of 2813 amino acid residues (SEQ ID NO:22).
During secretion, the signal peptide from amino acid residue 1 to
22 and the propeptide from amino acid residue 23 to 763 are cleaved
off, leaving a mature VWF of 2050 amino acid residues. The amino
acid numbering is thus often based on the prepro VWF and amino acid
S764 is thus the first amino acid in the mature molecule. The
mature molecule is believed to contain 12 Asn-linked and 10 Thr/Ser
linked oligosaccharide side chains. Furthermore this molecule can
form dimers, trimers etc. with multimer molecule weight of up to
several million Daltons. Different allelic VWF variants are found
in human beings and it is thus understood that VWF fragments
according to the present invention can be derived from any one of
these naturally occurring variants.
[0037] The glycosylation heterogeneity, together with the multimer
forming properties, of the full length molecule makes it quite
challenging to construct an expression system and a downstream
purification procedure for a pharmaceutical composition of VWF.
[0038] The understanding of the organization and the boundaries of
domains in VWF is not yet complete. Only the so-called A domains
are well characterized and their crystal structures determined. The
chemical assignments of di-sulfides within VWF are limited.
However, recent studies on homologies of domains in VWF to domains
in and other proteins suggest that several disulfide bonds may be
formed. The domain definition of VWF described in Zhou et al. Blood
2012; 120, 449-458 is used herein.
[0039] The present invention relates to VWF fragments that are
preferably easier to produce than the full length molecule. VWF
fragments according to the invention furthermore preferably have
the ability to increase bioavailability of s.c. co-administered
FVIII. VWF fragments according to the present invention comprise
the at least the 15 N-terminal amino acids of the TIL'
domain/subdomain (spanning amino acids 764-778 of SEQ ID NO:22) or
the TIL' domain/subdomain (spanning amino acids 764-828 of SEQ ID
NO:22 or amino acids 764-829 of SEQ ID NO:22) or the TIL'/E'
domain/sub-domains (spanning amino acids 764-865 of SEQ ID NO:22)
and have a size of less than 1500 amino acids, preferably less than
1400 amino acids, preferably less than 1300 amino acids, preferably
less than 1200 amino acids, preferably less than 1100 amino acids,
preferably less than 1000 amino acids, preferably less than 900
amino acids, preferably less than 800 amino acids, preferably less
than 700 amino acids, preferably less than 600 amino acids,
preferably less than 500 amino acids, preferably less than 400
amino acids, preferably less than 300 amino acids, preferably less
than 275 amino acids, preferably less than 250 amino acids,
preferably less than 225 amino acids preferably less than 200 amino
acids, preferably less than 175 amino acids, preferably less than
150 amino acids, preferably less than 125 amino acids, preferably
less than 100 amino acids, preferably less than 95 amino acids,
preferably less than 90 amino acids, preferably less than 85 amino
acids, or preferably less than 80 amino acids, or preferably less
than 75 amino acids, or preferably less than 70 amino acids, or
preferably less than 65 amino acids, or preferably less than 60
amino acids, or preferably less than 55 amino acids, or preferably
less than 50 amino acids, or preferably less than 45 amino acids,
or preferably less than 40 amino acids, or preferably less than 35
amino acids, or preferably less than 30 amino acids, or preferably
less than 25 amino acids, or preferably less than 20 amino acids,
or preferably less than 15 amino acids. VWF fragments according to
the invention preferably comprise the TIL'/E'/D3 domains (where D3
is divided into subdomains VWD3-C8-3-TIL-3-E3) spanning amino acids
764-1250 or amino acids 764-1261 or amino acids 764-1268 of SEQ ID
NO:22 of SEQ ID NO:22.VWF fragments according to the invention
preferably comprise at least the 15 N-terminal amino acids of TIL',
TIL' or TIL'/E' domains (amino acids 764-778, 764-828 or amino
acids 764-865 of SEQ ID NO:22). VWF fragments according to the
invention may furthermore contain fewer potentially antigenic
regions. The molecular weight of VWF fragment dimers according to
the present invention may--naturally--be about twice as high as for
the monomeric fragments (Dimers according to the present invention
may thus comprise up to about 2400 amino acids if the monomer size
is 1200 amino acids).
[0040] Preferably, the VWF fragments according to the present
invention comprise at least amino acids 764-828 (SEQ ID NO:4), or
at least amino acids 764-865 (SEQ ID NO:5), or at least amino acids
764-1035 (SEQ ID NO:6), or at least amino acids 764-1041 (SEQ ID
NO:7), or at least amino acids 764-1045 (SEQ ID NO:8), or at least
amino acids 764-1128 (SEQ ID NO:9), or at least amino acids
764-1198 (SEQ ID NO:10), or at least amino acids 764-1250 (SEQ ID
NO:11), or at least amino acids 764-1261 (SEQ ID NO:14), or at
least amino acids 764-1268 (SEQ ID NO:16).
[0041] In an embodiment, the C1099 and/or the C1142 cysteines may
be mutated in the VWF fragments according to the present invention.
These cysteine residues are believed to be responsible for the
oligomerization/dimerization of the VWF protein. VWF fragments with
both cysteines intact may form dimers and homo-oligomers. Modifying
both of these cysteines may lead to a product composed of monomer
VWF fragments, whereas deletion of one or the other may lead to
dimer VWF fragments or potentially to oligomer VWF fragments. Both
of the above scenarios may lead to a simpler product purification
procedure as compared to the full-length protein.
[0042] In another embodiment, both of the C1099 and C1142 cysteines
are kept intact which may lead to a preferentially dimeric VWF
fragment. There may be a safety advantage associated with the
native sequences incl. the C1099 and the C1142 cysteines.
[0043] Surprisingly, co-formulation of FVIII and VWF fragments
according to the invention demonstrate improved bioavailability
compared to co-formulation of FVIII with a full length VWF
molecule. The co-formulations according to the invention show
increased bioavailability of Factor VIII when injected
subcutaneously. VWF fragments according to the present invention
comprise the D' domain (spanning amino acids 764-865/866 of SEQ ID
NO: 22) which is thought to be the primary FVIII binding site where
FVIII may dock onto D' by electrostatic dipole-dipole like
interactions. VWF fragments according to the invention preferably
comprise the D' domain and/or the D3-domain (the D3 domain spans
amino acids 865/866-1250/1261/1268 of SEQ ID NO: 16). Based on the
findings herein, it is possible that both the D' and the D'D3
domains have the ability to bind to FVIII. VWF fragments according
to the invention do not to any significant degree (i.e. preferably
less than 5%, more preferably less than 4%, preferably less than
3%, preferably less than 2%, more preferably less than 1%) form
multimers (i.e., having more than two units, such as e.g.
oligomers) because the cysteines (C1099 and C1142) essential for
multimer assembly are not present or have been mutated/substituted.
Some VWF fragments according to the present invention do
furthermore not form dimers to any significant degree--in
particular those wherein the C1099 and/or C1142 cysteines are not
present.
[0044] In some cases, VWF fragments forming dimers may, however,
also be useful in connection with the present invention--the
TIL'/E'/D3/A1 dimer has e.g. been shown to have a higher FVIII
affinity than the monomer. VWF fragment dimers may furthermore be a
relatively homogenous product that can be produced relatively
easily.
[0045] One advantage of the VWF fragments according to the
invention is that it is easier to produce such compounds on an
industrial scale as a relatively homogenous product due to the low
degree of multimerization and due to the fact that the compounds
are smaller compounds with fewer posttranslational modifications
compared to full length VWF. This means that a high expression
level is easier to obtain and/or purification will be less complex
due to a less complex molecule. Also, production of recombinant
peptides and proteins in simple organisms such as e.g. yeast is a
faster and more inexpensive production method compared to
production in mammalian cell lines--some VWF fragments according to
the present invention can be produced in yeast.
[0046] VWF fragments according to the present invention can be in
the form of one single VWF fragment (such as e.g. the entire
TIL'/E'/D3/A1 region spanning amino acids 764-1459 in SEQ ID NO:22)
or alternatively in the form of multiple groups of sequential amino
acids from VWF fused together and thus deleting intermediary
fragments (such as e.g. a "fusion" of the TIL' and the TIL'/E'
domain spanning amino acids 764-828+764-865 in SEQ ID NO:22).
Another example could be amino acids 764-828+1127-1197 in SEQ ID
NO:22. VWF fragments according to the invention may alternatively
be in the form of the repetitive elements. Homologous or
heterologous "spacer" sequences may be introduced between the fused
VWF fragments/elements (such as e.g. a multiple fusion of TIL'/E'
domains such as e.g. TIL'/E'TIL'/E'TIL'/E'). VWF fragments
according to the invention may also comprise one or more amino acid
alternations (e.g. substitutions, deletions, additions) in the VWF
derived sequence(s).
[0047] Bioavailability of FVIII in connection with extravascular
co-administration of FVIII and VWF fragments according to the
invention may be further improved by conjugating FVIII with at
least one half life extending moiety. It thus follows, that
extra-vascular co-administration of VWF fragments comprising the
TIL' and/or the TIL'/E' domains with a FVIII molecule conjugated
with at least one half life extending moiety is associated with a
relatively high FVIII bioavailability.
[0048] Examples of VWF fragments according to the present invention
(using the domain annotation from Zhou et al.) are shown below in
SEQ ID NOs: 4-21. TIL'/E'/VWD3 I, TIL'/E'/VWD3 II and TIL'/E'/VWD3
III denote three versions (different lengths) of TIL'/E'/VWD3.
TABLE-US-00003 SEQ ID NO: 4: amino acids 764-828 (TIL'):
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL ERCP
SEQ ID NO: 5: amino acids 764-865 (TIL'/E'):
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQG KEYAPGETVK IGCNTCVCQDRKWNCTDHVCDA SEQ ID NO: 6: amino
acids 764-1035 (TIL'/E'/VWD3 I):
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQ VEEDPVDFGN SWKVSSQCADTR SEQ ID NO: 7: amino acids
764-1041 (TIL'/E'/VWD3 II):
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQ VEEDPVDFGN SWKVSSQCADTRKVPLDS SEQ ID NO: 8: amino acids
764-1045 (TIL'/E'/VWD3 III):
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQ VEEDPVDFGN SWKVSSQCADTRKVPLDSSPAT SEQ ID NO: 9: amino
acids 764-1128 (TIL'/E'/VWD3/C8-3) - Cysteine 1099 is marked with
bold. This cysteine can be substituted to another amino acid, e.g.
Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKD ETH
FEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTS
DVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQ SEQ
ID NO: 10: amino acids 764-1198 (TIL'/E'/VWD3/C8-3/TIL-3) -
Cysteines 1099 and 1142 are marked with bold. One or both of these
cysteines can be substituted to another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPV SEQ ID NO: 11: amino acids 764-1250 (TIL'/E'/D3 I)
- Cysteines 1099 and 1142 are marked with bold. One or both of
these cysteines can be substituted to another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDA SEQ ID NO: 12: amino acids 864-1250 (D3 I) - Cysteines 1099
and 1142 are marked with bold. One or both of these cysteines can
be substituted to another amino acid, e.g. Ser:
ATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK
RVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTY
QEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNI
MKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQH
GKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHA
HCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQ
EPGGL VVPPTDA SEQ ID NO: 13: amino acids 864-1268 (D3 II) -
Cysteines 1099 and 1142 are marked with bold. One or both of these
cysteines can be substituted to another amino acid, e.g. Ser:
ATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK
RVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTY
QEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNI
MKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQH
GKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHA
HCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQ
EPGGL VVPPTDAPVSPTTLYVEDISEPPLHD SEQ ID NO: 14: amino acids
764-1261(TIL'/E'/D3 II) - Cysteines 1099 and 1142 are marked with
bold. One or both of these cysteines can be substituted to another
amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVED SEQ ID NO: 15: amino acids 764-1264 (TIL'/E'/D3
III) - Cysteines 1099 and 1142 are marked with bold. One or both of
these cysteines can be substituted to another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEP SEQ ID NO: 16: amino acids 764-1268
(TIL'/E'/D3 IV) - Cysteines 1099 and 1142 are marked with bold. One
or both of these cysteines can be substituted to another amino
acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHD SEQ ID NO: 17: amino acids 764-1459
(TIL'/E'/D3/A1 I) - Cysteines 1099 and 1142 are marked with bold.
One or both of these cysteines can be substituted to another amino
acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHDFYCS RLLDLVFLLD GSSRLSEAEF EVLKAFVVDM
MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRI ASQVKYAGSQVASTSEVLKY
TLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLK
QIRLIEKQAPENKAFVLSSVDELEQQRDEI VSYLCD SEQ ID NO: 18: amino acids
764-1463 (TIL'/E'/D3/A1 II) - Cysteines 1099 and 1142 are marked
with bold. One or both of these cysteines can be substituted to
another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHDFYCS RLLDLVFLLD GSSRLSEAEF EVLKAFVVDM
MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRI ASQVKYAGSQVASTSEVLKY
TLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLK
QIRLIEKQAPENKAFVLSSVDELEQQRDEI VSYLCDLAPE SEQ ID NO: 19: amino
acids 764-1464 (TIL'/E'/D3/A1 III) - Cysteines
1099 and 1142 are marked with bold. One or both of these cysteines
can be substituted to another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHDFYCS RLLDLVFLLD GSSRLSEAEF EVLKAFVVDM
MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKY
TLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLK
QIRLIEKQAPENKAFVLSSVDELEQQRDEI VSYLCDLAPEA SEQ ID NO: 20: amino
acids 764-1683 (TIL'/E'/D3/A1/A2) - Cysteines 1099 and 1142 are
marked with bold. One or both of these cysteines can be substituted
to another amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHDFYCS RLLDLVFLLD GSSRLSEAEF EVLKAFVVDM
MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRI ASQVKYAGSQVASTSEVLKY
TLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLK
QIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTL
GPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYP
FSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASD
EIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGE
GLQIPTLSPA SEQ ID NO: 21: amino acids 764-1873
(TIL'/E'/D3/A1/A2/A3) - Cysteines 1099 and 1142 are marked with
bold. One or both of these cysteines can be substituted to another
amino acid, e.g. Ser:
SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVAL
ERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFP
GECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV
KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ
NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL
TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA
TLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKIL
DELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQ ICHCDVVNLTCEACQEPGGL
VVPPTDAPVSPTTLYVEDISEPPLHDFYCS RLLDLVFLLD GSSRLSEAEF EVLKAFVVDM
MERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRI ASQVKYAGSQVASTSEVLKY
TLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEK
QAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRN
SMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQ
SKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRL
PGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAP
DCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVL
QYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGAS
KAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLP
TMVTLGNSFLHKLCS SEQ ID NO: 22: wild-type human VWF according to the
UniProtKB/ Swiss-Prot database (entry P04275) - cysteine residues
at positions 1099 and 1142 are marked with bold:
MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGG
CQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYK
LSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSW
ALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKT
LCECAGGLECACPALLEYARTCAQEGMVLYGVVTDHSACSPVCPAGMEYRQCVSPCARTCQS
LHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQ
WICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVC
TRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDW
DGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQ
KQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGA
LASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEG
CFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLL
PDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLC
PPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIG
MAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIE
LFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNF
DGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCR
ILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATL
CPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELL
QTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTD
APVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKW
VRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEAS
RITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVD
ELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSD
KIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQ
GGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNA
NVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGS
SSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQRE
GGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIG
DRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGD
VWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTG
SSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVEL
HSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFAS
KTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQV
LLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCP
PSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQH
QFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVC
DPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCC
DEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVC
TCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQS
SWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSC
RCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNT
GECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEH
KCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSID
INDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK
[0049] FVIII Molecules/Variants/Derivatives/Analogues:
[0050] The term "FVIII" as used herein, is intended to designate
any FVIII molecule having FVIII activity, incl. wt FVIII, B domain
deleted/truncated FVIII molecules, variants of FVIII exhibiting
substantially the same or improved biological activity relative to
wt FVIII and FVIII-related polypeptides, in which one or more of
the amino acids of the parent peptide have been chemically
modified, e.g. by protein:protein fusion, alkylation, PEGylation,
HESylation, PASylation, PSAylation, acylation, ester formation or
amide formation or the like (conjugated to a half-life extending
moiety).
[0051] Half-Life Extending Moieties/Protractive Groups:
[0052] The term "half-life extending moieties" is herein understood
to refer to one or more chemical groups, e.g. a hydrophilic
polymer, such as e.g. PEG and/or a polysaccharide covalently
attached to FVIII via e.g. --SH, --OH, --COOH, --CONH2, --NH2, or
one or more N- and/or O-glycan structures that can increase in vivo
circulatory half life when conjugated to these proteins. Examples
of protractive groups/half-life extending moieties suitable for
being conjugated to FVIII in connection with the present invention
include: Biocompatible fatty acids and derivatives thereof, Hydroxy
Alkyl Starch (HAS) e.g. Hydroxy Ethyl Starch (HES), Poly Ethylene
Glycol (PEG), Poly (Glyx-Sery)n (HAP), Hyaluronic acid (HA),
Heparosan polymers (HEP), Phosphorylcholine-based polymers (PC
polymer), Fleximers, Dextran, Poly-sialic acids (PSA), an Fc
domain, an Fc receptor, Transferrin, Albumin, Elastin like
peptides, XTEN polymers, Albumin binding peptides, a CTP peptide,
and any combination thereof. In general, conjugation of FVIII with
one or more half-life extending moieties (such as e.g. hydrophilic
polymers) generally have a better bioavailability in connection
with s.c./intradermal co-administration with VWF fragments
according to the invention as compared with FVIII with no half life
extending moieties.
[0053] PEGylated FVIII molecules in connection with the present
invention may have one or more polyethylene glycol (PEG) molecules
attached to any part of the FVIII protein including any amino acid
residue or carbohydrate moiety. Chemical and/or enzymatic methods
can be employed for conjugating PEG or other polymeric groups (half
life extending moieties) to a glycan on FVIII. An example of an
enzymatic conjugation process is described e.g. in WO03031464. The
glycan may be naturally occurring or it may be inserted via e.g.
insertion of an N-linked and/or O-linked glycan using methods well
known in the art. "Cysteine-PEGylated FVIII" according to the
present invention have one or more PEG molecules conjugated to a
sulfhydryl group of a cysteine present in FVIII. "Cysteine-acylated
FVIII" according to the present invention have one or more
hydrophobic half-life extending moieties (e.g. fatty acids)
conjugated to a sulfhydryl group of a cysteine in FVIII--this
cysteine residue may be introduced by genetic engineering or a part
of the native amino acid sequence. It is furthermore possible to
link half-life extending moieties to other amino acid residues.
[0054] Fusion Proteins:
[0055] Fusion proteins according to the present invention are
proteins created through the in-frame joining of two or more DNA
sequences which originally encoded FVIII and the fusion partner.
Translation of the fusion protein DNA sequence will result in a
single protein sequence which may have functional properties
derived from each of the original proteins or peptides. DNA
sequences encoding fusion proteins may be created artificially by
standard molecular biology methods such as overlapping PCR or DNA
ligation and the assembly is performed excluding the stop codon in
the first 5'-end DNA sequence while retaining the stop codon in the
3'end DNA sequence. The resulting fusion protein DNA sequence may
be inserted into an appropriate expression vector that supports the
heterologous fusion protein expression in a standard host
organism.
[0056] Fusion proteins may contain a linker or spacer peptide
sequence that separates the protein or peptide parts which define
the fusion protein. The linker or spacer peptide sequence may
facilitate the correct folding of the individual protein or peptide
parts and may make it more likely for the individual protein or
peptide parts to retain their individual functional properties.
Linker or spacer peptide sequences may be inserted into fusion
protein DNA sequences during the in frame assembly of the
individual DNA fragments that make up the complete fusion protein
DNA sequence i.e. during overlapping PCR or DNA ligation. Examples
of fusion proteins comprising FVIII and a fusion partner are shown
in WO2011101284.
[0057] Fc Fusion Protein:
[0058] The term "Fc fusion protein" is herein meant to encompass
FVIII fused to an Fc domain that can be derived from any antibody
isotype. An IgG Fc domain will often be preferred due to the
relatively long circulatory half-life of IgG antibodies. The Fc
domain may furthermore be modified in order to modulate certain
effector functions such as e.g. complement binding and/or binding
to certain Fc receptors. Fusion of FVIII with an Fc domain, which
has the capacity to bind to FcRn receptors, will generally result
in a prolonged in vivo circulatory half-life. Mutations in
positions 234, 235 and 237 in an IgG Fc domain will generally
result in reduced binding to the Fc.gamma.RI receptor and possibly
also the Fc.gamma.RIIa and the Fc.gamma.RIII receptors. These
mutations do not alter binding to the FcRn receptor, which promotes
a long circulatory in vivo half-life by an endocytic recycling
pathway. Preferably, a modified IgG Fc domain of a fusion protein
according to the invention comprises one or more of the following
mutations that will result in decreased affinity to certain Fc
receptors (L234A, L235E, and G237A) and in reduced C1q-mediated
complement fixation (A330S and P331S), respectively. Alternatively,
the Fc domain may be an IgG4 Fc domain, preferably comprising the
S241P/S228P mutation.
[0059] Bioavailability (of FVIII): The term "Bioavailability"
describes the percentage of compound absorbed to the blood after
extravascular is calculated as the Area under the concentration
time curves after extravascular dosing of the compound. This is
calculated from the Area under the concentration curves of FVIII
after s.c. administration divided by the dose, relatively to the
area under the concentrations curve divided by the dose of the same
FVIII compound, dosed i.v. According to the present invention, the
bioavailability of FVIII molecules (in connection with
subcutaneous/intradermal co-administration of FVIII and VWF
fragments according to the invention) is at least 3%, preferably at
least 5%, preferably at least 6%, preferably at least 7%,
preferably at least 8%, preferably at least 9%, preferably at least
10%, preferably at least 11%, preferably at least 12%, preferably
at least 13%, preferably at least 14%, preferably at least 15%,
preferably at least 16%, preferably at least 17%, preferably at
least 18%, preferably at least 19%, preferably at least 20%,
preferably at least 21%, preferably at least 22%, preferably at
least 23%, preferably at least 24%, preferably at least 25%,
preferably at least 26%, preferably at least 27%, preferably at
least 28%, preferably at least 29%, preferably at least 30%,
preferably at least 31%, preferably at least 32%, preferably at
least 33%, preferably at least 34%, preferably at least 35%,
preferably at least 36%, preferably at least 37%, preferably at
least 38%, preferably at least 39%, preferably at least 40%,
preferably at least 41%, preferably at least 42%, preferably at
least 43%, preferably at least 44%, preferably at least 45%,
preferably at least 46%, preferably at least 47%, preferably at
least 48%, preferably at least 49%, preferably at least 50%,
preferably at least 55%, preferably at least 60%, preferably at
least 65%, preferably at least 70%, and most preferably at least
75%. Bioavailability can be measured as described herein.
Preferably, the FVIII bioavailability (FVIII antigen and/or
activity) of formulations according to the invention will be high
enough to exert prophylactic effects under conditions with normal
activity when such formulations are administered extravascularly
(e.g. subcutaneously or intra-dermally) e.g. once or twice a day or
once, twice or three times a week. Preferably, FVIII dosages are
comparable with those used in connection with I.V. administration
of FVIII, preferably twice as high, and more preferably three times
as high, more preferably four times as high, more preferably about
10 times as high, more preferably about 15 times as high, more
preferably about 20 times as high, and most preferably about 25
times as high. Safety and cost considerations may be considered in
connection with dosage determinations.
[0060] Saturation of FVIII with VWF fragments according to the
invention: saturation of FVIII with VWF fragment/the relative
amount of FVIII bound to or in complex with VWF/the amount of FVIII
bound to VWF divided by the total amount of FVIII. This calculation
is based on the KD value of the binding between FVIII and the
protein. For FVIII binding to VWF fragments, the measured KI values
are used as KD.
[0061] The following (quadratic) equations can be used to calculate
the concentration of bound FVIII (A) to another protein (B) from
the total concentrations [A]t [B]t.
K D = [ A ] .times. [ B ] [ AB ] [ A ] = [ A ] t - [ AB ] [ B ] = [
B ] t - [ AB ] [ AB ] 2 - ( K D + [ A ] t + [ B ] t ) .times. [ AB
] + [ A ] t .times. [ B ] t = 0 ##EQU00001## .alpha. .times. [ AB ]
2 + .beta. .times. [ AB ] + .delta. = 0 ##EQU00001.2## .alpha. = 1
, .beta. = - ( K D + [ A ] t + [ B ] t ) , .delta. = [ A ] t
.times. [ B ] t [ AB ] = - .beta. .+-. .beta. 2 - 4 .times. .alpha.
.times. .delta. 2 .times. .alpha. ##EQU00001.3##
[0062] Pharmaceutical Compositions:
[0063] The present invention provides compositions comprising VWF
fragments and preferably also FVIII. Accordingly, one object of the
invention is to provide a pharmaceutical composition comprising a
FVIII molecule present in a concentration from 40 IU/ml to 25,000
IU/ml, and wherein said composition has a pH from 2.0 to 10.0. In a
preferred embodiment, the FVIII molecules are co-administered
together with VWF fragments. Pharmaceutical compositions according
to the invention may thus comprise FVIII in a concentration of from
40 IU/ml to 25,000 IU/ml, such as e.g. from 50-25,000 IU/ml,
100-25,000 IU/ml, 250-25,000 IU/ml, 500-25,000 IU/ml, 1000-25,000
IU/ml, 2000-25,000 IU/ml, 3000-25,000 IU/ml, 4000-25,000 IU/ml,
5000-25,000 IU/ml, 6000-25,000, 7000-25,000, 8000-25,000,
9000-25,000, 10,000-25,000 IU/ml, 50-20,000 IU/ml, 100-20,000
IU/ml, 250-20,000 IU/ml, 500-20,000 IU/ml, 1000-20,000 IU/ml,
2000-20,000 IU/ml, 3000-20,000 IU/ml, 4000-20,000 IU/ml,
5000-20,000 IU/ml, 6000-20,000 IU/ml, 7000-20,000 IU/ml,
8000-20,000 IU/ml, 9000-20,000 IU/ml, 10,000-20,000 IU/ml,
50-15,000 IU/ml, 100-15,000 IU/ml, 250-15,000 IU/ml, 500-15,000
IU/ml, 1000-15,000 IU/ml, 2000-15,000 IU/ml, 3000-15,000 IU/ml,
4000-15,000 IU/ml, 5000-15,000 IU/ml, 6000-15,000 IU/ml,
7000-15,000 IU/ml, 8000-15,000 IU/ml, 9000-15,000 IU/ml,
10,000-15,000 IU/ml, 50-10,000 IU/ml, 100-10,000 IU/ml, 250-10,000
IU/ml, 500-10,000 IU/ml, 1000-10,000 IU/ml, 2000-10,000 IU/ml,
3000-10,000 IU/ml, 4000-10,000 IU/ml, 5000-10,000 IU/ml, 50-5000
IU/ml, 100-5000 IU/ml, 250-5000 IU/ml, 500-5000 IU/ml, and
1000-5000 IU/ml. Compositions according to the invention may
further comprise one or more pharmaceutically acceptable excipients
such as e.g. a buffer system, a preservative, a tonicity agent, a
chelating agent, a stabilizer, or a surfactant, as well as various
combinations thereof. The use of preservatives, isotonic agents,
chelating agents, stabilizers and surfactants in pharmaceutical
compositions is well-known to the skilled person. Reference may be
made to Remington: The Science and Practice of Pharmacy, 19th
edition, 1995.
[0064] In one embodiment, the pharmaceutical composition is an
aqueous composition. Such a composition is typically a solution or
a suspension, but may also include colloids, dispersions,
emulsions, and multi-phase materials. The term "aqueous
composition" is defined as a composition comprising at least 50%
w/w water. Likewise, the term "aqueous solution" is defined as a
solution comprising at least 50% w/w water, and the term "aqueous
suspension" is defined as a suspension comprising at least 50% w/w
water.
[0065] In another embodiment, the pharmaceutical composition is a
freeze-dried composition, to which the physician or the patient
adds solvents and/or diluents prior to use.
[0066] In a further aspect, the pharmaceutical composition
comprises an aqueous solution of such an antibody, and a buffer,
wherein the antibody is present in a concentration from 1 mg/ml or
above, and wherein said composition has a pH from about 2.0 to
about 10.0.
[0067] Pharmaceutical compositions according to the present
invention are preferably suitable for extravascular administration
(e.g. s.c. or intradermal administration) in
prophylactic/therapeutic treatment of blood clotting diseases.
[0068] "Ratio of FVIII:VWF":
[0069] According to the present invention, preferred ratios of
FVIII and VWF/VWF fragment include FVIII/VWF ratios (molar ratios)
from 0.5:1 to 1:50, such as e.g. 1:1 to 1:50, such as e.g. 1:1 to
1:25, such as e.g. 1:1 to 1:20, or 1:1 to 1:15, or 1:1 to 1:10, or
1:1 to 1:7,5, or 1:7 to 1:8, or 1:6 to 1:8, or 1:6 to 1:9, or 1:5
to 1:10. Preferred ratios thus include: 1:1, 1:2, 1:3, 1:4, 1:5,
1:5,5; 1:6; 1:6,5, 1:7; 1:7,1; 1:7,2; 1:7,3; 1:7,4; 1:7,5; 1:7,6;
1:7,7; 1:7,8; 1:7,9, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35,
1:40, 1:45, and 1:50. Preferred ratios include: 0.5:1; 0.6:1;
0.7:1; 0.8:1; 0.9:1; 1:1; 1.1:1; 1.2:1; 1.3:3; 1.4:1, and 1.5:1. A
molar ratio close to 1:1 generally has the advantage of minimizing
the required amount of active substance. The optimal ratio between
FVIII and VWF fragment in a co-formulation mixture may be
determined by calculating the amount of bound FVIII:VWF at certain
protein concentrations based on the binding affinity to the VWF
variant for the FVIII species in question. The binding affinity can
be determined e.g. by ELISA, SPR or by ITC.
[0070] "Haemophilia":
[0071] Haemophilia/hemophilia/blood clotting diseases is a group of
hereditary genetic disorders that impair the body's ability to
control blood clotting or coagulation ("bleeding disorders"), which
is used to stop bleeding when a blood vessel is broken. Haemophilia
A (clotting factor VIII deficiency) is the most common form of the
disorder, present in about 1 in 5,000-10,000 male births. In
connection with the present invention, the term "haemophilia"
encompasses von Willebrand disease.
List of Embodiments
[0072] 1. A VWF fragment comprising up to 1500, 1400, 1300, or
1200, wherein said VWF fragment comprises the TIL' domain. Said
fragment may comprise different or repetitive VWF sequences joined
by peptide bonds. [0073] 2. A VWF fragment according to the
invention, wherein said fragment comprises the TIL' and the E'
domains. [0074] 3. A VWF fragment consisting of the TIL' or the
TIL'/E' domains. [0075] 4. A VWF fragment (according to the
invention), wherein said fragment comprises the amino acid sequence
according to any one of SEQ ID NO: 4, 5, 6, 7, 8. 9. 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or 21. [0076] 5. A VWF fragment
according to the invention, wherein said VWF fragment does not
comprise cysteine residues at position(-s) 1099 and/or 1142 of SEQ
ID NO: 22. These cysteine residue(-s) can be deleted by amino acid
substitution and/or deletion. [0077] 6. A VWF fragment according to
the invention, wherein said fragment comprises SEQ ID NO: 9,
wherein the 1099 Cysteine residue is substituted with another amino
acid, such as e.g. Histidine, Alanine, Isoleucine Arginine,
Leucine, Asparagine, Lysine, Aspartic acid, Methionine,
Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan,
Glycine, Valine, Proline, Serine, Taurine, and Tyrosine. [0078] 7.
A VWF fragment according to the invention, wherein the 1099
cysteine residue is substituted with Serine. [0079] 8. A VWF
fragment according to the invention, wherein said fragment
comprises an amino acid sequence selected from the list consisting
of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, wherein the 1099 and
the 1142 cysteine residues are substituted with another amino acid,
such as e.g. Histidine, Alanine, Isoleucine Arginine, Leucine,
Asparagine, Lysine, Aspartic acid, Methionine, Phenylalanine,
Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine,
Proline, Serine, Taurine, and/or Tyrosine. [0080] 9. A VWF fragment
according to the invention, wherein the 1099 and the 1142 cysteine
residues are substituted with serine. [0081] 10. A pharmaceutical
composition comprising a VWF fragment according to the invention,
wherein less than 10%, preferably less than 9%, preferably less
than 8%, preferably less than 7%, preferably less than 6%,
preferably less than 5%, preferably less than 4%, preferably less
than 3%, preferably less than 2%, preferably less than 1% of said
VWF fragment are in the form of oligomers and/or multimers. [0082]
11. A VWF fragment according to the invention, wherein said VWF
fragment is part of a dimer. The percentage of dimer formation may
be at least 5%, preferably at least 10%, preferably at least 15%,
preferably at least 20%, preferably at least 25%, preferably at
least 30%, preferably at least 35%, preferably at least 40%,
preferably at least 45%, preferably at least 50%, preferably at
least 55%, preferably at least 60%, preferably at least 65%,
preferably at least 70%, preferably at least 75%, preferably at
least 80%, preferably at least 85%, preferably at least 90%, most
preferably at least 95%. [0083] 12. A pharmaceutical composition
comprising FVIII and a VWF fragment, wherein FVIII bioavailability
is at least 5% following extravascular (e.g.
sub-cutaneous/intradermal) administration of said pharmaceutical
formulation. [0084] 13. A pharmaceutical composition comprising
FVIII and a VWF fragment, wherein FVIII bioavailability is at least
5% following extravascular (e.g. sub-cutaneous/intra-dermal)
administration of said pharmaceutical formulation, wherein the
ratio of FVIII and VWF fragment is about 0.5:1-1:50. Preferably
said ratio is about 0.5:1, 1:1, or 1:2. [0085] 14. A VWF fragment,
wherein the amino acid sequence of said VWF fragment comprises or
consists of an amino acid sequence selected from the list
consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
[0086] 15. A pharmaceutical composition comprising: (i) a VWF
fragment according to the invention; and (ii) FVIII, preferably
recombinant FVIII. Alternatively, said composition may comprise
two, three, four, five or more different VWF fragments according to
the invention and/or two, three, four, or five different FVIII
molecules. [0087] 16. A pharmaceutical composition according to the
invention, wherein said FVIII molecule comprises a truncated B
domain at a size of 5-700 amino acids, such as e.g. 5-500, 5-400,
5-300, 5-200, 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 10-700, 10-500,
10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-700,
20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-25, 50-700,
50-500, 50-400, 50-300, 50-200, 50-100, 100-700, 100-500, 100-400,
100-300, 100-200, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 45, 50, 75, or 100 amino acids. [0088] 17. A
pharmaceutical composition according to the invention, wherein the
amino acid sequence of said truncated B domain is derived from the
wt FVIII B domain amino acid sequence. [0089] 18. A pharmaceutical
composition according to the invention, wherein said FVIII molecule
is a B domain truncated FVIII molecule, wherein said B domain
comprises an O-glycan linked to the Ser 750 amino acid residue as
set forth in SEQ ID NO:1. Preferably, said FVIII molecule comprises
one O-linked glycan in the truncated B domain, wherein said
O-linked glycan is attached to the Ser 750 residue as set forth in
SEQ ID NO:1. [0090] 19. A pharmaceutical composition according to
the invention, wherein said FVIII molecule comprises a B domain
having the amino acid sequence as set forth in SEQ ID NO:2.
Alternatively, one or more amino acids in the B domain are deleted
from SEQ ID NO:2, such as e.g. the N-terminal Ser residue and/or
the C-terminal Arg residue. [0091] 20. A pharmaceutical composition
according to the invention, wherein the amino acid sequence of the
FVIII B domain comprises or consists of an amino acid sequence
selected from the group consisting of: amino acids
741-857+1637-1648; amino acids 741-914+1637-1648; amino acids
741-954+1637-1648; amino acids 741-965+1637-1648; amino acids
741-965+1637-1648; amino acids 741-1003+1637-1648; amino acids
741-1003+1637-1648; amino acids 741-1020+1637-1648; amino acids
741-1079+1637-1648; amino acids 741-1206+1637-1648; amino acids
741-1261+1637-1648; amino acids 741-1309+1637-1648; amino acids
741-914+1637-1648; amino acids 741-954+1637-1648; amino acids
741-968+1637-1648; amino acids 741-1003+1637-1648; amino acids
741-1018+1637-1648; amino acids 741-1070+1637-1648; amino acids
741-1230+1637-1648; amino acids 741-1301+1637-1648; amino acids
741-965+1637-1648; amino acids 741-965+1637-1648; amino acids
741-965+1637-1648; and amino acids 741-965+1637-1648 as set forth
in SEQ ID NO:1. [0092] 21. A pharmaceutical composition according
to the invention, wherein said FVIII molecule is conjugated with at
least one half-life extending moiety. Preferably, said half life
extending moiety is a water soluble polymer. Preferably a PEG
and/or a polysaccharide. [0093] 22. A pharmaceutical composition
according to the invention, wherein at least one water soluble
polymer is covalently attached to a glycan present in the B domain,
preferably an O-glycan, preferably an O-glycan attached to the
Ser750 amino acid residue as set forth in SEQ ID NO:1. [0094] 23. A
pharmaceutical composition according to the invention, wherein said
water soluble polymer is selected from the group consisting of:
PEG, PSA, HES, HEP and HSA. [0095] 24. A pharmaceutical composition
according to the invention, wherein said FVIII molecule is produced
using an expression vector encoding a FVIII molecule comprising the
FVIII B domain is as set forth in SEQ ID NO:2. [0096] 25. A
pharmaceutical composition according to the invention, wherein the
bioavailability of said FVIII molecule is at least 2, 3, 4, 5, 6,
7, 8, 9, or 10%. Preferably, the bioavailability is measured as the
area under the curve of the plasma levels of FVIII after
subcutaneous administration using either an antigen assay or a
clotting assay. [0097] 26. A pharmaceutical composition according
to the invention, wherein the ratio between FVIII and VWF is 1:50,
1:34, 1:25, 1:20: 1:15, 1:10, 1:7,5, preferably 0.5:1, 1:1, or 1:2.
[0098] 27. A pharmaceutical formulation according to the invention,
wherein the concentration of FVIII is at least about 100, 150, 200,
250, 300, 350, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000,
17,000, 18,000, 19,000, 20,000, 20,000, 21,000, 22,000, 23,000,
24,000, 25,000, 26,000, 27,000, 28,000, 29,000, or 30,000 IU/ml.
[0099] 28. A pharmaceutical formulation according to the invention,
wherein the amount of FVIII bound to VWF fragment is at least 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of
the total amount of FVIII in said formulation. [0100] 29. Use of a
compound according to the invention, or a pharmaceutical
composition according to the invention, for treatment of
haemophilia by extravascular, preferably subcutaneous,
administration. The pharmaceutical composition according to the
invention can also be administered by intradermal administration.
The pharmaceutical composition according to the invention can
furthermore be administered by intravenous administration. [0101]
30. A method of treatment of a haemophilia, wherein said method
comprises subcutaneous administration of a therapeutically
effective amount of a compound according to the present invention,
or a pharmaceutical composition according to the present invention,
to a patient in need thereof. [0102] 31. A method of increasing
bioavailability of FVIII, wherein said method comprises a step of
extravascular (e.g. subcutaneous/intradermal) co-administration of
FVIII and a VWF fragment according to the invention, wherein the
ratio of said FVIII and said VWF fragment is about 1:1-1:50,
preferably 0.5:1, 1:1, 1:2, 1:10, 1:20 or 1:34. [0103] 32. A DNA
molecule encoding a VWF fragment according to the invention. [0104]
33. An expression vector comprising a DNA molecule according to the
invention. [0105] 34. A host cell comprising an expression vector
according to the invention. [0106] 35. A method for making a VWF
fragment according to the invention, wherein said method comprises
incubation of a host cell in a suitable medium under suitable
conditions and subsequently recovering said recombinant VWF
fragment. [0107] 36. A pharmaceutical composition according to the
invention, wherein said composition comprises one or more VWF
fragments according to the invention. [0108] 37. A pharmaceutical
composition comprising one or more VWF fragments according to the
invention. [0109] 38. A method of treatment of von willebrand
disease, wherein said method comprises extravascular (e.g.
subcutaneous) administration of a therapeutically effective amount
of a pharmaceutical composition according to the present invention,
to a patient in need thereof. [0110] 39. A VWF fragment or VWF-like
polypeptide comprising the 15 N terminal amino acids of the TIL'
sequence 764-778, or more. Relatively small VWF fragments according
to the present invention may form part of, or be "embedded" in or
grafted onto a scaffold polypeptide sequence of any origin,
including non-VWF origin. [0111] 40. A VWF fragment according to
the invention, wherein said VWF fragment interacts with/binds to
residues C1858-Q1874, S2063-D2074 AND V2125-A2146 of the FVIII
amino acid sequence as set forth in SEQ ID NO:1. [0112] 41. A VWF
fragment according to the invention, wherein said fragment is
conjugated with a half life extending moiety. [0113] 42. A VWF
fragment according to the invention, wherein said fragment is
conjugated with a half life extending moiety via a N- and/or
O-linked glycan. [0114] 43. A VWF fragment according to the
invention, wherein said VWF fragment reduced uptake of FVIII by
antigen presenting cells in connection with binding of said VWF
fragment to FVIII.
[0115] It is understood that all aspects and embodiments of the
invention can be combined and that they are not to be understood in
any limiting way.
EXAMPLES
[0116] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
Example 1
Subcutaneous Administration in FVIII Knockout Mice (1)
[0117] Two test compounds were prepared: [0118] a) GlycoPEGylated
FVIII, i.e. "N8-GP" (prepared essentially as disclosed in example
1+2 in WO2009108806) 2000 U FVIII/ml determined by chromogenic
activity equivalent to 1.2 .mu.M based on protein content. [0119]
b) GlycoPEGylated FVIII i.e. N8-GP (2000 U FVIII/ml or 1.2 .mu.M,
co-formulated with 0.74 mg/ml VWF fragment TIL'/E'/D3/A1
(equivalent to 9.3 .mu.M)
[0120] Both test compounds were formulated in 18 mg/ml NaCl, 3
mg/ml saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80,
0.25 mg/ml CaCl.sub.2, pH 7.3.
[0121] 12 FVIII KO mice, exon 16 knock-out in a mixed background of
C57BI/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J)
with an approximate weight of 22 g were dosed subcutaneously in the
flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test
compound.
[0122] Blood was sampled at 1, 3, 7, 17, 24, 30, 48, 72 and 96 h
post administration. The mice were anaesthetized by
Isoflurane/O.sub.2/N.sub.2O prior to blood sampling via the
retroorbital plexus. Three samples were taken from each mouse.
Blood (45 .mu.l) was stabilised with 5 .mu.l of sodium-citrate
(0.13 M) and added 200 .mu.l FVIII coatest SP buffer (50 mM
TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After
centrifugation at 4000 g for 5 minutes at room temperature, the
supernatants were immediately frozen on dry ice before storage at
-80.degree. C. prior to analysis.
[0123] Samples were analysed with regards to FVIII activity in a
chromogenic assay as described by Ovlisen K et al. J. Thromb.
Haemost, 2008, 6: 969-975 and by FVIII antigen analysis using two
FVIII light chain antibodies (4F45 and 4F11) in a FVIII LOCI assay
(Luminescence oxygen channelling immunoassay).
[0124] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phoenix (Pharsight
Corporation) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using a previous i.v. pharmacokinetic
study of N8-GP in FVIII KO mice.
[0125] The circulating profiles of FVIII activity are shown
graphically in FIG. 1, the circulating concentrations of FVIII
antigen are shown in FIG. 2.
[0126] In this experiment, the bioavailability of GlycoPEGylated
FVIII alone was calculated to be 27% based on activity and 19%
based on antigen. The co-formulation with VWF increased the
bioavailability to 40 and 47%, respectively.
Example 2
Subcutaneous Administration in FVIII Knockout Mice (2)
[0127] Two test compounds were prepared: [0128] a) GlycoPEGylated
FVIII (500 IU FVIII/ml determined by chromogenic activity
equivalent to 0.3 .mu.M) [0129] b) GlycoPEGylated FVIII (500 IU
FVIII/ml or 0.3 .mu.M, co-formulated with 0.185 mg/ml VWF fragment
TIL'/E'/D3/A1 (equivalent to 2.3 .mu.M)
[0130] Based on a measured IC50 of 1.5 nM of the VWF fragment to
FVIII and assuming that the measured IC50 equals K.sub.d, 99% of
the FVIII should be bound to VWF in this composition.
[0131] Both test compounds were formulated in 18 mg/ml NaCl, 3
mg/ml saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80,
0.25 mg/ml CaCl2, pH.about.7.3.
[0132] 12 FVIII KO mice, exon 16 knock-out in a mixed background of
C57BI/6 and SV129, bred at Taconic M&B (B6.12954-F8tm1Kaz/J)
with an approximate weight of 22 g were dosed subcutaneously in the
flank with 2500 IU/kg FVIII or FVIII/VWF, 6 mice with each test
compound.
[0133] Blood was sampled at 1, 3, 7, 17, 24, 30, 48, 72 and 96 h
post administration. The mice were anaesthetized by
Isoflurane/O.sub.2/N.sub.2O prior to blood sampling via the
retroorbital plexus. Three samples were taken from each mouse. 45
.mu.l of blood was stabilised with 5 .mu.l of sodium-citrate (0.13
M) and added 200 .mu.l FVIII coatest SP buffer (50 mM TRIS-HCl, 1%
BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g
for 5 minutes at room temperature, the samples were immediately
frozen on dry ice before storage at -80.degree. C. prior to
analysis.
[0134] Samples were analysed with regards to FVIII activity in a
chromogenic assay as described by Ovlisen K et al. J. Thromb.
Haemost, 2008, 6: 969-975 and by FVIII antigen analysis using two
FVIII light chain antibodies (4F45 and 4F11) in a FVIII LOCI assay
(Luminescence oxygen channelling immunoassay).
[0135] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phonix (Pharsight
Corporaton) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using a previous i.v. pharmacokinetic
study of N8-GP in FVIII KO mice.
[0136] The circulating profiles of FVIII activity are shown
graphically in FIG. 3, the circulating concentrations of FVIII
antigen are shown in FIG. 4.
[0137] In this experiment, the bioavailability of GlycoPEGylated
FVIII alone was calculated to be 29% based on activity and 14%
based on antigen. The co-formulation with VWF increased the
bioavailability to 36% (antigen measurement).
Example 3
Haemostatic Efficacy of s.c. Administrated Co-Formulations of FVIII
Compounds with VWF Compounds
Study Outline:
[0138] Animals: FVIII k/o mice, 8-18 weeks old, male and females
[0139] Tail bleeding: n=6-12 per timepoint/group [0140]
Thrombo-elastography: n=2-4 per timepoint/group [0141]
Administration route: s.c. in the neck or flank (i.v. in the tail
vein for control groups) [0142] Dose volumes 1-10 ml/kg
Groups:
[0143] Vehicle controls dosed 24 hr prior to injury
[0144] i.v. controls dosed 5 min prior to injury
[0145] FVIII compounds co-formulated with VWF compounds dosed s.c.
5 min, 1, 3, 5, 12, 24, 48, 72, 96, 120, 144 or 168 hr prior to
injury.
Procedures:
[0146] Compounds of interest are prepared in buffer (10 mM
L-Histidine, 8.8 mM Sucrose, 0.01% Polysorbate 80, 308 mM NaCl, 1.7
mM CaCl2 (dihydrate), 0.37 mM L-Methionine, pH 6.9) to a
concentration between 40 and 10000 U/ml and stored at -80 C until
use.
[0147] Before tail transection, the mice are anaesthetised with
isoflurane and placed on a heating pad.
[0148] The tails are placed in pre-heated saline at 37.degree. C.
for 10 min.
[0149] I.v. controls are injected 5 min, 24 or 48 hr prior to
injury.
[0150] The tail is transected 4 mm from the tip.
[0151] Immediately before tail cut a 20 .mu.l blood sample is drawn
from the peri-orbital plexus for FVIII determination.
[0152] Blood is collected over 30 min and the haemoglobin
concentration determined by spectrophotometry at 550 nm.
[0153] Parallel animals are used for blood sampling and subsequent
analysis of their clotting parameters (ex vivo efficacy).
[0154] Results:
[0155] The prophylactic effect of the co-formulation is determined
from comparing the blood loss during the 30 min study period at a
certain time after s.c. administration (5 min until 168 hr) to that
of 1, a vehicle control and 2, an i.v. control group with FVIII or
glycoPEGylated FVIII. FIG. 10 shows that glycoPEGylated FVIII are
haemostatic effective 24 hr after s.c. administration of 2500 U/kg
as shown by reduction of blood loss and shortening of clot time ex
vivo. Similar effect is seen for FVIII co-formulated with a VWF
fragment.
Example 4
Evaluation of Bioavailability of FVIII
[0156] Bioavailability of co-compositions of FVIII and VWF/VWF
fragments according to the invention can be determined from
evaluations of the effect on bioavailability in PK experiments as
those described in examples 1 and 2 as well as evaluations of the
prophylactic effect as described in example 3.
[0157] The bioavailability of a FVIII compound co-formulated with a
concentration of VWF fragment that enables the majority of FVIII to
be bound to a VWF fragment compound in the injection composition
can be determined from the concentration of FVIII compound in the
composition and from experiments evaluating the binding affinity of
the VWF fragment compound to the FVIII compound such as e.g.
surface plasmon resonance experiments.
Example 5
Titration of Dosis of FVIII: VWF Co-Composition
[0158] Dose titration can be carried out as disclosed in examples
1-3. Briefly, plasma concentration of FVIII will be evaluated after
s.c. administration of doses of 70, 100, 150, 280, 500, 1000 and
2500 IU/kg (FVIII units) alone or together with a VWF fragment in
FVIII k/o mice.
Example 6
Titration of Ratio Between FVIII Compound and VWF Compound
[0159] Titration of ratios between FVIII and VWF can be carried out
as disclosed in experiments similar to that in examples 1 and 2 as
well as that described in example 3.
[0160] For PK evaluation, doses of 280, 500, 1000 or 2500 IU/kg
FVIII compound will be co-formulated with VWF fragments at a molar
ratio of 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:7.7 or up to 1:100
(FVIII to VWF fragment) and plasma concentration of FVIII evaluated
in FVIII k/o mice after s.c. administration. The maximum molar
surplus of VWF fragment to FVIII will be determined from binding
affinities of the fragment to the FVIII compound in question; the
highest molar surplus used will be the one that should result in at
least 99% of the FVIII used bound to a VWF fragment.
[0161] For prophylactic effect, the candidate compositions from the
PK experiments will be evaluated in efficacy models, such as the
tail bleeding described in example 3.
Example 7
Effect of VWF on Immunogenicity of FVIII
[0162] The immuno-modulatory effect of VWF co-formulated with a
FVIII compound is evaluated in comparison to wild type FVIII and
FVIII compounds alone.
[0163] In vivo, the relative immunogenicity is evaluated from the
titer of FVIII binding antibodies and the determination of the
level of neutralizing antibodies (inhibitors) at certain time
points after administration. The assay for detection of FVIII
binding antibodies is a radioimmunoassay (RIA). Briefly, anti-FVIII
antibodies from a sample bind to radioactive .sup.125I-labelled
rFVIII. Immunoglobulin and immune complexes bind to protein
G-sepharose and is precipitated by centrifugation. The
radioactivity in the precipitate is measured and this is
proportional to the amount of anti-FVIII antibodies in the sample.
The result is expressed in percent of the total amount of added
radioactivity. i.e. as % bound/total (% B/T).
[0164] Samples positive for anti-FVIII antibodies are analysed for
the presence of FVIII neutralizing antibodies using a chromogenic
assay. Briefly, samples are incubated with 1 IU/ml FVIII for 1 hr.
The remaining FVIII activity is determined by addition of FIX, FX,
thrombin, CaCl.sub.2 and phospholipids. After incubation the amount
of generated FXa is determined by addition of the chromogenic
substrate S-2760 and the change in optical density (OD) is
measured. The OD change is proportional to FVIII activity in the
samples, and is compared to samples containing a known amount of
FVIII and no inhibitors. The % remaining activity of the test
sample is calculated compared to the reference samples without
inhibitors/anti-FVIII antibodies added. Furthermore, the presence
of anti-VWF antibodies is measured by ELISA using monoclonal or
polyclonal anti-human VWF antibodies which does not cross react
with murine VWF. If a strong anti-VWF response is detected, this
can be expected to interfere with the binding of VWF to FVIII and
the in vivo analysis is repeated using murine VWF fragments.
[0165] The appearance of anti-drug antibodies is evaluated after
repeated (e.g. once weekly for 4 weeks or once daily for three
weeks) s.c. administration of the compounds in naive mice, in FVIII
k/o mice as well as in mice tolerized to human FVIII. The readout
is the ratio of animals with positive titres at certain time points
after the first and/or the last administration (e.g. 1, 2, 3, 4, 5,
6, 7 or 8 weeks). FVIII k/o mice are injected weekly e.g. with 1000
IU/kg FVIII alone or in combination with VWF in a molar ratio
ensuring that at least e.g. 87% of FVIII is bound to VWF. For daily
administration, the FVIII dose is lower and based upon the
bioavailability of the FVIII-VWF complex. Mice tolerized to hFVIII
are injected weekly for e.g. eight weeks s.c. with e.g. 1000 IU/kg
FVIII with or without VWF and in some experiments including
additional challenge with complete Freund's adjuvant (CFA) for the
first injection followed by weekly challenges by incomplete
Freund's adjuvant (IFA).
[0166] Relative immunogenicity of VWF versus VWF fragments and of
wild type FVIII versus a FVIII compound co-formulated with VWF is
furthermore evaluated in vitro in a human CD4+ T-cell assay. This
is done using peripheral blood mononuclear cells (PBMCs) depleted
of CD8+ T-cells. FVIII is added to the cell culture e.g. for eight
days. T-cell proliferation is evaluated during the course of the
assay by pulsing for e.g. 18 h with .sup.3H-thymidine in
sub-samples from the cultures and subsequently measuring
.sup.3H-thymidine incorporation. Interleukin 2 production is
measured at the end of the assay using an ELISPOT IL-2 kit e.g.
from R&D Systems, following the manufacturer's instructions.
The data obtained in the assays are converted to a "stimulation
index" describing the ratio between compound-stimulated versus
un-stimulated cells.
[0167] The HLA-binding capacity of VWF has been evaluated using in
silico analysis of HLA-binding properties. Strong binding to a
sequence in a modified VWF may indicate novel T-cell epitopes,
although the in silico analysis tool is predicting epitopes that
may not be processed by the naturally occurring proteases. In order
to predict if the Cys->Ser mutation will induce a risk of
induced immunogenicity in the VWF-mutants, the VWF protein
sequences are applied to an in silico peptide/HLA-II binding
prediction software. The peptide/HLA-II binding prediction software
is based on two different algorithms, NetMHCIIpan 2.1
(NetMHCIIpan-2.0--Improved pan-specific HLA-DR predictions using a
novel concurrent alignment and weight optimization training
procedure. Nielsen M, Lundegaard C, Justesen S, Lund O, and Buus S.
Immunome Res. 2010 Nov. 13; 6(1):9) performing pan-specific HLA-DR
predictions--and NetMHCII 2.0 (NN-align--A neural network-based
alignment algorithm for MHC class II peptide binding prediction.
Nielsen M and Lund O. BMC Bioinformatics. 2009 Sep. 18; 10:296)
performing HLA-DP/DQ predictions.
[0168] Twenty-three amino acid long peptides with the point of
mutation in position 12 are used as input to the algorithms. The
optimal processed peptide is assumed to be a 15'mer peptide with a
nine amino acid core peptide binding to the HLA-II. The output is
15 amino acid long peptides with 9 amino acid long core peptides
(in contact with HLA-II) and the predicted binding affinities in
nanomolar.
[0169] The predicted binding affinities of the VWF mutant peptides
are in the same range as the binding affinities of the wild type
sequences (data not shown)--and because the peptides are predicted
to bind with relatively poor affinity to the HLA-II molecules, the
risk of inducing novel CD4+T-cell epitopes is considered to be very
low.
[0170] Of note, the in silico peptide/HLA-II binding predictions
are based on experimental peptide/HLA-II binding data where it is
very challenging to test cysteine-rich peptides (due to the nature
of the peptides). Thus, cysteine-rich peptides are underrepresented
in data sets used to train the different prediction algorithms.
Therefore, the peptide/HLA-II binding predictions of these
cysteine-rich VWF peptides are uncertain and should be analysed
further using other immunogenicity prediction platforms (etc. in
vitro peptide/HLA-II binding assays or ex vivo T-cell assays).
Example 8
Subcutaneous Administration in FVIII Knockout Mice (3)
[0171] Two test compounds were prepared: [0172] a) B-domain
truncated FVIII ("turoctocog alfa"/"N8"--produced essentially as
disclosed in example 1 in WO2009108806) (4000 IU FVIII/ml
determined by chromogenic activity assay and equivalent to 2.4
.mu.M) [0173] b) B-domain truncated FVIII (turoctocog alfa) (1000
IU FVIII/ml determined by chromogenic activity assay and equivalent
to 0.6 .mu.M) co-formulated with 0.37 mg/ml VWF fragment
TIL'/E'/D3/A1 (equivalent to 4.6 .mu.M)
[0174] Based on a measured binding affinity of 1.5 nM of the VWF
fragment to FVIII, 99% of the FVIII should be bound to VWF in this
composition.
[0175] Both test compounds were formulated in 18 mg/ml NaCl, 3
mg/ml saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80,
0.25 mg/ml CaCl.sub.2, pH .about.7.3.
[0176] 12 FVIII KO mice, exon 16 knock-out in a mixed background of
C57BI/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J)
with an approximate weight of 22 g were dosed subcutaneously in the
flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test
compound.
[0177] Blood was sampled at 1, 3, 7, 17, 24, 30, 48, 72 and 96 h
post administration. The mice were anaesthetized by
Isoflurane/O.sub.2/N.sub.2O prior to blood sampling via the
retroorbital plexus. Three samples were taken from each mouse. 45
.mu.l of blood was stabilised with 5 .mu.l of sodium-citrate (0.13
M) and added 200 .mu.l FVIII coatest SP buffer (50 mM TRIS-HCl, 1%
BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g
for 5 minutes at room temperature, the supernatants were
immediately frozen on dry ice before storage at -80.degree. C.
prior to analysis.
[0178] Samples were analysed with regards to FVIII activity in a
chromogenic assay as described by Ovlisen K et al. J. Thromb.
Haemost, 2008, 6: 969-975 and by FVIII antigen analysis using two
FVIII light chain antibodies (4F45 and 4F11) in a FVIII LOCI assay
(Luminescence oxygen channelling immunoassay).
[0179] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phoenix (Pharsight
Corporaton) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using a previous i.v. pharmacokinetic
study of N8-GP in FVIII KO mice.
[0180] The circulating profiles of FVIII activity are shown
graphically in FIG. 5 and antigen levels are shown in FIG. 6.
[0181] In this experiment, the bioavailability of B-domain
truncated FVIII alone was calculated to be 0.9% based on activity.
The co-formulation with the VWF fragment increased the
bioavailability to 11%.
Example 9
Subcutaneous Administration in FVIII Knockout Mice (4)
[0182] Two test compounds were prepared: [0183] a) 226 amino acid B
domain variant (1000 IU FVIII/ml determined by chromogenic activity
assay and equivalent to 2.4 .mu.M) [0184] b) 226 amino acid B
domain variant (1000 IU FVIII/ml determined by chromogenic activity
assay and equivalent to 0.6 .mu.M) co-formulated with 0.37 mg/ml
VWF fragment TIL'/E'/D3/A1 (equivalent to 4.6 .mu.M)
[0185] Based on a measured binding affinity of 1.5 nM of the VWF
fragment to FVIII, 99% of the FVIII should be bound to VWF in this
composition.
[0186] Both test compounds were formulated in 18 mg/ml NaCl, 3
mg/ml saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80,
0.25 mg/ml CaCl.sub.2, pH .about.7.3.
[0187] 12 FVIII KO mice, exon 16 knock-out in a mixed background of
C57BI/6 and SV129, bred at Taconic M&B (B6.129S4-F8tmlKaz/J)
with an approximate weight of 22 g were dosed subcutaneously in the
flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test
compound.
[0188] Blood was sampled at 1, 3, 7, 17, 24, 30, 48, 72 and 96 h
post administration. The mice were anaesthetized by
Isoflurane/O.sub.2/N.sub.2O prior to blood sampling via the
retro-orbital plexus. Three samples were taken from each mouse. 45
.mu.l of blood was stabilised with 5 .mu.l of sodium-citrate (0.13
M) and added 200 .mu.l FVIII coatest SP buffer (50 mM TRIS-HCl, 1%
BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g
for 5 minutes at room temperature, the supernatants were
immediately frozen on dry ice before storage at -80.degree. C.
prior to analysis.
[0189] Samples were analysed with regards to FVIII activity in a
chromogenic assay as described by Ovlisen K et al. J. Thromb.
Haemost, 2008, 6: 969-975 and by FVIII antigen analysis using two
FVIII light chain antibodies (4F45 and 4F11) in a FVIII LOCI assay
(Luminescence oxygen channelling immunoassay).
[0190] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phonix (Pharsight
Corporaton) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using a previous i.v. pharmacokinetic
study of N8-GP in FVIII KO mice.
[0191] In this experiment, the bioavailability of the 226 amino
acid B domain FVIII variant alone was similar to that obtained with
co-formulation with VWF. Hence, for this variant with a longer
B-domain, VWF did not increase the bioavailability.
Example 10
Construction of Expression Vectors Encoding FVIII Molecules
[0192] Plasmid with insert encoding the F8-500 FVIII molecule
(F8-500 equals turoctocog alfa/N8 encoding sequence) was used for
production of FVIII. Starting at the N-terminus, the F8-500 vector
encodes the FVIII heavy chain without the B domain (amino acids
1-740), a 21 amino acid linker (SFSQNSRHPSQNPPVLKRHQR--SEQ ID
NO:2), and the FVIII light chain (amino acids 1649-2332 of
full-length wild-type human FVIII). The sequence of the 21 amino
acid linker is derived from the FVIII B domain and consists of
amino acids 741-750 and 1638-1648 of full length wild-type human
FVIII. Fragments of FVIII cDNA were amplified from full length
FVIII cDNA and inserted into F8-500 coding plasmid giving rise to
DNA constructs encoding the BDD FVIII.
[0193] Contructs encoding F8-500D-HIS-C2-linked-(GGGS)6-hFc(IgG1),
F8-500D-HIS-C2-linked-(GGGS)6-mFc(IgG2A), and
F8-500D-HIS-C2-linked-(GGGS)6-albumin were established as described
in the following. The internal BamHI site (aa 604-606) in F8-500
coding DNA was eliminated by site-directed mutagenesis and DNA
encoding the flexible (GGGS).sub.6 linker was inserted 3' to the
coding region. A new BamHI site was introduced in the 3' end of the
linker-coding DNA in order to ease cloning of C-terminal fusion
partners between BamHI and NotI sites. Thus, a construct encoding
F8-500-C2-linked-(GGGS)6 was generated. DNA encoding human Fc
(IgG1), mouse Fc (IgG2a), and human serum albumin was
amplified.
[0194] The PCR products were inserted between the BamHI and Not I
sites of the F8-500-C2-linked-(GGGS)6 coding vector giving rise to
constructs encoding F8-500-C2-linked-(GGGS)6-hFc(IgG1),
F8-500-C2-linked-(GGGS)6-mFc(IgG2A), and
F8-500-C2-linked-(GGGS)6-albumin. A SphI/ClaI restriction fragment
from the latter constructs were transferred to a F8-500D-His coding
constructs in order to generate
F8-500D-HIS-C2-linked-(GGGS)6-hFc(IgG1)-,
F8-500D-HIS-C2-linked-(GGGS)6-mFc(IgG2A)-, and
F8-500D-HIS-C2-linked-(GGGS)6-albumin coding constructs.
[0195] For transient expression as described in Example 11, DNA
constructs consisting of the mammalian expression vector pTT5 with
insert encoding BDD FVIII were utilized. For generation of stable
cell lines producing BDD FVIII, the vector pTSV7 is utilized. This
vector encodes dihydrofolate reductase allowing selection of
transfected cells with the dihydrofolate reductase system. A
SpeI/AgeI restriction fragment from a pTT5-derived vector encoding
F8-500D-His was transferred to a pTSV7-derived vector encoding
F8-500 leading to construct #1917 consisting of pTSV7 with insert
encoding F8-500D-His.
Example 11
Transient Expression of FVIII
[0196] HKB11 cells at a density of 0.9-1.1.times.10.sup.6 were
transfected with a complex of plasmid (0.7 mg/l or 1.0 mg/l) and
the transfection agent, 293Fectin (Invitrogen) (1.0 ml/l or 1.4
ml/l). The transfection complex was prepared by diluting the
plasmid and the transfection separately, mixing the two solutions,
and incubating the mixture at room temperature for 20 minutes. The
complex mixture was added to the cell suspension and the suspension
was incubated in shaker incubator for 4 or 5 days at 36.5.degree.
C. or 37.degree. C. and at 5% or 8% CO.sub.2. Cell culture harvests
were analysed by chromogenic FVIII assay as described in Example 14
and/or filtered through a 0.22 .mu.m membrane filter and utilized
for purification of FVIII as described in Example 13.
Example 12
Stable Cell Line Expressing FVIII
[0197] Serum-free adapted CHO-DUKX-B11 cells were transfected with
the expression plasmid construct #1917 described in Example 10 and
encoding the FVIII F8-500D-His. Transfected cells were selected
with the dihydrofolate reductase system and cloned by limiting
dilution. Clones were screened for FVIII production by ELISA and
chromogenic activity assay. The clone GedT019A was selected for
upscaling. The cells were transferred to a bioreactor. The
F8-500D-His protein was purified from cell culture harvests as
described in Example 13
Example 13
Purification of FVIII
[0198] A column was packed with the resin VIIISelect (GE
Healthcare), with the dimensions 1.6 cm in diameter and 4 cm in bed
height giving 8 mL, and was equilibrated with 20 mM Imidazole+10 mM
CaCl.sub.2+0.01% Tween80+250 mM NaCl, pH7.3 at 500 cm/h. The
culture filtrate prepared as described in Example 3 was applied to
the column, and the column was subsequently washed with first
equilibration buffer and then 20 mM Imidazole+10 mM
CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3. The bound FVIII was
eluted isocratic at 90 cm/h with 20 mM Imidazole+10 mM
CaCl.sub.2+0.01% Tween80+1M Ammoniumacetate+6.5M Propylenglycol,
pH7.3. The fractions containing FVIII were pooled and diluted 1:10
with 20 mM Imidazole+10 mM CaCl.sub.2+0.01% Tween80, pH7.3 and
applied to a column packed with F25-Sepharose (Thim et al.,
Haemophilia, 2009). The column dimension was 1.6 cm in diameter and
2 cm in bed height giving 4 mL in column volume. The column was
equilibrated at 180 cm/h with 20 mM Imidazole+10 mM
CaCl.sub.2+0.01% Tween80+150 mM NaCl+1M Glycerol, pH7.3 prior to
application. After application the column was washed first with
equilibration buffer and then 20 mM Imidazole+10 mM
CaCl.sub.2+0.01% Tween80+650 mM NaCl, pH7.3. The bound FVIII was
isocratic eluted with 20 mM Imidazole+10 mM CaCl.sub.2+0.01%
Tween80+2.5M NaCl+50% (v/v) Ethylenglycol, pH7.3 at 30 cm/h. The
fractions containing FVIII were pooled and diluted 1:15 with 20 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80, pH7.3, except
FVIII-variants with deletions of the a3 domain which were diluted
1:45 in the same buffer. The diluted pool was applied to a column
packed with Poros 50HQ (PerSeptive Biosystem), with the column
dimensions 0.5 cm in diameter and 5 cm in bed height giving 1 mL in
column volume. The column was equilibrated at 300 cm/h with 20 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80+50 mM NaCl+1M Glycerol,
pH7.3 prior to application. The column was washed with
equilibration buffer before the elution using a linear gradient
over 5 column volumes from equilibration buffer to 20 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80+1M NaCl+1M Glycerol,
pH7.3. The fractions containing FVIII were pooled and the pool was
stored at -80.degree. until use.b
[0199] The FVIII molecules with HIS-tag were purified essentially
as described above, however the second purification step
(F25-sepharose) was exchanged to Chelating Sepharose FF (GE
Healtcare) charged with 2 column volumes of 1M NiSO.sub.4. The
column dimension was 0.5 cm in diameter and 5 cm bed height giving
1 mL column volume. The column was equilibrated with 30 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3 at 180
cm/h prior to application. After application the column was washed
with 30 column volumes of equilibration buffer prior to elution
using a linear gradient over 5 column volumes to 250 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3. The
fractions containing FVIII were pooled and diluted 1:30 with 20 mM
Imidazole+10 mM CaCl.sub.2+0.01% Tween80, pH7.3. The final
purification step (Poros 50HQ) was performed as described
above.
Example 14
FVIII Activity in Cell Culture Harvests Measured by Chromogenic
Assay
[0200] The FVIII activity (FVIII:C) of the rFVIII compound was
evaluated in a chromogenic FVIII assay using Coatest SP reagents
(Chromogenix) as follows: rFVIII samples and a FVIII standard
(Coagulation reference, Technoclone) were diluted in Coatest assay
buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with
preservative). Fifty .mu.l of samples, standards, and buffer
negative control were added to 96-well microtiter plates
(Spectraplates MB, Perkin Elmer). All samples were tested diluted
1:100, 1:400, 1:1600, and 1:6400. The factor IXa/factor X reagent,
the phospholipid reagent and CaCl.sub.2 from the Coatest SP kit
were mixed 5:1:3 (vol:vol:vol) and 75 .mu.l of this added to the
wells. After 15 min incubation at room temperature, 50 .mu.l of the
factor Xa substrate 5-2765/thrombin inhibitor 1-2581 mix was added
and the reactions were incubated 5 min at room temperature before
25 .mu.l 1 M citric acid, pH 3, was added. The absorbance at 405 nm
was measured on an Envision microtiter plate reader (Perkin Elmer)
with absorbance at 620 nm used as reference wavelength. The value
for the negative control was subtracted from all samples and a
calibration curve prepared by linear regression of the absorbance
values plotted vs. FVIII concentration. The yields of the present
FVIII relative to that of the F8-500 protein are shown in Table
1.
Example 15
FVIIIactivity in Purified Samples Measured by Chromogenic Assay
[0201] The FVIII activity (FVIII:C) of the rFVIII compound was
evaluated in a chromogenic FVIII assay using Coatest SP reagents
(Chromogenix) as follows: rFVIII samples and a FVIII standard (e.g.
purified wild-type rFVIII calibrated against the 7th international
FVIII standard from NIBSC) were diluted in Coatest assay buffer (50
mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty
.mu.l of samples, standards, and buffer negative control were added
to 96-well microtiter plates (Nunc) in duplicates. The factor
IXa/factor X reagent, the phospholipid reagent and CaCl.sub.2 from
the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 .mu.l of
this added to the wells. After 15 min incubation at room
temperature 50 .mu.l of the factor Xa substrate S-2765/thrombin
inhibitor 1-2581 mix was added and the reactions incubated 10 min
at room temperature before 25 .mu.l 1 M citric acid, pH 3, was
added. The absorbance at 415 nm was measured on a Spectramax
microtiter plate reader (Molecular Devices) with absorbance at 620
nm used as reference wavelength. The value for the negative control
was subtracted from all samples and a calibration curve prepared by
linear regression of the absorbance values plotted vs. FVIII
concentration. The specific activity was calculated by dividing the
activity of the samples with the protein concentration determined
by HPLC. For HPLC, the concentration of the sample was determined
by integrating the area under the peak in the chromatogram
corresponding to the light chain and compare with the area of the
same peak in a parallel analysis of a wild-type rFVIII, where the
concentration was determined by amino acid analyses. The results
are shown in Table 1.
Example 16
FVIII Activity in Purified Samples Measured by One-Stage Clot
Assay
[0202] FVIII activity (FVIII:C) of the rFVIII compounds was further
evaluated in a one-stage FVIII clot assay as follows: rFVIII
samples and a FVIII standard (e.g. purified wild-type rFVIII
calibrated against the 7th international FVIII standard from NIBSC)
were diluted in HBS/BSA buffer (20 mM hepes, 150 mM NaCl, pH 7.4
with 1% BSA) to approximately 10 U/ml followed by 10-fold dilution
in FVIII-deficient plasma containing VWF (Dade Behring or Siemens).
The samples were subsequently diluted in HBS/BSA buffer. The APTT
clot time was measured on an ACL300R or an ACL9000 instrument
(Instrumentation Laboratory) using the single factor program.
FVIII-deficient plasma with VWF (Dade Behring or Siemens) was used
as assay plasma and SynthASil, (HemosIL.TM., Instrumentation
Laboratory) as aPTT reagent. In the clot instrument, the diluted
sample or standard is mixed with FVIII-deficient plasma, aPTT
reagents at 37.degree. C. Calcium chloride is assed and time until
clot formation is determined by turbidity. The FVIII activity in
the sample is calculated based on a standard curve of the clot
formation times of the dilutions of the FVIII standard. The results
are shown in table 1.
TABLE-US-00004 TABLE 1 Yields and specific activities of different
BDD FVIII molecules ("His-tagged" for easier purification). Yield
by transient Specific activity Specific activity transfection
measured by measured by one- B domain amino (relative to F8-
chromogenic stage clot assay Compound acids 500) assay (IU/mg)
(IU/mg) F8-500E-His 741-857 + 1637-1648 0.7 10501 9122 F8-500L-His
741-914 + 1637-1648 0.6 10330 8282 F8-500M-His 741-954 + 1637-1648
0.6 12404 10259 F8-500D-His 741-965 + 1637-1648 0.3 9015 9579
F8-500G-His 741-965 + 1637-1648 0.7 11507 9822 Amino acid
replacements: N757Q-N784Q- N828Q-N900Q- N943Q-N963Q F8-500N-His
741-1003 + 1637-1648 0.4 -- -- F8-500H-His 741-1020 + 1637-1648 0.7
10027 10541 F8-500I-His 741-1079 + 1637-1648 0.7 -- -- F8-500J-His
741-1206 + 1637-1648 0.6 -- -- F8-500F-His 741-1261 + 1637-1648 0.3
5691 4855 F8-500K-His 741-1309 + 1637-1648 0.4 -- -- F8-500-His2-4N
741-914 + 1637-1648 0.6 -- -- F8-500-His2-5N 741-954 + 1637-1648
0.7 -- -- F8-500-His2-6N 741-968 + 1637-1648 0.6 14088 12784
F8-500-His2-7N 741-1003 + 1637-1648 0.5 7211 7542 F8-500-His2-8N
741-1018 + 1637-1648 0.7 8664 7481 F8-500-His2-10N 741-1070 +
1637-1648 0.6 12391 8253 F8-500-His2-11N 741-1230 + 1637-1648 0.5
-- -- F8-500-His2-15N 741-1301 + 1637-1648 0.4 -- -- F8-500D-His-
741-965 + 1637-1648 0.5 15282 9729 D519V-E1984A F8-500D-His-C2
741-965 + 1637-1648 0.6 -- -- linked-(GGGS)6- hFc(IgG1)
F8-500D-His-C2 741-965 + 1637-1648 0.6 13509 8858 linked-(GGGS)6-
mFc(IgG2a) F8-500D-His-C2 741-965 + 1637-1648 0.7 12226 5852
linked-(GGGS)6- albumin
Example 17
Construction of Expression Vectors Encoding VWF Fragments
[0203] DNA fragments encoding the VWF signal peptide, followed by
different C-terminally truncated versions, the VWF D' domain and
the VWF D3 domain, an Ala-Leu-Ala spacer and a HPC4 tag were
generated by polymerase chain reaction (PCR) using plasmid pLC095
as template (Plasmid pLLC095 is described in Example 26. The primer
JP1000 was used as forward primer in all PCR reactions in
combination with the reverse primers JP1001-JP1008 shown in Table
2.
TABLE-US-00005 TABLE 2 Forward primer Forward primer Sequence
(5'-3') JP1000 VWF-HindIII S
CTAAGCGTAAGCTTGCCACCATGATTCCTGCCAGATTTGC CGG (SEQ ID NO: 23)
Reverse primer Reverse primer Sequence (5'-3') JP1001 VWF 764-828
TGGTCCTCAGCTAGCGCGGGACACCTTTCCAGGGCCACA C (SEQ ID NO: 24) JP1002
VWF 764-865 TGGTCCTCAGCTAGCGCGGCATCACACACATGGTCTGTG C (SEQ ID NO:
25) JP1003 VWF 764-1035 TGGTCCTCAGCTAGCGCTCTGGTGTCAGCACACTGCGAG CTC
(SEQ ID NO: 26) JP1004 VWF 764-1041
TGGTCCTCAGCTAGCGCTGAGTCCAGAGGCACTTTTCTGG (SEQ ID NO: 27) JP1005 VWF
764-1045 TGGTCCTCAGCTAGCGCGGTGGCAGGGGATGAGTCCAGA G (SEQ ID NO: 28)
JP1006 VWF 764-1250 TGGTCCTCAGCTAGCGCGGCATCTGTGGGAGGCACCACC (SEQ ID
NO: 29) JP1007 VWF 764-1261 TGGTCCTCAGCTAGCGCGTCCTCCACATACAGAGTGGTG
(SEQ ID NO: 30) JP1008 VWF 764-1268
TGGTCCTCAGCTAGCGCATCGTGCAACGGCGGTTCCGAG (SEQ ID NO: 31)
[0204] The PCR products were digested with HindIII and NheI and
were subsequently cloned into a HindIII and NheI digested pJSV164
vector using Rapid DNA Ligation kit (Roche Diagnostics GmbH,
Mannheim, Germany). pJSV164 is a pTT5 based expression vector (Yves
Durocher, CNRC, Montreal, Canada) containing a CD33 signal peptide
and a HPC4 tag. Digestion of pJSV164 with HindIII and NheI removes
the CD33 signal peptide and allows cloning of the gene of interest
in frame with the HPC4 tag to generate an expression cassette
encoding a C-terminally HPC4 tagged gene of interest in which the
gene of interest and the HPC4 tag is separated by an Ala-Leu-Ala
linker peptide. The ligation reactions were transformed into Top10
cells (Life Technologies, Carlsbad, Calif., USA).
[0205] The resulting eight plasmids were named as shown in Table 3.
The amino acid sequences of the generated proteins are outlined in
SEQ ID NO's: 4, 5, 6, 7, 8, 11 and 16.
TABLE-US-00006 TABLE 3 Vector name Insert pJSV343 VWF 764-828-HPC4
(SEQ ID NO: 4) pJSV344 VWF 764-865-HPC4 (SEQ ID NO: 5) pJSV345 VWF
764-1035-HPC4 (SEQ ID NO: 6) pJSV346 VWF 764-1041-HPC4 (SEQ ID NO:
7) pJSV347 VWF 764-1045-HPC4 (SEQ ID NO: 8) pJSV348 VWF
764-1250-C1099/1142S-HPC4 (SEQ ID NO: 11) pJSV349 VWF
764-1261-C1099/1142S-HPC4 (SEQ ID NO: 14) pJSV350 VWF
764-1268-C1099/1142S-HPC4 (SEQ ID NO: 16)
Example 18
Construction of Expression Vectors Encoding VWF Fragments (2)
[0206] Three additional HPC4 tagged, truncated variants of VWF were
generated by Ligation independent cloning (LIC) using pJSV348 (see
Example 17) as template. Three independent PCR reactions were
set-up on pJSV438 using the primers shown in Table 4.
TABLE-US-00007 TABLE 4 Fragment Primer name Primer sequence (5'-3')
VWF(864-1250)- VWF(864-1250)- GGGACCCTTTGTGATGCCACGTGCTCCACGATCGG
HPC4 HPC4 S (SEQ ID NO: 32) (SEQ ID NO 12) VWF(864-1250)-
GCACGTGGCATCACAAAGGGTCCCTGGCAAAATGA HPC4 AS G (SEQ ID NO: 33)
VWF(764-1128)- VWF(764-1128)- TTGTGCCCCCAGGAGGACCAAGTAGATCCGCGGCT
HPC4 HPC4 S C (SEQ ID NO: 34) (SEQ ID NO: 9) VWF(764-1129)-
TACTTGGTCCTCCTGGGGGCACAATGTGGCCGTC HPC4 AS (SEQ ID NO: 35)
VWF(764-1198)- VWF(764-1198)- GACTGTCCAGTGGAGGACCAAGTAGATCCGCGG
HPC4 HPC4 S (SEQ ID NO: 36) (SEQ ID NO: 10) VWF(764-1198)-
TTGGTCCTCCACTGGACAGTCTTCAGGGTCAA (SEQ HPC4 AS ID NO: 37)
[0207] The three PCR fragments VWF(864-1250)-HPC4,
VWF(764-1128)-HPC4 and VWF(764-1198)-HPC4 were 5685/5610/5817 by in
size respectively. The PCR fragments were DpnI treated to remove
methylated template DNA. The PCR fragments were subsequently
purified from gel and were self-ligated by LIC using the In-Fusion
HD Cloning Kit (Clontech, Mountain View, Calif.,
USA) to generate circular DNA fragments and subsequently
transformed into Top10 cells (Life Technologies, Carlsbad, Calif.,
USA).
[0208] The resulting three plasmids were named as shown in Table 5.
The amino acid sequences of the generated proteins are outlined in
SEQ ID NOs:12, 9, and 10.
TABLE-US-00008 TABLE 5 Vector name Insert pJSV405
VWF(864-1250)-C1099/1142S-HPC4 monomer (SEQ ID NO: 12) pJSV406
VWF(764-1128)-C1099S-HPC4 monomer (SEQ ID NO: 9) pJSV407
VWF(764-1198)-C1099/1142S-HPC4 monomer (SEQ ID NO: 10)
Example 19
Transient Expression of VWF Fragments
[0209] Human embryonic kidney 293 6E suspension cells at a density
of 0.9-1.1.times.10.sup.6 cells/ml were transfected with a complex
of VWF fragment coding plasmid (0.7 mg/l or 1.0 mg/l) and the
transfection agent 293Fectin (Invitrogen) (1.0 ml/l or 1.4 ml/l).
The transfection complex was prepared by diluting the plasmid and
the transfection separately, mixing the two solutions, and
incubating the mixture at room temperature for 20 minutes. The
complex mixture was added to the cell suspension and the suspension
was incubated in shaker incubator for 5 days at 36.5.degree. C. or
37.degree. C. and at 5% or 8% CO.sub.2. Cell culture harvests were
filtered through a 0.22 .mu.m membrane filter and utilized for
purification of VWF fragment as described in Example 22.
Example 20
Preparation of Dimer Forms of VWF Fragments
[0210] In the native full length VWF molecule (SEQ ID NO:22) two
cysteine residues in the N-terminal part of the molecule are
supposed to participate in the dimerization and/or multimerization
of VWF: Cys1099 and Cys1142.
[0211] In all of the monomeric fragments of the sequences (SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
SEQ ID NO:20, and SEQ ID NO:21) two cysteine residues (Cys1099 and
Cys1142) are mutated to other amino acid residues so that the
expressed molecule is not able to form dimers/multimers. A
monomeric fragment of SEQ ID N0:9 is generated by mutating Cys 1099
to another amino acid residue.
[0212] In some cases, a dimeric form of the VWF fragments is
wanted. This can be accomplished in several ways:
[0213] One method to accomplish dimer formation is to keep the two
residues at position 1099 and position 1142 as cysteines. In order
to make a recombinant dimeric molecule, the cDNA encoding the
desired VWF fragment is including the presequence of VWF e.g the D1
D2 sequence of VWF (amino acid residues 23-763 of SEQ ID NO:22).
This will, during processing in the golgi apparatus align two
monomers of a given VWF fragment in a configuration allowing a
dimeric molecule to be formed with two disulphide bonds in which
Cys1099 in monomer 1 is connected to a Cys1099 in monomer 2 and
Cys1142 in monomer 1 is connected to Cys1142 in monomer 2.
[0214] Another method to accomplish dimer formation is to avoid the
inclusion of the presequence (amino acid residues 23-763 of SEQ ID
NO:22) and simply let a recombinant VWF fragment with Cys in
position 1099 and 1142 form a dimeric molecule. This can in
principle result in a series of different dimers e.g.:
[0215] Cys1099-Cys1099/Cys1142-Cys1142 (two disulphide bonds--like
above)
[0216] Cys1099-Cys1142/Cys1099-Cys1142 (two disulphide bonds)
[0217] Cys1099-Cys1099 (one disulphide bond)
[0218] Cys1142-Cys1142 (one disulphide bond)
[0219] Cys1099-Cys1142 (one disulphide bond)
[0220] Yet another method to accomplish dimer formation may be toto
replace one of the cysteine residues 1099 or 1142 with other amino
acid residues (e.g. Serine, Arginine).
[0221] If Cys1099 is replaced with a non-Cysteine residue, the
molecule may form a dimer by establishment of a disulphide bond
between Cys1142 in monomer 1 with Cys1142 in monomer 2.
[0222] If Cys1142 is replaced with a non-Cysteine residue, the
molecule may form a dimer by establishment of a disulphide bond
between Cys1099 in monomer 1 with Cys1099 in monomer 2.
[0223] The dimeric forms mentioned above may be constructed either
with or without the D1 D2 presequence of VWF (amino acid residues
23-763 of SEQ ID N0:22).
[0224] The different monomeric and dimeric forms will have
different properties with regards to their binding to FVIII, their
ease of production and their effect on bioavailability of FVIII
when injected subcutaneously as a co-formulation.
Example 21
Evaluation of Binding of VWF and VWF Fragments to FVIII Using a
Competition ELISA
[0225] In order to investigate the binding of the different VWF
fragments to FVIII the following method is used. Briefly, human VWF
is coated in a microtiterplate and incubated overnight at 4.degree.
C. After blocking, a solution with pre-incubated FVIII (1 nM) and
VWF/VWF-fragment is added to the plate, followed by detection with
biotinylated anti FVIII antibody and streptavidin-peroxidase S-POD
(1:20000). The absorbance is measured at 450/620 nm. The IC50
values are shown in Table 6.
TABLE-US-00009 TABLE 6 Derived Compound from SEQ number
Domain/comment VWF fragment sequence ID NOs: IC50 (Ki) 2304 TIL'E'
VWF(764-865)-ALA-HPC4 monomer 5 2.0 .mu.M 2306 TIL'/E'/VWD3 II
VWF(764-1041)-ALA-HPC4 monomer 7 2.2 .mu.M 2307 TIL'/E'/VWD3 III
VWF(764-1045)-ALA-HPC4 monomer 8 2.0 .mu.M 2308 TIL'/E'/D3 I
VWF(764-1250)-C1099/1142S-ALA-HPC4 monomer 11 12 nM 2309 TIL'/E'/D3
II VWF(764-1261)-C1099/1142S-ALA-HPC4 monomer 14 10 mM 2310
TIL'/E'/D3 III VWF(764-1268)-C1099/1142S-ALA-HPC4 monomer 16 15 nM
0170 TIL'/E'/D3/A1 III VWF(764-1464)-C1099/1142S-HPC4 monomer 19 12
nM 0194 TIL'/E'/D3/A1 III VWF(764-1464)-C1099S-HPC4 monomer 19 8.0
nM 0240 TIL'/E'/D3/A1 VWF(764-1464)-HPC4 dimer 19 0.7 nM IIIdimer
0001 D3 I VWF(864-1250)-C1099/1142S-ALA-HPC4 monomer 12 20 .mu.M
0003 TIL'/E'/VWD3/C8- VWF(764-1198)-C1099/1142S-ALA-HPC4 monomer 10
28 nM 3/TIL-3 0314 Plasma derived full VWF (764-2813) 22 1.1 nM
length VWF
[0226] These differences in FVIII binding between different
fragments could indicate different effects in a subcutaneously
administered FVIII co-formulation. The 1050 values are also being
used to determine the optimal VWF and FVIII concentrations in the
co-formulation mixtures.
Example 22
Purification and Characterisation of HPC4-Tagged VWF Fragments
[0227] Some VWF fragments are cloned and expressed with a
C-terminal HPC4 tag: EDQVDPRLIDGK (SEQ ID NO:38). Sometimes an
additional linker with the sequence of ALA is introduced between
the VWF fragment and the HPC4 tag. After cloning, expression and
cell culturing the cell media is added CaCl.sub.2 to a final
concentration of 1 mM. The media is passed over an anti-HPC4
column. The column is equilibrated with 20 mM HEPES, 100 mM NaCl, 1
mM CaCl.sub.2, pH=7.5. After application of the cell media, the
column is washed with 20 mM HEPES, 1M NaCl, 1 mM CaCl.sub.2, pH=7.5
and the HPC4-tagged VWF fragment is subsequently eluted with 20 mM
HEPES, 100 mM NaCl, 5 mM EDTA, pH=7.5. The pool from the anti-HPC4
column is added 3 volumes of water to reduce the conductivity and
applied onto a Mono Q column. Prior to the application the Mono Q
column is equilibrated with 20 mM HEPES, 100 mM NaCl, 5 mM EDTA,
pH=7.5. The Mono Q column is washed with 20 mM HEPES, 100 mM NaCl,
pH=7.5 and the VWF fragment is eluted with a gradient from 100 mM
NaCl to 2M NaCl in 20 mM HEPES, 10 mM CaCl.sub.2, pH=7.5.
[0228] The purified protein is characterised by 1) SDS-gel
electrophoreses, 2) analytical HPLC and 3) amino acid sequence
analysis.
[0229] Purification and Characterisation of Non-Tagged VWF
Fragments.
[0230] After cloning, expression and cell culturing the cell media
is passed over an anti-VWF column. The anti-VWF antibody recognise
amino acid residue number 764-865 of VWF (SEQ ID NO:5). The column
is equilibrated with 20 mM HEPES, 100 mM NaCl, pH=7.5. After
application of the cell media, the column is washed with 20 mM
HEPES, 1M NaCl, pH=7.5 and the VWF fragment is subsequently eluted
with 50 mM acetic acid, 100 mM NaCl, pH=4.0. The pool from the
anti-VWF column is adjusted to pH=7.5 and applied onto a Mono Q
column. Prior to the application the Mono Q column is equilibrated
with 20 mM HEPES, 100 mM NaCl, pH=7.5. The Mono Q column is washed
with 20 mM HEPES, 100 mM NaCl, pH=7.5 and the VWF fragment is
eluted with a gradient from 100 mM NaCl to 2M NaCl in 20 mM HEPES,
pH=7.5.
[0231] The purified VWF fragment is characterised by 1) SDS-gel
electrophoreses, 2) analytical HPLC and 3) amino acid sequence
analysis.
Example 23
Evaluation of VWF Fragments Binding to FVIII by Using Isothermal
Titration Calorimetry
[0232] All protein samples are dialyzed in 50 mM Hepes pH 7.4, 150
mM NaCl, 10 mM CaCl.sub.2 buffer. Each iTC experiment involves
filling the iTC cell with FVIII (approximately 250 .mu.L) and the
syringe with VWF variants (approximately 40 .mu.L). Temperature is
set as required and the protein sample is allowed to equilibrate
under given experimental conditions (approximately 10 minutes).
Typically 17-20 injections (of 2-2.5 .mu.L) of VWF variants into
cell, containing FVIII, are performed. The first injection is
always of 0.2 .mu.L and is discarded from the final data analysis
in order to account for diffusion during equilibration step.
Stirring speed is set between 700-1000 rpm. Filter period for data
collection is 5 sec with a high feedback mode setting. Each
titration is spaced by 120 sec. Appropriate control experiments are
performed. Raw data is processed to set baseline and integrated to
obtain a final isotherm. This binding isotherm is fit to a
single-site model to yield K.sub.d, stoichiometry (n), .DELTA.H,
and .DELTA.S values to complete characterization of VWF variant
binding to FVIII. An example binding isotherm is shown in FIG. 9.
These data are being used for determining the optimal
concentrations of the FVIII and the VWF fragment in co-formulations
intended for subcutaneous administrations.
Example 24
Subcutaneous Administration in FVIII Knockout Mice
[0233] Test compounds were prepared as follows: Test compounds were
formulated in 18 mg/ml NaCl, 3 mg/ml saccharose, 1.5 mg/ml
L-histidine, 0.1 mg/ml polysorbate 80, 0.25 mg/ml CaCl2, pH
.about.7.3. For test formulations containing VWF or VWF fragments
the % FVIII bound by VWF in the co-formulation was calculated using
the available IC50 (Ki) values as described above in example 21
(table 6) assuming K.sub.i=K.sub.d or the K.sub.d values obtained
as described in example 23.
[0234] FVIII KO mice, exon 16 knock-out in a mixed background of
C57B1/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J)
with an approximate weight of 22 g were dosed subcutaneously in the
flank with FVIII in combination with various proteins, 6-9 mice
with each test compound. The dose volume was 5 ml/kg or 0.25 ml/kg
if indicated in table 7.
[0235] Blood was sampled at 9 time points from 0-96 h, n=2-3
mice/time point, 3 blood samples from each mice in a sparse
sampling regime. The mice were anaesthetized by
Isoflurane/O.sub.2/N.sub.2O prior to blood sampling via the
retroorbital plexus. 45 .mu.l of blood was stabilised with 5 .mu.l
of sodium-citrate (0.13 M) and added 200 .mu.l FVIII Coatest SP
buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3).
After centrifugation at 4000 g for 5 minutes at room temperature,
the supernatants were immediately frozen on dry ice before storage
at -80.degree. C. prior to analysis.
[0236] Samples were analysed with regards to FVIII chromogenic
activity as described by Ovlisen K et al. J. Thromb. Haemost, 2008,
6: 969-975 and by FVIII antigen analysis using two FVIII light
chain antibodies (4F45 and 4F11) in a FVIII LOCI assay
(Luminescence oxygen channelling immunoassay).
[0237] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phoenix (Pharsight
Corporaton) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using a previous i.v. pharmacokinetic
study of N8 or N8-GP in the FVIII KO mouse strain.
[0238] The s.c. FVIII bioavailabilities of the test compounds are
shown in table 7 below and in FIGS. 7 and 8.
TABLE-US-00010 TABLE 7 Bioavailability values of a series of
different FVIII molecules and FVIII/VWF fragment co-formulations
obtained with s.c. administration in FVIII k/o mice. The left
column "FVIII" denotes the FVIII compound used in the experiment.
The column labelled "FVIII dose" denotes the FVIII dose (IU/kg)
used in the experiment, the column labelled "co-formulation
protein" denotes the co-formulated protein (if any) used in the
experiment. The column labelled "Molar ratio" denotes the molar
ratio to FVIII of the protein in the co-formulation. The column
labelled "FVIII Saturation" denotes the calculated fraction of
FVIII that is binding the co-formulated protein at the
concentrations used in the experiment. The column labelled "F %"
denotes the bioavailability of FVIII in the experiment. FVIII
Co-Formulation FVIII FVIII Dose Protein Molar Ratio Saturation F %
Turoctocog alfa 5000 (764-1464) 1 87% 7.3 monomer VWF rFVIII
derived 2500 (764-1464) 1 82% 7.4 from the full- Dimer VWF length
sequence (Kogenate .RTM.) Turoctocog alfa 2500 (764-1250) 1 82% 7.6
Monomer VWF Turoctocog alfa 2500 (764-1041) 34 82% 7.8 Monomer VWF
Turoctocog alfa 2500 (764-828) 1 12% 1.4 Monomer VWF Turoctocog
alfa 2500 (764-865) 1 12% 2.7 Monomer VWF Turoctocog alfa 2500
(764-1045) 1 12% 2.0 Monomer VWF Turoctocog alfa 2500 (764-865) 34
83.3% 4.3 Monomer VWF Turoctocog alfa 2500/ (764-1041) 3x 5.5% 5.03
0.25 ml/kg Monomer VWF Turoctocog alfa 2500/ (764-865) 3x 86.5% 1.9
0.25 ml/kg Monomer VWF Turoctocog alfa 2500/ (764-1464) 1x 99% 8.4
0.25 ml/kg Dimer VWF Turoctocog alfa 2500 (764-1464) 1 82% 5.6
Murine monomer VWF Turoctocog alfa 2500 Human serum 611 Not 3.7
Albumin applicable Turoctocog alfa 2500 plasma derived 1 99% 0.0
full length VWF Turoctocog alfa 5000 (764-1464) 7.7 99% 8.2 monomer
VWF Turoctocog alfa 5000 (764-1464) 3 99% 6.7 monomer VWF
Turoctocog alfa 5000 (764-1464) 1 87% 7.3 monomer VWF Turoctocog
alfa 5000 None Not Not 2.3 applicable applicable FVIII with a 226
5000 None Not Not 4.3 aa B domain applicable applicable FVIII with
a - 226 5000 (764-1464) 7.7 0.99 7.0 aa B domain monomer VWF N8-GP
2500 (764-1464) 1 0.82 27 monomer VWF N8-GP 10000 (764-1464) 7.7
0.99 47 monomer VWF N8-GP 2500 (764-1464) 7.7 0.99 36 monomer VWF
N8-GP 2500 (764-1464) 1 0.99 33 Dimer VWF FVIII-K1804- 2500
(764-1464) 1 0.82 50 Hep157 monomer VWF FVIII-K1804- 2500 None Not
Not 27 Hep157 applicable applicable PSA40Kd-O- 2500 (764-1464) 1
0.82 8.8 Glycan-N8 monomer VWF PSA40Kd-O- 2500 None Not Not 6.1
Glycan-N8 applicable applicable 40kDa-PEG- 10000 None Not Not 20
FVIII- applicable applicable K2092A + F2093A N8-GP 10000 4F30 FVIII
5 0.99 11 reduced uptake antibody N8-GP 1000 Hirudin 0.5 mg/kg Not
7.6 applicable N8-GP 10000 Hyaluronidase 0.5 activity Not 8.4 ratio
applicable N8-GP 20000 None Not Not 28 applicable applicable N8-GP
10000 None Not Not 19 applicable applicable N8-GP 2500 None Not Not
14 applicable applicable N8-GP 1000 None Not Not 17 applicable
applicable
[0239] The s.c. bioavailability of FVIII co-formulated with a VWF
fragment appear to depend on the saturation of the FVIII VWF
binding sites in the co-formulation rather than on the VWF fragment
length. The shortest VWF fragment, wherein a >80% saturation of
FVIII was achieved, was 764-865--this formulation displayed a FVIII
bioavailability of 4.3% (34 molar excess of N8/turoctocog alfa over
VWF fragment). The longest VWF fragment tested, under similar
conditions with respect to saturation, was the 764-1464 fragment
which resulted in a FVIII bioavailability of 7.3%. The dimer form
of the 764-1464 dosed in a lower volume of 0.25 ml/kg resulted in a
FVIII bioavailability of 8.4%.
[0240] Fragments shorter than 764-1250, which do not contain the
entire D3 region, bind FVIII with a higher IC50 (K.sub.i) than
longer fragments. Thus, 1 to 1 molar formulation of FVIII and VWF
fragments shorter than 764-1250 displayed lower FVIII
bioavailabilities, i.e. less than 4%.
[0241] The s.c. FVIII bioavailability-improving effect of VWF
fragments according to the invention may thus be obtained by
saturation of the FVIII VWF binding sites with VWF-fragment. Short
VWF fragments with relatively low FVIII binding affinity should
thus be used in higher ratios compared to longer VWF fragments with
better binding FVIII binding properties in order to obtain a high
degree of bioavailability.
[0242] FVIII derived from the full-length sequence (Kogenate.RTM.)
displayed the same degree of bioavailability as FVIII with a
truncated B domain (turoctocog alfa/N8) when co-formulated with the
764-1464 VWF fragment. This indicates that high FVIII
bioavailability is not dependent on co-formulation with turoctocog
alfa/N8 but is dependent on presence of the VWF fragment.
Co-formulation of FVIII (turoctocog alfa/N8) with full-length
plasma-derived human VWF resulted in FVIII bioavailability of about
0% thus demonstrating that only fragments of VWF are able to
enhance bioavailability of FVIII. The reason for the lack of effect
of the full-length VWF may be due to the presence of collagen
binding site in the A3 domain which may result in binding and
entrapment of. Preferrred VWF fragments according to the present do
thus not comprise the A3 domain. Alternatively or additionally, the
multimerisation capabilities of full-length VWF produces large
multimers that restricts systemic absorption due to size of the
complex. The data indicates that also longer VWF fragments
(prefeably without the A3 domain) than those tested in table 7 will
have the same beneficial effect on FVIII bioavailability.
[0243] Serum albumin did not improve the s.c. bioavailability of
FVIII (turoctocog alfa/N8). Thus, presence of additional protein in
a FVIII formulation does not appear to increase the s.c.
bioavailability of FVIII--unless this protein is a VWF fragment
according to the present invention.
[0244] VWF dose was not critical for FVIII s.c. bioavailability as
seen for molar ratios between 1:1 and 1:7.7 of FVIII:VWF fragment.
The critical factor for achieving a high FVIII bioavailability thus
appear to be a high degree of FVIII saturation (binding) with VWF
fragment. All compositions in these experiments comprising a
calculated saturation of N8 of at least 86.8% thus resulted in
similar bioavailabilities. VWF fragments according to the invention
may thus protect FVIII at the s.c. injection site.
[0245] FVIII with a 226 amino acid (aa) B domain (SEQ ID NO:3),
displayed a higher s.c. FVIII bioavailability than turoctocog
alfa/N8. However, bioavailability of this FVIII with a 226 aa
B-domain was comparable to turoctocog alfa/N8 in connection with
s.c. co-administration with the VWF-fragment 764-1464
(TIL'/E'/D3/A1) monomer. It may thus be speculated that the
additional amino acids in the 226 aa B-domain (compared to
turoctocog alfa/N8) may protect clearance sites of FVIII in
connection with extravascular administration thereof, meaning that
such FVIII molecules might be used for s.c. administration with or
without VWF according to the present invention.
[0246] FVIIIK1804C-HEP157, displayed a bioavailability of 50% dosed
in co-administration with the VWF-fragment 764-1464 (TIL'/E'/D3/A1)
monomer and a bioavailability of 27% dosed alone.
PSA40Kd-O-Glycan-N8, displayed a bioavailability of 8.8% dosed in
co-administration with the VWF-fragment 764-1464 (TIL'/E'/D3/A1)
monomer and 6.11% dosed alone. It may thus be speculated that
conjugation of FVIII molecules with Heparosan polymers and/or
Polysialic acid polymers either protects FVIII against
breakdown/uptake in the sub cutis or enhances s.c. absorption.
Heparosan appear to be more effective than Sialic acid polymers in
enhancing the s.c. bioavailability. Both FVIII variants displayed
higher bioavailability's when dosed together with VWF fragment.
[0247] N8-GP and FVIIIK1804C-HEP157+764-1464 (TIL'/E'/D3/A1)
monomer and dimer, resulted in the highest bioavailability
obtained. Bioavailability of N8-GP may thus be increased by
increasing the dose or the concentration in the co-formulation.
Dose volume was 5 ml/kg in all dosing's, thus the N8-GP
concentration in the dosing solution was 2 times higher in the
20000 IU/kg dosing than in the 10000 IU/kg dosing. This resulted in
28% and 19% bioavailability respectively.
[0248] The 764-1464 dimer VWF fragment does not contain any
mutations. The 764-1464 dimer VWF fragment binds stronger to
Turoctocog alfa and N8-GP (table 6) but result in a similar
bioavailability of FVIII as the monomer version of the fragment.
This indicates that substituting Cys1099 and/or Cys1142 in the VWF
fragments according to the invention does not influence the
bioavailability of FVIII. Also, the binding affinity of VWF
fragments to N8-GP does not influence the effect on bioavailability
of N8-GP as long as more than 80% of the FVIII molecules are in
complex with VWF fragment in co-formulation. Additionally, since
the dimer version of VWF fragment 764-1464 improves the
bioavailability, the maximum molecular weight of a desired VWF
fragment may be equal to or larger than 158.8 KDa.
[0249] Co-formulation of N8-GP with hyaluronidase did not increase
the FVIII bioavailability, indicating that the Hyaluron network in
the extracellular matrix in the subcutis is not hindering the
passage of FVIII into the bloodstream. Likewise, Hirudin dosed to a
level that inhibits thrombin activity in vivo did not affect
bioavailability of N8-GP. Thrombin activation of FVIII does thus
not appear to affect s.c. FVIII bioavailability.
[0250] The antibody 4F30 (further characterised in WO2012035050),
which bind to C1 and inhibits cellular uptake of FVIII, did not
improve the bioavailability of N8-GP. In this formulation, 2000
IU/ml N8-GP was co-formulated with 1 mg/ml of 4F30 which means that
99.6% of FVIII was bound to the mAb also after in vivo dilution
assuming a K.sub.d of 0.6 nM, an in vivo dilution of 20.times., a
molecular weight for FVIII (turoctocog alfa/N8) of 170000 g/mol, a
specific activity of 10000 IU/mg for turoctocog alfa/N8, and a
molecular weight for 4F30 of 150000 g/mol. Also, the PEGylated
FVIII with K2092A+F2093A mutations displayed decreased uptake in
cells but the mutations did not improve the bioavailability
compared to N8-GP. Inhibition of cellular FVIII uptake does thus
not appear to be the mechanism by which co-formulated VWF fragments
result in increased s.c. bioavailability of FVIII.
Example 25
Subcutaneous Administration in New Zealand White Rabbits
[0251] Test compounds were formulated in 18 mg/ml NaCl, 3 mg/ml
saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80, 0.25
mg/ml CaCl.sub.2, pH .about.7.3. For test formulations containing
VWF or VWF fragments the % FVIII bound by VWF was calculated using
the available IC50 values (table 6) assuming
1C50=K.sub.i=K.sub.d.
[0252] Female New Zealand white rabbits weighing approximately 2-3
kg were used for the study. The animals were allowed free access to
feed and water. The rabbits were dosed subcutaneously over the
thigh with FVIII in combination with various proteins, 4-5 rabbits
with each test compound. The dose volume was 0.2 ml/kg or 1
ml/kg.
[0253] Blood was sampled at 11 time points from 0 to 96 h with
n=4-5 rabbits/time point. At each sampling time point, 1 ml blood
was sampled from an ear artery by use of a 21 G needle and EDTA
coated tubes. The tubes were centrifuged within 10 minutes after
blood drawing at 4000 G for 5 minutes and plasma separatedThe
samples were immediately frozen on dry ice before storage at
-80.degree. C. prior to analysis. The samples were analysed by
FVIII antigen analysis using two FVIII light chain antibodies (4F45
and 4F11) in a FVIII LOCI assay (Luminescence oxygen channelling
immunoassay).
[0254] Mean plasma concentration versus time data were analysed by
non-compartmental analysis using WnNonlin Phoenix (Pharsight
Corporation) estimating the given pharmacokinetic parameters. The
bioavailability was estimated using pharmacokinetics of FVIII
(turoctocog alfa/N8) and N8-GP administered i.v. to rabbits.
[0255] The obtained bioavailabilities are shown in table 8.
TABLE-US-00011 TABLE 8 Saturation FVIII Molar ratio co- FVIII with
co- Dose/dose co formulation formulation formulated FVIII volume
protein protein:FVIII protein (%) F % FVIII (turoctocog 2000/0.2
ml/kg TIL'/E'/D3/A1 3 99 6.2 alfa/N8) + VWF N8-GP 700/0.2 ml/kg --
-- -- 40 N8-GP + VWF 700/0.2 ml/kg TIL'/E'/D3/A1 3 99 59 N8-GP +
VWF 500/1 ml/kg TIL'/E'/D3/A1 3 82 34
[0256] The s.c. bioavailability in rabbits of N8-GP and N8-GP
co-formulated with VWF fragment TIL'/E'/D3/A1 dosed in a dosing
volume of 0.2 ml/kg was 40 and 59%, respectively. The
bioavailability of N8-GP+VWF dosed in a dosing volume of 1 ml/kg
was 34%. The bioavailability of N8-GP may thus be influenced either
by the species or by the differences in dosing volumes (5 ml/kg in
mice and 0.2 ml/kg or 1 ml/kg in rabbits). 0.2 ml/kg is closest to
a dosing volume relevant for humans. FVIII (turoctocog alfa/N8)
dosed together with VWF fragment TIL'/E'/D3/A1 displayed a similar
bioavailability in rabbits compared to mice despite the higher
dosing concentration.
Example 26
Construction of Expression Vectors Encoding VWF Fragments
[0257] Plasmid #796 consisting of the pZEMHygro vector with insert
consisting of wild-type human VWF cDNA was utilized as the starting
point for generating DNA constructs for the expression of truncated
human VWF proteins.
[0258] DNA encoding the VWF signal peptide, followed by the VWF
TIL'E' domain, the VWF D3 domain, the VWF A1 domain, and a HPC4 tag
was generated by polymerase chain reaction (PCR) using plasmid #796
as template, forward primer oLLC089 VWF forward, and reverse primer
oLLC092 VWF A1 HPC4 reverse. These primers contain a Nhe I and a
Not I restriction site, respectively. The resulting PCR product was
inserted into the pCR2.1-TOPO vector
[0259] (Invitrogen). From here the VWF(TIL'/E'/D3/A1)-HPC4 coding
DNA was excised with the Nhe I and a Not I restriction enzymes and
inserted into pZEM219b digested with the same restriction enzymes.
Thus, the pLLC089 construct was established consisting of pZEM219b
with insert encoding VWF(TIL'/E'/D3/A1)-HPC4.
[0260] Nucleotide substitutions leading to the amino acid
replacements C1099/1142S in the VWF VWF(TIL'/E'/D3/A1)-HPC4 protein
encoded by pLLC089 were introduced by site-directed mutagenesis of
pLCC089 using the QuikChange XL Site-directed Mutagenesis kit
(Stratagene) and the oLLC101-f, oLLC102-r, oLLC103-f, and oLLC104-r
mutagenesis primers. The site.directed mutagenesis gave rise to the
pLLC095 vector consisting of pZEM219b with insert encoding VWF
(TIL'/E'/D3/A1)C1099/1142S-HPC4.
TABLE-US-00012 TABLE 9 Oligonucleotide primers used for generating
VWF fragment coding DNA contructs Primer name Primer sequence
(5'-3') oLLC089 VWF CCGCTAGCCCATGATTCCTGCCAGATTTGCCGGGGT forward
GCTGCTTGCTCTGGCCCTCATTTTGCCAGGGACCCT TTGTAGCCTATCCTGTCGGCCCCCCATG
(SEQ ID NO: 39) oLLC092 VWF A1 GATGCGGCCGCCTACTACTATTTGCCATCAATCAG
HPC4 reverse ACGCGGATCCACCTGATCTTCGGCTTCAGGGGCAA GGTCACAGAGGTAGC
(SEQ ID NO: 40) oLLC101-f CATTGGGGACTGCGCCTCCTTCTGCGACACCATTGC TGCC
(SEQ ID NO: 41) oLLC102-r GGCAGCAATGGTGTCGCAGAAGGAGGCGCAGTCCCC AATG
(SEQ ID NO: 42) oLLC103-f CGGGAGAACGGGTATGAGTCTGAGTGGCGCTATAAC
AGCTGTGC (SEQ ID NO: 43) oLLC104-r
GCACAGCTGTTATAGCGCCACTCAGACTCATACCCG TTCTCCCG (SEQ ID NO: 44)
Example 27
Stable Cell Lines Expressing VWF Fragments
[0261] Baby hamster kidney (BHK) cells grown in Dulbecco's modified
Eagle's medium with 10% fetal calf serum were transfected with
pLL095 using Genejuice transfection reagent (Merck). A pool of
transfected cells was generated by selection with 1.5 .quadrature.M
methotrexate giving rise to a non-clonal BHK cell line producing
VWF (TIL7E7D3/A1)C1099/11425-HPC4. The cells were seeded in a
biofermentor and the VWF (TIL'/E'/D3/A1)C1099/1142S-HPC4 protein
was purified from the cell culture supernatant as described in
Example 22.
[0262] CHO-DUKX-B11 suspension cells grown in suspension were
transfected with pLLC095 by electroporation. A pool of transfected
cells was generated by adaptation to growth in medium without
nucleosides. Subsequently, the pool was adapted to growth in the
presence of 100 mM methotrexate giving rise to the VWF
(TIL'/E'/D3/A1)C1099/1142S-HPC4 producing non-clonal CHO-DUKX-B11
cell line MBML001. The cells were seeded in a biofermentor and the
VWF (TIL7E7D3/A1)C1099/11425-HPC4 protein was purified from the
cell culture supernatant as described in Example 22.
Example 28
VWF Fragments Protects FVIII Against Cellular Uptake
[0263] The effect of plasma-derived (pd) VWF and fragments of VWF
on FVIII cellular uptake is evaluated in human monocyte-derived
macrophages or dendritic cells, which both are antigen presenting
cells, or U87 MG cells. U87 MG cells are obtained from ATCC
(HTB-14). The cells are cultured in fibronectin-coated 24-well
plates for 48 hours in EMEM supplemented with 10% heat inactivated
FCS at 37.degree. C. in 5% CO.sub.2The cells are carefully washed
with buffer A (10 mM HEPES, 150 mM NaCl, 4 KCl, 11 mM Glucose, pH
7.4) and incubated for 15 min with buffer B (buffer A supplemented
with 5 mM CaCl.sub.2 and 1 mg/ml BSA). Radioactively labelled FVIII
(.sup.125I-FVIII, final concentration 1 nM) is incubated alone or
premixed with different concentrations of pdVWF (American
Diagnostica, final concentration 0.001 nM-50 nM based on monomer
content) or TIL'/E'/D3/A1 (final concentration 0.25 nM-500 nM or
1000 nM) 10 min prior to addition to the U87 MG cells and incubated
with the cells 1 hour at 37.degree. C. to allow binding and
internalization. Cells are subsequently washed three times with
ice-cold buffer B. Surface bound proteins are cleaved off by
incubating the cells in PBS containing 100 .mu.g/ml trypsin, 50
.mu.g/ml proteinase K, 5 mM EDTA (pH 7.4) for 1 hour on ice. The
detached cells are transferred to tubes and centrifuged to pellet
the cells. The supernatant representing the cell bound FVIII is
transferred to new tubes. The radioactivity in tubes with the
supernatants (bound FVIII) and cell pellets (internalized FVIII)
are quantified in a gamma counter, and values calculated in FVIII
concentration by using a standard curve based on .sup.125I-FVIII.
Bound .sup.125I-FVIII in the absence of VWF are set to 100%.
[0264] Dendritic cells and macrophages are differentiated from
monocytes isolated from buffy coats by magnetic separation using
magnetic anti-CD14-beads (Miltenyi Biotec) and a MACS column
(Miltenyi Biotec) according to the manufactures instructions.
Monocytes (0.5.times.10.sup.6 cells/ml) are seeded in T-75 tissue
culture flasks and cultured in IMDM media (GIBCO) containing 10%
FBS, 1% penicillin/streptomycin and 3.3 ng/ml M-CSF (R&D
Systems) in order to differentiate the cells into macrophages.
Additional 3.3 ng/ml M-CSF is added after three days of culturing.
The monocytes can alternatively be differentiated into dendritic
cells by stimulating with 40 ng/ml GM-CSF (R&D Systems) and 40
ng/ml IL-4 for five days. Dendritic cells are washed in buffer B
and transferred to low binding Nunc tubes with 0.5.times.10.sup.6
cells/tube. Fluorescently labelled FVIII, e.g. Oregon-Green FVIII
(e.g. 30 and 100 nM) are added and incubated 1 hour at 37.degree.
C. Cells are washed once and analysed by flow cytometry using a LRS
Fortessa instrument (BD). The macrophages are after six days
culturing washed with PBS and incubated 10-20 min at 4.degree. C.
with 2.5 mM EDTA in PBS with 5% FCS to detach cells. Macrophages
(7.times.10.sup.6/well) are seeded on fibronectin-coated 96-well
glass bottom tissue culture plates (Perkin Elmer ViewPlate Black).
24 hours post seeding the cells are washed once with buffer B
before addition of 30 nM fluorescently-labelled FVIII (e.g.
OregonGreen-FVIII) alone or in the presence of increasing
concentrations (15-240 nM) of pdVWF (American Diagnostica) or
TIL'/E'/D3/A1. Macrophages are incubated for 1 hour at 37.degree.
C. Subsequently, cells are washed twice with buffer B to remove
non-internalized material before addition of PBS containing 2.5
.mu.g/ml Hoechst33342 (Molecular Probes) to visualize the cell
nuclei. The plate is then immediately imaged on the Operetta.RTM.
High Content Screening system (Perkin Elmer, Hamburg) in widefield
fluorescence mode using the 20.times. high NA objective. Ten fields
per well are imaged and analysed. The approach to image analysis in
the Harmony.RTM. software is based on counting nuclei (Hoechst
channel), followed by texture analysis (FVIII channel) using the
"find particle" method to detect vesicular FVIII. Dead or apoptotic
cells are excluded from the analysis based on nuclei fragmentation
and/or excessive binding of FVIII to the plasma membrane. In order
to quantify the internalized FVIII the integrated fluorescent
intensity of the vesicular FVIII signal is calculated and plotted
against time.
[0265] IC50 values for inhibition of FVIII binding and
internalization in U87 MG cells and macrophages are shown in table
10. Both pdVWF and TIL'/E'/D3/A1 are able to inhibit FVIII cell
binding/uptake in both cell types providing sufficient high
concentrations are used.
[0266] As uptake in antigen presenting cells is the initial step in
presenting FVIII to the immune system, the data may indicate that a
reduced immune response can be achieved upon co-formulation of
FVIII with a VWF fragment.
TABLE-US-00013 TABLE 10 Effect of pdVWF and TIL'/E'/D3/A1 fragment
on FVIII binding and internalization in U87 MG cells and uptake in
macrophages. IC50 (nM) Maximal inhibition (%) TIL'/E'/ TIL'/E'/
Cell type pdVWF D3/A1 pdVWF D3/A1 U87 (n = 3-4) 1.2 .+-. 0.9 17.6
.+-. 13.0 34.3 .+-. 4.2 39.8 .+-. 7.8 Binding U87 (n = 3-4) 1.3
.+-. 1.2 22.1 .+-. 19.2 32.2 .+-. 7.0 41.2 .+-. 11.5
Internalization Macrophages 15.6 .+-. 3.5 31.5 .+-. 6.1 32.6 .+-.
11.4 47.2 .+-. 11.7 (n = 3)
Example 29
Efficacy of FVIII Compounds Co-Formulated with VWF Variants after
Subcutaneous Dosing
[0267] FVIII deficient, FVIII-KO mice, 12-16 weeks old, male and
females are divided into 3 groups of 12 animals. In each group,
eight animals are subjected to tail bleeding and 4 animals are used
in parallel for ex vivo efficacy testing using ROTEM analysis.
[0268] GlycoPEGylated FVIII or vehicle is dosed s.c. 24 hr prior to
tail transection. As a positive control glycoPEGylated FVIII is
dosed i.v. 5 min prior to injury. The s.c injection is performed in
the neck and the i.v. injection in a lateral tail vein. The dose
volume is 5 ml/kg.
[0269] GlycoPEGylated FVIII is prepared in buffer (10 mM
L-Histidine, 8.8 mM Sucrose, 0.01% Polysorbate 80, 308 mM NaCl, 1.7
mM CaCl.sub.2 (dihydrate), 0.01% Polysorbate 80 0.1 mg/ml, pH 6.9)
to a concentration of 40 and 500 U/ml and stored at -80.degree. C.
until use.
[0270] Before tail transection, the mice are anaesthetised with
isoflurane and placed on a heating pad. The tails are placed in
pre-heated saline at 37.degree. C. for 10 min. The tail is
transected 4 mm from the tip.
[0271] Immediately before tail transection a 20 .mu.l blood sample
is drawn from the peri-orbital plexus for FVIII determination.
[0272] Blood is collected over 30 min and the haemoglobin
concentration determined by spectrophotometry at 550 nm.
[0273] Parallel animals are used for blood sampling and subsequent
analysis of their clotting parameters (ex vivo efficacy). A blood
sample is taken from the peri-orbital plexus with 20 .mu.L
capillary tubes without additive. The blood sample is diluted 1:10
in 0.13M sodium citrate and carefully mixed and stored at rum
temperature for immediate thromboelastography by ROTEM. The blood
sample is re-calcified by adding 7 .mu.L CaCl.sub.2 to a mini
curvet (StarTEM). Thereafter, 105 .mu.L of blood is added to the
mini curvet and mixed. The analysis is performed until the maximum
amplitude is reached.
[0274] Results:
[0275] The prophylactic effect of s.c. administered FVIII is
determined by comparing the blood loss during the 30 min study
period at 24 hr after s.c. administration to that of 1) a vehicle
control group and 2) an i.v. control group with glycoPEGylated
FVIII. The blood loss in the group dosed s.c. with glycoPEGylated
FVIII is comparable to the blood loss in the group dosed i.v. (FIG.
10, left panel). The blood loss data are supported by the ex vivo
efficacy parallel study of the examined clotting parameters, e.g.
clot time (FIG. 10, right panel).
[0276] In conclusion, subcutaneously administered FVIII appear to
be hemostatically active based on the PK profile and the results
from the ex vivo activity. Therefore, subcutaneously administered
FVIII co-formulated with a VWF fragment is also believed to be
hemostatically active as can be predicted from its pharmacokinetic
profile.
Example 30
Effect of s.c. Administered FVIII.+-.VWF Fragments in
FVIII-Deficient Mice
[0277] Test Compounds:
[0278] Test compounds are prepared in 10 mM L-Histidine (1.55
mg/ml), 8.8 mM Sucrose (3.0 mg/ml), 308 mM NaCl (18 mg/ml), 1.7 mM
CaCl2 dihydrate (0.25 mg/ml), 0.01% Polysorbate 80 (0.1 mg/ml), pH
7.3.
[0279] Animals: Experiments are performed using groups of F8
knockout (FVIII k/o) mice (129/C57BL/6 or C57BL/6, exon 16
disrupted). Animals are included in experiments when 12-18 weeks
old at which time they are weighing roughly 18-25 grams. Twelve to
15 animals are included per group.
[0280] Administration of test compounds: Test compounds are
administered subcutaneously (or intravenously for controls) using a
dose volume of maximally 10 ml/kg (or 5 ml/kg for controls).
[0281] Bleeding Model:
[0282] A tail vein transection (TVT) bleeding model is conducted
with the mice under full isoflurane anaesthesia. Briefly, following
anaesthesia the bleeding challenge comprises a template-guided
transection of a lateral tail vein at a tail diameter of 2.7 mm.
The tail is immersed in saline at 37.degree. C. allowing visual
recording of the bleeding for 60 min, where after the blood is
isolated and the blood loss determined by measuring the haemoglobin
concentration as described in "Example 3". When feasible and
justified, blood is sampled for assessment of FVIII activity
(FVIII:C) in plasma as described above.
[0283] Dose Response:
[0284] Different doses of FVIII or FVIII co-formulated with VWF
fragments (e.g. N8-GP/VWF) are injected subcutaneously at defined
time point(s) prior to TVT. Vehicle and intravenous
control/treatment groups are included for no effect and maximal
effect, respectively.
[0285] Duration of Action:
[0286] FVIII or FVIII/VWF is injected s.c. to identify prolonged
effect, i.e. improved bleeding phenotype after treatment. TVT is
performed at several time points, e.g. 24, 48, 72, 96, after
dosing.
[0287] Repeated Dose:
[0288] FVIII or FVIII/VWF fragment is dosed s.c. once daily for
several days. TVT is performed at different time points to assess
any improvement in the bleeding phenotype.
[0289] Data processing and analyses: Data are physically recorded
throughout the experiment. Hereafter, data are aggregated for
analysis using MS Excel (Microsoft, WA, USA) before being analysed
in GraphPad Prism version 5 (GraphPad Software, Inc, CA, USA).
Example 31
Effect of s.c. FVIII.+-.VWF Fragments in Other FVIII-Deficient
Species
[0290] Additional pharmacodynamic experiments are conducted in
other species to verify effect after subcutaneous administration in
non-murine animal models of haemophilia A, e.g. rat and dog. FVIII
or FVIII/VWF are injected subcutaneously before assessing ex vivo
effect, before inducing a bleeding challenge, or as a means to
treat or prevent spontaneous bleeds.
[0291] Test Compounds:
[0292] Test compounds are prepared in 10 mM L-Histidine (1.55
mg/ml), 8.8 mM Sucrose (3.0 mg/ml), 308 mM NaCl (18 mg/ml), 1.7 mM
CaCl2 dihydrate (0.25 mg/ml), 0.01% Polysorbate 80 (0.1 mg/ml), pH
7.3.
[0293] Animals: Experiments are performed in adolescent rats (-12
weeks old) or dogs (6+ months old) with haemophilia A.
[0294] Administration of test compounds: Test compounds are
administered subcutaneously (or intravenously for controls) using a
dose volume of maximally 10 ml/kg (or 5 ml/kg for controls).
[0295] Dog effect model: In dogs with haemophilia A the effect is
assessed ex vivo using surrogate markers, e.g. thrombelastography
as previously described (Knudsen et al, 2011; Haemophilia, 17,
962-970), or in vivo, e.g. using a standardized bleeding challenge
monitored by acoustic force radiation force impulse (ARFI)
ultrasound as described (Scola et al, 2011; Ultrasound in Med.
& Biol., 37(12), 2126-2132). Capacity allowing, test compound
are administered to treat spontaneously bleeding dogs. Effect is
monitored by assessing the resolution of clinical manifestation in
comparison with historic data on i.v. treatment.
[0296] Rat effect model: In rats with haemophilia A the effect is
assessed ex vivo using surrogate markers, e.g. thrombelastography
as described above for mice and dogs, or in vivo, e.g. using a
standardized bleeding challenge as described for mice. Capacity
allowing, test compound are administered to treat spontaneously
bleeding rats. Effect is monitored by assessing the resolution of
clinical manifestation in comparison with historic data on i.v.
treatment.
[0297] Additional pharmacodynamic experiments are conducted in
other species to verify effect after subcutaneous administration in
non-murine animal models of haemophilia A, e.g. rat and dog.
Example 32
Construction of Expression Vectors Encoding VWF Fragments
[0298] A nucleotide substitution leading to the amino acid
replacement S1142C in the VWF(764-1250)-C1099/1142S-ALA-HPC4
protein encoded by pJSV348 described in Example 17 was introduced
by PCR-based site-directed mutagenesis using the VWF 1099C S and
VWF 1099C AS primers (Table P). This gave rise to the pGB237 vector
consisting of pTT5 with insert encoding
VWF(764-1250)-C1099S-ALA-HPC4 (SEQ ID NO:11). The cysteine at
position 1142 allows dimerization of the protein as described in
Example 20.
[0299] Likewise, a nucleotide substitution leading to the amino
acid replacement S1099C in the VWF(764-1250)-C1099/1142S-ALA-HPC4
protein encoded by pJSV348 described in Example 17 was introduced
by PCR-based site-directed mutagenesis using the VWF 1142C S and
VWF 1142C AS primers (Table P). This gave rise to the pGB238 vector
consisting of pTT5 with insert encoding
VWF(764-1250)-C1142S-ALA-HPC4 (SEQ ID NO:11). The cysteine at
position 1099 allows dimerization of the protein as described in
Example 20.
[0300] In a similar manner, the S1099C amino acid replacement was
introduced in the VWF(764-1128)-C1099S-HPC4 protein encoded by
pJSV406 described in Example 18, giving rise to the pGB249 vector
consisting of pTT5 with insert encoding VWF(764-1128)-HPC4 (SEQ ID
NO:9). The cysteine at position 1099 allows dimerization of the
protein as described in Example 20.
[0301] cDNA encoding amino acid 1-1250 of human VWF was amplified
by PCR using plasmid #796 (described in Example 26) as template,
forward primer JP1000 VWF-HindIII S (Table 2), and reverse primer
JP1006 VWF764-1250 (Table 2). Primer JP1006 VWF764-1250 contains a
Nhe I site. The resulting PCR product was inserted into the
pCR4BLUNT-TOPO vector (Invitrogen) downstream of Pme I restriction
site. From here, the vWF(1-1250) coding DNA was excised with the
Pme I and a Nhe I restriction enzymes and inserted into pJSV164
described in Example 17 generating the pGB242 vector consisting of
pTT5 with insert encoding vWF(1-1250)-ALA-HPC4. The cysteines at
position 1099 and 1142 allow dimerization of the protein as
described in Example 20, and proteolytic removal of the presequence
will generate vWF(764-1250)-ALA-HPC4 (SEQ ID NO:11).
[0302] DNA sequences of pJSV348 (described in Example 17) and
construct #796 (described in Example 26) were inverse amplified by
PCR using overlapping primers. The pJSV348 sequence was amplified
using primer 2764pJSV348 and 1202pJSV348R (Table P), while the
construct #796 sequence was amplified using primer 221#796F and
3537#796R (Table P). The amplification products from pJSV348
(recipient) and construct #796 (donor) were excised from an agarose
gel and joined by ligation independent cloning (LIC) using the
In-Fusion HD Cloning Kit (Clontech) to generate circular DNA and
subsequently transformed into Stellar competent cells (Clontech).
The resulting expression vector, named pGB252 consists of PTT5 with
insert encoding VWF(1-1128)-ALA-HPC4. The cystein at position 1099
allows dimerization of the protein as described in Example 20, and
proteolytic removal of the presequence will generate
vWF(764-1128)-ALA-HPC4 (SEQ ID NO:9).
[0303] Likewise, amplification using pJSV348 (described in Example
17) as template with the primers 2764pJSV348 and 1202pJSV348R
(Table P) and amplification using #796 (described in Example 26) as
template with the primers 221#796F and 3747#796R (Table P)
generated pJSV348 (recipient) and construct #796 (donor)
amplification products that were also excised from an agarose gel
and joined by ligation independent cloning (LIC) using the
In-Fusion HD Cloning Kit (Clontech) to generate circular DNA and
subsequently transformed into Stellar competent cells (Clontech).
The resulting expression vector, named pGB253 consists of PTT5 with
insert encoding VWF(1-1198)-ALA-HPC4. The cysteines at position
1099 and 1142 allow dimerization of the protein as described in
Example 20, and proteolytic removal of the presequence will
generate vWF(764-1198)-ALA-HPC4 (SEQ ID NO:10).
[0304] In a similar manner, DNA sequences of pJSV348 (described in
Example 17) and construct #796 (described in Example 26) were
inverse amplified by PCR using overlapping primers. The pJSV348
sequence was amplified using primer 2764pJSV348 and 2420pJSV348R
(Table 11), while the construct #796 sequence was amplified using
primer 3666#796F and 5203#796R (Table P). The amplification
products from pJSV348 (recipient) and construct #796 (donor) were
excised from an agarose gel and joined by ligation independent
cloning (LIC) using the In-Fusion HD Cloning Kit (Clontech) to
generate circular DNA and subsequently transformed into Stellar
competent cells (Clontech). The resulting expression vector, named
pGB250 consists of PTT5 with insert encoding
VWF(764-1873)-C1099/1142C-ALA-HPC4 (SEQ ID NO:20).
[0305] Human VWF cDNA sequences amplified from construct #796
(described in Example 26) were combined generating the pLLC122
vector consisting of pZEM219b with insert encoding vWF
(1-1464)-HPC4. The cysteines at position 1099 and 1142 allow
dimerization of the protein as described in Example 20, and
proteolytic removal of the presequence will generate
vWF(764-1464)-HPC4 (SEQ ID NO:19).
TABLE-US-00014 TABLE 11 Oligonucleotide primers used for generating
VWF fragment coding DNA constructs Primer name Primer sequence
(5'-3') VWF 1099C S GGGGACTGCGCCTGCTTCTGCGACACC (SEQ ID NO: 45) VWF
1099C AS GGTGTCGCAGAAGCAGGCGCAGTCCCC (SEQ ID NO: 46) VWF 1142C S
GAACGGGTATGAGTGTGAGTGGCGCTATA (SEQ ID NO: 47) VWF 1142C AS
TATAGCGCCACTCACACTCATACCCGTTC (SEQ ID NO: 48) 2764pJSV348F
GCGCTAGCTGAGGACCAAGTAGATCCGCGGCTCATTGATGGG (SEQ ID NO: 49)
1202pJSV348R GGGCCAGAGCAAGCAGCACCCCGGCAAATCTGGCAGG (SEQ ID NO: 50)
221#796F CCTGCCAGATTTGCCGGGGTGCTGCTTGCTCTGGCCC (SEQ ID NO: 51)
3537#796R TACTTGGTCCTCAGCTAGCGCCTGGGGGCACAATGTGGCCGTCCTCC (SEQ ID
NO: 52) 3747#796R TACTTGGTCCTCAGCTAGCGCCACTGGACAGTCTTCAGGGTCAACGC
(SEQ ID NO: 53) 2420pJSV348R GGCTCAGGGTGCTGACACGTGACTTGACAGGCAGGTGC
(SEQ ID NO: 54) 3666#796F GCACCTGCCTGTCAAGTCACGTGTCAGCACCCTGAGCC
(SEQ ID NO: 55) 5203#796R
TACTTGGTCCTCAGCTAGCGCTGCAGGGGAGAGGGTGGGGATCTGC (SEQ ID NO: 56)
Example 33
VWF Fragments Inhibit FVIII Uptake by Human Dendritic Cells
[0306] Human monocyte-derived dendritic cells were prepared as
described in example 28. Expression of the dendritic cell markers
CD209 and CD86 were controlled by flow cytometry using a LRS
Fortessa instrument (BD). Fluorescent labelled FVIII (Oregon
green-FVIII, 30 nM final concentration) was premixed with different
concentrations of plasma-derived VWF or VWF fragments before
incubating 1 h at 37.degree. C. with dendritic cells. Live/Dead
cell kit (Invitrogen # L10119, APC-Cy7) was used for gating on live
dendritic cells, and FVIII uptake within this cell population was
quantified. Data was normalized for each individual experiment. The
signal in samples without VWF was defined as 100% FVIII uptake, and
the signal in the sample with the highest concentration of
plasma-derived VWF (240 nM based on monomer content) was defined as
0%. Values from 3-5 experiments were combined and IC50 values
calculated using non-linear regression in Prism software
(log(inhibitor) vs. response--Variable slope (four parameters)).
The resulting IC50 values are shown in table 12. The data show that
all tested VWF fragments were able to inhibit FVIII uptake by the
dendritic cells provided sufficiently high concentrations are used.
As FVIII uptake by antigen-presenting cells is the initial step in
presenting FVIII to the immune system the data suggests that
co-formulation of FVIII with sufficiently high concentration of VWF
fragment may have a potential in reducing immunogenicity of
FVIII.
TABLE-US-00015 TABLE 12 Effect of plasma derived VWF and VWF
fragments on FVIII uptake in dendritic cells. Domain/comment VWF
fragment sequence IC50 (nM)* TIL'/E'/VWD3 VWF(764-1041)-ALA-HPC4
570 (400-820) monomer TIL'/E'/D3 VWF(764-1250)-C1099/ 31 (25-39)
1142S-ALA-HPC4 monomer TIL'E'/D3/A1 monomer VWF(764-1464)-C1099/ 31
(18-52) 1142S-HPC4 monomer TIL'E'/D3/A1 dimer VWF(764-1464)-HPC4 16
(11-22) dimer** Plasma-derived VWF VWF (764-2813) 9.8 (7.6-13)
*Best fit value and 95% confidence intervals of data from 3-5
experiments **IC50 value based on molar concentration of the dimer,
i.e. multiply IC50 with 2 to reflect IC50 value based on content of
VWF monomer fragment.
Example 34
Effect of s.c. FVIII.+-.VWF Fragments in Animals with Inhibiting
Antibodies Against FVIII
[0307] The objective is to evaluate the potential of pharmaceutical
compositions to treat haemophilia A patients with inhibitors
against FVIII. We dose FVIII alone or co-formulated with
VWF-fragments subcutaneously to naive FVIII-KO mice or FVIII-KO
mice where inhibitors are induced by repeated subcutaneous or
intravenous administrations of FVIII prior to treatment with the
compositions, or by injecting a polyclonal or monoclonal anti-FVIII
antibody. The effect of the treatments is evaluated in
anaesthetized mice after transection of a lateral tail vein. The
tail is placed in pre-warmed saline at 37.degree. C. and the
bleeding is observed for 60 minutes. The blood loss during the
experiment is a measure of the effect of the composition.
Example 35
Administration of VWF Fragments to VWF Knockout Mice
[0308] Test compound:
[0309] Murine VWF fragment TIL'/E'/D3/A1 1.829 nmol/ml, 0.015
mg/ml
[0310] The test compound was formulated in 20 mM imidazol 150 mM
NaCl, 0.02% Tween 80, 1.1M Glycerol, 10 mM CaCl2, pH 7.3
[0311] 6 VWF knockout mice, with an approximate weight of 25 g were
dosed intravenously in the tail with 9.48 nmol/kg Murine VWF
fragment TIL'/E'/D3/A1.
[0312] Blood was sampled pre-dose and at 0.08, 0.33, 0.5, 1, 2, 4,
7, 18 and 24 h post administration in a sparse sample design with 2
mice sampled per time point. The mice were anaesthetized by
Isoflurane/O2/N2O prior to blood sampling via the retroorbital
plexus. Three samples were taken from each mouse. Blood (45 .mu.l)
was stabilised with 5 .mu.l of sodium-citrate (0.13 M) and added
200 .mu.l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA,
Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for
5 minutes at room temperature, the supernatants were immediately
frozen on dry ice before storage at -80.degree. C. prior to
analysis.
[0313] Samples were analysed with regards to FVIII concentration in
an antigen LOCI assay (Luminescence oxygen channelling
immunoassay).
[0314] Mean plasma concentration versus time data were analysed
relatively to the predose values.
[0315] The relative mean FVIII concentration in time after dosing
is shown in table 13
TABLE-US-00016 TABLE 13 Effect of Murine D'D3A1 IV on FVIII blood
concentration in VWF KO mice. Time (h) FVIII increase (% of
predose) 0.08 174 0.33 190 0.5 176 1 163 2 274 4 250 7 330 18 225
24 207
[0316] FVIII concentration increased gradually in time after dosing
of VWF fragment intravenously with a Tmax after 7 hours. This
finding supports the potential for VWF fragments for the treatment
of VWF disease as well as haemophilic disorders.
Example 36
Interaction Mapping by HX-MS of vWF Fragments TIL'/E'/D3/A1,
TIL'/E'/D3, TICE', and TIC/E'NWD3 on Turoctocog alfa (FVIII) and
Turoctocog alfa (FVIII) on vWF fragment TIL'/E'/D3/A1
[0317] Introduction to HX-MS
[0318] The HX-MS technology exploits that hydrogen exchange (HX) of
a protein can readily be followed by mass spectrometry (MS). By
replacing the aqueous solvent containing hydrogen with aqueous
solvent containing deuterium, incorporation of a deuterium atom at
a given site in a protein will give rise to an increase in mass of
1 Da. This mass increase can be monitored as a function of time by
mass spectrometry in quenched samples of the exchange reaction. The
deuterium labelling information can be sub-localized to regions in
the protein by pepsin digestion under quench conditions and
following the mass increase of the resulting peptides.
[0319] One use of HX-MS is to probe for sites involved in molecular
interactions by identifying regions of reduced hydrogen exchange
upon protein-protein complex formation. Usually, binding interfaces
will be revealed by marked reductions in hydrogen exchange due to
steric exclusion of solvent. Protein-protein complex formation may
be detected by HX-MS simply by measuring the total amount of
deuterium incorporated in either protein members in the presence
and absence of the respective binding partner as a function of
time. The HX-MS technique uses the native components, i.e., protein
and antibody or Fab fragment, and is performed in solution. Thus
HX-MS provides the possibility for mimicking the in vivo conditions
(for a recent review on the HX-MS technology, see Wales and Engen,
Mass Spectrom. Rev. 25, 158 (2006)).
[0320] Materials
[0321] Protein batches used were:
[0322] FVIII protein batches used were:
[0323] FVIII (N8, Turoctocog alfa, SEQ ID NO:2) Batch
0155-0000-0004-37A
[0324] vWF Fragments
[0325] D'D3A1 (SEQ ID NO:19; Cys1099Ser; Cys1142Ser) Batch
0129-0000-0170-6B; 2304 (SEQ ID NO:5) Batch 0129-0000-2304-1B; 2307
(SEQ ID NO:8) Batch 0129-0000-2307-1B; 2308 (SEQ ID NO:11) Batch
0129-0000-2308 2B.
[0326] All proteins were buffer exchanged into 20 mM Imidazole, 500
mM NaCl, 10 mM CaCl2, adjusted to pH 7.3 before experiments.
[0327] Methods: HX-MS Experiments
[0328] Instrumentation and Data Recording
[0329] The HX experiments were performed on a nanoACQUITY UPLC
System with HDX Technology (Waters Inc.) coupled to a Synapt G2
mass spectrometer (Waters Inc.). The Waters HDX system contained a
Leap robot (H/D-x PAL; Waters Inc.) operated by the LeapShell
software (Leap Technologies Inc/Waters Inc.), which performed
initiation of the deuterium exchange reaction, reaction time
control, quench reaction, injection onto the UPLC system and
digestion time control. The Leap robot was equipped with two
temperature controlled stacks maintained at 20.degree. C. for
buffer storage and HX reactions and maintained at 2.degree. C. for
storage of protein and quench solution, respectively. The Waters
HDX system furthermore contained a temperature controlled chamber
holding the pre- and analytical columns, and the LC tubing and
switching valves at 1.degree. C. A separately temperature
controlled chamber holds the pepsin column at 25.degree. C. For the
inline pepsin digestion, 100 .mu.L quenched sample containing 100
pmol hIL-21 was loaded and passed over a Poroszyme.RTM. Immobilized
Pepsin Cartridge (2.1.times.30 mm (Applied Biosystems)) placed at
25.degree. C. using a isocratic flow rate of 100 .mu.L/min (0.1%
formic acid:CH.sub.3CN 95:5). The resulting peptides were trapped
and desalted on a VanGuard pre-column BEH C18 1.7 .mu.m
(2.1.times.5 mm (Waters Inc.)). Subsequently, the valves were
switched to place the pre-column in-line with the analytical
column, UPLC-BEH C18 1.7 .mu.m (1.times.100 mm (Waters Inc.)), and
the peptides separated using a 8 min gradient of 8-45% B delivered
at 120 .mu.l/min from the nanoAQUITY UPLC system (Waters Inc.). The
mobile phases consisted of A: 0.1% formic acid and B: 0.1% formic
acid in CH.sub.3CN. The ESI MS data and the separate elevated
energy (MS.sup.E) experiments were acquired in positive ion mode
using a Synapt G2 mass spectrometer (Waters Inc.).
Leucine-enkephalin was used as the lock mass ([M+H].sup.+ ion at
m/z 556.2771) and data was collected in continuum mode (For further
description, see Andersen and Faber, Int. J. Mass Spec., 302,
139-148(2011)).
[0330] Data Analysis
[0331] Peptic peptides were identified in separate experiments
using standard MS.sup.E methods where the peptides and fragments
are further aligned utilizing the ion mobility properties of the
Synapt G2 (Waters Inc.). MS.sup.E data were processed using
ProteinLynx Global Server version version 2.5 (Waters Inc.). The
HX-MS raw data files were processed in the DynamX software (Waters
Inc.). DynamX automatically performs the lock mass-correction and
deuterium incorporation determination, i.e., centroid determination
of deuterated peptides. Furthermore, all peptides were inspected
manually to ensure correct peak and deuteration assignment by the
software.
[0332] Epitope Mapping Experiment
[0333] Amide hydrogen/deuterium exchange (HX) was initiated by a
10-fold dilution of FVIII in the presence or absence of vWF
fragment, i.e., D'D3A1, 2308, 2307, or -2304 at time 0 into 20 mM
Imidazole, 150 mM NaCl, 10 mM CaCl2, pH 7.3 (uncorrected value) at
later time points into the corresponding deuterated buffer (i.e. 20
mM Imidazole, 150 mM NaCl, 10 mM CaCl2 prepared in D.sub.2O, 98%
D.sub.2O final, pH 7.3 (uncorrected value)). All HX reactions were
carried out at 20.degree. C. and contained 3 .mu.M FVIII in the
absence or presence of 4.5 .mu.M vWF fragment thus giving a 1.5
fold molar excess of vWF fragment binding partner. At appropriate
time intervals ranging from 10 sec to 240 sec, 50 .mu.l aliquots of
the HX reaction were quenched by 50 .mu.l ice-cold quenching buffer
(1.36 M TCEP, 2 M urea) resulting in a final pH of 2.5 (uncorrected
value).
[0334] Results and Discussion
[0335] Interaction Mapping of 2304 and 2307 on FVIII
[0336] The HX time-course of 191 peptides, covering 83% of the
primary sequence of FVIII were monitored in the absence or presence
of the vWF fragments 2304 or 2307 for i.e., 10, 20, 30, 40, 60,
120, and 240 sec.
[0337] The vWF fragments 2304 and 2307 both induce identical
alterations in the exchange profile of FVIII and will be described
together here. The observed exchange pattern in the time points
(i.e., 10, 20, 30, 40, 60, 120, and 240 sec) in the presence or
absence of 2304/2307 can be divided into different groups: One
group of peptides display an exchange pattern that is unaffected by
the binding of 2304/2307. In contrast, another group of peptides in
FVIII show protection from exchange upon 2304/2307 binding.
[0338] The regions displaying protection upon 2304/2307 binding
encompass peptides covering residues 1855-1875, 1857-1875,
2062-2070, 2125-2147, 2125-2148, 2127-2147, 2275-2291, 2275-2302,
2275-2305, 2292-2305, and 2293-2312 (Table 14). However, by
comparing the relative amounts of exchange protection within each
peptide upon binding 2304/2307 and the lack of epitope effects in
overlapping and adjacent peptides in these regions, the regions
that display reduced deuterium incorporation can be narrowed to
residues 1862-1875, 2062-2070, 2125-2147, and 2285-2299.
[0339] Interaction Mapping of D'D3A1 and 2308 on FVIII
[0340] The HX time-course of 185 peptides, covering 79% of the
primary sequence of FVIII were monitored in the absence or presence
of the vWF fragments D'D3A1 or 2308 for 10, 20, 30, 40, 60, 120,
and 240 sec.
[0341] The vWF fragments D'D3A1 and 2308 both induce identical
alterations in the exchange profile of FVIII and will be described
together here.
[0342] The regions displaying protection upon D'D3A1 or 2308
binding encompass peptides covering residues 1669-1680, 1738-1765,
1743-1765, 1856-1869, 1870-1874, 2061-2074, 2063-2074, 2123-2146,
and 2260-2280 (Table 15).
[0343] However, by comparing the relative amounts of exchange
protection within each peptide upon binding of D'D3A1 or 2308 and
the lack of epitope effects in overlapping and adjacent peptides in
these regions, the regions that display reduced deuterium
incorporation can be narrowed to residues 1671-1680, 1745-1754,
1858-1874, 2063-2074, 2125-2146, 2262-2280.
[0344] Interaction Mapping of FVIII on D'D3A1
[0345] The HX time-course of 82 peptides, covering 58% of the
primary sequence of vWF fragment D'D3A1 were monitored in the
absence or presence of FVIII for 10, 20, 40, 60, 120, and 240
sec.
[0346] The region displaying exchange protection upon FVIII binding
encompass the peptide covering residues 768-778 (Table 16).
[0347] However, by comparing the relative amounts of exchange
protection within each peptide upon binding FVIII and the lack of
epitope effects in overlapping and adjacent peptides in these
regions, the regions that display reduced deuterium incorporation
can be narrowed to residues 770-778.
Conclusion
[0348] Upon binding of either 2304 or 2307 all regions of FVIII
showed similar responses. The same group of peptides were affected
by vWF fragment binding in the early time-points.
[0349] Furthermore, these affected regions identified for 2304/2307
binding were found to show overlap with affected regions upon
binding to D'D3A1/2308 within domain A3 and C1 of FVIII.
[0350] Due to lacking sequence coverage of the peptic peptide map
conducted to the HX-MS time course of 2304/2307 binding it was not
possible to exchange characteristics for residues 1671-1680. Thus
it was not possible to verify if 2304/2307 binding induces exchange
protection to this region as it was identified upon D'D3A1/2308
binding.
[0351] Upon binding of FVIII the regions covering residues 770-778
of D'D3A1 showed exchange protection. The obtained sequence
coverage of 58% of D'D3A1 afforded by the peptic peptides conducted
to HXMS analysis of FVIII binding, does not allow to leave out that
more interaction site are present within D'D3A1/2308.
[0352] Conclusion
[0353] The identified regions of FVIII showing protection upon
binding to vWF fragments D'D3A1, 2308, 2304, or 2307 are
structurally situated at remote distances when mapping on to the
crystal structure PDB: 2R7E. This makes it highly unlikely that
they can all be assigned to protection induced by binding interface
between FVIII and the vWF fragments D'D3A1, 2308, 2304, or 2307.
The HX-MS analysis is unable to distinguish between exchange
protection induced by binding interface with exchange protections
induced by rapid conformational changes.
[0354] Thus it is plausible that the observed regions showing
exchange protection upon binding to vWF fragments D'D3A1, 2308,
2304, or 2307 are induced by both binding interface and
conformational changes of FVIII.
[0355] The HXMS study of FVIII binding to vWF fragments D'D3A1,
2308, 2304, or 2307 revealed overlapping regions within domains A3
and C1, and therefore the complex binding to this part of FVIII is
identical for the vWF fragments investigated.
[0356] The observed discrepancy in domain C2 hints that this part
of FVIII undergoes conformational changes upon complex formation
with the vWF-fragments. Furthermore, the obtained results hint that
the truncation differences between D'D3A1/2308 and 2304/2307
induces different conformational changes of domain C2. In contrast
the truncation difference between 2304 and 2307 does not seem to
affect the conformational orientation of C2, since identical
exchange profiles of domain C2 were observed for binding to these
vWF-fragment species.
[0357] It is well known that the domains C1 and C2 are essential
for the membrane binding affinity of FVIII. It can be speculated
that conformational changes of these part of FVIII will reduce the
membrane binding ability of FVIII. The conformational position of
domains C1 and C2 of FVIII complex bound to the vWF fragments might
be unfavourable for membrane binding affinity of FVIII.
Furthermore, it is highly likely that the fragments in complex with
FVIII will shield for the membrane binding affinity of FVIII as it
has been established for the membrane binding characteristics of
FVIII complex bound to endogenous vWF. A reduced membrane binding
affinity of FVIII complex bound to the vWF fragments in comparison
to free FVIII would lead to a reduced binding of FVIII to cell
membranes of the immune system, e.g. antigen presenting cells. This
could decrease presentation of FVIII-derived peptides on MHC class
II and it can therefore be speculated that FVIII complex bound to
vWF fragments will be less immunogenic than free FVIII.
TABLE-US-00017 TABLE 14 HXMS analysis of FVIII (Turoctocog alfa;
seq. no. using wt FVIII) (SEQ ID NO: 2) binding to the vWF
fragments 2304 (SEQ ID NO: 5) or 2307 (SEQ ID NO: 8). After
deuterium exchange reaction. FVIII is digested with pepsin yielding
the present peptic peptides identified to show exchange protection
in the presence of 2304 or 2307. Sequence Domain 2304 2307
L1855-E1875 A3 EX EX V1857-E1875 A3 EX EX W2062-W2070 A3 EX EX
V2125-R2147 C1 EX EX V2125-Y2148 C1 EX EX F2127-R2147 C1 EX EX
F2275-T2291 C2 EX EX F2275-L2302 C2 EX EX F2275-Y2305 C2 EX EX
P2292-Y2305 C2 EX EX V2293-S2312 C2 EX EX EX: exchange protection
of FVIII residues upon 2304 or 2307 binding indicating interaction
region (40 sec incubation in D2O, >0.4 Da).
TABLE-US-00018 TABLE 15 HXMS analysis of FVIII (Turoctocog alfa;
seq. no. using wt FVIII) (SEQ ID NO: 2) binding to the vWF
fragments D'D3A1 (SEQ ID NO: 19; Cys1099Ser; Cys1142Ser) or 2308
(SEQ ID NO: 11; Cys1099Ser; Cys1142Ser). After deuterium exchange
reaction. FVIII is digested with pepsin yielding the present peptic
peptides identified to show exchange protection in the presence of
D'D3A1 or 2308. Sequence Domain D'D3A1 2308 S1669-Y1680 a3 EX EX
F1738-E1765 A3 EX EX F1743-E1765 A3 EX EX L1856-R1869 A3 EX EX
Q1870-Q1874 A3 EX EX A2061-D2074 C1 EX EX S2063-D2074 C1 EX EX
L2123-A2146 C1 EX EX F2260-V2280 C2 EX EX EX: exchange protection
of FVIII residues upon D'D3A1 or 2308 binding indicating
interaction region (40 sec incubation in D2O, >0.4 Da).
TABLE-US-00019 TABLE 16 HXMS analysis of vWF fragment D'D3A1 (SEQ
ID NO: 19; Cys1099Ser; Cys1142Ser) binding to the FVIII (Turoctocog
alfa (SEQ ID NO: 2). After deuterium exchange reaction. D'D3A1 is
digested with pepsin yielding the present peptic peptide identified
to show exchange protection in the presence of FVIII. Sequence
Domain FVIII R768-A778 D' EX EX: exchange protection of D'D3A1
residues upon FVIII binding indicating interaction region (40 sec
incubation in D2O, >0.4 Da).
Example 37
Complex formation of FVIII (SEQ ID NO:2) with TIC/E7D3/A1 III (SEQ
ID NO:19; Cys1099Ser; Cys1142Ser) and of FVIII (SEQ ID NO:2) with
TIL'/E'/D3 II (SEQ ID NO:14; Cys1099Ser; Cys1142Ser) analysed by
SEC-UV
[0358] Materials
[0359] Protein batches used were:
[0360] FVIII protein batches used were:
[0361] FVIII (N8, Turoctocog alfa, SEQ ID NO:2) Batch
0155-0000-0004-37A; TIL'/E'/D3/A1 III (SEQ ID NO:19; Cys1099Ser;
Cys1142Ser) Batch 0129-0000-0170-6B; TIL'/E'/D3 II (SEQ ID NO:14;
Cys1099Ser; Cys1142Ser) Batch 0129-0000-2309-1B.
[0362] Methods
[0363] Size-exclusion chromatography was performed on a Waters
Biosuite, 4.6.times.300 mm column using a flow rate of 0.3 ml/min
and a running buffer of 155 mM NaCl, 10 mM Calciumacetat, 10%
Isopropanol at 25.degree. C. The absorbance of the effluent was
monitored by a UV detector at 280 nm. SEC-UV characterization were
performed of FVIII, TIL'/E'/D3/A1 III, TIL'/E'/D3 II, and 1:2
complexes of FVIII-TIL'/E'/D3/A1 III and of FVIII-TIL'/E'/D3 II.
Samples of FVIII 10 .mu.M, TIL'/E'/D3/A1 III 20 .mu.M, TIL'/E'/D3
II 20 .mu.M, and in complex were prepared and 15 .mu.L were loaded
on to the column.
[0364] Results and Conclusion
[0365] SEC-UV of the mixtures of FVIII-TIL'/E'/D3/A1 III and
FVIII-TIL'/E'/D3 II showed significant fractions of the complex to
elute intact from the column. The complex would be expected to
elute a little earlier than FVIII; this was also observed in both
cases.
Sequence CWU 1
1
5612332PRTHomo sapiens 1Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu
Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro
Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro
Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu
Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro
Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr
Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90
95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp
Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val
Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu
Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp
Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu
Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe
Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His
Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215
220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His
Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His
Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr
Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe
Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala
Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340
345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser
Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr
Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys
Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys
Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe
Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly
Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465
470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly
Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile
Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr
Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe
Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro
Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly
Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser
Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585
590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe
Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr
Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu
Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val
Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr
Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly
Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu
Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710
715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn
Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro
Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu
Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr
Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu
Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser
Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp
Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835
840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu
Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys
Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro
Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu
Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp
Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser
Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys
Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955
960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu
Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser
Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly
Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln
Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr
Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala
Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr
Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075
1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys
1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg
Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro
Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys Ser
Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys Val
Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly Leu
Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe Leu
Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185 Asn
Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195
1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr
1205 1210 1215 Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser
Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr
Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser
Thr Asn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys
Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln
Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg
Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln
Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315
1320 Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr
1325 1330 1335 Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr
Leu Thr 1340 1345 1350 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala
Ile Thr Gln Ser 1355 1360 1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser
His Ser Ile Pro Gln Ala 1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile
Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr
Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410 Ser His Leu
Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln
Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435
1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu
1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr
Tyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp
Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys
Val His Ile Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr
Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly
Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn
Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val
Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555
1560 Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu
1565 1570 1575 Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala
Phe Lys 1580 1585 1590 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys
Glu Ser Asn His 1595 1600 1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln
Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val Thr Trp Ala Lys Gln Gly
Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu
Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln
Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser
Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675
1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr
1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met
Ser Ser 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser
Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu
Phe Thr Asp Gly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly
Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile
Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg
Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu
Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795
1800 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys
1805 1810 1815 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp
Cys Lys 1820 1825 1830 Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
Lys Asp Val His 1835 1840 1845 Ser Gly Leu Ile Gly Pro Leu Leu Val
Cys His Thr Asn Thr Leu 1850 1855 1860 Asn Pro Ala His Gly Arg Gln
Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875 Phe Phe Thr Ile Phe
Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890 Asn Met Glu
Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905 Asp
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915
1920 Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln
1925 1930 1935 Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu
Asn Ile 1940 1945 1950 His Ser Ile His Phe Ser Gly His Val Phe Thr
Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn
Leu Tyr Pro Gly Val Phe 1970 1975 1980 Glu Thr Val Glu Met Leu Pro
Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly
Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010 Phe Leu Val
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025 Ser
Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035
2040 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser
2045 2050 2055 Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile
Lys Val 2060 2065 2070 Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile
Lys Thr Gln Gly 2075 2080 2085 Ala Arg Gln Lys Phe Ser Ser Leu Tyr
Ile Ser Gln Phe Ile Ile 2090 2095 2100 Met Tyr Ser Leu Asp Gly Lys
Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115 Ser Thr Gly Thr Leu
Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130 Gly Ile Lys
His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145 Ile
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155
2160 Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu
2165 2170 2175 Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr
Ala Ser 2180 2185 2190 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser
Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn
Ala Trp Arg Pro Gln Val 2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu
Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235 Lys Val Thr Gly Val
Thr Thr
Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys
Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265 His Gln Trp
Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280 Gln
Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290
2295 Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
2300 2305 2310 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys
Glu Ala 2315 2320 2325 Gln Asp Leu Tyr 2330 221PRTArtificial
SequenceLinker sequence 2Ser Phe Ser Gln Asn Ser Arg His Pro Ser
Gln Asn Pro Pro Val Leu 1 5 10 15 Lys Arg His Gln Arg 20
31667PRTArtificial Sequence226 amino acid B domain FVIII variant
3Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1
5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro
Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe
Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly
Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr
Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val
Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp
Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe
Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135
140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser
145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu
Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val
Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser
Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro
Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 Ser Leu
Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260
265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu
Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile Ser Ser His
Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val Asp Ser
Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu Glu
Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp
Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln
Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385
390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn
Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe
Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile
Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu
Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn Gln Ala Ser
Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr Asp Val
Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 His
Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505
510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp
Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys
Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp
Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg
Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn
Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser
Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu
Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630
635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly
Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu
Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe
Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys
Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu
Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu
Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755
760 765 Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln
Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser
Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser Asp Leu Gln Glu
Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser Pro Gly Ala
Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His Phe Arg
Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro Glu
Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr
Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875
880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro
Val His 900 905 910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys
Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu
Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu
Met Asn Ser Gln Glu Ser Ser Trp 945 950 955 960 Gly Lys Asn Val Ser
His His His His His His Ser Gln Asn Pro Pro 965 970 975 Val Leu Lys
Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser 980 985 990 Asp
Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys 995
1000 1005 Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser
Pro 1010 1015 1020 Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile
Ala Ala Val 1025 1030 1035 Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser
Ser Pro His Val Leu 1040 1045 1050 Arg Asn Arg Ala Gln Ser Gly Ser
Val Pro Gln Phe Lys Lys Val 1055 1060 1065 Val Phe Gln Glu Phe Thr
Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1070 1075 1080 Arg Gly Glu Leu
Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1085 1090 1095 Arg Ala
Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1100 1105 1110
Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1115
1120 1125 Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val
Lys 1130 1135 1140 Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln
His His Met 1145 1150 1155 Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
Ala Trp Ala Tyr Phe 1160 1165 1170 Ser Asp Val Asp Leu Glu Lys Asp
Val His Ser Gly Leu Ile Gly 1175 1180 1185 Pro Leu Leu Val Cys His
Thr Asn Thr Leu Asn Pro Ala His Gly 1190 1195 1200 Arg Gln Val Thr
Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1205 1210 1215 Asp Glu
Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1220 1225 1230
Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1235
1240 1245 Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp
Thr 1250 1255 1260 Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile
Arg Trp Tyr 1265 1270 1275 Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
His Ser Ile His Phe 1280 1285 1290 Ser Gly His Val Phe Thr Val Arg
Lys Lys Glu Glu Tyr Lys Met 1295 1300 1305 Ala Leu Tyr Asn Leu Tyr
Pro Gly Val Phe Glu Thr Val Glu Met 1310 1315 1320 Leu Pro Ser Lys
Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1325 1330 1335 Glu His
Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1340 1345 1350
Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1355
1360 1365 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala
Pro 1370 1375 1380 Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn
Ala Trp Ser 1385 1390 1395 Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
Asp Leu Leu Ala Pro 1400 1405 1410 Met Ile Ile His Gly Ile Lys Thr
Gln Gly Ala Arg Gln Lys Phe 1415 1420 1425 Ser Ser Leu Tyr Ile Ser
Gln Phe Ile Ile Met Tyr Ser Leu Asp 1430 1435 1440 Gly Lys Lys Trp
Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1445 1450 1455 Met Val
Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1460 1465 1470
Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1475
1480 1485 Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met
Gly 1490 1495 1500 Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met
Glu Ser Lys 1505 1510 1515 Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser
Ser Tyr Phe Thr Asn 1520 1525 1530 Met Phe Ala Thr Trp Ser Pro Ser
Lys Ala Arg Leu His Leu Gln 1535 1540 1545 Gly Arg Ser Asn Ala Trp
Arg Pro Gln Val Asn Asn Pro Lys Glu 1550 1555 1560 Trp Leu Gln Val
Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1565 1570 1575 Thr Thr
Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1580 1585 1590
Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1595
1600 1605 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln
Asp 1610 1615 1620 Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro
Leu Leu Thr 1625 1630 1635 Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
Val His Gln Ile Ala 1640 1645 1650 Leu Arg Met Glu Val Leu Gly Cys
Glu Ala Gln Asp Leu Tyr 1655 1660 1665 465PRTArtificial SequencevWF
fragment amino acids 764-828 (TIL') 4Ser Leu Ser Cys Arg Pro Pro
Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu
Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu
Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro
Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55
60 Pro 65 5102PRTArtificial SequencevWF fragment amino acids
764-865 (TIL'/E') 5Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val
Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr
Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met Gly
Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg His
Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe His
Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile Gly
Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr 85 90 95
Asp His Val Cys Asp Ala 100 6272PRTArtificial SequencevWF fragment
amino acids 764-1035 (TIL'/E'/VWD3 I) 6Ser Leu Ser Cys Arg Pro Pro
Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu
Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu
Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro
Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55
60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn
Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly
Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe
Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly
Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly
Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr
Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu
Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185
190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys
Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe
Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu
Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys
Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 7 278PRTArtificial
SequencevWF fragment amino
acids 764-1041 (TIL'/E'/VWD3 II) 7Ser Leu Ser Cys Arg Pro Pro Met
Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly
Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys
Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly
Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65
70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn
Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly
Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe
Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly
Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly
Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr
Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu
Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185
190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys
Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe
Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu
Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys
Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp
Ser 275 8 282PRTArtificial SequencevWF fragment amino acids
764-1045 (TIL'/E'/VWD3 III) 8Ser Leu Ser Cys Arg Pro Pro Met Val
Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu
Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met
Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met
Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro
Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70
75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys
Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met
Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro
Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser
Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys
Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile
Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val
Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195
200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln
Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp
Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln
Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val
Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser
Ser Pro Ala Thr 275 280 9 365PRTArtificial SequencevWF fragment
amino acids 764-1128 (TIL'/E'/VWD3/C8-3) 9Ser Leu Ser Cys Arg Pro
Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala
Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu
Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50
55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val
Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp
Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile
Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu
Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys
Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys
Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val
Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180
185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu
Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu
Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn
Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn
Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp
Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu
Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln
Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305
310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys
Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys
Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu
Cys Pro Gln 355 360 365 10435PRTArtificial SequencevWF fragment
amino acids 764-1198 (TIL'/E'/VWD3/C8-3/TIL-3) 10Ser Leu Ser Cys
Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu
Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35
40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg
Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu
Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg
Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser
Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys
Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp
Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly
Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165
170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu
Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys
Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val
Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys
Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser
Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn
Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val
Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290
295 300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp
Val 305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly
Asp Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His
Val Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala
Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu
Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro
Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala
Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410
415 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430 Cys Pro Val 435 11487PRTArtificial SequencevWF fragment
amino acids 764-1250 (TIL'/E'/D3 I 11Ser Leu Ser Cys Arg Pro Pro
Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu
Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu
Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro
Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55
60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn
Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly
Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe
Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly
Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly
Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr
Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu
Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185
190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys
Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe
Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu
Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys
Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp
Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr
Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe
Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310
315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala
Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala
Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys
Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr
Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln
Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val
Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys
Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435
440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His
Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro
Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala 485
12386PRTArtificial SequencevWF fragment amino acids 864-1250 (D3 I)
12Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly 1
5 10 15 Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln
Asp 20 25 30 Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val
Gly Asn Lys 35 40 45 Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
Arg Val Thr Ile Leu 50 55 60 Val Glu Gly Gly Glu Ile Glu Leu Phe
Asp Gly Glu Val Asn Val Lys 65 70 75 80 Arg Pro Met Lys Asp Glu Thr
His Phe Glu Val Val Glu Ser Gly Arg 85 90 95 Tyr Ile Ile Leu Leu
Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 100 105 110 His Leu Ser
Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 115 120 125 Cys
Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 130 135
140 Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser
145 150 155 160 Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val
Pro Leu Asp 165 170 175 Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
Lys Gln Thr Met Val 180 185 190 Asp Ser Ser Cys Arg Ile Leu Thr Ser
Asp Val Phe Gln Asp Cys Asn 195 200 205 Lys Leu Val Asp Pro Glu Pro
Tyr Leu Asp Val Cys Ile Tyr Asp Thr 210 215 220 Cys Ser Cys Glu Ser
Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile 225 230 235 240 Ala Ala
Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp 245 250 255
Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg 260
265 270 Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala Pro
Ala 275 280 285 Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys
Pro Val Gln 290 295 300 Cys Val Glu Gly Cys His Ala His Cys Pro Pro
Gly Lys Ile Leu Asp 305 310 315 320 Glu Leu Leu Gln Thr Cys Val Asp
Pro Glu Asp Cys Pro Val Cys Glu 325 330 335 Val Ala Gly Arg Arg Phe
Ala Ser Gly Lys Lys Val Thr Leu Asn Pro 340 345 350 Ser Asp Pro Glu
His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu 355 360 365 Thr Cys
Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr 370 375 380
Asp Ala 385 13405PRTArtificial SequencevWF fragment amino acids
864-1268 (D3 II)
13Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly 1
5 10 15 Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln
Asp 20 25 30 Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val
Gly Asn Lys 35 40 45 Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
Arg Val Thr Ile Leu 50 55 60 Val Glu Gly Gly Glu Ile Glu Leu Phe
Asp Gly Glu Val Asn Val Lys 65 70 75 80 Arg Pro Met Lys Asp Glu Thr
His Phe Glu Val Val Glu Ser Gly Arg 85 90 95 Tyr Ile Ile Leu Leu
Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 100 105 110 His Leu Ser
Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 115 120 125 Cys
Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 130 135
140 Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser
145 150 155 160 Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val
Pro Leu Asp 165 170 175 Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
Lys Gln Thr Met Val 180 185 190 Asp Ser Ser Cys Arg Ile Leu Thr Ser
Asp Val Phe Gln Asp Cys Asn 195 200 205 Lys Leu Val Asp Pro Glu Pro
Tyr Leu Asp Val Cys Ile Tyr Asp Thr 210 215 220 Cys Ser Cys Glu Ser
Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile 225 230 235 240 Ala Ala
Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp 245 250 255
Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg 260
265 270 Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala Pro
Ala 275 280 285 Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys
Pro Val Gln 290 295 300 Cys Val Glu Gly Cys His Ala His Cys Pro Pro
Gly Lys Ile Leu Asp 305 310 315 320 Glu Leu Leu Gln Thr Cys Val Asp
Pro Glu Asp Cys Pro Val Cys Glu 325 330 335 Val Ala Gly Arg Arg Phe
Ala Ser Gly Lys Lys Val Thr Leu Asn Pro 340 345 350 Ser Asp Pro Glu
His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu 355 360 365 Thr Cys
Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr 370 375 380
Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu 385
390 395 400 Pro Pro Leu His Asp 405 14498PRTArtificial SequencevWF
fragment amino acids 764-1261(TIL'/E'/D3 II) 14Ser Leu Ser Cys Arg
Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg
Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp
Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40
45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr
Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys
Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr
Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr
Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr
Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn
Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg
Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170
175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala
Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val
Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly
Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser
Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser
Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro
Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys
Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295
300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp
Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val
Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr
Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn
Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala
Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys
Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420
425 430 Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys
Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile
Cys His Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln
Glu Pro Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala Pro
Val Ser Pro Thr Thr Leu Tyr Val 485 490 495 Glu Asp
15502PRTArtificial SequencevWF fragment amino acids 764-1264
(TIL'/E'/D3 III) 15Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val
Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr
Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met Gly
Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg His
Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe His
Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile Gly
Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr 85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100
105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln
Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr
Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser
Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu Gly
Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys Arg
Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser Gly
Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val Val
Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln 225
230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp
Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys
Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser Ser Pro Ala Thr
Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met Val Asp Ser Ser
Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln Asp Cys Asn Lys
Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315 320 Cys Ile Tyr
Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335 Phe
Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345
350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg
Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His
Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu Gly
Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu Leu
Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys Glu
Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr Leu
Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460 Asp
Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465 470
475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr
Val 485 490 495 Glu Asp Ile Ser Glu Pro 500 16506PRTArtificial
SequencevWF fragment amino acids 764-1268 (TIL'/E'/D3 IV) 16Ser Leu
Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20
25 30 Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys
Pro 35 40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu
Glu Arg Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro
Gly Glu Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln
Asp Arg Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr
Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly
Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val
Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu
Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150
155 160 Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp
Gly 165 170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His
Phe Glu Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu
Gly Lys Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile
Ser Val Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly
Leu Cys Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu
Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe
Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275
280 285 Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp
Val 290 295 300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr
Leu Asp Val 305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser
Ile Gly Asp Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr
Ala His Val Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg
Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu
Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys
Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395
400 Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro
Glu Asp 420 425 430 Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala
Ser Gly Lys Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His
Cys Gln Ile Cys His Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu
Ala Cys Gln Glu Pro Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr
Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val 485 490 495 Glu Asp Ile
Ser Glu Pro Pro Leu His Asp 500 505 17696PRTArtificial SequencevWF
fragment amino acids 764-1459 (TIL'/E'/D3/A1 I) 17Ser Leu Ser Cys
Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu
Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35
40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg
Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu
Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg
Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser
Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys
Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp
Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly
Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165
170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu
Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys
Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val
Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys
Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser
Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn
Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val
Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290
295 300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp
Val 305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly
Asp Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His
Val Cys Ala Gln His Gly 340 345 350 Lys Val Val
Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu
Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375
380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His
Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys
Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys Glu Val Ala Gly Arg
Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp
Pro Glu His Cys Gln Ile Cys His Cys 450 455 460 Asp Val Val Asn Leu
Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465 470 475 480 Val Val
Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val 485 490 495
Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu 500
505 510 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu
Ala 515 520 525 Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met
Glu Arg Leu 530 535 540 Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val
Val Glu Tyr His Asp 545 550 555 560 Gly Ser His Ala Tyr Ile Gly Leu
Lys Asp Arg Lys Arg Pro Ser Glu 565 570 575 Leu Arg Arg Ile Ala Ser
Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 580 585 590 Ser Thr Ser Glu
Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys 595 600 605 Ile Asp
Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser 610 615 620
Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly 625
630 635 640 Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly
Pro His 645 650 655 Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln
Ala Pro Glu Asn 660 665 670 Lys Ala Phe Val Leu Ser Ser Val Asp Glu
Leu Glu Gln Gln Arg Asp 675 680 685 Glu Ile Val Ser Tyr Leu Cys Asp
690 695 18700PRTArtificial SequencevWF fragment amino acids
764-1463 (TIL'/E'/D3/A1 II) 18Ser Leu Ser Cys Arg Pro Pro Met Val
Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu
Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met
Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met
Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro
Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70
75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys
Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met
Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro
Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser
Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys
Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile
Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val
Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195
200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln
Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp
Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln
Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val
Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser
Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met
Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln
Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315
320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln
His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro
Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu
Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val
Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln
Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile
Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys
Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440
445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly
Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro
Thr Thr Leu Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro Pro Leu His
Asp Phe Tyr Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val Phe Leu Leu
Asp Gly Ser Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe Glu Val Leu
Lys Ala Phe Val Val Asp Met Met Glu Arg Leu 530 535 540 Arg Ile Ser
Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 545 550 555 560
Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 565
570 575 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val
Ala 580 585 590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile
Phe Ser Lys 595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu
Leu Leu Met Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met Ser Arg Asn
Phe Val Arg Tyr Val Gln Gly 625 630 635 640 Leu Lys Lys Lys Lys Val
Ile Val Ile Pro Val Gly Ile Gly Pro His 645 650 655 Ala Asn Leu Lys
Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665 670 Lys Ala
Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 675 680 685
Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu 690 695 700
19701PRTArtificial SequencevWF fragment amino acids 764-1464
(TIL'/E'/D3/A1 III) 19Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu
Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys
Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met
Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg
His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe
His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile
Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr 85 90
95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys
Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly
Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro
Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu
Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys
Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser
Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val
Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215
220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu
Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln
Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser Ser Pro Ala
Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met Val Asp Ser
Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln Asp Cys Asn
Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315 320 Cys Ile
Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340
345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys
Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp
Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln
His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu
Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu
Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys
Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr
Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465
470 475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu
Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr
Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser
Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe Glu Val Leu Lys Ala Phe
Val Val Asp Met Met Glu Arg Leu 530 535 540 Arg Ile Ser Gln Lys Trp
Val Arg Val Ala Val Val Glu Tyr His Asp 545 550 555 560 Gly Ser His
Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 565 570 575 Leu
Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 580 585
590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys
595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met
Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg
Tyr Val Gln Gly 625 630 635 640 Leu Lys Lys Lys Lys Val Ile Val Ile
Pro Val Gly Ile Gly Pro His 645 650 655 Ala Asn Leu Lys Gln Ile Arg
Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665 670 Lys Ala Phe Val Leu
Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 675 680 685 Glu Ile Val
Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala 690 695 700
20920PRTArtificial SequencevWF fragment amino acids 764-1683
(TIL'/E'/D3/A1/A2) 20Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu
Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys
Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met
Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg
His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe
His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile
Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr 85 90
95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys
Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly
Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro
Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu
Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys
Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser
Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val
Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215
220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu
Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln
Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser Ser Pro Ala
Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met Val Asp Ser
Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln Asp Cys Asn
Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315 320 Cys Ile
Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340
345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys
Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp
Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln
His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu
Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu
Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys
Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr
Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465
470 475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu
Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr
Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser
Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe Glu Val Leu Lys
Ala Phe Val Val Asp Met Met Glu Arg Leu 530 535 540 Arg Ile Ser Gln
Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 545 550 555 560 Gly
Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 565 570
575 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala
580 585 590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe
Ser Lys 595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu
Leu Met Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met Ser Arg Asn Phe
Val Arg Tyr Val Gln Gly 625 630 635 640 Leu Lys Lys Lys Lys Val Ile
Val Ile Pro Val Gly Ile Gly Pro His 645 650 655 Ala Asn Leu Lys Gln
Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665 670 Lys Ala Phe
Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 675 680 685 Glu
Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro 690 695
700 Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu
705 710 715 720 Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met Val
Leu Asp Val 725 730 735 Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly
Glu Ala Asp Phe Asn 740 745 750 Arg Ser Lys Glu Phe Met Glu Glu Val
Ile Gln Arg Met Asp Val Gly 755 760 765 Gln Asp Ser Ile His Val Thr
Val Leu Gln Tyr Ser Tyr Met Val Thr 770 775 780 Val Glu Tyr Pro Phe
Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln 785 790 795 800 Arg Val
Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly 805 810 815
Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly 820
825 830 Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn
Pro 835 840 845 Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln
Val Val Pro 850 855 860 Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu
Leu Glu Arg Ile Gly 865 870 875 880 Trp Pro Asn Ala Pro Ile Leu Ile
Gln Asp Phe Glu Thr Leu Pro Arg 885 890 895 Glu Ala Pro Asp Leu Val
Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu 900 905 910 Gln Ile Pro Thr
Leu Ser Pro Ala 915 920 21 1110PRTArtificial SequencevWF fragment
amino acids 764-1873 (TIL'/E'/D3/A1/A2/A3) 21Ser Leu Ser Cys Arg
Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg
Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp
Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40
45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr
Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys
Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr
Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr
Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr
Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn
Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg
Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170
175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala
Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val
Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly
Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser
Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser
Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro
Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys
Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295
300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp
Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val
Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr
Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn
Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala
Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys
Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420
425 430 Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys
Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile
Cys His Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln
Glu Pro Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala Pro
Val Ser Pro Thr Thr Leu Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro
Pro Leu His Asp Phe Tyr Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val
Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe
Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu 530 535 540
Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 545
550 555 560 Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro
Ser Glu 565 570 575 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly
Ser Gln Val Ala 580 585 590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu
Phe Gln Ile Phe Ser Lys 595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg
Ile Thr Leu Leu Leu Met Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met
Ser Arg Asn Phe Val Arg Tyr Val Gln Gly 625 630 635 640 Leu Lys Lys
Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His 645 650 655 Ala
Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665
670 Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp
675 680 685 Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro
Pro Pro 690 695 700 Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly
Pro Gly Leu Leu 705 710 715 720 Gly Val Ser Thr Leu Gly Pro Lys Arg
Asn Ser Met Val Leu Asp Val 725 730 735 Ala Phe Val Leu Glu Gly Ser
Asp Lys Ile Gly Glu Ala Asp Phe Asn 740 745 750 Arg Ser Lys Glu Phe
Met Glu Glu Val Ile Gln Arg Met Asp Val Gly 755 760 765 Gln Asp Ser
Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr 770 775 780 Val
Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln 785 790
795 800 Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr
Gly 805 810 815 Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val
Ser Gln Gly 820 825 830 Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met
Val Thr Gly Asn Pro 835 840 845 Ala Ser Asp Glu Ile Lys Arg Leu Pro
Gly Asp Ile Gln Val Val Pro 850 855 860 Ile Gly Val Gly Pro Asn Ala
Asn Val Gln Glu Leu Glu Arg Ile Gly 865 870 875 880 Trp Pro Asn Ala
Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg 885 890 895 Glu Ala
Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu 900 905 910
Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp 915
920 925 Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr
Phe 930 935 940 Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser Lys
Ala Asn Ile 945 950 955 960 Gly Pro Arg Leu Thr Gln Val Ser Val Leu
Gln Tyr Gly Ser Ile Thr 965 970 975 Thr Ile Asp Val Pro Trp Asn Val
Val Pro Glu Lys Ala His Leu Leu 980 985 990 Ser Leu Val Asp Val Met
Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly 995 1000 1005 Asp Ala Leu
Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His 1010 1015 1020 Gly
Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr 1025 1030
1035 Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg
1040 1045 1050 Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp
Arg Tyr 1055 1060 1065 Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly Pro
Ala Gly Asp Ser 1070 1075 1080 Asn Val Val Lys Leu Gln Arg Ile Glu
Asp Leu Pro Thr Met Val 1085 1090 1095 Thr Leu Gly Asn Ser Phe Leu
His Lys Leu Cys Ser 1100 1105 1110 222813PRTHomo sapiens 22Met Ile
Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile 1 5 10 15
Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20
25 30 Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp
Gly 35 40 45 Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu
Ala Gly Gly 50 55 60 Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp
Phe Gln Asn Gly Lys 65 70 75 80 Arg Val Ser Leu Ser Val Tyr Leu Gly
Glu Phe Phe Asp Ile His Leu 85 90 95 Phe Val Asn Gly Thr Val Thr
Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110 Tyr Ala Ser Lys Gly
Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125 Leu Ser Gly
Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140 Asn
Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150
155 160 Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr
Gln 165 170 175 Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn
Ser Trp Ala 180 185 190 Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala
Ser Pro Pro Ser Ser 195 200 205 Ser Cys Asn Ile Ser Ser Gly Glu Met
Gln Lys Gly Leu Trp Glu Gln 210 215 220 Cys Gln Leu Leu Lys Ser Thr
Ser Val Phe Ala Arg Cys His Pro Leu 225 230 235 240 Val Asp Pro Glu
Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255 Cys Ala
Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270
Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275
280 285 Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln
Cys 290 295 300 Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile
Asn Glu Met 305 310 315 320 Cys Gln Glu Arg Cys Val Asp Gly Cys Ser
Cys Pro Glu Gly Gln Leu 325 330 335 Leu Asp Glu Gly Leu Cys Val Glu
Ser Thr Glu Cys Pro Cys Val His 340 345 350 Ser Gly Lys Arg Tyr Pro
Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365 Thr Cys Ile Cys
Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380 Pro Gly
Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp 385 390 395
400 Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg
405 410 415 Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val
Gln Cys 420 425 430 Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val
Thr Val Arg Leu 435 440 445 Pro Gly Leu His Asn Ser Leu Val Lys Leu
Lys His Gly Ala Gly Val 450 455 460 Ala Met Asp Gly Gln Asp Val Gln
Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480 Arg Ile Gln His Thr
Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495 Asp Leu Gln
Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510 Ser
Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520
525 Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro
530 535 540 Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp
Cys Gln 545 550 555 560 Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala
Leu Asn Pro Arg Met 565 570 575 Thr Arg Phe Ser Glu Glu Ala Cys Ala
Val Leu Thr Ser Pro Thr Phe 580 585 590 Glu Ala Cys His Arg Ala Val
Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605 Arg Tyr Asp Val Cys
Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620 Ala Leu Ala
Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val 625 630 635 640
Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645
650 655 Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser
Leu 660 665 670 Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu
Gly Cys Phe 675 680 685 Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly
Asp Cys Val Pro Lys 690 695 700 Ala Gln Cys Pro Cys Tyr Tyr Asp Gly
Glu Ile Phe Gln Pro Glu Asp 705 710 715 720 Ile Phe Ser Asp His His
Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735 His Cys Thr Met
Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750 Leu Ser
Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765
Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770
775 780 Gly Leu Glu Cys Thr
Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met 785 790 795 800 Ser Met
Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815
His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820
825 830 Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn
Thr 835 840 845 Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr Asp His
Val Cys Asp 850 855 860 Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
Leu Thr Phe Asp Gly 865 870 875 880 Leu Lys Tyr Leu Phe Pro Gly Glu
Cys Gln Tyr Val Leu Val Gln Asp 885 890 895 Tyr Cys Gly Ser Asn Pro
Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910 Gly Cys Ser His
Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925 Val Glu
Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940
Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg 945
950 955 960 Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp
Asp Arg 965 970 975 His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr
Gln Glu Lys Val 980 985 990 Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile
Gln Asn Asn Asp Leu Thr 995 1000 1005 Ser Ser Asn Leu Gln Val Glu
Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020 Ser Trp Lys Val Ser
Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035 Leu Asp Ser
Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050 Thr
Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060
1065 Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
1070 1075 1080 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp
Cys Ala 1085 1090 1095 Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His
Val Cys Ala Gln 1100 1105 1110 His Gly Lys Val Val Thr Trp Arg Thr
Ala Thr Leu Cys Pro Gln 1115 1120 1125 Ser Cys Glu Glu Arg Asn Leu
Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140 Trp Arg Tyr Asn Ser
Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155 His Pro Glu
Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170 His
Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180
1185 Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly
1190 1195 1200 Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro
Ser Asp 1205 1210 1215 Pro Glu His Cys Gln Ile Cys His Cys Asp Val
Val Asn Leu Thr 1220 1225 1230 Cys Glu Ala Cys Gln Glu Pro Gly Gly
Leu Val Val Pro Pro Thr 1235 1240 1245 Asp Ala Pro Val Ser Pro Thr
Thr Leu Tyr Val Glu Asp Ile Ser 1250 1255 1260 Glu Pro Pro Leu His
Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu 1265 1270 1275 Val Phe Leu
Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe 1280 1285 1290 Glu
Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg 1295 1300
1305 Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp
1310 1315 1320 Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg
Pro Ser 1325 1330 1335 Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr
Ala Gly Ser Gln 1340 1345 1350 Val Ala Ser Thr Ser Glu Val Leu Lys
Tyr Thr Leu Phe Gln Ile 1355 1360 1365 Phe Ser Lys Ile Asp Arg Pro
Glu Ala Ser Arg Ile Thr Leu Leu 1370 1375 1380 Leu Met Ala Ser Gln
Glu Pro Gln Arg Met Ser Arg Asn Phe Val 1385 1390 1395 Arg Tyr Val
Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro 1400 1405 1410 Val
Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile 1415 1420
1425 Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val
1430 1435 1440 Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr
Leu Cys 1445 1450 1455 Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu
Pro Pro Asp Met 1460 1465 1470 Ala Gln Val Thr Val Gly Pro Gly Leu
Leu Gly Val Ser Thr Leu 1475 1480 1485 Gly Pro Lys Arg Asn Ser Met
Val Leu Asp Val Ala Phe Val Leu 1490 1495 1500 Glu Gly Ser Asp Lys
Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys 1505 1510 1515 Glu Phe Met
Glu Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp 1520 1525 1530 Ser
Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr Val 1535 1540
1545 Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln
1550 1555 1560 Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr
Asn Thr 1565 1570 1575 Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser
Phe Leu Val Ser 1580 1585 1590 Gln Gly Asp Arg Glu Gln Ala Pro Asn
Leu Val Tyr Met Val Thr 1595 1600 1605 Gly Asn Pro Ala Ser Asp Glu
Ile Lys Arg Leu Pro Gly Asp Ile 1610 1615 1620 Gln Val Val Pro Ile
Gly Val Gly Pro Asn Ala Asn Val Gln Glu 1625 1630 1635 Leu Glu Arg
Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp 1640 1645 1650 Phe
Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg 1655 1660
1665 Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu Ser Pro Ala
1670 1675 1680 Pro Asp Cys Ser Gln Pro Leu Asp Val Ile Leu Leu Leu
Asp Gly 1685 1690 1695 Ser Ser Ser Phe Pro Ala Ser Tyr Phe Asp Glu
Met Lys Ser Phe 1700 1705 1710 Ala Lys Ala Phe Ile Ser Lys Ala Asn
Ile Gly Pro Arg Leu Thr 1715 1720 1725 Gln Val Ser Val Leu Gln Tyr
Gly Ser Ile Thr Thr Ile Asp Val 1730 1735 1740 Pro Trp Asn Val Val
Pro Glu Lys Ala His Leu Leu Ser Leu Val 1745 1750 1755 Asp Val Met
Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly Asp Ala 1760 1765 1770 Leu
Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His Gly Ala 1775 1780
1785 Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr Asp Val
1790 1795 1800 Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg
Ser Asn 1805 1810 1815 Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp
Arg Tyr Asp Ala 1820 1825 1830 Ala Gln Leu Arg Ile Leu Ala Gly Pro
Ala Gly Asp Ser Asn Val 1835 1840 1845 Val Lys Leu Gln Arg Ile Glu
Asp Leu Pro Thr Met Val Thr Leu 1850 1855 1860 Gly Asn Ser Phe Leu
His Lys Leu Cys Ser Gly Phe Val Arg Ile 1865 1870 1875 Cys Met Asp
Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp Val Trp 1880 1885 1890 Thr
Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro Asp Gly 1895 1900
1905 Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg Gly Leu
1910 1915 1920 Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val
Glu Glu 1925 1930 1935 Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val
Cys Thr Gly Ser 1940 1945 1950 Ser Thr Arg His Ile Val Thr Phe Asp
Gly Gln Asn Phe Lys Leu 1955 1960 1965 Thr Gly Ser Cys Ser Tyr Val
Leu Phe Gln Asn Lys Glu Gln Asp 1970 1975 1980 Leu Glu Val Ile Leu
His Asn Gly Ala Cys Ser Pro Gly Ala Arg 1985 1990 1995 Gln Gly Cys
Met Lys Ser Ile Glu Val Lys His Ser Ala Leu Ser 2000 2005 2010 Val
Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly Arg Leu 2015 2020
2025 Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn Val Tyr
2030 2035 2040 Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly
His Ile 2045 2050 2055 Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln
Leu Gln Leu Ser 2060 2065 2070 Pro Lys Thr Phe Ala Ser Lys Thr Tyr
Gly Leu Cys Gly Ile Cys 2075 2080 2085 Asp Glu Asn Gly Ala Asn Asp
Phe Met Leu Arg Asp Gly Thr Val 2090 2095 2100 Thr Thr Asp Trp Lys
Thr Leu Val Gln Glu Trp Thr Val Gln Arg 2105 2110 2115 Pro Gly Gln
Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys Leu Val 2120 2125 2130 Pro
Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu Phe Ala 2135 2140
2145 Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala Ile Cys
2150 2155 2160 Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val
Ile Ala 2165 2170 2175 Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val
Cys Val Asp Trp 2180 2185 2190 Arg Thr Pro Asp Phe Cys Ala Met Ser
Cys Pro Pro Ser Leu Val 2195 2200 2205 Tyr Asn His Cys Glu His Gly
Cys Pro Arg His Cys Asp Gly Asn 2210 2215 2220 Val Ser Ser Cys Gly
Asp His Pro Ser Glu Gly Cys Phe Cys Pro 2225 2230 2235 Pro Asp Lys
Val Met Leu Glu Gly Ser Cys Val Pro Glu Glu Ala 2240 2245 2250 Cys
Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln Phe Leu 2255 2260
2265 Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys Thr Cys
2270 2275 2280 Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys
Pro Thr 2285 2290 2295 Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val
Ala Arg Leu Arg 2300 2305 2310 Gln Asn Ala Asp Gln Cys Cys Pro Glu
Tyr Glu Cys Val Cys Asp 2315 2320 2325 Pro Val Ser Cys Asp Leu Pro
Pro Val Pro His Cys Glu Arg Gly 2330 2335 2340 Leu Gln Pro Thr Leu
Thr Asn Pro Gly Glu Cys Arg Pro Asn Phe 2345 2350 2355 Thr Cys Ala
Cys Arg Lys Glu Glu Cys Lys Arg Val Ser Pro Pro 2360 2365 2370 Ser
Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gln Cys 2375 2380
2385 Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser Thr Val
2390 2395 2400 Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn
Asp Cys 2405 2410 2415 Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys
Val Cys Val His 2420 2425 2430 Arg Ser Thr Ile Tyr Pro Val Gly Gln
Phe Trp Glu Glu Gly Cys 2435 2440 2445 Asp Val Cys Thr Cys Thr Asp
Met Glu Asp Ala Val Met Gly Leu 2450 2455 2460 Arg Val Ala Gln Cys
Ser Gln Lys Pro Cys Glu Asp Ser Cys Arg 2465 2470 2475 Ser Gly Phe
Thr Tyr Val Leu His Glu Gly Glu Cys Cys Gly Arg 2480 2485 2490 Cys
Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro Arg Gly 2495 2500
2505 Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp Ala Ser
2510 2515 2520 Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val
Lys Glu 2525 2530 2535 Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys
Pro Gln Leu Glu 2540 2545 2550 Val Pro Val Cys Pro Ser Gly Phe Gln
Leu Ser Cys Lys Thr Ser 2555 2560 2565 Ala Cys Cys Pro Ser Cys Arg
Cys Glu Arg Met Glu Ala Cys Met 2570 2575 2580 Leu Asn Gly Thr Val
Ile Gly Pro Gly Lys Thr Val Met Ile Asp 2585 2590 2595 Val Cys Thr
Thr Cys Arg Cys Met Val Gln Val Gly Val Ile Ser 2600 2605 2610 Gly
Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro Cys Pro 2615 2620
2625 Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys Gly Arg
2630 2635 2640 Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly
Gln Ile 2645 2650 2655 Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp
Gly Cys Asp Thr 2660 2665 2670 His Phe Cys Lys Val Asn Glu Arg Gly
Glu Tyr Phe Trp Glu Lys 2675 2680 2685 Arg Val Thr Gly Cys Pro Pro
Phe Asp Glu His Lys Cys Leu Ala 2690 2695 2700 Glu Gly Gly Lys Ile
Met Lys Ile Pro Gly Thr Cys Cys Asp Thr 2705 2710 2715 Cys Glu Glu
Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu Gln Tyr 2720 2725 2730 Val
Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp Ile His 2735 2740
2745 Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser Ile Asp
2750 2755 2760 Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro
Thr Arg 2765 2770 2775 Thr Glu Pro Met Gln Val Ala Leu His Cys Thr
Asn Gly Ser Val 2780 2785 2790 Val Tyr His Glu Val Leu Asn Ala Met
Glu Cys Lys Cys Ser Pro 2795 2800 2805 Arg Lys Cys Ser Lys 2810
2343DNAArtificial SequenceSynthetic Primer 23ctaagcgtaa gcttgccacc
atgattcctg ccagatttgc cgg 432440DNAArtificial SequenceSynthetic
Primer 24tggtcctcag ctagcgcggg acacctttcc agggccacac
402540DNAArtificial SequenceSynthetic Primer 25tggtcctcag
ctagcgcggc atcacacaca tggtctgtgc 402642DNAArtificial
SequenceSynthetic Primer 26tggtcctcag ctagcgctct ggtgtcagca
cactgcgagc tc 422740DNAArtificial SequenceSynthetic Primer
27tggtcctcag ctagcgctga gtccagaggc acttttctgg 402840DNAArtificial
SequenceSynthetic Primer 28tggtcctcag ctagcgcggt ggcaggggat
gagtccagag 402939DNAArtificial SequenceSynthetic Primer
29tggtcctcag ctagcgcggc atctgtggga ggcaccacc 393039DNAArtificial
SequenceSynthetic Primer 30tggtcctcag ctagcgcgtc ctccacatac
agagtggtg 393139DNAArtificial SequenceSynthetic Primer 31tggtcctcag
ctagcgcatc gtgcaacggc ggttccgag 393235DNAArtificial
SequenceSynthetic Primer 32gggacccttt gtgatgccac gtgctccacg atcgg
353336DNAArtificial SequenceSynthetic Primer 33gcacgtggca
tcacaaaggg tccctggcaa aatgag 363436DNAArtificial SequenceSynthetic
Primer 34ttgtgccccc aggaggacca agtagatccg cggctc
363534DNAArtificial SequenceSynthetic Primer
35tacttggtcc tcctgggggc acaatgtggc cgtc 343633DNAArtificial
SequenceSynthetic Primer 36gactgtccag tggaggacca agtagatccg cgg
333732DNAArtificial SequenceSynthetic Primer 37ttggtcctcc
actggacagt cttcagggtc aa 323812PRTArtificial SequenceHPC4 tag 38Glu
Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys 1 5 10
39100DNAArtificial SequenceSynthetic Primer 39ccgctagccc atgattcctg
ccagatttgc cggggtgctg cttgctctgg ccctcatttt 60gccagggacc ctttgtagcc
tatcctgtcg gccccccatg 1004085DNAArtificial SequenceSynthetic Primer
40gatgcggccg cctactacta tttgccatca atcagacgcg gatccacctg atcttcggct
60tcaggggcaa ggtcacagag gtagc 854140DNAArtificial SequenceSynthetic
Primer 41cattggggac tgcgcctcct tctgcgacac cattgctgcc
404240DNAArtificial SequenceSynthetic Primer 42ggcagcaatg
gtgtcgcaga aggaggcgca gtccccaatg 404344DNAArtificial
SequenceSynthetic Primer 43cgggagaacg ggtatgagtc tgagtggcgc
tataacagct gtgc 444444DNAArtificial SequenceSynthetic Primer
44gcacagctgt tatagcgcca ctcagactca tacccgttct cccg
444527DNAArtificial SequenceSynthetic Primer 45ggggactgcg
cctgcttctg cgacacc 274627DNAArtificial SequenceSynthetic Primer
46ggtgtcgcag aagcaggcgc agtcccc 274729DNAArtificial
SequenceSynthetic Primer 47gaacgggtat gagtgtgagt ggcgctata
294829DNAArtificial SequenceSynthetic Primer 48tatagcgcca
ctcacactca tacccgttc 294942DNAArtificial SequenceSynthetic Primer
49gcgctagctg aggaccaagt agatccgcgg ctcattgatg gg
425037DNAArtificial SequenceSynthetic Primer 50gggccagagc
aagcagcacc ccggcaaatc tggcagg 375137DNAArtificial SequenceSynthetic
Primer 51cctgccagat ttgccggggt gctgcttgct ctggccc
375247DNAArtificial SequenceSynthetic Primer 52tacttggtcc
tcagctagcg cctgggggca caatgtggcc gtcctcc 475347DNAArtificial
SequenceSynthetic Primer 53tacttggtcc tcagctagcg ccactggaca
gtcttcaggg tcaacgc 475438DNAArtificial SequenceSynthetic Primer
54ggctcagggt gctgacacgt gacttgacag gcaggtgc 385538DNAArtificial
SequenceSynthetic Primer 55gcacctgcct gtcaagtcac gtgtcagcac
cctgagcc 385646DNAArtificial SequenceSynthetic Primer 56tacttggtcc
tcagctagcg ctgcagggga gagggtgggg atctgc 46
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