U.S. patent application number 13/734129 was filed with the patent office on 2013-05-30 for single domain vhh antibodies against von willebrand factor.
This patent application is currently assigned to Ablynx N.V.. The applicant listed for this patent is Karen Silence. Invention is credited to Karen Silence.
Application Number | 20130136736 13/734129 |
Document ID | / |
Family ID | 37431613 |
Filed Date | 2013-05-30 |
United States Patent
Application |
20130136736 |
Kind Code |
A1 |
Silence; Karen |
May 30, 2013 |
Single Domain VHH Antibodies Against Von Willebrand Factor
Abstract
The present invention relates to improved Nanobodies.TM. against
von Willebrand Factor (vWF), as well as to polypeptides comprising
or essentially consisting of one or more of such Nanobodies. The
invention also relates to nucleic acids encoding such Nanobodies
and polypeptides; to methods for preparing such Nanobodies and
polypeptides; to host cells expressing or capable of expressing
such Nanobodies or polypeptides; to compositions comprising such
Nanobodies, polypeptides, nucleic acids or host cells; and to uses
of such Nanobodies, such polypeptides, such nucleic acids, such
host cells or such compositions, in particular for prophylactic,
therapeutic or diagnostic purposes, such as the prophylactic,
therapeutic or diagnostic purposes.
Inventors: |
Silence; Karen; (Overijse,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Silence; Karen |
Overijse |
|
BE |
|
|
Assignee: |
Ablynx N.V.
Zwijnaarde
BE
|
Family ID: |
37431613 |
Appl. No.: |
13/734129 |
Filed: |
January 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12822729 |
Jun 24, 2010 |
8372398 |
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13734129 |
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11920793 |
Nov 20, 2007 |
7807162 |
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PCT/EP2006/004773 |
May 19, 2006 |
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12822729 |
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60683474 |
May 20, 2005 |
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Current U.S.
Class: |
424/133.1 ;
424/158.1; 530/387.3; 530/389.3; 530/391.7 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/92 20130101; C07K 2317/22 20130101; A61P 43/00 20180101;
A61P 7/02 20180101; A61P 9/14 20180101; C07K 2317/76 20130101; Y02A
50/30 20180101; A61P 9/10 20180101; A61K 38/36 20130101; A61P 41/00
20180101; C07K 2317/569 20130101; C07K 16/36 20130101; C07K 2317/24
20130101; A61P 7/00 20180101 |
Class at
Publication: |
424/133.1 ;
530/389.3; 530/387.3; 530/391.7; 424/158.1 |
International
Class: |
C07K 16/36 20060101
C07K016/36 |
Claims
1. Nanobody against Von Willebrand Factor (vWF), said Nanobody
consisting of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: i) CDR1 comprises an amino acid sequence selected from the
group consisting of: TABLE-US-00100 [SEQ ID NO: 15] NYGMG [SEQ ID
NO: 16] SYTLG [SEQ ID NO: 17] NYNMG [SEQ ID NO: 18] SSAMA [SEQ ID
NO: 19] YYNTG [SEQ ID NO: 20] IGAMG [SEQ ID NO: 21] IGTMG [SEQ ID
NO: 22] YNPMG
and amino acid sequences that have 2 or only 1 "amino acid
difference(s)" with SEQ ID NOS: 15-22, and in which: ii) CDR2
comprises an amino acid sequence selected from the group consisting
of: TABLE-US-00101 [SEQ ID NO: 23] SISWSGTYTAYSDNVKG [SEQ ID NO:
24] GISWSGVSTDYAEFAKG [SEQ ID NO: 25] TSISWSGSYTAYADNVKG [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG [SEQ ID NO: 27] TITSGGRTSYADSVKG [SEQ ID
NO: 28] AISWSGGLTYYADSVKG [SEQ ID NO: 29] TITSGGSTNYADPVKG [SEQ ID
NO: 30] TITSGGSTNYADSVKG [SEQ ID NO: 31] AISRTGGSTYYARSVEG [SEQ ID
NO: 32] AISRTGGSTYYPDSVEG
and amino acid sequences that have at least 80%, sequence identity
with SEQ ID NOS: 23-32, and in which: iii) CDR3 comprises an amino
acid sequence selected from the group consisting of: TABLE-US-00102
[SEQ ID NO: 33] QSRYRSNYYDHDDKYAY [SEQ ID NO: 34] LGRYRSNWRNIGQYDY
[SEQ ID NO: 35] QSRYSSNYYDHDDKYAY [SEQ ID NO: 36] SNRYRTHTTQAMYNY
[SEQ ID NO: 37] VVDGKRAP [SEQ ID NO: 38] NRRQKTVQMGERAYDY [SEQ ID
NO: 39] NLKQGSYGYRFNDY [SEQ ID NO: 40] NLKQGDYGYRFNDY [SEQ ID NO:
41] AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 42] AGVRAEDGRVRTLPSEYTF [SEQ ID
NO: 43] AGVRAEDGRVRSLPSEYTF
and amino acid sequences that have at least 80%, sequence identity
with SEQ ID NOS: 33-43.
2. The Nanobody of claim 1, wherein a) CDR1 comprises the amino
acid sequence YNPMG [SEQ ID NO: 22]; or an amino acid sequences
that has 2 or only 1 amino acid difference(s) with the amino acid
sequence YNPMG [SEQ ID NO: 22]; and b) CDR2 comprises the amino
acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or an amino acid
sequence that has at least 80% sequence identity with the amino
acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; and c) CDR3
comprises the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO:
42]; or an amino acid sequence that has at least 80% sequence
identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID
NO: 42].
3. Nanobody according to claim 2, in which CDR1 comprises the amino
acid sequence YNPMG [SEQ ID NO: 22]; CDR2 comprises the amino acid
sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32] and CDR3 comprises the
amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].
4. Nanobody according to claim 1, wherein any amino acid
substitution is a conservative amino acid substitution.
5. Nanobody according to claim 1, wherein the Nanobody is a
KERE-class Nanobody.
6. Humanized variant of the Nanobody of claim 1.
7. Nanobody which has at least 80% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID NO's
60-73 and SEQ ID NO's 86-97.
8. The Nanobody of claim 7, wherein the Nanobody has at least 80%
sequence identity with the Nanobody 12A2H1 (SEQ ID NO: 90).
9. Humanized variant of the Nanobody of claim 7.
10. Nanobody 12A2H1 (SEQ ID NO: 90).
11. A polypeptide which comprises the Nanobody of claim 1.
12. A polypeptide which comprises the Nanobody of claim 7.
13. A polypeptide which comprises at least two Nanobodies of claim
1.
14. A polypeptide which comprises at least two Nanobodies of claim
7.
15. The polypeptide according to claim 13, wherein the at least two
Nanobodies are linked to each other via a linker.
16. The polypeptide according to claim 14, wherein the at least two
Nanobodies are linked to each other via a linker.
17. A polypeptide which has at least 80% sequence identity an amino
acid sequence selected from the group consisting of SEQ ID NOS:
74-82 and SEQ ID NOS 98-106.
18. The polypeptide of claim 17, wherein the polypeptide has at
least 80% sequence identity with SEQ ID NO: 98.
19. Pharmaceutical composition comprising the Nanobody of claim 1
and a pharmaceutically acceptable carrier.
20. Pharmaceutical composition comprising the Nanobody of claim 7
and a pharmaceutically acceptable carrier.
21. Pharmaceutical composition comprising the polypeptide of claim
11 and a pharmaceutically acceptable carrier.
22. Pharmaceutical composition comprising the polypeptide of claim
12 and a pharmaceutically acceptable carrier.
23. A method of treating a disease or disorder related to
platelet-mediated aggregation, the method comprising: administering
the pharmaceutical composition of claim 19 to treat the disease or
disorder related to platelet-mediated aggregation.
24. A method of treating a disease or disorder related to
platelet-mediated aggregation, the method comprising: administering
the pharmaceutical composition of claim 20 to treat the disease or
disorder related to platelet-mediated aggregation.
25. A method of treating a disease or disorder related to
platelet-mediated aggregation, the method comprising: administering
the pharmaceutical composition of claim 21 to treat the disease or
disorder related to platelet-mediated aggregation.
26. A method of treating a disease or disorder related to
platelet-mediated aggregation, the method comprising: administering
the pharmaceutical composition of claim 22 to treat the disease or
disorder related to platelet-mediated aggregation.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/822,729, filed Jun. 24, 2010, which is a continuation U.S.
application Ser. No. 11/920,793, filed Nov. 20, 2007, which is a
national stage filing under 35 U.S.C. .sctn.371 of international
application PCT/EP2006/004773, filed May 19, 2006, and claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. provisional
application Ser. No. 60/683,474, filed May 20, 2005.
[0002] The present invention relates to improved Nanobodies.TM.
against von Willebrand Factor (vWF), as well as to polypeptides
comprising or essentially consisting of one or more of such
Nanobodies. [Note: Nanobody.TM., Nanobodies.TM. and Nanoclone.TM.
are trademarks of Ablynx N.V.]
[0003] The invention also relates to nucleic acids encoding such
Nanobodies and polypeptides; to methods for preparing such
Nanobodies and polypeptides; to host cells expressing or capable of
expressing such Nanobodies or polypeptides; to compositions
comprising such Nanobodies, polypeptides, nucleic acids or host
cells; and to uses of such Nanobodies, such polypeptides, such
nucleic acids, such host cells or such compositions, in particular
for prophylactic, therapeutic or diagnostic purposes, such as the
prophylactic, therapeutic or diagnostic purposes mentioned
below.
[0004] Other aspects, embodiments, advantages and applications of
the invention will become clear from the further description
hereinbelow.
[0005] WO 04/062551 of Applicant relates to Nanobodies against Von
Willebrand Factor (vWF) and to the preparation and use thereof, in
particular for the prevention and/or treatment of diseases and
disorders relating to platelet-mediated aggregation.
[0006] The anti-vWF Nanobodies according to WO 04/062551 may be
humanized and may be monovalent or multivalent, the latter of which
leads to increased affinity for vWF. The anti-vWF Nanobodies
according to WO 04/062551 may also be multispecific, and may in
particular be in the form of a multispecific construct comprising
two or more Nanobodies against vWF and a further Nanobody directed
against a serum protein such as human serum albumin, which leads to
an increased half-life in vivo.
[0007] The anti-vWF Nanobodies described in WO 04/062551 may be
directed against any epitope or conformation of vWF (such as the A1
domain or A3 domain), but are preferably directed against the A1
domain, and in particular against the activated conformation of the
A1 domain.
[0008] WO 04/062551 also describes the preparation of the anti-vWF
Nanobodies, nucleotide sequences encoding the anti-vWF Nanobodies,
as well as pharmaceutical compositions comprising the anti-vWF
Nanobodies.
[0009] The anti-vWF Nanobodies and compositions described in WO
04/062551 may be used for the prevention and treatment of diseases
and disorders related to platelet-mediated aggregation, such as the
formation of a non-occlusive thrombus, the formation of an
occlusive thrombus, arterial thrombus formation, acute coronary
occlusion, peripheral arterial occlusive disease, restenosis and
disorders arising from coronary by-pass graft, coronary artery
valve replacement and coronary interventions such angioplasty,
stenting or atherectomy, hyperplasia after angioplasty, atherectomy
or arterial stenting, occlusive syndrome in a vascular system or
lack of patency of diseased arteries, thrombotic thrombocytopenic
purpura (TTP), transient cerebral ischemic attack, unstable or
stable angina pectoris, cerebral infarction, HELLP syndrome,
carotid endarterectomy, carotid artery stenosis, critical limb
ischaemia, cardioembolism, peripheral vascular disease, restenosis
and myocardial infarction.
[0010] The pharmaceutical compositions described in WO 04/062551
may be suitable for intravenous, subcutaneous, oral, sublingual,
topical, nasal, vaginal or rectal administration, or for
administration by inhalation; and may also comprise a trombolytic
agent, such as staphylokinase, tissue plasminogen activator,
streptokinase, single chain streptokinase, urokinase and acyl
plasminogen streptokinase complex. The anti-vWF Nanobodies
described in WO 04/062551 may also be used for diagnostic purposes
(optionally in the form of a kit-of-parts) or in coatings for
medical devices such as stents
[0011] It is a general object of the present invention to provide
Nanobodies against vWF, in particular against human vWF.
[0012] In particular, it is an object of the present invention to
provide Nanobodies against vWF, in particular against human vWF,
and to provide proteins or polypeptides comprising the same, that
are suitable for therapeutic and/or diagnostic use, and in
particular for the prevention, treatment and/or diagnosis of one or
more diseases and disorders associated with and/or mediated by vWF
such as those mentioned above, and/or that can be used in the
preparation of a pharmaceutical composition for the prevention
and/or treatment of one or more diseases associated with and/or
mediated by vWF, such as those mentioned above.
[0013] More in particular, it is an object of the invention to
provide Nanobodies against vWF, and to provide proteins and
polypeptides comprising the same, that are either an alternative to
the Nanobodies and polypeptides against vWF described in WO
04/062551 and/or that have one or more improved properties or
characteristics, compared to the Nanobodies and polypeptides
against vWF described in WO 04/062551.
[0014] More in particular, it is an object of the invention to
provide Nanobodies against vWF, and to provide proteins or
polypeptides comprising the same, that are improved compared to the
Nanobodies and polypeptides against vWF described in WO 04/062551
with respect to one or more of the following properties or
characteristics: [0015] increased affinity for vWF, either in a
monovalent format, in a multivalent format (for example in a
bivalent format) and/or in a multispecific format (for example one
of the multispecific formats described in WO 04/062551 or
hereinbelow); [0016] better suitability for formatting in a
multivalent format (for example in a bivalent format); [0017]
better suitability for formatting in a multispecific format (for
example one of the multispecific formats described in WO 04/062551
or hereinbelow); [0018] improved suitability or susceptibility for
"humanizing" substitutions (as defined herein); and/or [0019] less
immunogenicity, either in a monovalent format, in a multivalent
format (for example in a bivalent format) and/or in a multispecific
format (for example one of the multispecific formats described in
WO 04/062551 or hereinbelow) in a monovalent format; [0020]
increased stability, either in a monovalent format, in a
multivalent format (for example in a bivalent format) and/or in a
multispecific format (for example one of the multispecific formats
described in WO 04/062551 or hereinbelow) in a monovalent format;
[0021] increased specificity towards vWF, either in a monovalent
format, in a multivalent format (for example in a bivalent format)
and/or in a multispecific format (for example one of the
multispecific formats described in WO 04/062551 or hereinbelow) in
a monovalent format; [0022] decreased or where desired increased
cross-reactivity with vWF from different species; and/or [0023] one
or more other improved properties desirable for pharmaceutical use
(including prophylactic use and/or therapeutic use) and/or for
diagnostic use (including but not limited to use for imaging
purposes), either in a monovalent format, in a multivalent format
(for example in a bivalent format) and/or in a multispecific format
(for example one of the multispecific formats described in WO
04/062551 or hereinbelow).
[0024] These objects are achieved by the Nanobodies against vWF and
by the polypeptides described herein. The Nanobodies against vWF
and polypeptides described herein are in particular directed
against human vWF, but it is included within the scope of the
invention that some of the anti-vWF Nanobodies and polypeptides of
the invention may show cross-reactivity with vWF from other
vertebrate animals, in particular from other warm-blooded animals,
more in particular from other mammals, and in particular from other
species of primates, such as the baboons used in the Examples
below. However, as with anti-vWF Nanobodies described in WO
04/062551, the present invention in its broadest sense is not
particularly limited to or defined by a specific epitope, domain or
confirmation of vWF against which the Nanobodies and polypeptides
of the invention are directed. However, it is generally assumed and
preferred that the Nanobodies and polypeptides of the invention are
directed against the A1 domain of vWF, either in its activated or
non-activated confirmation.
[0025] Thus, in a first aspect, the invention relates to a Nanobody
(as defined herein), against vWF, which consist of 4 framework
regions (FR1 to FR4 respectively) and 3 complementarity determining
regions (CDR1 to CDR3 respectively), in which: [0026] i) CDR1
comprises or essentially consists of an amino acid sequence chosen
from the group consisting of:
TABLE-US-00001 [0026] [SEQ ID NO: 15] NYGMG [SEQ ID NO: 16] SYTLG
[SEQ ID NO: 17] NYNMG [SEQ ID NO: 18] SSAMA [SEQ ID NO: 19] YYNTG
[SEQ ID NO: 20] IGAMG [SEQ ID NO: 21] IGTMG [SEQ ID NO: 22]
YNPMG
[0027] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0028] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0029] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0030] and/or from
the group consisting of amino acid sequences that have 2 or only 1
"amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0031] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0032] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0033] ii) CDR2 comprises or essentially
consist of an amino acid sequence chosen from the group consisting
of:
TABLE-US-00002 [0033] [SEQ ID NO: 23] SISWSGTYTAYSDNVKG [SEQ ID NO:
24] GISWSGVSTDYAEFAKG [SEQ ID NO: 25] TSISWSGSYTAYADNVKG [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG [SEQ ID NO: 27] TITSGGRTSYADSVKG [SEQ ID
NO: 28] AISWSGGLTYYADSVKG [SEQ ID NO: 29] TITSGGSTNYADPVKG [SEQ ID
NO: 30] TITSGGSTNYADSVKG [SEQ ID NO: 31] AISRTGGSTYYARSVEG [SEQ ID
NO: 32] AISRTGGSTYYPDSVEG
[0034] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0035] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0036] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0037] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0038] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0039] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0040] iii) CDR3 comprises or
essentially consists of an amino acid sequence chosen from the
group consisting of:
TABLE-US-00003 [0040] [SEQ ID NO: 33] QSRYRSNYYDHDDKYAY [SEQ ID NO:
34] LGRYRSNWRNIGQYDY [SEQ ID NO: 35] QSRYSSNYYDHDDKYAY [SEQ ID NO:
36] SNRYRTHTTQAMYNY [SEQ ID NO: 37] VVDGKRAP [SEQ ID NO: 38]
NRRQKTVQMGERAYDY [SEQ ID NO: 39] NLKQGSYGYRFNDY [SEQ ID NO: 40]
NLKQGDYGYRFNDY [SEQ ID NO: 41] AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 42]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 43] AGVRAEDGRVRSLPSEYTF
[0041] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0042] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0043] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0044] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0045] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0046] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s).
[0047] In another aspect, the invention relates to a Nanobody (as
defined herein), against vWF, which consist of 4 framework regions
(FR1 to FR4 respectively) and 3 complementarity determining regions
(CDR1 to CDR3 respectively), in which: [0048] i) CDR1 is an amino
acid sequence chosen from the group consisting of:
TABLE-US-00004 [0048] [SEQ ID NO: 15] NYGMG [SEQ ID NO: 16] SYTLG
[SEQ ID NO: 17] NYNMG [SEQ ID NO: 18] SSAMA [SEQ ID NO: 19] YYNTG
[SEQ ID NO: 20] IGAMG [SEQ ID NO: 21] IGTMG [SEQ ID NO: 22]
YNPMG
[0049] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0050] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0051] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0052] and/or from
the group consisting of amino acid sequences that have 2 or only 1
"amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0053] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0054] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0055] ii) CDR2 is an amino acid
sequence chosen from the group consisting of:
TABLE-US-00005 [0055] [SEQ ID NO: 23] SISWSGTYTAYSDNVKG [SEQ ID NO:
24] GISWSGVSTDYAEFAKG [SEQ ID NO: 25] TSISWSGSYTAYADNVKG [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG [SEQ ID NO: 27] TITSGGRTSYADSVKG [SEQ ID
NO: 28] AISWSGGLTYYADSVKG [SEQ ID NO: 29] TITSGGSTNYADPVKG [SEQ ID
NO: 30] TITSGGSTNYADSVKG [SEQ ID NO: 31] AISRTGGSTYYARSVEG [SEQ ID
NO: 32] AISRTGGSTYYPDSVEG
[0056] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0057] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0058] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0059] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0060] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0061] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0062] iii) CDR3 is an amino acid
sequence chosen from the group consisting of:
TABLE-US-00006 [0062] [SEQ ID NO: 33] QSRYRSNYYDHDDKYAY [SEQ ID NO:
34] LGRYRSNWRNIGQYDY [SEQ ID NO: 35] QSRYSSNYYDHDDKYAY [SEQ ID NO:
36] SNRYRTHTTQAMYNY [SEQ ID NO: 37] VVDGKRAP [SEQ ID NO: 38]
NRRQKTVQMGERAYDY [SEQ ID NO: 39] NLKQGSYGYRFNDY [SEQ ID NO: 40]
NLKQGDYGYRFNDY [SEQ ID NO: 41] AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 42]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 43] AGVRAEDGRVRSLPSEYTF
[0063] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0064] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0065] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0066] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0067] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0068] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s).
[0069] The Nanobodies against vWF as described above and as further
described hereinbelow are also referred to herein as Nanobodies of
the invention.
[0070] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[0071] In another aspect, the invention relates to a Nanobody
against vWF, which consist of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), which is chosen from the group consisting of
Nanobodies with the one of the following combinations of CDR1, CDR2
and CDR3, respectively:
TABLE-US-00007 CDR1: [SEQ ID NO: 15] NYGMG; CDR2: [SEQ ID NO: 23]
SISWSGTYTAYSDNVKG; CDR3: [SEQ ID NO: 33] QSRYRSNYYDHDDKYAY; CDR1:
[SEQ ID NO: 16] SYTLG; CDR2: [SEQ ID NO: 24] GISWSGVSTDYAEFAKG;
CDR3: [SEQ ID NO: 34] LGRYRSNWRNIGQYDY; CDR1: [SEQ ID NO: 15]
NYGMG; CDR2: [SEQ ID NO: 25] TSISWSGSYTAYADNVKG; CDR3: [SEQ ID NO:
35] QSRYSSNYYDHDDKYAY CDR1: [SEQ ID NO: 17] NYNMG; CDR2: [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG; CDR3: [SEQ ID NO: 36] SNRYRTHTTQAMYNY
CDR1: [SEQ ID NO: 18] SSAMA; CDR2: [SEQ ID NO: 27]
TITSGGRTSYADSVKG; CDR3: [SEQ ID NO: 37] VVDGKRAP; CDR1: [SEQ ID NO:
19] YYNTG; CDR2: [SEQ ID NO: 28] AISWSGGLTYYADSVKG; CDR3: [SEQ ID
NO: 38] NRRQKTVQMGERAYDY CDR1: [SEQ ID NO: 20] IGAMG; CDR2: [SEQ ID
NO: 29] TITSGGSTNYADPVKG; CDR3: [SEQ ID NO: 39] NLKQGSYGYRFNDY
CDR1: [SEQ ID NO: 20] IGAMG; CDR2: [SEQ ID NO: 30]
TITSGGSTNYADSVKG; CDR3: [SEQ ID NO: 39] NLKQGSYGYRFNDY CDR1: [SEQ
ID NO: 20] IGAMG; CDR2: [SEQ ID NO: 30] TITSGGSTNYADSVKG; CDR3:
[SEQ ID NO: 40] NLKQGDYGYRFNDY CDR1: SEQ ID NO: 21 IGTMG; CDR2:
[SEQ ID NO: 30] TITSGGSTNYADSVKG; CDR3: [SEQ ID NO: 40]
NLKQGDYGYRFNDY CDR1: [SEQ ID NO: 22] YNPMG; CDR2: [SEQ ID NO: 31]
AISRTGGSTYYARSVEG; CDR3: [SEQ ID NO: 41] AGVRAEDGRVRTLPSEYNF CDR1:
[SEQ ID NO: 22] YNPMG; CDR2: [SEQ ID NO: 32] AISRTGGSTYYPDSVEG;
CDR3: [SEQ ID NO: 42] AGVRAEDGRVRTLPSEYTF CDR1: [SEQ ID NO: 22]
YNPMG; CDR2: [SEQ ID NO: 32] AISRTGGSTYYPDSVEG; CDR3: [SEQ ID NO:
43] AGVRAEDGRVRSLPSEYTF
[0072] In the Nanobodies of the invention that comprise the
combinations of CDR's mentioned above, each CDR can be replaced by
a CDR chosen from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the mentioned CDR's; in which [0073] (1) any
amino acid substitution is preferably a conservative amino acid
substitution (as defined herein); and/or [0074] (2) said amino acid
sequence preferably only contains amino acid substitutions, and no
amino acid deletions or insertions, compared to the above amino
acid sequence(s); and/or chosen from the group consisting of amino
acid sequences that have 3, 2 or only 1 (as indicated in the
preceding paragraph) "amino acid difference(s)" (as defined herein)
with the mentioned CDR(s) one of the above amino acid sequences, in
which: [0075] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0076] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s).
[0077] However, of the Nanobodies of the invention that comprise
the combinations of CDR's mentioned above, Nanobodies comprising
one or more of the CDR's listed above are particularly preferred;
Nanobodies comprising two or more of the CDR's listed above are
more particularly preferred; and Nanobodies comprising three of the
CDR's listed above are most particularly preferred.
TABLE-US-00008 TABLE I Preferred combinations of CDR's, of CDR's
and framework sequences, and of CDR's and humanized FR's CLONE ID
FR1 ID CDR1 FR2 ID CDR1 ID FR3 CDR3 ID FR4 12A5 122 AVQLVESGGG 148
IGAMG 174 MYRQAPGK 200 TITSGGSTNY 226 RFTISRDGPKNTVYLQ 252
NLKQGSYGY 278 WGQGTQ LVQPGGSLRL QRELVA ADPVKG MNSLKPEDTAVYYCYA
RFNDY VTVSS SCLASGRIFS 12B1 123 QVQLVESGGG 149 NYGMG 175 WFRQAPGK
201 SISWSGTYT 227 RFTISRDNAKNTVYLQ 253 QSRYRSNYYDH 279 WGQGTQ
LVQAGGSLRL EREFVT AYSDNVKG MDSLKPEDTAVYYCAA DDKYAY VTVSS SCAASGRTFS
12B6 124 QVQLVESGGG 150 YNPMG 176 WFRQAPGK 202 AISRTGGST 228
RFTISRDNAKRMVYLQ 254 AGVRAEDGRVR 280 WGQGTQ LVQAGGALRL ERDVVA
YYARSVEG MNALKPEDTAVYYCAA TLPSEYNF VTVSS SCAASGRTFS 12D11 125
AVQLVDSGGG 151 SSYTLG 177 WFRQAPGK 203 GISWSGVST 229 RFTISRDHAANT
255 LGRYRSNWRNI 281 WGQGTQ LVQAGGSLRL EREFVG DYAEFAKG
VYLEMNSLKPEDTAV GQYDY VTVSS SCTASERTTF YYCAA 12-E3 126 EVQLVESGGG
152 NYGMG 178 WFRQAPGK 204 SISWSGSYT 230 RFTISRDNAKNTVYLQ 256
QSRYSSNYYDH 282 WGQGTQ LVQAGGSLRL EREFVT AYADNVKG MDSLKPGDTAVYYCAA
DDKYAY VTVSS SCAASGRTFN 12C9 127 AVQLVESGGG 153 SSAMA 179 WYRQASGK
205 TITSGGRTSY 231 RFTISRDNAKNTVYLQ 257 VVDGKRAP 283 WGQGTQ
LVQPGGSLKL QRELVA ADSVKG MNSLKPEDTAVYDCNF VTVSS SCATSGSIFS 14F8 128
AVQLVESGGG 154 YYNTG 180 WFRQAPGK 206 AISWSGGLT 232
RFTISRDNAKDMVYLQ 258 NRRQKTVQMGE 284 WGQGTQ LVQAGESLRL EREFVA
YYADSVKG MASLKPEDTAVYYCAA RAYDY VTVSS SCTSSGRAFS 12B4 129
QVQLVESGGG 155 IGAMG 181 LYRQAPGK 207 TITSGGSTNY 233
RFTISRDGPKNTVYLQ 259 NLKQGSYGYR 285 WGQGTQ LVQPGGSLRL QRELVA ADSVKG
MNSLKPEDTAVYYCYA FN DY VTVSS SCLASGRIFS 12-E8 130 AVQLEESGGG 156
IGAMG 182 LYRQAPGK 208 TITSGGSTNY 234 RFTISRDGAKNTVYLQ 260
NLKQGDYGYR 286 WGQGTQ LVQPGGSLRL QRELVA ADSVKG MNSLKPEDTAVYYCYA FN
DY VTVSS SCLASGRIFS 12A6 131 QVQLVESGGG 157 IGTMG 183 LYRQAPGK 209
TITSGGSTNY 235 RFTISRDGAKNTVYLQ 261 NLKQGDYGYR 287 WGQGTQ
LVQPGGSLRL QRELVA ADSVKG MNSLRPEDTAVYYCYA FN DY VTVSS SCLASGRIFS
12D8 132 AVQLVESGGG 158 IGTMG 184 LYRQAPGK 210 TITSGGSTNY 236
RFTISRDGAKNTVYLQ 262 NLKQGDYGY 288 WGQGTQ LVQPGGSLRL QRELVA ADSVKG
MNSLRPEDTAVYYCYA RFNDY VTVSS SCLASGRIFS 12A2 133 QVKLEESGGG 159
YNPMG 185 WFRQAPGK 211 AISRTGGST 237 RFTISRDNAKRMVYLQ 263
AGVRAEDGRVR 289 WGQGTQ LVQAGGALRL ERDLVA YYPDSVEG MNNLKPEDTAVYYCAA
TLPSEYTF VTVSS SCAASGRTFS 12F2 134 QVKLVESGGG 160 YNPMG 186
WFRQAPGR 212 AISRTGGST 238 RFTISRDNAKRMVYLQ 264 AGVRAEDGRVR 290
WGQGTQ LVQAGGALRL ERDVVA YYPDSVEG MNNLKPEDTAVYYCAA SLPSEYTF VTVSS
SCAASGRTFS 14H10 135 QVKLEESGGG 161 YNPMG 187 WFRQAPGK 213
AISRTGGST 239 RFTISRDNAKRMVYLE 265 AGVRAEDGRVR 291 WGQGTQ
LVQAGGALRL ERDVVA YYPDSVEG MNNLKPDDTAVYYCAA TLPSEYTF VTVSS
SCAASGRTFS 12B6H1 136 EVQLVESGGG 162 YNPMG 188 WFRQAPGK 214
AISRTGGST 240 RFTISRDNAKRMVYLQ 266 AGVRAEDGRVR 292 WGQGTQ
LVQPGGSLRL GRDVVA YYARSVEG MNSLRAEDTAVYYCAA TLPSEYNF VTVSS
SCAASGRTFS 12B6H2 137 EVQLVESGGG 163 YNPMG 189 WFRQAPGK 215
AISRTGGST 241 RFTISRDNAKRMVYLQ 267 AGVRAEDGRVR 293 WGQGTQ
LVQPGGSLRL GREVVA YYARSVEG MNSLRAEDTAVYYCAA TLPSEYNF VTVSS
SCAASGRTFS 12B6H3 138 EVQLVESGGG 164 YNPMG 190 WFRQAPGK 216
AISRTGGST 242 RFTISRDNAKNMVYLQ 268 AGVRAEDGRVR 294 WGQGTQ
LVQPGGSLRL GRDVVA YYARSVEG MNSLRAEDTAVYYCAA TLPSEYNF VTVSS
SCAASGRTFS 12B6H4 139 EVQLVESGGG 165 YNPMG 191 WFRQAPGK 217
AISRTGGST 243 RFTISRDNAKRSVYLQ 269 AGVRAEDGRVR 295 WGQGTQ
LVQPGGSLRL GRDVVA YYARSVEG MNSLRAEDTAVYYCAA TLPSEYNF VTVSS
SCAASGRTFS 12A2H1 140 EVQLVESGGG 166 YNPMG 192 WFRQAPGK 218
AISRTGGST 244 RFTISRDNAKRMVYLQ 270 AGVRAEDGRVR 296 WGQGTQ
LVQPGGSLRL GRELVA YYPDSVEG MNSLRAEDTAVYYCAA TLPSEYTF VTVSS
SCAASGRTFS 12A2H3 141 EVQLVESGGG 167 YNPMG 193 WFRQAPGK 219
AISRTGGST 245 RFTISRDNAKNMVYLQ 271 AGVRAEDGRVR 297 WGQGTQ
LVQPGGSLRL GRELVA YYPDSVEG MNSLRAEDTAVYYCAA TLPSEYTF VTVSS
SCAASGRTFS 12A2H4 142 EVQLVESGGG 168 YNPMG 194 WFRQAPGK 220
AISRTGGST 246 RFTISRDNAKRSVYLQ 272 AGVRAEDGRVR 298 WGQGTQ
LVQPGGSLRL GRELVA YYPDSVEG MNSLRAEDTAVYYCAA TLPSEYTF VTVSS
SCAASGRTFS 12A2H11 143 EVQLVESGGG 169 YNPMG 195 WFRQAPGK 221
AISRTGGST 247 RFTISRDNAKRMVYLQ 273 AGVRAEDGRVR 299 WGQGTQ
LVQPGGSLRL GRELVA YYPDSVEG MNSLRAEDTAVYYCAA TLPSEYTF VTVSS
SCAASGFTFS 12A2H13 144 EVQLVESGGG 170 YNPMG 196 WFRQAPGK 222
AISRTGGST 248 RFTISRDNAKNSVYLQ 274 AGVRAEDGRVR 300 WGQGTL
LVQPGGSLRL GRELVA YYPDSVEG MNSLRAEDTAVYYCAA TLPSEYTF VTVSS
SCAASGFTFS 12A5H1 145 EVQLVESGGG 171 IGAMG 197 MYRQAPGK 223
TITSGGSTNY 249 RFTISRDGPKNTVYLQ 275 NLKQGSYGYR 301 WGQGTQ
LVQPGGSLRL GRELVA ADPVKG MNSLRAEDTAVYYCYA FNDY VTVSS SCAASGRIFS
12A5H2 146 EVQLVESGGG 172 IGAMG 198 MYRQAPGK 224 TITSGGSTNY 250
RFTISRDGAKNTVYLQ 276 NLKQGSYGYR 302 WGQGTQ LVQPGGSLRL GRELVA ADPVKG
MNSLRAEDTAVYYCYA FNDY VTVSS SCAASGRIFS 12A5H3 147 EVQLVESGGG 173
IGAMG 199 MYRQAPGK 225 TITSGGSTNY 251 RFTISRDNAKNTVYLQ 277
NLKQGSYGYR 303 WGQGTQ LVQPGGSLRL GRELVA ADPVKG MNSLRAEDTAVYYCYA
FNDY VTVSS SCAASGRIFS
[0078] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I; and/or from the group consisting
of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2
or only 1 "amino acid difference(s)" (as defined herein) with at
least one of the CDR1, CDR2 and CDR3 sequences, respectively,
listed in Table I. In this context, by "suitably chosen" is meant
that, as applicable, a CDR1 sequence is chosen from suitable CDR1
sequences (i.e. as defined herein), a CDR2 sequence is chosen from
suitable CDR2 sequences (i.e. as defined herein), and a CDR3
sequence is chosen from suitable CDR3 sequence (i.e. as defined
herein), respectively.
[0079] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table I or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table I; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table I.
[0080] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table I;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table I.
[0081] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table I or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table I, respectively; and at least one of the CDR1 and
CDR2 sequences present is suitably chosen from the group consisting
of the CDR1 and CDR2 sequences, respectively, listed in Table I or
from the group of CDR1 and CDR2 sequences, respectively, that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the CDR1 and CDR2 sequences, respectively, listed in
Table I; and/or from the group consisting of the CDR1 and CDR2
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table I.
[0082] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I or from the group of CDR1, CDR2 and
CDR3 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the CDR1, CDR2 and
CDR3 sequences, respectively, listed in Table I; and/or from the
group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, that have 3, 2 or only 1 amino acid difference(s)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I.
[0083] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table I. Preferably, in this
embodiment, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences, respectively,
listed in Table I; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table I.
[0084] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table I. Preferably, in this
embodiment, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with the CDR1 and CDR2
sequences, respectively, listed in listed in Table I; and/or from
the group consisting of the CDR1 and CDR2 sequences, respectively,
that have 3, 2 or only 1 amino acid difference(s) with at least one
of the CDR1 and CDR2 sequences, respectively, listed in Table
I.
[0085] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table I. Preferably, in this
embodiment, the remaining CDR sequence present are suitably chosen
from the group of CDR sequences that that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
corresponding CDR sequences listed in Table I; and/or from the
group consisting of CDR sequences that have 3, 2 or only 1 amino
acid difference(s) with at least one of the corresponding sequences
listed in Table I.
[0086] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table I, and either the CDR1 sequence
or the CDR2 sequence is suitably chosen from the group consisting
of the CDR1 and CDR2 sequences, respectively, listed in Table I.
Preferably, in this embodiment, the remaining CDR sequence present
are suitably chosen from the group of CDR sequences that that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences listed in Table I;
and/or from the group consisting of CDR sequences that have 3, 2 or
only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table I.
[0087] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I.
[0088] Also, generally, the combinations of CDR's listed in Table I
(i.e. those mentioned on the same line in Table I) are preferred.
Thus, it is generally preferred that, when a CDR in a Nanobody of
the invention is a CDR sequence mentioned in Table I or is suitably
chosen from the group of CDR sequences that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with a CDR sequence
listed in Table I; and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with a
CDR sequence listed in Table I, that at least one and preferably
both of the other CDR's are suitably chosen from the CDR sequences
that belong to the same combination in Table I (i.e. mentioned on
the same line in Table I) or are suitably chosen from the group of
CDR sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with the CDR sequence(s) belonging to the same combination
and/or from the group consisting of CDR sequences that have 3, 2 or
only 1 amino acid difference(s) with the CDR sequence(s) belonging
to the same combination. The other preferences indicated in the
above paragraphs also apply to the combinations of CDR's mentioned
in Table I.
[0089] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table I, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table I (but
belonging to a different combination), and a CDR3 sequence.
[0090] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table I; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table I (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
I (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table I; a CDR2 sequence, and one of
the CDR3 sequences listed in Table I; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table I; and a CDR3 sequence that has
3, 2 or 1 amino acid differences with the CDR3 sequence mentioned
in Table I that belongs to the same combination as the CDR2
sequence.
[0091] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
I; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table I that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table I that belongs
to the same combination; (2) a CDR1 sequence; a CDR 2 listed in
Table I and a CDR3 sequence listed in Table I (in which the CDR2
sequence and CDR3 sequence may belong to different
combinations).
[0092] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
I; the CDR2 sequence listed in Table I that belongs to the same
combination; and a CDR3 sequence mentioned in Table I that belongs
to a different combination; or (2) a CDR1 sequence mentioned in
Table I; a CDR2 sequence that has 3, 2 or 1 amino acid differences
with the CDR2 sequence mentioned in Table I that belongs to the
same combination; and more than 80% sequence identity with the CDR3
sequence listed in Table I that belongs to same different
combination.
[0093] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table I, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table I that belongs to the same combination;
and the CDR3 sequence mentioned in Table I that belongs to the
same.
[0094] In the most preferred in the Nanobodies of the invention,
the CDR1, CDR2 and CDR3 sequences present are suitably chosen from
the one of the combinations of CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table I.
[0095] Preferably, when a CDR sequence is suitably chosen from the
group of CDR sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with one of the CDR
sequences listed in Table I; and/or when a CDR sequence is suitably
chosen from the group consisting of CDR sequences that have 3, 2 or
only 1 amino acid difference(s) with one of the CDR sequences
listed in Table I: [0096] i) any amino acid substitution is
preferably a conservative amino acid substitution (as defined
herein); and/or [0097] ii) said amino acid sequence preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the CDR sequence listed in Table I.
[0098] According to a non-limiting but preferred embodiment of the
invention, the CDR sequences in the Nanobodies of the invention are
as defined above and are also such that the Nanobody of the
invention binds to vWF with an dissociation constant (K.sub.D) of
10.sup.-5 to 10.sup.-12 moles/liter (M) or less, and preferably
10.sup.-7 to 10.sup.-12 moles/liter (M) or less and more preferably
10.sup.-8 to 10.sup.-12 moles/liter (M), and/or with an association
constant (K.sub.A) of at least 10.sup.7 M.sup.-1, preferably at
least 10.sup.8 M.sup.-1, more preferably at least 10.sup.9
M.sup.-1, such as at least 10.sup.12 M.sup.-1; and in particular
with a K.sub.D less than 500 nM, preferably less than 200 nM, more
preferably less than 10 nM, such as less than 500 pM. The K.sub.D
and K.sub.A values of the Nanobody of the invention against vWF can
be determined in a manner known per se, for example using the assay
described herein. More generally, the Nanobodies described herein
preferably have a dissociation constant with respect to vWF that is
as described in this paragraph.
[0099] In another aspect, the invention relates to a Nanobody with
an amino acid sequence that is chosen from the group consisting of
SEQ ID NO's: 60 to 73 and SEQ ID NO's: 86 to 97 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more "sequence identity" (as defined herein) with one or
more of the amino acid sequences of SEQ ID NO's: 60 to 73 and SEQ
ID NO's: 86 to 97, which amino acid sequences most preferably have
framework sequences that are as further defined below under the
general description of the framework sequences of Nanobodies.
[0100] According to a specific, but non-limiting embodiment, the
latter amino acid sequences have been "humanized", as further
described below.
[0101] Most preferably, the Nanobodies of the invention are chosen
from the group consisting of SEQ ID NO's: 60 to 73 and SEQ ID NO's:
86 to 97, of which the "humanized" Nanobodies of SEQ ID NO's: 86 to
97 may be particularly preferred.
[0102] Nanobodies that are particular preferred according to the
invention is Nanobody 12B6 (SEQ ID NO: 62) and homologues and
variants thereof, and in particular humanized variants thereof.
Some particularly preferred, but non-limiting homologues and
(humanized) variants are for example Nanobodies 12A2 (SEQ ID NO:
71); 12F2 (SEQ ID NO: 72); 14H10 (SEQ ID NO: 73) and humanized
variants thereof, such as 12B6H1 (SEQ ID NO: 86); 12B6H2 (SEQ ID
NO: 87); 12B6H3 (SEQ ID NO: 88); 12B6H4 (SEQ ID NO: 89); 12A2H1
(SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92);
12A2H11 (SEQ ID NO: 93) and 12A2H13 (SEQ ID NO: 94).
[0103] Particularly preferred in the invention is Nanobody 12A2
(SEQ ID NO: 71) and homologues and variants thereof, and in
particular humanized variants thereof. Some particularly preferred,
but non-limiting homologues and (humanized) variants are for
example Nanobodies 12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91);
12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and 12A2H13 (SEQ ID
NO: 94), of which Nanobody 12A2H1 (SEQ ID NO: 90) is in particular
preferred.
[0104] Thus, one preferred but non-limiting aspect of the invention
relates to aNanobody against Von Willebrand Factor (vWF), said
Nanobody consisting of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which:
[0105] a) CDR1 comprises or essentially consists of: [0106] the
amino acid sequence YNPMG [SEQ ID NO: 22]; or [0107] an amino acid
sequences that has 2 or only 1 amino acid difference(s) with the
amino acid sequence YNPMG [SEQ ID NO: 22];
[0108] and
[0109] b) CDR2 comprises or essentially consists of: [0110] the
amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or [0111] an
amino acid sequence that has at least 80%, preferably at least 90%,
more preferably at least 95%, even more preferably at least 99%
sequence identity with the amino acid sequence AISRTGGSTYYPDSVEG
[SEQ ID NO: 32]; or [0112] an amino acid sequences that has 2 or
only 1 amino acid difference(s) with the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32];
[0113] and
[0114] c) CDR3 comprises or essentially consists of: [0115] the
amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or [0116]
an amino acid sequence that has at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with the amino acid sequence
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or [0117] an amino acid
sequences that has only 1 amino acid difference with the amino acid
sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].
[0118] In particular, the invention relates to such a Nanobody, in
which: [0119] CDR1 comprises or essentially consists of the amino
acid sequence YNPMG [SEQ ID NO: 22]; or in which: [0120] CDR2
comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or in which [0121] CDR3
comprises or essentially consists of the amino acid sequence
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].
[0122] For example, the invention relates to such Nanobodies, in
which: [0123] CDR1 comprises or essentially consists of the amino
acid sequence YNPMG [SEQ ID NO: 22]; and CDR3 comprises or
essentially consists of the amino acid sequence AGVRAEDGRVRTLPSEYTF
[SEQ ID NO: 42]; or in which: [0124] CDR1 comprises or essentially
consists of the amino acid sequence YNPMG [SEQ ID NO: 22]; and CDR2
comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or in which: [0125] CDR2
comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; and CDR3 comprises or
essentially consists of the amino acid sequence AGVRAEDGRVRTLPSEYTF
[SEQ ID NO: 42]
[0126] In one aspect, the invention relates to such a Nanobody, in
which CDR1 comprises or essentially consists of the amino acid
sequence YNPMG [SEQ ID NO: 22]; and CDR3 comprises or essentially
consists of the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO:
42].
[0127] The invention also relates to humanized variants of such a
Nanobody. Some preferred, but non-limiting humanizing substitutions
will be described herein, or will be clear to the skilled person by
comparing the corresponding non-humanized and humanized Nanobodies
disclosed herein. Some particularly useful humanizing substitutions
are one or more of those present in the humanized variants of 12A2
(as will be clear to the skilled person from a comparison of the
sequences of 12A2H1 (SEQ ID NO: 90) with the corresponding
humanized sequences of 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO:
92); 12A2H11 (SEQ ID NO: 93) and 12A2H13 (SEQ ID NO: 94).
[0128] Anotherone preferred but non-limiting aspect of the
invention relates to a Nanobody against Von Willebrand Factor
(vWF), said Nanobody consisting of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which:
[0129] d) CDR1 is: [0130] the amino acid sequence YNPMG [SEQ ID NO:
22]; or [0131] an amino acid sequences that has 2 or only 1 amino
acid difference(s) with the amino acid sequence YNPMG [SEQ ID NO:
22];
[0132] and
[0133] e) CDR2 is: [0134] the amino acid sequence AISRTGGSTYYPDSVEG
[SEQ ID NO: 32]; or [0135] an amino acid sequence that has at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with the amino acid
sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or [0136] an amino acid
sequences that has 2 or only 1 amino acid difference(s) with the
amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32];
[0137] and
[0138] f) CDR3 is: [0139] the amino acid sequence
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or [0140] an amino acid
sequence that has at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID
NO: 42]; or [0141] an amino acid sequences that has only 1 amino
acid difference with the amino acid sequence AGVRAEDGRVRTLPSEYTF
[SEQ ID NO: 42].
[0142] In particular, the invention relates to such a Nanobody, in
which: [0143] CDR1 is the amino acid sequence YNPMG [SEQ ID NO:
22]; or in which: [0144] CDR2 is the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or in which [0145] CDR3 is the
amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].
[0146] For example, the invention relates to such Nanobodies, in
which: [0147] CDR1 is the amino acid sequence YNPMG [SEQ ID NO:
22]; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ
ID NO: 42]; or in which: [0148] CDR1 is the amino acid sequence
YNPMG [SEQ ID NO: 22]; and CDR2 is the amino acid sequence
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or in which: [0149] CDR2 is the
amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; and CDR3 is
the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
[0150] In one aspect, the invention relates to such a Nanobody, in
which CDR1 is the amino acid sequence YNPMG [SEQ ID NO: 22]; and
CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO:
42].
[0151] The invention also relates to humanized variants of such a
Nanobody. Some preferred, but non-limiting humanizing substitutions
will be described herein, or will be clear to the skilled person by
comparing the corresponding non-humanized and humanized Nanobodies
disclosed herein. Some particularly useful humanizing substitutions
are one or more of those present in the humanized variants of 12A2
(as will be clear to the skilled person from a comparison of the
sequences of 12A2H1 (SEQ ID NO: 90) with the corresponding
humanized sequences of 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO:
92); 12A2H11 (SEQ ID NO: 93) and 12A2H13 (SEQ ID NO: 94).
[0152] The Nanobodies described herein may be GLEW-class
Nanobodies, "103 P, R or S"-class Nanobodies or "KERE-class
Nanobodies" (all as described herein). In particular, the
Nanobodies described herein may be KERE-class Nanobodies, although
the invention is not limited thereto.
[0153] In another aspect, the invention relates to a Nanobody which
has at least 80%, or at least 90%, or at least 95%, or at least 99%
sequence identity (as defined herein) with at least one of the
Nanobodies from the group consisting of SEQ ID NO's 60-73 and SEQ
ID NO's 86-97.
[0154] In particular, the invention relates to a Nanobody which has
at least 80%, or at least 90%, or at least 95%, or at least 99%
sequence identity (as defined herein) with at least one of the
Nanobodies 12B6 (SEQ ID NO: 62); 12A2 (SEQ ID NO: 71); 12F2 (SEQ ID
NO: 72); 14H10 (SEQ ID NO: 73); 12B6H1 (SEQ ID NO: 86); 12B6H2 (SEQ
ID NO: 87); 12B6H3 (SEQ ID NO: 88); 12B6H4 (SEQ ID NO: 89); 12A2H1
(SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92);
12A2H11 (SEQ ID NO: 93) and/or 12A2H13 (SEQ ID NO: 94).
[0155] More in particular, the invention relates to a Nanobody
which has at least 80%, or at least 90%, or at least 95%, or at
least 99% sequence identity (as defined herein) with at least one
of the Nanobodies 12A2 (SEQ ID NO: 71); 12A2H1 (SEQ ID NO: 90);
12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO:
93) and/or 12A2H13 (SEQ ID NO: 94).
[0156] Even more in particular, the invention relates to a Nanobody
which has at least 80%, or at least 90%, or at least 95%, or at
least 99% sequence identity (as defined herein) with the Nanobody
12A2H1 (SEQ ID NO: 90).
[0157] The invention also relates to humanized variants of such
Nanobodies. Some preferred, but non-limiting humanizing
substitutions will be described herein, or will be clear to the
skilled person by comparing the corresponding non-humanized and
humanized Nanobodies disclosed herein. Some particularly useful
humanizing substitutions are one or more of those present in the
humanized variants of 12A2 (as will be clear to the skilled person
from a comparison of the sequences of 12A2H1 (SEQ ID NO: 90) with
the corresponding humanized sequences of 12A2H3 (SEQ ID NO: 91);
12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and 12A2H13 (SEQ ID
NO: 94).
[0158] The invention also relates to a Nanobody that is chosen from
the group consisting of the Nanobodies of SEQ ID NO's 60-73 and SEQ
ID NO's 86-97.
[0159] In particular, the invention relates to a Nanobody that is
chosen from the group consisting of the Nanobodies 12B6 (SEQ ID NO:
62); 12A2 (SEQ ID NO: 71); 12F2 (SEQ ID NO: 72); 14H10 (SEQ ID NO:
73); 12B6H1 (SEQ ID NO: 86); 12B6H2 (SEQ ID NO: 87); 12B6H3 (SEQ ID
NO: 88); 12B6H4 (SEQ ID NO: 89); 12A2H1 (SEQ ID NO: 90); 12A2H3
(SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93)
and/or 12A2H13 (SEQ ID NO: 94).
[0160] More in particular, the invention relates to a Nanobody that
is chosen from the group consisting of the Nanobodies 12A2 (SEQ ID
NO: 71); 12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4
(SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and/or 12A2H13 (SEQ ID NO:
94). A particularly useful Nanobody is Nanobody 12A2H1 (SEQ ID
NO:90).
[0161] The Nanobodies described herein preferably have framework
sequences that are as further described herein. Some particularly
preferred framework sequences (FR1, FR2, FR3 and FR4, respectively)
are those of Nanobody 12A2 and its humanized variants; and
framework sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with one of said
framework sequences; and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of said framework sequences (in which any
amino acid substitution is preferably a conservative amino acid
substitution; and/or in which said amino acid sequence preferably
contains amino acid substitutions and no more than 3 amino acid
deletions or no more than 3 amino acid insertions). Nanobodies
against vWF with such framework sequences form a further aspect of
the invention.
[0162] In particular, the invention relates to a Nanobody against
vWF, in which FR1 is SEQ ID NO: 140; FR2 is SEQ ID NO: 192; FR3 is
SEQ ID 244; and FR4 is SEQ ID NO: 296; or framework sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of said framework sequences; and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of said
framework sequences (in which any amino acid substitution is
preferably a conservative amino acid substitution; and/or in which
said amino acid sequence preferably contains amino acid
substitutions and no more than 3 amino acid deletions or no more
than 3 amino acid insertions).
[0163] More in particular, the invention relates to a Nanobody
against vWF, in which FR1 is SEQ ID NO: 140; FR2 is SEQ ID NO: 192;
FR3 is SEQ ID 244; and FR4 is SEQ ID NO: 296.
[0164] In another aspect, the invention relates to a polypeptide
that comprises or essentially consists of at least one Nanobody
against vWF as defined herein. Such polypeptides are also referred
to herein as "polypeptides of the invention" and may be as further
described hereinbelow and/or as generally described in WO 02/062551
for the Nanobodies disclosed therein, and may for example be
multivalent polypeptides or multispecific polypeptides, again as
further described hereinbelow.
[0165] Preferably, a polypeptide of the invention is either
bivalent or trivalent (i.e. comprising two or three Nanobodies of
the invention, respectively, optionally linked via one or two
linkers as defined herein, respectively) or a multispecific
polypeptide, comprising one or two, and preferably two, Nanobodies
of the invention and at least one Nanobody directed against a serum
protein, and in particular against a human serum protein, such as
against human serum albumin.
[0166] In one preferred, but non-limiting embodiments, the
Nanobodies of the invention present in the polypeptides of the
invention are chosen from the group consisting of SEQ ID NO's: 60
to 73 and SEQ ID NO's: 86 to 97, and in particular from the
"humanized" Nanobodies of SEQ ID NO's 86 to 97. The Nanobodies
against human serum albumin present in the polypeptides of the
invention are preferably as defined herein, and are more preferably
chosen from the group consisting of SEQ ID NO's: 107 to 121, and in
particular from the "humanized" Nanobodies against human serum
albumin of SEQ ID NO's 114-121.
[0167] Some preferred, but non-limiting examples of polypeptides of
the invention are the polypeptides of SEQ ID NO's: 74 to 82 and the
polypeptides of SEQ ID NO's 98-106. Other polypeptides of the
invention may for example be chosen from the group consisting of
amino acid sequences that have more than 80%, preferably more than
90%, more preferably more than 95%, such as 99% or more "sequence
identity" (as defined herein) with one or more of the amino acid
sequences of SEQ ID NO's: 74 to 82 and/or SEQ ID NO's 98 to 106, in
which the Nanobodies comprised within said amino acid sequences are
preferably as defined herein.
[0168] According to one aspect of the invention, the Nanobodies,
proteins and polypeptides described herein have essentially no
influence on the cleavage of ULvWF by ADAMTS-13. In particular,
when the Nanobodies, proteins and polypeptides described herein are
used at the doses described herein, the cleavage of ULvWF by
ADAMTS-13 (either in vivo upon administration and/or as measured
using a suitable assay, such as the assay described herein),
essentially does not reduce or inhibit the cleavage of ULvWF by
ADAMTS-13, i.e. by not more than 50%, preferably not more than 20%,
even more preferably not more than 10%, such as less than 5% or
essentially not at all). Thus, one further aspect of the invention
relates to a Nanobody, protein or polypeptide, and in particular a
Nanobody, protein or polypeptide as described herein, that
essentially does not reduce or inhibit the cleavage of ULvWF by
ADAMTS-13.
[0169] In another aspect, the invention relates to a nucleic acid
that encodes a Nanobody of the invention and/or a polypeptide of
the invention. Such a nucleic acid will also be referred to below
as a "nucleic acid of the invention" and may for example be in the
form of a genetic construct, as defined herein.
[0170] In another aspect, the invention relates to host or host
cell that expresses or is capable of expressing a Nanobody of the
invention and/or a polypeptide of the invention; and/or that
contains a nucleic acid encoding a Nanobody of the invention and/or
a polypeptide of the invention. Such a host or a host cell may also
be analogous to the hosts and host cells described in WO 02/062551,
but expressing or capable of expressing a Nanobody of the invention
and/or a polypeptide of the invention and/or containing a nucleic
acid as described herein.
[0171] The invention further relates to a product or composition
containing or comprising a Nanobody of the invention, a polypeptide
of the invention; and/or a nucleic acid of the invention. Such a
product or composition may for example be a pharmaceutical
composition (as described below) or a product or composition for
diagnostic use (as also described below). Such a product or
composition may also be analogous to the products and compositions
described in WO 02/062551, but containing or comprising a Nanobody
of the invention, a polypeptide of the invention or a nucleic acid
of the invention.
[0172] The invention further relates to methods for preparing or
generating the Nanobodies, polypeptides, nucleic acids, host cells,
products and compositions as described herein, which methods are as
further described below. Also, generally, the Nanobodies,
polypeptides, nucleic acids, host cells, products and compositions
described herein may also be prepared and used in a manner
analogous to the manner described in WO 02/062551.
[0173] The invention further relates to applications and uses of
the above Nanobodies, polypeptides, nucleic acids, host cells,
products and compositions described herein, which applications and
uses include, but are not limited to, the applications and uses
described hereinbelow and/or the further uses and applications for
Nanobodies against vWF and/or for polypeptides containing the same
in WO 02/062551.
[0174] Other aspects, embodiments, advantages and applications of
the invention will become clear from the further description
hereinbelow.
DESCRIPTION OF THE DRAWINGS
[0175] FIG. 1: Binding of nanobodies to vWF in ELISA
[0176] FIG. 2: Alignment of 12A5 homologue nanobody sequences
(12A5, SEQ ID NO:60; 12B4, SEQ ID NO:67; 12E8, SEQ ID NO:68; 12A6,
SEQ ID NO:69; 12D8, SEQ ID NO:70)
[0177] FIG. 3: Alignment of 12B6 homologue nanobody sequences
(12B6, SEQ ID NO:62; 12A2, SEQ ID NO:71; 12F2, SEQ ID NO:72; 14H10,
SEQ ID NO:73)
[0178] FIG. 4: Binding of 12A5 homologue nanobodies to vWF in
BIACORE
[0179] FIG. 5: Binding of 12B6 homologue nanobodies to vWF in
BIACORE
[0180] FIG. 6: Platelet adhesion at different concentrations of
12B6, 12A2 and 12A5 nanobodies
[0181] FIG. 7a: Binding in ELISA to vWF for 12B6 nanobody after
heating at increasing temperatures
[0182] FIG. 7b: Binding in ELISA to vWF for 12A2 nanobody after
heating at increasing temperatures
[0183] FIG. 7c: Binding in ELISA to vWF for 12A5 nanobody after
heating at increasing temperatures
[0184] FIG. 8a: Binding of vWF from different species to 12B6
nanobody in ELISA
[0185] FIG. 8b: Binding of vWF from different species to 12A2
nanobody in ELISA
[0186] FIG. 8c: Binding of vWF from different species to 12A5
nanobody in ELISA
[0187] FIG. 9: Binding of bivalent 12B6 nanobodies to vWF in
BIACORE
[0188] FIG. 10: Binding of bivalent 12A2 nanobodies to vWF in
BIACORE
[0189] FIG. 11: Binding of bivalent 12A5 nanobodies to vWF in
BIACORE
[0190] FIG. 12: Binding in ELISA to vWF of bivalent 12B6 nanobodies
after heating at increasing temperatures
[0191] FIG. 13: Binding in ELISA to vWF of bivalent 12A2 nanobodies
after heating at increasing temperatures
[0192] FIG. 14: Binding in ELISA to vWF of bivalent 12A5 nanobodies
after heating at increasing temperatures
[0193] FIG. 15: Alignment of humanised 12B6 nanobody sequences
(12B6, SEQ ID NO:62; 12B6H1, SEQ ID NO:86; 12B6H2, SEQ ID NO:87;
12B6H3, SEQ ID NO:88; 12B6H4, SEQ ID NO:89)
[0194] FIG. 16: Binding in ELISA to vWF of wild type and humanised
12B6 nanobody
[0195] FIG. 17: Alignment of humanised 12A2 nanobody sequences
(12A2, SEQ ID NO:71, 12A2H1, SEQ ID NO:90, 12A2H3, SEQ ID NO:91;
12A2H4, SEQ ID NO:92; 12A2H11, SEQ ID NO:93; 12A2H13, SEQ ID
NO:94)
[0196] FIG. 18: Binding in ELISA to vWF of humanised 12A2
nanobodies, after heating at increasing temperatures
[0197] FIG. 19: Binding in ELISA to vWF of humanised 12A2
nanobodies
[0198] FIG. 20: Alignment of humanised 12A5 nanobody sequences
(12A5, SEQ ID NO:60; 12A5H1, SEQ ID NO:95; 12A5H2, SEQ ID NO:96;
12A5H3, SEQ ID NO:97)
[0199] FIG. 21: Binding in ELISA to vWF of wild type and humanised
12A5 nanobody
[0200] FIG. 22: Alignment of nanobodies selected for bivalent form
(12A2H1, SEQ ID NO:90; 12A2H4, SEQ ID NO:92; 12B6H2, SEQ ID
NO:87)
[0201] FIG. 23: Platelet adhesion at different concentrations of
bivalent (humanised) nanobodies
[0202] FIG. 24: Blood flow pattern for Folts model in baboons
[0203] FIG. 25: Experimental setup for Folts model in baboons
[0204] FIG. 26: Folts study of baboon control group. The blood flow
in function of time is shown, indicating the CFRs (representative
of 2 independent experiments)
[0205] FIG. 27: Folts study of baboon group treated with Aspegic.
The blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0206] FIG. 28: study of baboon group treated with Heparin. The
blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0207] FIG. 29: Folts study of baboon group treated with Plavix.
The blood flow in function of time is shown, indicating the CFRs
(representative of 4 independent experiments)
[0208] FIG. 30: Folts study of baboon group treated with Reopro.
The blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0209] FIG. 31: Folts study of baboon group treated with ALX-0081
(SEQ ID NO:98). The blood flow in function of time is shown,
indicating the CFRs (representative of 8 independent
experiments)
[0210] FIG. 32: Flow read out from baboon ID 6 treated with a
combination of Aspegic, Heparin, Plavix and ALX-0081
[0211] FIG. 33: Averages of relative blood loss in function of
different doses of Plavix, Reopro and ALX-0081
[0212] FIG. 34: Average length of CFRs and average relative amount
of blood loss for animals treated with Plavix in function of
increasing drug dose.
[0213] FIG. 35: Average length of CFRs and average relative amount
of blood loss for animals treated with Reopro in function of
increasing drug dose
[0214] FIG. 36: Average length of CFRs and average relative amount
of blood loss for animals treated with ALX-0081 in function of
increasing drug dose
[0215] FIG. 37: ristocetin-induced aggregation (%, .box-solid.) and
length of CFRs (s, .diamond-solid.) for each baboon treated with
ALX-0081 in function of all doses
[0216] FIG. 38: Concentration of ALX-0081 in plasma versus the
length of CFRs for all baboons treated with ALX-0081
[0217] FIG. 39: Concentration of ALX-0081 in plasma versus relative
amount of blood loss from the gauzes
[0218] FIG. 40: Folts study of baboon 1 treated with ALX-0081 and
vWF. The blood flow in function of time is shown, indicating the
CFRs
[0219] FIG. 41: strings (arrows) of adhered platelets on ULvWF
secreted from stimulated endothelial cells
[0220] FIG. 42: Absence of strings when platelets are perfused over
ULvWF in the presence of ALX-0081
[0221] FIG. 43: Control perfusion experiment: ULvWF strings before
(panel A, indicated with red arrows) and during (panel B) perfusion
with normal plasma. In panel B, ULvWF strings being cleaved by
ADAMTS-13 are indicated with a blue and red arrow for a piece of an
ULvWF string moving away or for largely cleaved ULvWF strings
respectively
[0222] FIG. 44: Perfusion experiment in presence of ALX-0081.
Microscopic image of a field before (panel A) and of the same field
after (panel B) perfusion with normal plasma. An UlvWF string is
indicated in panel A with a red arrow which is absent in panel B
due to cleavage of the ULvWF by ADAMTS-13.
[0223] FIG. 45: cleavage of A1-A2-A3 by ADAMTS-13 present in normal
pool plasma (NPP) in the absence and presence of ALX-0081.
DETAILED DESCRIPTION OF THE INVENTION
[0224] The above and other aspects and embodiments of the invention
will become clear from the further description hereinbelow, in
which:
a) Unless indicated or defined otherwise, all terms used have their
usual meaning in the art, which will be clear to the skilled
person. Reference is for example made to the standard handbooks,
such as Sambrook et al, "Molecular Cloning: A Laboratory Manual"
(2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989);
F. Ausubel et al, eds., "Current protocols in molecular biology",
Green Publishing and Wiley Interscience, New York (1987); Roitt et
al., "Immunology" (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); and
Janeway et al., "Immunobiology" (6.sup.th Ed.), Garland Science
Publishing/Churchill Livingstone, New York (2005), as well as to
the general background art cited above; b) Unless indicated
otherwise, the term "immunoglobulin sequence"--whether it used
herein to refer to a heavy chain antibody or to a conventional
4-chain antibody--is used as a general term to include both the
full-size antibody, the individual chains thereof, as well as all
parts, domains or fragments thereof (including but not limited to
antigen-binding domains or fragments such as V.sub.HH domains or
V.sub.H/V.sub.L domains, respectively). In addition, the term
"sequence" as used herein (for example in terms like
"immunoglobulin sequence", "antibody sequence", "variable domain
sequence", "V.sub.HH sequence" or "protein sequence"), should
generally be understood to include both the relevant amino acid
sequence as well as nucleic acid sequences or nucleotide sequences
encoding the same, unless the context requires a more limited
interpretation; c) Unless indicated otherwise, all methods, steps,
techniques and manipulations that are not specifically described in
detail can be performed and have been performed in a manner known
per se, as will be clear to the skilled person. Reference is for
example again made to the standard handbooks, to the general
background art referred to above and to the further references
cited therein; d) Amino acid residues will be indicated according
to the standard three-letter or one-letter amino acid code, as
mentioned in Table 1;
TABLE-US-00009 TABLE 1 one-letter and three-letter amino acid code
Nonpolar, Alanine Ala A uncharged Valine Val V (at pH
6.0-7.0).sup.(3) Leucine Leu L Isoleucine Ile I Phenylalanine Phe F
Methionine.sup.(1) Met M Tryptophan Trp W Proline Pro P Polar,
Glycine.sup.(2) Gly G uncharged Serine Ser S (at pH 6.0-7.0)
Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q
Tyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH
6.0-7.0) Histidine.sup.(4) His H Aspartate Asp D Glutamate Glu E
Notes: .sup.(1)Sometimes also considered to be a polar uncharged
amino acid. .sup.(2)Sometimes also considered to be a nonpolar
uncharged amino acid. .sup.(3)As will be clear to the skilled
person, the fact that an amino acid residue is referred to in this
Table as being either charged or uncharged at pH 6.0 to 7.0 does
not reflect in any way on the charge said amino acid residue may
have at a pH lower than 6.0 and/or at a pH higher than 7.0; the
amino acid residues mentioned in the Table can be either charged
and/or uncharged at such a higher or lower pH, as will be clear to
the skilled person. .sup.(4)As is known in the art, the charge of a
His residue is greatly dependant upon even small shifts in pH, but
a His residu can generally be considered essentially uncharged at a
pH of about 6.5.
e) For the purposes of comparing two or more nucleotide sequences,
the percentage of "sequence identity" between a first nucleotide
sequence and a second nucleotide sequence may be calculated by
dividing [the number of nucleotides in the first nucleotide
sequence that are identical to the nucleotides at the corresponding
positions in the second nucleotide sequence] by [the total number
of nucleotides in the first nucleotide sequence] and multiplying by
[100%], in which each deletion, insertion, substitution or addition
of a nucleotide in the second nucleotide sequence--compared to the
first nucleotide sequence--is considered as a difference at a
single nucleotide (position). Alternatively, the degree of sequence
identity between two or more nucleotide sequences may be calculated
using a known computer algorithm for sequence alignment such as
NCBI Blast v2.0, using standard settings.
[0225] Some other techniques, computer algorithms and settings for
determining the degree of sequence identity are for example
described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318,
WO 00/78972, WO 98/49185 and GB 2 357 768-A.
[0226] Usually, for the purpose of determining the percentage of
"sequence identity" between two nucleotide sequences in accordance
with the calculation method outlined hereinabove, the nucleotide
sequence with the greatest number of nucleotides will be taken as
the "first" nucleotide sequence, and the other nucleotide sequence
will be taken as the "second" nucleotide sequence;
f) For the purposes of comparing two or more amino acid sequences,
the percentage of "sequence identity" between a first amino acid
sequence and a second amino acid sequence may be calculated by
dividing [the number of amino acid residues in the first amino acid
sequence that are identical to the amino acid residues at the
corresponding positions in the second amino acid sequence] by [the
total number of nucleotides in the first amino acid sequence] and
multiplying by [100%], in which each deletion, insertion,
substitution or addition of an amino acid residue in the second
amino acid sequence--compared to the first amino acid sequence--is
considered as a difference at a single amino acid residue
(position), i.e. as an "amino acid difference" as defined
herein.
[0227] Alternatively, the degree of sequence identity between two
amino acid sequences may be calculated using a known computer
algorithm, such as those mentioned above for determining the degree
of sequence identity for nucleotide sequences, again using standard
settings.
[0228] Usually, for the purpose of determining the percentage of
"sequence identity" between two amino acid sequences in accordance
with the calculation method outlined hereinabove, the amino acid
sequence with the greatest number of amino acid residues will be
taken as the "first" amino acid sequence, and the other amino acid
sequence will be taken as the "second" amino acid sequence.
[0229] Also, in determining the degree of sequence identity between
two amino acid sequences, the skilled person may take into account
so-called "conservative" amino acid substitutions, which can
generally be described as amino acid substitutions in which an
amino acid residue is replaced with another amino acid residue of
similar chemical structure and which has little or essentially no
influence on the function, activity or other biological properties
of the polypeptide. Such conservative amino acid substitutions are
well known in the art, for example from WO 04/037999, GB-A-2 357
768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred)
types and/or combinations of such substitutions may be selected on
the basis of the pertinent teachings from WO 04/037999 as well as
WO 98/49185 and from the further references cited therein.
[0230] Such conservative substitutions preferably are substitutions
in which one amino acid within the following groups (a)-(e) is
substituted by another amino acid residue within the same group:
(a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser,
Thr, Pro and Gly; (b) polar, negatively charged residues and their
(uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively
charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar
residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:
Phe, Tyr and Trp.
[0231] Particularly preferred conservative substitutions are as
follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or
into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp;
Gly into Ala or into Pro; His into Asn or into Gln; Be into Leu or
into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into
Glu; Met into Leu, into Tyr or into Be; Phe into Met, into Leu or
into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;
and/or Phe into Val, into Be or into Leu.
[0232] Any amino acid substitutions applied to the polypeptides
described herein may also be based on the analysis of the
frequencies of amino acid variations between homologous proteins of
different species developed by Schulz et al., Principles of Protein
Structure, Springer-Verlag, 1978, on the analyses of structure
forming potentials developed by Chou and Fasman, Biochemistry 13:
211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis
of hydrophobicity patterns in proteins developed by Eisenberg et
al., Proc. Nad. Acad. Sci. USA 81: 140-144, 1984; Kyte &
Doolittle; J. Molec. Biol. 157: 105-132, 198 1, and Goldman et al.,
Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein
in their entirety by reference. Information on the primary,
secondary and tertiary structure of Nanobodies given in the
description below and in the general background art cited above.
Also, for this purpose, the crystal structure of a V.sub.HH domain
from a llama is for example given by Desmyter et al., Nature
Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural
Structural Biology (1996); 3, 752-757; and Decanniere et al.,
Structure, Vol. 7, 4, 361 (1999);
g) amino acid sequences and nucleic acid sequences are said to be
"exactly the same" if they have 100% sequence identity (as defined
herein) over their entire length; h) when comparing two amino acid
sequences, the term "amino acid difference" refers to an insertion,
deletion or substitution of a single amino acid residue on a
position of the first sequence, compared to the second sequence; it
being understood that two amino acid sequences can contain one, two
or more such amino acid differences; i) a nucleic acid sequence or
amino acid sequence is considered to be "(in) essentially isolated
(form)"--for example, compared to its native biological source
and/or the reaction medium or cultivation medium from which it has
been obtained--when it has been separated from at least one other
component with which it is usually associated in said source or
medium, such as another nucleic acid, another protein/polypeptide,
another biological component or macromolecule or at least one
contaminant, impurity or minor component. In particular, a nucleic
acid sequence or amino acid sequence is considered "essentially
isolated" when it has been purified at least 2-fold, in particular
at least 10-fold, more in particular at least 100-fold, and up to
1000-fold or more. A nucleic acid sequence or amino acid sequence
that is "in essentially isolated form" is preferably essentially
homogeneous, as determined using a suitable technique, such as a
suitable chromatographical technique, such as
polyacrylamide-gelelectrophoresis; j) The term "domain" as used
herein generally refers to a globular region of an antibody chain,
and in particular to a globular region of a heavy chain antibody,
or to a polypeptide that essentially consists of such a globular
region. Usually, such a domain will comprise peptide loops (for
example 3 or 4 peptide loops) stabilized, for example, as a sheet
or by disulfide bonds. k) The term `antigenic determinant` refers
to the epitope on the antigen recognized by the antigen-binding
molecule (such as a Nanobody or a polypeptide of the invention) and
more in particular by the antigen-binding site of said molecule.
The terms "antigenic determinant" and "epitope" may also be used
interchangeably herein. l) An amino acid sequence (such as a
Nanobody, an antibody, a polypeptide of the invention, or generally
an antigen binding protein or polypeptide or a fragment thereof)
that can bind to, that has affinity for and/or that has specificity
for a specific antigenic determinant, epitope, antigen or protein
(or for at least one part, fragment or epitope thereof) is said to
be "against" or "directed against" said antigenic determinant,
epitope, antigen or protein. m) The term "specificity" refers to
the number of different types of antigens or antigenic determinants
to which a particular antigen-binding molecule or antigen-binding
protein (such as a Nanobody or a polypeptide of the invention)
molecule can bind. The specificity of an antigen-binding protein
can be determined based on affinity and/or avidity. The affinity,
represented by the equilibrium constant for the dissociation of an
antigen with an antigen-binding protein (KD), is a measure for the
binding strength between an antigenic determinant and an
antigen-binding site on the antigen-binding protein: the lesser the
value of the KD, the stronger the binding strength between an
antigenic determinant and the antigen-binding molecule
(alternatively, the affinity can also be expressed as the affinity
constant (KA), which is 1/KD). As will be clear to the skilled
person (for example on the basis of the further disclosure herein),
affinity can be determined in a manner known per se, depending on
the specific antigen of interest. Avidity is the measure of the
strength of binding between an antigen-binding molecule (such as a
Nanobody or polypeptide of the invention) and the pertinent
antigen. Avidity is related to both the affinity between an
antigenic determinant and its antigen binding site on the
antigen-binding molecule and the number of pertinent binding sites
present on the antigen-binding molecule. Typically, antigen-binding
proteins (such as the Nanobodies and/or polypeptides of the
invention) will bind with a dissociation constant (KD) of 10.sup.-5
to 10.sup.-12 moles/liter (M) or less, and preferably 10.sup.-7 to
10.sup.-12 moles/liter (M) or less and more preferably 10.sup.-8 to
10.sup.-12 moles/liter, and/or with an association constant
(K.sub.A) of at least 10.sup.7 M.sup.-1, preferably at least
10.sup.8 M.sup.-1, more preferably at least 10.sup.9 M-1, such as
at least 10.sup.12 M.sup.-1. Any K.sub.D value greater than
10.sup.-4 M is generally considered to indicate non-specific
binding. Preferably, a Nanobody or polypeptide of the invention
will bind to the desired antigen with an K.sub.D less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM. Specific binding of an antigen-binding protein
to an antigen or antigenic determinant can be determined in any
suitable manner known per se, including, for example, Scatchard
analysis and/or competitive binding assays, such as
radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich
competition assays, and the different variants thereof known per se
in the art. n) as further described hereinbelow, the amino acid
sequence and structure of a Nanobody can be considered--without
however being limited thereto--to be comprised of four framework
regions or "FR's", which are referred to in the art and hereinbelow
as "Framework region 1" or "FR1"; as "Framework region 2" or"FR2";
as "Framework region 3" or "FR3"; and as "Framework region 4" or
"FR4", respectively; which framework regions are interrupted by
three complementary determining regions or "CDR's", which are
referred to in the art as "Complementarity Determining Region/" or
"CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as
"Complementarity Determining Region 3" or "CDR3", respectively; o)
as also further describe hereinbelow, the total number of amino
acid residues in a Nanobody can be in the region of 110-120, is
preferably 112-115, and is most preferably 113. It should however
be noted that parts, fragments or analogs (as further described
hereinbelow) of a Nanobody are not particularly limited as to their
length and/or size, as long as such parts, fragments or analogs
meet the further requirements outlined hereinbelow and are also
preferably suitable for the purposes described herein; p) the amino
acid residues of a Nanobody are numbered according to the general
numbering for V.sub.H domains given by Kabat et al. ("Sequence of
proteins of immunological interest", US Public Health Services, NIH
Bethesda, Md., Publication No. 91), as applied to V.sub.HH domains
from Camelids in the article of Riechmann and Muyldermans, referred
to above (see for example FIG. 2 of said reference). According to
this numbering, FR1 of a Nanobody comprises the amino acid residues
at positions 1-30, CDR1 of a Nanobody comprises the amino acid
residues at positions 31-36, FR2 of a Nanobody comprises the amino
acids at positions 36-49, CDR2 of a Nanobody comprises the amino
acid residues at positions 50-65, FR3 of a Nanobody comprises the
amino acid residues at positions 66-94, CDR3 of a Nanobody
comprises the amino acid residues at positions 95-102, and FR4 of a
Nanobody comprises the amino acid residues at positions 103-113.
[In this respect, it should be noted that--as is well known in the
art for V.sub.H domains and for V.sub.HH domains--the total number
of amino acid residues in each of the CDR's may vary and may not
correspond to the total number of amino acid residues indicated by
the Kabat numbering (that is, one or more positions according to
the Kabat numbering may not be occupied in the actual sequence, or
the actual sequence may contain more amino acid residues than the
number allowed for by the Kabat numbering). This means that,
generally, the numbering according to Kabat may or may not
correspond to the actual numbering of the amino acid residues in
the actual sequence. Generally, however, it can be said that,
according to the numbering of Kabat and irrespective of the number
of amino acid residues in the CDR's, position 1 according to the
Kabat numbering corresponds to the start of FR1 and visa versa,
position 36 according to the Kabat numbering corresponds to the
start of FR2 and visa versa, position 66 according to the Kabat
numbering corresponds to the start of FR3 and visa versa, and
position 103 according to the Kabat numbering corresponds to the
start of FR4 and visa versa.].
[0233] Alternative methods for numbering the amino acid residues of
V.sub.H domains, which methods can also be applied in an analogous
manner to V.sub.HH domains from Camelids and to Nanobodies, are the
method described by Chothia et al. (Nature 342, 877-883 (1989)),
the so-called "AbM definition" and the so-called "contact
definition". However, in the present description, claims and
figures, the numbering according to Kabat as applied to V.sub.HH
domains by Riechmann and Muyldermans will be followed, unless
indicated otherwise; and
q) the Figures, Sequence Listing and the Experimental Part/Examples
are only given to further illustrate the invention and should not
be interpreted or construed as limiting the scope of the invention
and/or of the appended claims in any way, unless explicitly
indicated otherwise herein.
[0234] For a general description of heavy chain antibodies and the
variable domains thereof, reference is inter alia made to the
following references, which are mentioned as general background
art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije
Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO
00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO
02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO
03/054016 and WO 03/055527 of the Vlaams Instituut voor
Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and
applicant; WO 01/90190 by the National Research Council of Canada;
WO 03/025020 (=EP 1 433 793) by the Institute of Antibodies; as
well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO
04/062551 by applicant and the further published patent
applications by applicant;
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Davies and Riechmann, FEBS Lett. 1994 Feb. 21; 339(3): 285-90;
Muyldermans et al., Protein Eng. 1994 September; 7(9): 1129-3;
Davies and Riechmann, Biotechnology (NY) 1995 May; 13(5): 475-9;
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Landbouw Univ. Gent. 1995; 60/4a part I: 2097-2100; Davies and
Riechmann, Protein Eng. 1996 June; 9(6): 531-7; Desmyter et al.,
Nat Struct Biol. 1996 September; 3(9): 803-11; Sheriff et al., Nat
Struct Biol. 1996 September; 3(9): 733-6; Spinelli et al., Nat
Struct Biol. 1996 September; 3(9): 752-7; Arbabi Ghahroudi et al.,
FEBS Lett. 1997 Sep. 15; 414(3): 521-6; Vu et al., Mol Immunol.
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Journal of Camel Practice and Research 1997; 4: 177-182; Nguyen et
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[0235] As mentioned above, the invention generally relates to
Nanobodies directed against vWF, as well as to polypeptides
comprising or essentially consisting of one or more of such
Nanobodies, that can be used for the prophylactic, therapeutic
and/or diagnostic purposes described below and in WO 04/062551.
[0236] As also mentioned above and further described below, the
invention further relates to nucleic acids encoding such Nanobodies
and polypeptides, to methods for preparing such Nanobodies and
polypeptides, to host cells expressing or capable of expressing
such Nanobodies or polypeptides, to uses of such Nanobodies,
polypeptides, nucleic acids or host cells, and to compositions
comprising such Nanobodies, polypeptides, nucleic acids or host
cells.
[0237] Generally, it should be noted that the term Nanobody as used
herein in its broadest sense is not limited to a specific
biological source or to a specific method of preparation. For
example, as will be discussed in more detail below, the Nanobodies
of the invention can be obtained (1) by isolating the V.sub.HH
domain of a naturally occurring heavy chain antibody; (2) by
expression of a nucleotide sequence encoding a naturally occurring
V.sub.HH domain; (3) by "humanization" (as described below) of a
naturally occurring V.sub.HH domain or by expression of a nucleic
acid encoding a such humanized V.sub.HH domain; (4) by
"camelization" (as described below) of a naturally occurring
V.sub.H domain from any animal species, in particular a species of
mammal, such as from a human being, or by expression of a nucleic
acid encoding such a camelized V.sub.H domain; (5) by
"camelisation" of a "domain antibody" or "Dab" as described by Ward
et al (supra), or by expression of a nucleic acid encoding such a
camelized V.sub.H domain; (6) using synthetic or semi-synthetic
techniques for preparing proteins, polypeptides or other amino acid
sequences; (7) by preparing a nucleic acid encoding a Nanobody
using techniques for nucleic acid synthesis, followed by expression
of the nucleic acid thus obtained; and/or (8) by any combination of
the foregoing. Suitable methods and techniques for performing the
foregoing will be clear to the skilled person based on the
disclosure herein and for example include the methods and
techniques described in more detail hereinbelow.
[0238] However, according to a specific embodiment, the Nanobodies
of the invention do not have an amino acid sequence that is exactly
the same as (i.e. as a degree of sequence identity of 100% with)
the amino acid sequence of a naturally occurring V.sub.H domain,
such as the amino acid sequence of a naturally occurring V.sub.H
domain from a mammal, and in particular from a human being.
[0239] One particularly preferred class of Nanobodies of the
invention comprises Nanobodies with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.HH domain, but that has been "humanized", i.e. by replacing
one or more amino acid residues in the amino acid sequence of said
naturally occurring V.sub.HH sequence by one or more of the amino
acid residues that occur at the corresponding position(s) in a
V.sub.H domain from a conventional 4-chain antibody from a human
being (e.g. indicated above). This can be performed in a manner
known per se, which will be clear to the skilled person, for
example on the basis of the further description below and the prior
art on humanization referred to herein. Again, it should be noted
that such humanized Nanobodies of the invention can be obtained in
any suitable manner known per se (i.e. as indicated under points
(1)-(8) above) and thus are not strictly limited to polypeptides
that have been obtained using a polypeptide that comprises a
naturally occurring V.sub.HH domain as a starting material.
[0240] Another particularly preferred class of Nanobodies of the
invention comprises Nanobodies with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.H domain that has been "camelized", i.e. by replacing one or
more amino acid residues in the amino acid sequence of a naturally
occurring V.sub.H domain from a conventional 4-chain antibody by
one or more of the amino acid residues that occur at the
corresponding position(s) in a V.sub.HH domain of a heavy chain
antibody. This can be performed in a manner known per se, which
will be clear to the skilled person, for example on the basis of
the further description below. Reference is also made to WO
94/04678. Such camelization may preferentially occur at amino acid
positions which are present at the V.sub.H-V.sub.L interface and at
the so-called Camelidae hallmark residues (see for example also WO
94/04678), as also mentioned below. Preferably, the V.sub.H domain
or sequence that is used as a starting material or starting point
for generating or designing the camelized Nanobody is preferably a
V.sub.H sequence from a mammal, more preferably the V.sub.H
sequence of a human being. However, it should be noted that such
camelized Nanobodies of the invention can be obtained in any
suitable manner known per se (i.e. as indicated under points
(1)-(8) above) and thus are not strictly limited to polypeptides
that have been obtained using a polypeptide that comprises a
naturally occurring V.sub.H domain as a starting material.
[0241] For example, again as further described below, both
"humanization" and "camelization" can be performed by providing a
nucleotide sequence that encodes such a naturally occurring
V.sub.HH domain or V.sub.H domain, respectively, and then changing,
in a manner known per se, one or more codons in said nucleotide
sequence such that the new nucleotide sequence encodes a humanized
or camelized Nanobody of the invention, respectively, and then
expressing the nucleotide sequence thus obtained in a manner known
per se so as to provide the desired Nanobody of the invention.
Alternatively, based on the amino acid sequence of a naturally
occurring V.sub.HH domain or V.sub.H domain, respectively, the
amino acid sequence of the desired humanized or camelized Nanobody
of the invention, respectively, can be designed and then
synthesized de novo using techniques for peptide synthesis known
per se. Also, based on the amino acid sequence or nucleotide
sequence of a naturally occurring V.sub.HH domain or V.sub.H
domain, respectively, a nucleotide sequence encoding the desired
humanized or camelized Nanobody of the invention, respectively, can
be designed and then synthesized de novo using techniques for
nucleic acid synthesis known per se, after which the nucleotide
sequence thus obtained can be expressed in a manner known per se so
as to provide the desired Nanobody of the invention.
[0242] Other suitable ways and techniques for obtaining Nanobodies
of the invention and/or nucleotide sequences and/or nucleic acids
encoding the same, starting from (the amino acid sequence of)
naturally occurring V.sub.H domains or preferably V.sub.HH domains
and/or from nucleotide sequences and/or nucleic acid sequences
encoding the same will be clear from the skilled person, and may
for example comprising combining one or more amino acid sequences
and/or nucleotide sequences from naturally occurring V.sub.H
domains (such as one or more FR's and/or CDR's) with one or more
one or more amino acid sequences and/or nucleotide sequences from
naturally occurring V.sub.HH domains (such an one or more FR's or
CDR's), in a suitable manner so as to provide (a nucleotide
sequence or nucleic acid encoding) a Nanobody of the invention.
[0243] According to one preferred, but non-limiting aspect of the
aspect of the invention, a Nanobody in its broadest sense can be
generally defined as a polypeptide comprising: [0244] a) an amino
acid sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 108 according to the
Kabat numbering is Q; and/or: [0245] b) an amino acid sequence that
is comprised of four framework regions/sequences interrupted by
three complementarity determining regions/sequences, in which the
amino acid residue at position 44 according to the Kabat numbering
is E and in which the amino acid residue at position 45 according
to the Kabat numbering is an R; and/or: [0246] c) an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 103 according to the
Kabat numbering is chosen from the group consisting of P, R and S,
and is in particular chosen from the group consisting of R and
S.
[0247] Thus, in a first preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [0248]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which [0249] i) the amino acid residue at
position 108 according to the Kabat numbering is Q; and/or in
which: [0250] ii) the amino acid residue at position 44 according
to the Kabat numbering is E and in which the amino acid residue at
position 45 according to the Kabat numbering is an R; and/or in
which: [0251] iii) the amino acid residue at position 103 according
to the Kabat numbering is chosen from the group consisting of P, R
and S, and is in particular chosen from the group consisting of R
and S; and in which: [0252] iv) CDR 1 is an amino acid sequence
that is chosen from the group consisting of the following amino
acid sequences:
TABLE-US-00010 [0252] [SEQ ID NO: 15] NYGMG [SEQ ID NO: 16] SYTLG
[SEQ ID NO: 17] NYNMG [SEQ ID NO: 18] SSAMA [SEQ ID NO: 19] YYNTG
[SEQ ID NO: 20] IGAMG [SEQ ID NO: 21] IGTMG [SEQ ID NO: 22]
YNPMG
[0253] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0254] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0255] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0256] and/or from
the group consisting of amino acid sequences that have 2 or only 1
"amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0257] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0258] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0259] v) CDR 2 is an amino acid
sequence that is chosen from the group consisting of the following
amino acid sequences:
TABLE-US-00011 [0259] [SEQ ID NO: 23] SISWSGTYTAYSDNVKG [SEQ ID NO:
24] GISWSGVSTDYAEFAKG [SEQ ID NO: 25] TSISWSGSYTAYADNVKG [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG [SEQ ID NO: 27] TITSGGRTSYADSVKG [SEQ ID
NO: 28] AISWSGGLTYYADSVKG [SEQ ID NO: 29] TITSGGSTNYADPVKG [SEQ ID
NO: 30] TITSGGSTNYADSVKG [SEQ ID NO: 31] AISRTGGSTYYARSVEG [SEQ ID
NO: 32] AISRTGGSTYYPDSVEG
[0260] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0261] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0262] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0263] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0264] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0265] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and in which: [0266] vi) CDR 3 is an amino acid
sequence that is chosen from the group consisting of the following
amino acid sequences:
TABLE-US-00012 [0266] [SEQ ID NO: 33] QSRYRSNYYDHDDKYAY [SEQ ID NO:
34] LGRYRSNWRNIGQYDY [SEQ ID NO: 35] QSRYSSNYYDHDDKYAY [SEQ ID NO:
36] SNRYRTHTTQAMYNY [SEQ ID NO: 37] VVDGKRAP [SEQ ID NO: 38]
NRRQKTVQMGERAYDY [SEQ ID NO: 39] NLKQGSYGYRFNDY [SEQ ID NO: 40]
NLKQGDYGYRFNDY [SEQ ID NO: 41] AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 42]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 43] AGVRAEDGRVRSLPSEYTF
[0267] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0268] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0269] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequence(s); [0270] and/or from
the group consisting of amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0271] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0272] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s).
[0273] Preferably, in the Nanobodies of the invention: [0274] when
CDR1 is chosen from the group consisting of (1) NYGMG [SEQ ID NO:
15]; (2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
SISWSGTYTAYSDNVKG [SEQ ID NO: 23]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) QSRYRSNYYDHDDKYAY [SEQ ID NO: 33];
(2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with said amino
acid sequence; [0275] when CDR1 is chosen from the group consisting
of (1) SYTLG [SEQ ID NO: 16]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) GISWSGVSTDYAEFAKG [SEQ ID NO: 24]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
LGRYRSNRRNIGQYDY [SEQ ID NO: 34]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; [0276] when CDR1 is
chosen from the group consisting of (1) NYGMG [SEQ ID NO: 15]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
TSISWSGSYTAYADNVKG [SEQ ID NO: 25]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) QSRYSSNYYDHDDKYAY [SEQ ID NO: 35];
(2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with said amino
acid sequence; [0277] when CDR1 is chosen from the group consisting
of (1) NYNMG [SEQ ID NO: 15]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) SISWSGMSTYYTDSVKG [SEQ ID NO: 26]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
SNRYRTHTTQAMYNY [SEQ ID NO: 36]; (2) amino acid sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; [0278] when CDR1 is
chosen from the group consisting of (1) SSAMA [SEQ ID NO: 18]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
TITSGGRTSYADSVKG [SEQ ID NO: 27]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) VVDGKRAP [SEQ ID NO: 37]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; [0279] when CDR1 is chosen from the group consisting of
(1) YYNTG [SEQ ID NO: 19]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) AISWSGGLTYYADSVKG [SEQ ID NO: 28]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
NRRQKTVQMGERAYDY [SEQ ID NO: 38]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; [0280] when CDR1 is
chosen from the group consisting of (1) IGAMG [SEQ ID NO: 20]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
TITSGGSTNYADPVKG [SEQ ID NO: 29]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) NLKQGSYGYRFNDY [SEQ ID NO: 39];
(2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; [0281] when CDR1 is chosen from the group consisting of
(1) IGAMG [SEQ ID NO: 20]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2) amino
acid sequences that have at least 80%, preferably at least 90%,
more preferably at least 95%, even more preferably at least 99%
sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
NLKQGSYGYRFNDY [SEQ ID NO: 39]; (2) amino acid sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; [0282] when CDR1 is
chosen from the group consisting of (1) IGAMG [SEQ ID NO: 20]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) NLKQGDYGYRFNDY [SEQ ID NO: 40];
(2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with said amino
acid sequence; [0283] when CDR1 is chosen from the group consisting
of (1) IGTMG [SEQ ID NO: 21]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2) amino
acid sequences that have at least 80%, preferably at least 90%,
more preferably at least 95%, even more preferably at least 99%
sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
NLKQGDYGYRFNDY [SEQ ID NO: 40]; (2) amino acid sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; [0284] when CDR1 is
chosen from the group consisting of (1) YNPMG [SEQ ID NO: 22]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 2 or only 1 "amino
acid difference(s)" (as defined herein) with said amino acid
sequence; then CDR2 is chosen from the group consisting of (1)
AISRTGGSTYYARSVEG [SEQ ID NO: 31]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; and CDR3 is chosen
from the group consisting of (1) AGVRAEDGRVRTLPSEYNF [SEQ ID NO:
41]; (2) amino acid sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity (as defined herein) with said amino
acid sequence; and (3) amino acid sequences that have 3, 2 or only
1 "amino acid difference(s)" (as defined herein) with said amino
acid sequence; [0285] when CDR1 is chosen from the group consisting
of (1) YNPMG [SEQ ID NO: 22]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; [0286] when CDR1 is chosen from the group consisting of
(1) YNPMG [SEQ ID NO: 22]; (2) amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with said amino acid sequence; and (3) amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with said amino acid sequence; then CDR2 is chosen from the
group consisting of (1) AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; (2)
amino acid sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity (as defined herein) with said amino acid
sequence; and (3) amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with said amino acid
sequence; and CDR3 is chosen from the group consisting of (1)
AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]; (2) amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with said amino acid sequence; and (3) amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with said amino acid sequence; in which [0287] (1)
any amino acid substitution is preferably a conservative amino acid
substitution (as defined herein); and/or [0288] (2) said amino acid
sequence preferably only contains amino acid substitutions, and no
amino acid deletions or insertions, compared to the above amino
acid sequence(s).
[0289] In particular, a Nanobody against vWF according to the
invention may have the structure: [0290]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which [0291] i) the amino acid residue at
position 108 according to the Kabat numbering is Q; and/or in
which: [0292] ii) the amino acid residue at position 44 according
to the Kabat numbering is E and in which the amino acid residue at
position 45 according to the Kabat numbering is an R; and/or in
which: [0293] iii) the amino acid residue at position 103 according
to the Kabat numbering is chosen from the group consisting of P, R
and S, and is in particular chosen from the group consisting of R
and S; and in which: [0294] iv) CDR 1 is an amino acid sequence
that is chosen from the group consisting of the following amino
acid sequences:
TABLE-US-00013 [0294] [SEQ ID NO: 15] NYGMG [SEQ ID NO: 16] SYTLG
[SEQ ID NO: 17] NYNMG [SEQ ID NO: 18] SSAMA [SEQ ID NO: 19] YYNTG
[SEQ ID NO: 20] IGAMG [SEQ ID NO: 21] IGTMG [SEQ ID NO: 22]
YNPMG
and in which: [0295] v) CDR 2 is an amino acid sequence that is
chosen from the group consisting of the following amino acid
sequences:
TABLE-US-00014 [0295] [SEQ ID NO: 23] SISWSGTYTAYSDNVKG [SEQ ID NO:
24] GISWSGVSTDYAEFAKG [SEQ ID NO: 25] TSISWSGSYTAYADNVKG [SEQ ID
NO: 26] SISWSGMSTYYTDSVKG [SEQ ID NO: 27] TITSGGRTSYADSVKG [SEQ ID
NO: 28] AISWSGGLTYYADSVKG [SEQ ID NO: 29] TITSGGSTNYADPVKG [SEQ ID
NO: 30] TITSGGSTNYADSVKG [SEQ ID NO: 31] AISRTGGSTYYARSVEG [SEQ ID
NO: 32] AISRTGGSTYYPDSVEG
and in which: [0296] vi) CDR 3 is an amino acid sequence that is
chosen from the group consisting of the following amino acid
sequences:
TABLE-US-00015 [0296] [SEQ ID NO: 33] QSRYRSNYYDHDDKYAY [SEQ ID NO:
34] LGRYRSNWRNIGQYDY [SEQ ID NO: 35] QSRYSSNYYDHDDKYAY [SEQ ID NO:
36] SNRYRTHTTQAMYNY [SEQ ID NO: 37] VVDGKRAP [SEQ ID NO: 38]
NRRQKTVQMGERAYDY [SEQ ID NO: 39] NLKQGSYGYRFNDY [SEQ ID NO: 40]
NLKQGDYGYRFNDY [SEQ ID NO: 41] AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 42]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 43] AGVRAEDGRVRSLPSEYTF
[0297] Preferably, in the Nanobodies of the invention according to
the latter aspect: [0298] When CDR1 is:NYGMG [SEQ ID NO: 15]; then
CDR2 is:SISWSGTYTAYSDNVKG [SEQ ID NO: 23]; and CDR3
is:QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] [0299] When CDR1 is:SYTLG [SEQ
ID NO: 16]; then CDR2 is:GISWSGVSTDYAEFAKG [SEQ ID NO: 24]; and
CDR3 is:LGRYRSNWRNIGQYDY [SEQ ID NO: 34] [0300] When CDR1 is:NYGMG
[SEQ ID NO: 15]; then CDR2 is:TSISWSGSYTAYADNVKG [SEQ ID NO: 25];
and CDR3 is:QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] [0301] When CDR1
is:NYNMG [SEQ ID NO: 17]; then CDR2 is: SISWSGMSTYYTDSVKG [SEQ ID
NO: 26]; and CDR3 is: SNRYRTHTTQAMYNY [SEQ ID NO: 36] [0302] When
CDR1 is:SSAMA [SEQ ID NO: 18]; then CDR2 is: TITSGGRTSYADSVKG [SEQ
ID NO: 27]; and CDR3 is:VVDGKRAP [SEQ ID NO: 37] [0303] When CDR1
is:YYNTG [SEQ ID NO: 19]; then CDR2 is: AISWSGGLTYYADSVKG [SEQ ID
NO: 28]; and CDR3 is: NRRQKTVQMGERAYDY [SEQ ID NO: 38] [0304] When
CDR1 is:IGAMG [SEQ ID NO: 20]; then CDR2 is: TITSGGSTNYADPVKG [SEQ
ID NO: 29]; and CDR3 is: NLKQGSYGYRFNDY [SEQ ID NO: 39] [0305] When
CDR1 is:IGAMG [SEQ ID NO: 20]; then CDR2 is: TITSGGSTNYADSVKG [SEQ
ID NO: 30]; and CDR3 is: NLKQGSYGYRFNDY [SEQ ID NO: 39] [0306] When
CDR1 is:IGAMG [SEQ ID NO: 20]; then CDR2 is: TITSGGSTNYADSVKG [SEQ
ID NO: 30]; and CDR3 is: NLKQGDYGYRFNDY [SEQ ID NO: 40] [0307] When
CDR1 is:IGTMG [SEQ ID NO: 21]; then CDR2 is: TITSGGSTNYADSVKG [SEQ
ID NO: 30]; and CDR3 is: NLKQGDYGYRFNDY [SEQ ID NO: 40] [0308] When
CDR1 is:YNPMG [SEQ ID NO: 22]; then CDR2 is: AISRTGGSTYYARSVEG [SEQ
ID NO: 31]; and CDR3 is: AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41] [0309]
When CDR1 is:YNPMG [SEQ ID NO: 22]; and CDR2 is: AISRTGGSTYYPDSVEG
[SEQ ID NO: 32]; and CDR3 is: AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
[0310] When CDR1 is:YNPMG [SEQ ID NO: 22]; CDR2: AISRTGGSTYYPDSVEG
[SEQ ID NO: 32]; and CDR3 is: AGVRAEDGRVRSLPSEYTF [SEQ ID NO:
43]
[0311] In particular, according to one preferred, but non-limiting
aspect of the aspect of the invention, a Nanobody can generally be
defined as a polypeptide comprising an amino acid sequence that is
comprised of four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which; [0312]
a-1) the amino acid residue at position 44 according to the Kabat
numbering is chosen from the group consisting of G, E, D, G, Q, R,
S, L; and is preferably chosen from the group consisting of G, E or
Q; and [0313] a-2) the amino acid residue at position 45 according
to the Kabat numbering is chosen from the group consisting of L, R
or C; and is preferably chosen from the group consisting of L or R;
and [0314] a-3) the amino acid residue at position 103 according to
the Kabat numbering is chosen from the group consisting of W, R or
S; and is preferably W or R, and is most preferably W; [0315] a-4)
the amino acid residue at position 108 according to the Kabat
numbering is Q; or in which: [0316] b-1) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of E and Q; and [0317] b-2) the amino acid residue
at position 45 according to the Kabat numbering is R; and [0318]
b-3) the amino acid residue at position 103 according to the Kabat
numbering is chosen from the group consisting of W, R and S; and is
preferably W; [0319] b-4) the amino acid residue at position 108
according to the Kabat numbering is chosen from the group
consisting of Q and L; and is preferably Q; or in which: [0320]
c-1) the amino acid residue at position 44 according to the Kabat
numbering is chosen from the group consisting of G, E, D, Q, R, S
and L; and is preferably chosen from the group consisting of G, E
and Q; and [0321] c-2) the amino acid residue at position 45
according to the Kabat numbering is chosen from the group
consisting of L, R and C; and is preferably chosen from the group
consisting of L and R; and [0322] c-3) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of P, R and S; and is in particular chosen from
the group consisting of R and S; and [0323] c-4) the amino acid
residue at position 108 according to the Kabat numbering is chosen
from the group consisting of Q and L; is preferably Q.
[0324] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [0325]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0326] i) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of G, E, D, G, Q, R, S, L; and is preferably
chosen from the group consisting of G, E or Q; and in which: [0327]
ii) the amino acid residue at position 45 according to the Kabat
numbering is chosen from the group consisting of L, R or C; and is
preferably chosen from the group consisting of L or R; and in
which: [0328] iii) the amino acid residue at position 103 according
to the Kabat numbering is chosen from the group consisting of W, R
or S; and is preferably W or R, and is most preferably W; and in
which [0329] iv) the amino acid residue at position 108 according
to the Kabat numbering is Q; and in which: [0330] v) CDR1, CDR2 and
CDR3 are as defined herein, and are preferably as defined according
to one of the preferred definitions above, and are more preferably
as defined according to one of the more preferred definitions
above.
[0331] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [0332]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0333] i) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of E and Q; and in which: [0334] ii) the amino
acid residue at position 45 according to the Kabat numbering is R;
and in which: [0335] iii) the amino acid residue at position 103
according to the Kabat numbering is chosen from the group
consisting of W, R and S; and is preferably W; and in which: [0336]
iv) the amino acid residue at position 108 according to the Kabat
numbering is Q; and in which: [0337] vi) CDR1, CDR2 and CDR3 are as
defined herein, and are preferably as defined according to one of
the preferred definitions above, and are more preferably as defined
according to one of the more preferred definitions above.
[0338] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [0339]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0340] i) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of G, E, D, Q, R, S and L; and is preferably
chosen from the group consisting of G, E and Q; and in which:
[0341] ii) the amino acid residue at position 45 according to the
Kabat numbering is chosen from the group consisting of L, R and C;
and is preferably chosen from the group consisting of L and R; and
in which: [0342] iii) the amino acid residue at position 103
according to the Kabat numbering is chosen from the group
consisting of P, R and S; and is in particular chosen from the
group consisting of R and S; and in which: [0343] iv) the amino
acid residue at position 108 according to the Kabat numbering is
chosen from the group consisting of Q and L; is preferably Q; and
in which: [0344] v) CDR1, CDR2 and CDR3 are as defined herein, and
are preferably as defined according to one of the preferred
definitions above, and are more preferably as defined according to
one of the more preferred definitions above.
[0345] Two particularly preferred, but non-limiting groups of the
Nanobodies of the invention are those according to a) above;
according to a-1) to a-4) above; according to b) above; according
to b-1) to b-4) above; according to c) above; and/or according to
c-1) to c-4) above, in which; [0346] a) the amino acid residues at
positions 44-47 according to the Kabat numbering form the sequence
GLEW (or a GLEW-like sequence as defined herein) and the amino acid
residue at position 108 is Q; or in which: [0347] b) the amino acid
residues at positions 43-46 according to the Kabat numbering form
the sequence KERE or KQRE (or a KERE-like sequence) and the amino
acid residue at position 108 is Q or L, and is preferably Q.
[0348] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [0349]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0350] i) the amino acid residues at
positions 44-47 according to the Kabat numbering form the sequence
GLEW (or a GLEW-like sequence as defined herein) and the amino acid
residue at position 108 is Q; and in which: [0351] ii) CDR1, CDR2
and CDR3 are as defined herein, and are preferably as defined
according to one of the preferred definitions above, and are more
preferably as defined according to one of the more preferred
definitions above.
[0352] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [0353]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0354] i) the amino acid residues at
positions 43-46 according to the Kabat numbering form the sequence
KERE or KQRE (or a KERE-like sequence) and the amino acid residue
at position 108 is Q or L, and is preferably Q; and in which:
[0355] ii) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred definitions
above, and are more preferably as defined according to one of the
more preferred definitions above.
[0356] In the Nanobodies of the invention in which the amino acid
residues at positions 43-46 according to the Kabat numbering form
the sequence KERE or KQRE, the amino acid residue at position 37 is
most preferably F. In the Nanobodies of the invention in which the
amino acid residues at positions 44-47 according to the Kabat
numbering form the sequence GLEW, the amino acid residue at
position 37 is chosen from the group consisting of Y, H, I, V or F,
and is most preferably F.
[0357] Thus, without being limited hereto in any way, on the basis
of the amino acid residues present on the positions mentioned
above, the Nanobodies of the invention can generally be classified
is on the basis of the following three groups: [0358] a) The
"GLEW-group": Nanobodies with the amino acid sequence GLEW at
positions 44-47 according to the Kabat numbering and Q at position
108 according to the Kabat numbering. As further described herein,
Nanobodies within this group usually have a V at position 37, and
can have a W, P, R or S at position 103, and preferably have a W at
position 103. The GLEW group also comprises some GLEW-like
sequences such as those mentioned in Table 2 below; [0359] b) The
"KERE-group": Nanobodies with the amino acid sequence KERE or KQRE
or at positions 43-46 according to the Kabat numbering and Q or L
at position 108 according to the Kabat numbering. As further
described herein, Nanobodies within this group usually have a F at
position 37, an L or F at position 47; and can have a W, P, R or S
at position 103, and preferably have a W at position 103; [0360] c)
The "103 P, R, S-group": Nanobodies with a P R or S at position
103. These Nanobodies can have either the amino acid sequence GLEW
at positions 44-47 of the Kabat numbering or the amino acid
sequence KERE or KQRE at positions 43-46 according to the Kabat
numbering, the latter most preferably in combination with an F at
position 37 and an L or an F at position 47 (as defined for the
KERE-group); and can have Q or L at position 108 according to the
Kabat numbering, and preferably have Q.
[0361] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the
GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred definitions above, and are more preferably as
defined according to one of the more preferred definitions
above.
[0362] In another preferred, but non-limiting aspect, a Nanobody of
the invention may be a Nanobody belonging to the KERE-group (as
defined herein), and in which CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred definitions above, and are more preferably as defined
according to one of the more preferred definitions above.
[0363] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the 103 P,
R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred definitions above, and are more preferably as
defined according to one of the more preferred definitions
above.
[0364] Also, more generally and in addition to the 108Q, 43E/44R
and 103P, R, S residues mentioned above, the Nanobodies of the
invention can contain, at one or more positions that, in a
conventional V.sub.H domain, would form (part of) the
V.sub.H/V.sub.L interface, contain one or more amino acid residues
that are more highly charged than the amino acid residues that
naturally occur at the same position(s) in the corresponding
naturally occurring V.sub.H or V.sub.HH domain, and in particular
one or more charged amino acid residues (as mentioned in Table
1).
[0365] Such substitutions include, but are not limited to the
GLEW-like sequences mentioned in Table 2 below; as well as the
substitutions that are described in the International Application
WO 00/29004 for so-called "microbodies", e.g. a Q at position 108
and KLEW at positions 44-47.
[0366] In the Nanobodies of the invention, the amino acid residue
at position 83 is chosen from the group consisting of L, M, S, V
and W; and is preferably L.
[0367] Also, in the Nanobodies of the invention, the amino acid
residue at position 83 is chosen from the group consisting of R, K,
N, E, I and Q; and is most preferably either K or E (for Nanobodies
corresponding to naturally occurring V.sub.HH domains) or R (for
"humanized" Nanobodies, as described below). The amino acid residue
at position 84 is chosen from the group consisting of P, A, R, S, D
and V, and is most preferably P (for Nanobodies corresponding to
naturally occurring V.sub.HH domains) or R (for "humanized"
Nanobodies, as described below).
[0368] Furthermore, in the Nanobodies of the invention, the amino
acid residue at position 104 is chosen from the group consisting of
G and D; and is most preferably G.
[0369] Collectively, the amino acid residues at positions 11, 37,
44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are
as mentioned above, will also be referred to herein as the
"Hallmark Residues". The Hallmark Residues and the amino acid
residues at the corresponding positions of the most closely related
human VH domain, VH3, are summarized in Table 2.
[0370] Some especially preferred combinations of these Hallmark
Residues as occur in naturally occurring V.sub.HH domains are
mentioned in Table 3. For comparison, the corresponding amino acid
residues of the human V.sub.H3 called DP-47 have been indicated in
italics.
TABLE-US-00016 TABLE 2 Hallmark Residues in Nanobodies Position
Human V.sub.H3 Hallmark Residues 11 L, V; L, M, S, V, W; preferably
L predominantly L 37 V, I, F; F.sup.(1), Y, H, I or V, preferably
F.sup.(1) or Y usually V .sup. 44.sup.(8) G G.sup.(2), E.sup.(3),
D, Q, R, S, L; preferably G.sup.(2), E.sup.(3) or Q; most
preferably G.sup.(2) or E.sup.(3). .sup. 45.sup.(8) L L.sup.(2),
R.sup.(3), C, I, L, P, Q, V; preferably L.sup.(2) or R.sup.(3)
.sup. 47.sup.(8) W, Y W.sup.(2), L.sup.(1) or F.sup.(1), A, G, I,
M, R, S or Y; preferably W.sup.(2) , L.sup.(1), F.sup.(1) or R 83 R
or K; usually R R, K.sup.(5), N, E.sup.(5), I, M or Q; preferably K
or R; most preferably K 84 A, T, D; P.sup.(5), A, L, R, S, D, V;
preferably P predominantly A 103 W W.sup.(4), P.sup.(6) ,
R.sup.(6), S; preferably W 104 G G or D; preferably G 108 L, M or
T; Q, L.sup.(7) or R; preferably Q or L.sup.(7) predominantly L
Notes: .sup.(1)In particular, but not exclusively, in combination
with KERE or KQRE at positions 43-46. .sup.(2)Usually as GLEW at
positions 44-47. .sup.(3)Usually as KERE or KQRE at positions
43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG at positions
43-47. Alternatively, also sequences such as TERE (for example
TEREL), KECE (for example KECEL or KECER), RERE (for example
REREG), QERE (for example QEREG), KGRE (for example KGREG), KDRE
(for example KDREV) are possible. Some other possible, but less
preferred sequences include for example DECKL and NVCEL.
.sup.(4)With both GLEW at positions 44-47 and KERE or KQRE at
positions 43-46. .sup.(5)Often as KP or EP at positions 83-84 of
naturally occurring V.sub.HH domains. .sup.(6)In particular, but
not exclusively, in combination with GLEW at positions 44-47.
.sup.(7)With the proviso that when positions 44-47 are GLEW,
position 108 is always Q. .sup.(8)The GLEW group also contains
GLEW-like sequences at positions 44-47, such as for example GVEW,
EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and
ELEW.
TABLE-US-00017 TABLE 3 Some preferred combinations of Hallmark
Residues in naturally occurring Nanobodies. For humanization of
these combinations, reference is made to the specification. 11 37
44 45 47 83 84 103 104 108 DP-47 (human) M V G L W R A W G L "KERE"
group L F E R L K P W G Q L F E R F E P W G Q L F E R F K P W G Q L
Y Q R L K P W G Q L F L R V K P Q G Q L F Q R L K P W G Q L F E R F
K P W G Q "GLEW" group L V G L W K S W G Q M V G L W K P R G Q
[0371] In the Nanobodies, each amino acid residue at any other
position than the Hallmark Residues can be any amino acid residue
that naturally occurs at the corresponding position (according to
the Kabat numbering) of a naturally occurring V.sub.HH domain.
[0372] Such amino acid residues will be clear to the skilled
person. Tables 4-7 mention some non-limiting residues that can be
present at each position (according to the Kabat numbering) of the
FR1, FR2, FR3 and FR4 of naturally occurring V.sub.HH domains. For
each position, the amino acid residue that most frequently occurs
at each position of a naturally occurring V.sub.HH domain (and
which is the most preferred amino acid residue for said position in
a Nanobody) is indicated in bold; and other preferred amino acid
residues for each position have been underlined (note: the number
of amino acid residues that are found at positions 26-30 of
naturally occurring V.sub.HH domains supports the hypothesis
underlying the numbering Chothia (supra) that the residues at these
positions already form part of CDR1.)
[0373] In Tables 4-7, some of the non-limiting residues that can be
present at each position of a human V.sub.H3 domain have also been
mentioned. Again, for each position, the amino acid residue that
most frequently occurs at each position of a naturally occurring
human V.sub.H3 domain is indicated in bold; and other preferred
amino acid residues have been underlined.
TABLE-US-00018 TABLE 4 Non-limiting examples of amino acid residues
in FR1 (for the footnotes, see the footnotes to Table 2) Amino acid
residue(s): Pos. Human V.sub.H3 Camelid V.sub.HH's 1 E, Q Q, A, E,
D, H, R 2 V V, A, E, G, L, M, Q 3 Q Q, K, E, H, P, R, Y 4 L L, F,
P, R, V 5 V, L Q, E, L, V, M, P, A, I 6 E E, D, Q, A, H 7 S, T S,
F, H 8 G, R G, A, R 9 G G, E 10 G, V G, D, R, A, E, N, T, V 11
Hallmark residue: L, M, S, V, W, F, N, P, T, Y; preferably L 12 V,
I V, A, G, M 13 Q, K, R Q, E, K, D, G, A, H, L, N, P, R, T 14 P A,
Q, A, G, P, T, V, E, F, I, N, S 15 G G 16 G, R G, A, E, D, N, P, R,
S, V, W 17 S S, F, T, N, P, A, C 18 L L, V, M, Q, R 19 R, K R, K,
L, N, S, T, A, F, G, I, M, Q 20 L L, F, I, V, M, S 21 S S, F, T, G,
H, P, A 22 C C 23 A, T A, D, P, S, T, V, E, G, I, L, Q, R 24 A A,
I, S, T, V, C, E, F, G, L, N, P, Q, Y 25 S S, A, F, P, T, L, V 26 G
G, D, E, R, S, V, A, I, M, P, T 27 F S, F, R, L, P, G, N, A, D, E,
H, I, K, M, Q, T, V, Y 28 T N, T, E, D, S, I, R, A, G, R, F, Y, L,
M, P, V 29 F, V F, L, D, S, I, G, V, A, E, P, T, Y 30 S, D, G N, S,
E, G, A, D, M, T, H, I, P, R, V, W
TABLE-US-00019 TABLE 5 Non-limiting examples of amino acid residues
in FR2 (for the footnotes, see the footnotes to Table 2) Amino acid
residue(s): Pos. Human V.sub.H3 Camelid V.sub.HH's 36 W W 37
Hallmark residue: F.sup.(1), Y, H, I, A, L, P, S or V preferably
F.sup.(1) or Y 38 R R 39 Q Q, H, P, R, A, D, G, L, E 40 A A, F, G,
P, T, V, I, L, N, R, S, Y 41 P, S, T P, A, L, S, I, Q, T 42 G G, E,
D, R, T, V 43 K K, D, E, N, Q, R, T, V, A, L, M, S 44 Hallmark
residue: G.sup.(2), E.sup.(3), D, Q, R, S, L, A, F, K, M, N, P, V,
W, Y; preferably G.sup.(2), E.sup.(3) or Q; most preferably
G.sup.(2) or E.sup.(3). 45 Hallmark residue: L.sup.(2), R.sup.(3),
C, I, L, P, Q, V, D, E, G, H, K, T; preferably L.sup.(2) or
R.sup.(3). 46 E, V E, D, K, Q, V, A, G, N 47 Hallmark residue:
W.sup.(2), L.sup.(1) or F.sup.(1), A, G, I, M, R, S, D, E, H, K, Q,
T, V or Y; preferably W.sup.(2), L.sup.(1), F.sup.(1) or R 48 V V,
I, L, A, C, E, F, G, H, M, P, Q, R, S, T, V, W, Y 49 S, A, G A, S,
A, G, T, V, D, E, I, L, Q, R, Y
TABLE-US-00020 TABLE 6 Non-limiting examples of amino acid residues
in FR3 (for the footnotes, see the footnotes to Table 2) Amino acid
residue(s): Pos. Human V.sub.H3 Camelid V.sub.HH's 66 R R 67 F F,
L, V, A, D, I, S, Y 68 T T, A, S, D, F, G, I, K, N 69 I I, M, V, A,
F, L, R, S, T 70 S S, A, F, E, G, K, P, T, V 71 R R, G, I, K, Q, S,
T, W, A, F, L, M, N 72 D, E D, E, G, N, V, A, H, I, L, Q, S, T 73
N, D, G N, D, F, I, K, S, T, Y, A, G, H, L, M, R, V 74 A, S A, D,
G, N, P, S, T, F, H, I, L, R, V, Y 75 K K, A, E, K, L, N, Q, R, D,
G, I, M, S, T, V, W 76 N, S N, D, K, R, S, T, Y, E, G, H, I, Q 77
S, T, I T, A, E, I, M, S , K, L, N, R, V 78 L, A V, L, A, F, G, I,
M, E, N, Q, R, S, T, W 79 Y, H Y, A, D, F, H, S, T, C, E, I, L, N,
V, W 80 L L, F, V, M 81 Q Q, E, R, T, G, H, I, K, L, M, N 82 M M,
I, L, V, G, P, T 82a N, G N, D, G, H, S, T, A, E, I, K, R, V 82b S
S, N, D, G, R, A, C, E, F, I, K, M, P, T, V 82c L L, P, M, T, V 83
Hallmark residue: R, K.sup.(5), N, E.sup.(5), I, M, A, D, G, L, Q,
S, T or Q; preferably K or R; most preferably K 84 Hallmark
residue: P.sup.(5), A, L, R, S, D, V, F, G, H, N, T, Y; preferably
P 85 E, G E, D, G, Q, A, N, R, V, Y 86 D D, E, F, Y 87 T, M T, S,
A, C, M 88 A A, G, S, D, L, N, P 89 V, L V, A, D, I, L, M, N, R, T,
E, F, S 90 Y Y, F, E, H, N 91 Y, H Y, D, F, H, L, S, T, V, C, I, N,
R, W 92 C C 93 A, K, T A, N, G, H, K, R, S, T, V, Y, E, F, I, L, M,
Q 94 K, R, T A, V, C, F, G, I, L, R, S, D, E, K, M, N, P, Q, T, W,
Y T or K;
TABLE-US-00021 TABLE 7 Non-limiting examples of amino acid residues
in FR4 (for the footnotes, see the footnotes to Table 2) Amino acid
residue(s): Pos. Human V.sub.H3 Camelid V.sub.HH's 103 Hallmark
residue: W.sup.(4), P.sup.(6), R.sup.(6), S, F, G, K, L, N, Q, V,
Y; preferably W 104 Hallmark residue: G, A, R, S, T or D;
preferably G 105 Q, R Q, E, K, P, R, G, H, L, S, V 106 G G 107 T T,
A, I, N, P 108 Hallmark residue: Q, L.sup.(7), E, H, N, P, T or R;
preferably Q or L.sup.(7) 109 V V 110 T T, I, A 111 V V, A, I, G
112 S S, F, A, L, P, T, Y 113 S S, A, L, P, F, T
[0374] Thus, in another preferred, but not limiting aspect, a
Nanobody of the invention can have the structure [0375]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: [0376] i) the Hallmark residues are as
defined herein; and in which: [0377] ii) CDR1, CDR2 and CDR3 are as
defined herein, and are preferably as defined according to one of
the preferred definitions above, and are more preferably as defined
according to one of the more preferred definitions above.
[0378] In another preferred, but not limiting aspect, a Nanobody of
the invention can have the structure [0379]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: and in which [0380] i) FR1 is chosen
from the group consisting of the amino acid sequence:
TABLE-US-00022 [0380] [SEQ ID NO: 1] [1] QVQLQESGGGXVQAGGSLRLSCAASG
[26]
[0381] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0382]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0383] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); [0384] and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of the above amino acid sequences, in
which: [0385] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 4; and/or [0386] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and in which: [0387] ii) FR2 is chosen from
the group consisting of the amino acid sequence:
TABLE-US-00023 [0387] [SEQ ID NO: 2] [36] WXRQAPGKXXEXVA [49]
[0388] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0389]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 5; and/or [0390] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); [0391] and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of the above amino acid sequences, in
which: [0392] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 5; and/or [0393] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and in which: [0394] iii) FR3 is chosen
from the group consisting of the amino acid sequence:
TABLE-US-00024 [0394] [SEQ ID NO: 3] [66]
RFTISRDNAKNTVYLQMNSLXXEDTAVYYCAA [94]
[0395] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0396]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0397] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); [0398] and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of the above amino acid sequences, in
which: [0399] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 6; and/or [0400] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and in which: [0401] iv) FR4 is chosen from
the group consisting of the amino acid sequence:
TABLE-US-00025 [0401] [SEQ ID NO: 4] [103] XXQGTXVTVSS [113]
[0402] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0403]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0404] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); [0405] and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of the above amino acid sequences, in
which: [0406] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 6; and/or [0407] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and in which: [0408] v) CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred definitions above, and are more preferably as
defined according to one of the more preferred definitions above;
in which the Hallmark Residues are indicated by "X" and are as
defined hereinabove and in which the numbers between brackets refer
to the amino acid positions according to the Kabat numbering.
[0409] In another preferred, but not limiting aspect, a Nanobody of
the invention can have the structure [0410]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: and in which [0411] i) FR1 is chosen
from the group consisting of the amino acid sequence:
TABLE-US-00026 [0411] [SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG
[26]
[0412] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0413]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0414] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0415] (3) the Hallmark residue at position is as
indicated in the sequence above; [0416] and/or from the group
consisting of amino acid sequences that have 3, 2 or only 1 "amino
acid difference(s)" (as defined herein) with one of the above amino
acid sequences, in which: [0417] (1) any amino acid substitution at
any position other than a Hallmark position is preferably either a
conservative amino acid substitution (as defined herein) and/or an
amino acid substitution as defined in Table 4; and/or [0418] (2)
said amino acid sequence preferably only contains amino acid
substitutions, and no amino acid deletions or insertions, compared
to the above amino acid sequence(s); and [0419] (3) the Hallmark
residue at position is as indicated in the sequence above; and in
which: [0420] ii) FR2 is chosen from the group consisting of the
amino acid sequences:
TABLE-US-00027 [0420] [SEQ ID NO: 6] [36] WFRQAPGKERELVA [49] [SEQ
ID NO: 7] [36] WFRQAPGKEREFVA [49] [SEQ ID NO: 8] [36]
WFRQAPGKEREGA [49] [SEQ ID NO: 9] [36] WFRQAPGKQRELVA [49] [SEQ ID
NO: 10] [36] WFRQAPGKQREFVA [49] [SEQ ID NO: 11] [36] WYRQAPGKGLEWA
[49]
[0421] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0422] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 5; and/or [0423] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and [0424] (3) the Hallmark residues at
positions 37, 44, 45 and 47 are as indicated in each of the
sequences above; [0425] and/or from the group consisting of amino
acid sequences that have 3, 2 or only 1 "amino acid difference(s)"
(as defined herein) with one of the above amino acid sequences, in
which: [0426] (1) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 5; and/or [0427] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s); and [0428] (3) the Hallmark residues at
positions 37, 44, 45 and 47 are as indicated in each of the
sequences above; and in which: [0429] iii) FR3 is chosen from the
group consisting of the amino acid sequence:
TABLE-US-00028 [0429] [SEQ ID NO: 12] [66]
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]
[0430] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the above amino acid sequence; in which [0431]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0432] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0433] (3) the Hallmark residues at positions 83
and 84 are as indicated in each of the sequences above; [0434]
and/or from the group consisting of amino acid sequences that have
3, 2 or only 1 "amino acid difference(s)" (as defined herein) with
one of the above amino acid sequences, in which: [0435] (1) any
amino acid substitution at any position other than a Hallmark
position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0436] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0437] (3) the Hallmark residues at positions 83
and 84 are as indicated in each of the sequences above; and in
which: [0438] iv) FR4 is chosen from the group consisting of the
amino acid sequences:
TABLE-US-00029 [0438] [SEQ ID NO: 13] [103] WGQGTQVTVSS [113] [SEQ
ID NO: 14] [103] WGQGTLVTVSS [113]
[0439] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequence; in which
[0440] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0441] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0442] (3) the Hallmark residues at positions 103,
104 and 108 are as indicated in each of the sequences above; [0443]
and/or from the group consisting of amino acid sequences that have
3, 2 or only 1 "amino acid difference(s)" (as defined herein) with
one of the above amino acid sequences, in which: [0444] (1) any
amino acid substitution at any position other than a Hallmark
position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0445] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0446] (3) the Hallmark residues at positions 103,
104 and 108 are as indicated in each of the sequences above; and in
which: [0447] v) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred definitions
above, and are more preferably as defined according to one of the
more preferred definitions above.
[0448] In another preferred, but not limiting aspect, a Nanobody of
the invention can have the structure [0449]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: and in which [0450] i) FR1 is chosen
from the group consisting of the amino acid sequence:
TABLE-US-00030 [0450] [SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG
[26]
[0451] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0452] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0453] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0454] (3) the Hallmark residue at position is as
indicated in the sequence above; and in which: [0455] ii) FR2 is
chosen from the group consisting of the amino acid sequences:
TABLE-US-00031 [0455] [SEQ ID NO: 6] [36] WFRQAPGKERELVA [49] [SEQ
ID NO: 7] [36] WFRQAPGKEREFVA [49] [SEQ ID NO: 8] [36]
WFRQAPGKEREGA [49] [SEQ ID NO: 9] [36] WFRQAPGKQRELVA [49] [SEQ ID
NO: 10] [36] WFRQAPGKQREFVA [49]
[0456] and/or from the group consisting of amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0457] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 5; and/or [0458] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0459] (3) the Hallmark residues at positions 37,
44, 45 and 47 are as indicated in each of the sequences above; and
in which: [0460] iii) FR3 is chosen from the group consisting of
the amino acid sequence:
TABLE-US-00032 [0460] [SEQ ID NO: 12] [66]
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]
[0461] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0462] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0463] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0464] (3) the Hallmark residues at positions 83
and 84 are as indicated in each of the sequences above; and in
which: [0465] iv) FR4 is chosen from the group consisting of the
amino acid sequences:
TABLE-US-00033 [0465] [SEQ ID NO: 13] [103] WGQGTQVTVSS [113] [SEQ
ID NO: 14] [103] WGQGTLVTVSS [113]
[0466] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0467] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 7; and/or [0468] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0469] (3) the Hallmark residues at positions 103,
104 and 108 are as indicated in each of the sequences above; and in
which: [0470] v) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred definitions
above, and are more preferably as defined according to one of the
more preferred definitions above.
[0471] In another preferred, but not limiting aspect, a Nanobody of
the invention can have the structure [0472]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: and in which [0473] i) FR1 is chosen
from the group consisting of the amino acid sequence:
TABLE-US-00034 [0473] [SEQ ID NO: 5] [1] QVQLQESGGGLVQAGGSLRLSCAASG
[26]
[0474] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0475] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0476] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0477] (3) the Hallmark residue at position is as
indicated in the sequence above; and in which: [0478] ii) FR2 is
chosen from the group consisting of the amino acid sequence:
TABLE-US-00035 [0478] [SEQ ID NO: 11] [36] WYRQAPGKGLEWA [49]
[0479] and/or from the group consisting of amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0480] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 5; and/or [0481] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0482] (3) the Hallmark residues at positions 37,
44, 45 and 47 are as indicated in each of the sequences above; and
in which: [0483] iii) FR3 is chosen from the group consisting of
the amino acid sequence:
TABLE-US-00036 [0483] [SEQ ID NO: 12] [66]
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94]
[0484] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0485] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0486] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0487] (3) the Hallmark residues at positions 83
and 84 are as indicated in each of the sequences above; and in
which: [0488] iv) FR4 is chosen from the group consisting of the
amino acid sequence:
TABLE-US-00037 [0488] [SEQ ID NO: 13] [103] WGQGTQVTVSS [113]
[0489] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0490] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 7; and/or [0491] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequence(s); and [0492] (3) the Hallmark residues at positions 103,
104 and 108 are as indicated in each of the sequences above; and in
which: [0493] v) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred definitions
above, and are more preferably as defined according to one of the
more preferred definitions above.
[0494] In another preferred, but not limiting aspect, a Nanobody of
the invention can have the structure [0495]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and in which: and in which [0496] i) FR1 is chosen
from the group consisting of the FR1 sequences present in the
Nanobodies of SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97, and in
particular in the humanized Nanobodies of SEQ ID NO's 86 to 97,
[0497] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of said FR1 sequences; in which [0498] (1)
any amino acid substitution at any position other than a Hallmark
position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0499] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to said FR1 sequence; and
[0500] (3) the Hallmark residue at position is as indicated in said
FR1 sequence; [0501] and/or from the group consisting of amino acid
sequences that have 3, 2 or only 1 "amino acid difference(s)" (as
defined herein) with one of said FR1 sequences, in which: [0502]
(1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 4; and/or [0503] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to said FR1 sequence; and
[0504] (3) the Hallmark residue at position is as indicated in said
FR1 sequence; and in which: [0505] ii) FR2 is chosen from the group
consisting of the FR2 sequences present in the Nanobodies of SEQ ID
NO's 60 to 73 and SEQ ID NO's 86 to 97, and in particular in the
humanized Nanobodies of SEQ ID NO's 86 to 97, [0506] or from the
group consisting of amino acid sequences that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity (as defined herein) with
one of said FR2 sequences; in which [0507] (1) any amino acid
substitution at any position other than a Hallmark position is
preferably either a conservative amino acid substitution (as
defined herein) and/or an amino acid substitution as defined in
Table 5; and/or [0508] (2) said amino acid sequence preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to said FR2 sequence; and [0509] (3) the
Hallmark residues at positions 37, 44, 45 and 47 are as indicated
in said FR2 sequence; [0510] and/or from the group consisting of
amino acid sequences that have 3, 2 or only 1 "amino acid
difference(s)" (as defined herein) with one of said FR2 sequences,
in which: [0511] (1) any amino acid substitution at any position
other than a Hallmark position is preferably either a conservative
amino acid substitution (as defined herein) and/or an amino acid
substitution as defined in Table 5; and/or [0512] (2) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to said FR2
sequence; and [0513] (3) the Hallmark residues at positions 37, 44,
45 and 47 are as indicated in said FR2 sequence; and in which:
[0514] iii) FR3 is chosen from the group consisting of the FR3
sequences present in the Nanobodies of SEQ ID NO's 60 to 73 and SEQ
ID NO's 86 to 97, and in particular in the humanized Nanobodies of
SEQ ID NO's 86-97, [0515] or from the group consisting of amino
acid sequences that have at least 80%, preferably at least 90%,
more preferably at least 95%, even more preferably at least 99%
sequence identity (as defined herein) with one of said FR3
sequences; in which [0516] (1) any amino acid substitution at any
position other than a Hallmark position is preferably either a
conservative amino acid substitution (as defined herein) and/or an
amino acid substitution as defined in Table 6; and/or [0517] (2)
said amino acid sequence preferably only contains amino acid
substitutions, and no amino acid deletions or insertions, compared
to said FR3 sequence; and [0518] (3) the Hallmark residues at
positions 83 and 84 are as indicated in said FR3 sequence; [0519]
and/or from the group consisting of amino acid sequences that have
3, 2 or only 1 "amino acid difference(s)" (as defined herein) with
one of said FR3 sequences, in which: [0520] (1) any amino acid
substitution at any position other than a Hallmark position is
preferably either a conservative amino acid substitution (as
defined herein) and/or an amino acid substitution as defined in
Table 6; and/or [0521] (2) said amino acid sequence preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to said FR3 sequence; and [0522] (3) the
Hallmark residues at positions 83 and 84 are as indicated in said
FR3 sequence; and in which: [0523] iv) FR4 is chosen from the group
consisting of the FR4 sequences present in the Nanobodies of SEQ ID
NO's 60 to 73 and SEQ ID NO's 86 to 97, and in particular in the
humanized Nanobodies of SEQ ID NO's 86 to 97, [0524] or from the
group consisting of amino acid sequences that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity (as defined herein) with
one of said FR4 sequences; in which [0525] (1) any amino acid
substitution at any position other than a Hallmark position is
preferably either a conservative amino acid substitution (as
defined herein) and/or an amino acid substitution as defined in
Table 6; and/or [0526] (2) said amino acid sequence preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to said FR4 sequence; and [0527] (3) the
Hallmark residues at positions 103, 104 and 108 are as indicated in
said FR3 sequence; [0528] and/or from the group consisting of amino
acid sequences that have 3, 2 or only 1 "amino acid difference(s)"
(as defined herein) with one of said FR4 sequences, in which:
[0529] (1) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Table 6; and/or [0530] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to said FR4 sequence; and
[0531] (3) the Hallmark residues at positions 103, 104 and 108 are
as indicated in said FR4 sequence; and in which: [0532] v) CDR1,
CDR2 and CDR3 are as defined herein, and are preferably as defined
according to one of the preferred definitions above, and are more
preferably as defined according to one of the more preferred
definitions above.
[0533] Some particularly preferred Nanobodies of the invention can
be chosen from the group consisting of the amino acid sequences of
SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97, and in particular in
the humanized Nanobodies of SEQ ID NO's 86 to 97 or from the group
consisting of amino acid sequences that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity (as defined herein) with
one of the amino acid sequences of SEQ ID NO's 60 to 73 and SEQ ID
NO's 86 to 97 (and preferably of SEQ ID NO's 86 to 97); in which
[0534] (1) the Hallmark residues can be as indicated in Table 2
above; [0535] (2) any amino acid substitution at any position other
than a Hallmark position is preferably either a conservative amino
acid substitution (as defined herein) and/or an amino acid
substitution as defined in Tables 4-7; and/or [0536] (3) said amino
acid sequence preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the above
amino acid sequence(s).
[0537] Some even more particularly preferred Nanobodies of the
invention can be chosen from the group consisting of the amino acid
sequences of SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97, and in
particular in the humanized Nanobodies of SEQ ID NO's 86 to 97 or
from the group consisting of amino acid sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity (as defined
herein) with one of the amino acid sequences of SEQ ID NO's 60 to
73 and SEQ ID NO's 86 to 97 (and preferably of SEQ ID NO's 86 to
97); in which [0538] (1) the Hallmark residues are as indicated in
the pertinent sequence chosen from SEQ ID NO's 60 to 73 and SEQ ID
NO's 86 to 97 (and preferably from SEQ ID NO's 86 to 97); [0539]
(2) any amino acid substitution at any position other than a
Hallmark position is preferably either a conservative amino acid
substitution (as defined herein) and/or an amino acid substitution
as defined in Tables 4-7; and/or [0540] (3) said amino acid
sequence preferably only contains amino acid substitutions, and no
amino acid deletions or insertions, compared to the pertinent
sequence chosen from SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97
(and preferably from SEQ ID NO's 86 to 97).
[0541] Some of the most preferred Nanobodies of the invention can
be chosen from the group consisting of the amino acid sequences of
SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97, and in particular
from the humanized Nanobodies of SEQ ID NO's 86 to 97.
[0542] As will be clear from the above, the term Nanobodies of the
invention as used herein in its broadest sense also comprises
natural or synthetic mutants, variants, alleles, analogs and
orthologs (hereinbelow collectively referred to as "analogs") of
the Nanobodies mentioned in the SEQ ID NO's 60 to 73 and SEQ ID
NO's 86 to 97.
[0543] Generally, such analogs can for example comprise homologous
sequences, functional portions, or a functional portion of a
homologous sequence (as further defined below) of a Nanobody.
Generally, in such analogs, each amino acid residue (other than the
Hallmark Residue) in each of the framework regions can be replaced
by any other amino acid residue, provided that the total degree of
sequence identity of the framework regions remains as defined
herein. Preferably, however, in such analogs: [0544] one or amino
acid residues in the above framework sequences are replaced by one
or more amino acid residues that naturally occur at the same
position in a naturally occurring V.sub.HH domain. Some examples of
such substitutions are mentioned in Tables 4-7 above; and/or:
[0545] one or amino acid residues in the above framework sequences
are replaced by one or more amino acid residues that can be
considered a "conservative" amino acid substitution, as described
hereinabove; and/or: [0546] one or amino acid residues in the above
framework sequences are replaced by one or more amino acid residues
that naturally occur at the same position in a naturally occurring
V.sub.H domain of a human being. This is generally referred to as
"humanization" of the naturally occurring V.sub.HH/Nanobody in
general and of said position in particular, and will be discussed
in more detail hereinbelow; and: [0547] positions for which only
one amino acid residue is mentioned for both the V.sub.H domain and
the V.sub.HH domain in Tables 4-7 above are preferably not
replaced.
[0548] Also, although generally less preferred, in such analogs,
one or more amino acid residues may be deleted from the framework
regions and/or inserted into the framework regions (optionally in
addition to one or more amino acid substitutions as mentioned
above),
[0549] provided that the total degree of sequence identity of the
framework regions remains as defined herein. The Hallmark residues
should not be deleted. Also, most preferably, amino acid residues
for which only one amino acid residue is mentioned for both the
V.sub.H domain and the V.sub.HH domain in Tables 4-7 above are
preferably not deleted.
[0550] Preferably, such analogs should be such that they still can
bind to, have affinity for and/or have specificity for vWF, i.e.
with an affinity and/or a specificity which is at least 10%,
preferably at least 50%, more preferably at least 70%, even more
preferably at least 80%, such as at least 90%, at least 95%, at
least 99% or more, of the affinity and/or specificity of at least
one of the Nanobodies of SEQ ID No's 60 to 73 and SEQ ID NO's 86 to
97, as determined using a suitable assay, for example an assay to
determine binding of the analog to vWF, and in particular one of
the assays as used in the Examples below.
[0551] Generally, such analogs can for example be obtained by
providing a nucleic acid that encodes a naturally occurring
V.sub.HH domain, changing the codons for the one or more amino acid
residues that are to be humanized into the codons for the
corresponding human amino acid residue(s), expressing the nucleic
acid/nucleotide sequence thus obtained in a suitable host or
expression system; and optionally isolating and/or purifying the
analog thus obtained to provide said analog in essentially isolated
form (as defined hereinabove). This can generally be performed
using methods and techniques known per se, which will be clear to
the skilled person, for example from the handbooks and references
cited herein and/or from the further description hereinbelow.
Alternatively, and for example, a nucleic acid encoding an analog
can be synthesized in a manner known per se (for example using an
automated apparatus for synthesizing nucleic acid sequences with a
predefined amino acid sequence) and can be expressed in a suitable
host or expression system, upon which the analog thus obtained can
optionally be isolated and/or purified so as to provide said analog
in essentially isolated form (as defined hereinabove). Another way
to provide the analogs involves chemical synthesis of the pertinent
amino acid sequence using techniques for peptide synthesis known
per se, such as those mentioned hereinbelow.
[0552] It will be also generally be clear to the skilled person
that Nanobodies (including analogs thereof) can also be prepared
starting from human V.sub.H sequences (i.e. amino acid sequences or
the corresponding nucleotide sequences), such as for example human
V.sub.H3 sequences such as DP-47, DP-51 or DP-29, by changing one
or more amino acid residues in the amino acid sequence of said
human V.sub.H domain, so as to provide an amino acid sequence that
has (a) a Q at position 108; and/or (b) E at position 44 and/or R
at position 45, and preferably E at position 44 and R at position
45; and/or (c) P, R or S at position 103, as described above.
Again, this can generally be performed using the various methods
and techniques referred to in the previous paragraph, using an
amino acid sequence and/or nucleotide sequence for a human V.sub.H
domain as a starting point.
[0553] The term Nanobodies as used herein in its broadest sense
also comprises parts or fragments of the Nanobodies (including
analogs) of the invention as defined herein, which can again be as
further described below.
[0554] Generally, parts or fragments of the Nanobodies and/or
analogs have amino acid sequences in which, compared to the amino
acid sequence of the corresponding full length Nanobody or analog,
one or more of the amino acid residues at the N-terminal end, one
or more amino acid residues at the C-terminal end, one or more
contiguous internal amino acid residues, or any combination
thereof, have been deleted and/or removed. It is also possible to
combine one or more of such parts or fragments to provide a
Nanobody of the invention.
[0555] Preferably, the amino acid sequence of a Nanobody that
comprises one or more parts or fragments of a full length Nanobody
and/or analog should have a degree of sequence identity of at least
50%, preferably at least 60%, more preferably at least 70%, such as
at least 80%, at least 90% or at least 95%, with the amino acid
sequence of the corresponding full length Nanobody.
[0556] Also, the amino acid sequence of a Nanobody that comprises
one or more parts or fragments of a full length Nanobody and/or
analog is preferably such that is comprises at least 10 contiguous
amino acid residues, preferably at least 20 contiguous amino acid
residues, more preferably at least 30 contiguous amino acid
residues, such as at least 40 contiguous amino acid residues, of
the amino acid sequence of the corresponding full length
Nanobody.
[0557] Generally, such parts or fragments of the Nanobodies of the
invention will have amino acid sequences in which, compared to the
amino acid sequence of the corresponding full length Nanobody of
the invention, one or more of the amino acid residues at the
N-terminal end, one or more amino acid residues at the C-terminal
end, one or more contiguous internal amino acid residues, or any
combination thereof, have been deleted and/or removed. It is also
possible to combine one or more of such parts or fragments to
provide a Nanobody of the invention.
[0558] According to one preferred embodiment, a fragment as used
herein comprises at least one of the CDR's present in a full-sized
Nanobody of the invention, preferably at least two of the CDR's
present in a full-sized Nanobody of the invention, more preferably
at least CDR2 and CDR3 present in a full-sized Nanobody of the
invention, such as for example all three CDR's present in a
full-sized Nanobody of the invention.
[0559] According to another particularly preferred, but
non-limiting embodiment, such a part or fragment comprises at least
FR3, CDR3 and FR4 of the corresponding full length Nanobody of the
invention, i.e. as for example described in the International
application WO 03/050531 (Lasters et al.).
[0560] Preferably, such parts or fragments should be such that they
still can bind to, have affinity for and/or have specificity for
vWF, i.e. with an affinity and/or a specificity which is at least
10%, preferably at least 50%, more preferably at least 70%, even
more preferably at least 80%, such as at least 90%, at least 95%,
at least 99% or more, of the affinity and/or specificity of the
corresponding full-sized Nanobody of the invention, for example an
assay to determine binding of the analog to vWF, and in particular
one of the assays as used in the Examples below.
[0561] From the description hereinabove, it will be clear that the
amino acid sequences of the Nanobodies used herein differ at at
least one amino acid position in at least one of the framework
regions from the amino acid sequences of naturally occurring
V.sub.H domains, such as the amino acid sequences of naturally
occurring V.sub.H domains of antibodies from human beings. In
particular, it will be clear that the amino acid sequences of the
Nanobodies used herein differ at at least one of the Hallmark
Residues from amino acid sequences of naturally occurring V.sub.H
domains, such as the amino acid sequences of naturally occurring
V.sub.H domains from antibodies from Camelids and/or human
beings.
[0562] Thus, according to one specific embodiment, a Nanobody of
the invention has an amino acid sequence that differs at at least
one amino acid position in one of the framework regions from the
amino acid sequence of a naturally occurring V.sub.H domain.
According to a more specific, but non-limiting embodiment of the
invention, a Nanobody of the invention has an amino acid sequence
that differs at at least one of the Hallmark residues from the
amino acid sequence of a naturally occurring V.sub.H domain.
[0563] From the description hereinabove, it will also be clear that
the amino acid sequences of the some of the Nanobodies of the
invention, such as the humanized Nanobodies of the invention, will
differ at at least one amino acid position in at least one of the
framework regions (i.e. either at the position of a Hallmark
residue or at another position) from the amino acid sequences of
naturally occurring V.sub.HH domains. Thus, according to one
specific, but non-limiting embodiment, a Nanobody of the invention
has an amino acid sequence that differs at at least one amino acid
position in one of the framework regions from the amino acid
sequence of a naturally occurring V.sub.HH domain. According to a
more specific, but non-limiting embodiment of the invention, a
Nanobody of the invention has an amino acid sequence that differs
at at least one of the Hallmark residues from the amino acid
sequence of a naturally occurring V.sub.HH domain.
[0564] As mentioned above, the invention also relates to proteins
or polypeptides comprising at least one V.sub.HH domain (i.e. as
identified using the methods of the invention) or at least one
Nanobody based thereon.
[0565] According to one non-limiting embodiment of the invention,
such a polypeptide of the invention essentially consists of a
Nanobody. By "essentially consist of" is meant that the amino acid
sequence of the polypeptide of the invention either is exactly the
same as the amino acid sequence of a Nanobody (as mentioned above)
or corresponds to the amino acid sequence of a Nanobody in which a
limited number of amino acid residues, such as 1-10 amino acid
residues and preferably 1-6 amino acid residues, such as 1, 2, 3,
4, 5 or 6 amino acid residues, have been added to the amino
terminal end, to the carboxy terminal end, or both to the amino
terminal end and to the carboxy terminal end of the amino acid
sequence of the Nanobody.
[0566] Said amino acid residues may or may not change, alter or
otherwise influence the (biological) properties of the Nanobody and
may or may not add further functionality to the Nanobody. For
example, said amino acid residues may: [0567] a) form a "tag", i.e.
an amino acid sequence or residue that allows or facilitates the
purification of the Nanobody, for example using affinity techniques
directed against said sequence or residue. Thereafter, said
sequence or residue may be removed (e.g. by chemical or enzymatical
cleavage) to provide the nucleotide sequence of the invention (for
this purpose, the sequence or residue may optionally be linked to
the amino acid sequence of the invention via a cleavable linker
sequence). Some preferred, but non-limiting examples of such
residues are multiple histidine residues and glutatione residues,
[0568] b) can be a N-terminal Met residue, for example as result of
expression in a heterologous host cell or host organism. [0569] c)
may be one or more amino acid residues that can be provided with
functional groups and/or that have been functionalized, in a manner
known per se. For example, as is known in the art, amino acid
residues such as lysine and in particular cysteine allow for the
attachment of PEG groups, which may mask surface site on a protein
and thus for example decrease immunogenicity, improve half-life in
plasma and stabilize against proteolytic cleavage; [0570] d)
increase the half-life in serum of a Nanobody or polypeptide of the
invention Amino acid sequences that can be attached to and/or fused
with therapeutic proteins in order to increase their half-life in
vivo are well know to the skilled person and include human serum
proteins or fragments thereof (such as human serum albumin or a
part or fragment thereof), or even Fc portions of antibodies (in
particular of human antibodies). Also, as already described herein,
such an amino acid sequence for increasing the half-life may be an
amino acid sequence directed against a serum protein, such as a
Nanobody directed against a serum protein, for example against
human serum albumin.
[0571] With regard to pegylation, its should be noted that
generally, the invention also encompasses any Nanobody of the
invention and/or polypeptide of the invention that has been
pegylated at one or more amino acid positions, preferably in such a
way that said pegylation either (1) increases the half-life in
vivo; (2) reduces immunogenicity; (3) provides one or more further
beneficial properties known per se for pegylation; (4) does not
essentially affect the affinity of the Nanobody and/or polypeptide
for vWF (e.g. does not reduce said affinity by more than 90%,
preferably not by more than 50%, and more preferably not by more
than 10%, as determined by a suitable assay, such as those
described in the Examples below); and/or (4) does not affect any of
the other desired properties of the Nanobodies and/or polypeptides
of the invention. Suitable PEG-groups and methods for attaching
them, either specifically or non-specifically, will be clear to the
skilled person. Suitable kits and reagents for such pegylation can
for example be obtained from Nektar (CA, USA).
[0572] According to one non-limiting embodiment, one or more amino
acid residues can be added to, inserted in and/or substituted in
the amino acid sequence of a Nanobody or polypeptide of the
invention, so as to provide one or more specific amino acid
residues for attachment of a PEG-group.
[0573] The invention also encompasses any Nanobody of the invention
and/or polypeptide of the invention that has been glycosylated at
one or more amino acid positions, usually depending upon the host
used to express the Nanobody or polypeptide of the invention (as
further described below).
[0574] According to one non-limiting embodiment, one or more amino
acid residues can be added to, inserted in and/or substituted in
the amino acid sequence of a Nanobody or polypeptide of the
invention, so as to provide one or more specific amino acid
residues and/or a site that can be glycosylated by the host
organism used. By means of a preferred, but non-limiting example,
the N-residue on position 50 within CDR2 of a Nanobody of the
invention can for example be replaced by a Q, D or S residue so as
to provide a glycosylation site, e.g. for glycosylation by
Pichia.
[0575] According to another embodiment, a polypeptide of the
invention can comprise a the amino acid sequence of a Nanobody,
which is fused at its amino terminal end, at its carboxy terminal
end, or both at its amino terminal end and at its carboxy terminal
end with at least one further amino acid sequence.
[0576] Again, said further amino acid sequence(s) may or may not
change, alter or otherwise influence the (biological) properties of
the Nanobody and may or may not add further functionality to the
Nanobody.
[0577] For example, according to one preferred, but non-limiting
embodiment, said further amino acid sequence may comprise at least
one further Nanobody, so as to provide a polypeptide of the
invention that comprises at least two, such as three, four or five,
Nanobodies, in which said Nanobodies may optionally be linked via
one or more linker sequences (as defined herein).
[0578] Polypeptides of the invention comprising two or more
Nanobodies will also referred to herein as "multivalent"
polypeptides. For example a "bivalent" polypeptide of the Invention
comprises two Nanobodies, optionally linked via a linker sequence,
whereas a "trivalent" polypeptide of the invention comprises three
Nanobodies, optionally linked via two linker sequences; etc.
[0579] In a multivalent polypeptide of the invention, the two or
more Nanobodies may be the same or different. For example, the two
or more Nanobodies in a multivalent polypeptide of the invention:
[0580] may be directed against the same antigen, i.e. against the
same parts or epitopes of said antigen or against two or more
different parts or epitopes of said antigen; and/or: [0581] may be
directed against the different antigens; or a combination
thereof.
[0582] Thus, a bivalent polypeptide of the invention for example:
[0583] may comprise two identical Nanobodies; [0584] may comprise a
first Nanobody directed against a first part or epitope of an
antigen and a second Nanobody directed against the same part or
epitope of said antigen or against another part or epitope of said
antigen; [0585] or may comprise a first Nanobody directed against a
first antigen and a second Nanobody directed against a second
antigen different from said first antigen; whereas a trivalent
Polypeptide of the Invention for example: [0586] may comprises
three identical or different Nanobodies directed against the same
or different parts or epitopes of the same antigen; [0587] may
comprise two identical or different Nanobodies directed against the
same or different parts or epitopes on a first antigen and a third
Nanobody directed against a second antigen different from said
first antigen; or [0588] may comprise a first Nanobody directed
against a first antigen, a second Nanobody directed against a
second antigen different from said first antigen, and a third
Nanobody directed against a third antigen different from said first
and second antigen,
[0589] Polypeptides of the invention that contain at least two
Nanobodies, in which at least one Nanobody is directed against a
first antigen and at least one Nanobody is directed against a
second Nanobody different from the first antigen, will also be
referred to as "multispecific" Nanobodies. Thus, a "bispecific"
Nanobody is a Nanobody that comprises at least one Nanobody
directed against a first antigen and at least one further Nanobody
directed against a second antigen, whereas a "trispecific" Nanobody
is a Nanobody that comprises at least one Nanobody directed against
a first antigen, at least one further Nanobody directed against a
second antigen, and at least one further Nanobody directed against
a third antigen; etc.
[0590] Accordingly, in their simplest form, a bispecific
polypeptide of the invention is a bivalent polypeptide of the
invention (as defined herein), comprising a first Nanobody directed
against a first antigen and a second Nanobody directed against a
second antigen, in which said first and second Nanobody may
optionally be linked via a linker sequence (as defined herein);
whereas a trispecific polypeptide of the invention in its simplest
form is a trivalent polypeptide of the invention (as defined
herein), comprising a first Nanobody directed against a first
antigen, a second Nanobody directed against a second antigen and a
third Nanobody directed against a third antigen, in which said
first, second and third Nanobody may optionally be linked via one
or more, and in particular one and more in particular two, linker
sequences.
[0591] However, as will be clear from the description hereinabove,
the invention is not limited thereto, in the sense that a
multispecific polypeptide of the invention may comprise any number
of Nanobodies directed against two or more different antigens. For
multivalent and multispecific polypeptides containing one or more
V.sub.HH domains and their preparation, reference is also made to
Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, as well as
to EP 0 822 985.
[0592] Linkers for use in multivalent and multispecific
polypeptides will be clear to the skilled person, and for example
include gly-ser linkers, for example of the type
(gly.sub.xser.sub.y).sub.z, such as (for example
(gly.sub.4ser).sub.3 or (gly.sub.3ser.sub.2).sub.3, as described in
WO 99/42077, hinge like regions such as the hinge regions of
naturally occurring heavy chain antibodies or similar sequences.
For other suitable linkers, reference is also made to the general
background art cited above. Some particularly preferred linkers are
given in SEQ ID NO's 83 to 85, in which the linkers of SEQ ID NO's
84 and 85 are particularly preferred.
[0593] Linkers can also provide some functionality for the
multivalent or multispecific polypeptide. For example, linkers
containing one or more charged amino acid residues (see Table 1
above) can provide improved hydrophilic properties, whereas linkers
that form or contain small epitopes or tags can be used for the
purposes of detection, identification and/or purification.
[0594] As also further described herein, a multispecific
polypeptide of the invention directed against a desired antigen and
against at least one serum protein, such as the serum proteins
mentioned hereinbelow, and in particular against human serum
albumin, may show increased half-life in serum, compared to the
corresponding monovalent Nanobody.
[0595] As mentioned hereinabove, the methods described herein are
particularly suited for generating such multivalent of
multispecific polypeptides of the invention.
[0596] In a polypeptide of the invention, the at least one Nanobody
may also be linked to a conventional V.sub.H domain or to a natural
or synthetic analog of a V.sub.H domain, optionally via a linker
sequence.
[0597] In a polypeptide of the invention, the at least one Nanobody
may also be linked to a V.sub.L domain or to a natural or synthetic
analog of a V.sub.L domain, optionally via a linker sequence, so as
to provide a polypeptide of the invention that is in the form
analogous to a conventional scFv fragment, but containing a
Nanobody instead of a V.sub.H domain.
[0598] In a polypeptide of the invention, the at least one Nanobody
may also be linked to one or more of a CH1, CH2 and/or CH3 domain,
optionally via a linker sequence. For instance, a Nanobody linked
to a suitable CH1 domain could for example be used--together with
suitable light chains--to generate antibody fragments/structures
analogous to conventional Fab fragments or F(ab').sub.2 fragments,
but in which one or (in case of an F(ab').sub.2 fragment) one or
both of the conventional V.sub.H domains have been replaced by a
Nanobody. Such fragments may also be heterospecific or bispecific,
i.e. directed against two or more antigens. A Nanobody linked to
suitable CH2 and CH3 domains, for example derived from Camelids,
could be used to form a monospecific or bispecific heavy chain
antibody. Finally, a Nanobody linked to suitable CH1, CH2 and CH3
domains, for example derived from a human being, could be
used--together with suitable light chains--to form an antibody that
is analogous to a conventional 4-chain antibody, but in which one
or both of the conventional V.sub.H domains have been replaced by a
Nanobody.
[0599] Also, in addition to the one or more Nanobodies,
Polypeptides of the Invention can also contain functional groups,
moieties or residues, for example therapeutically active
substances, such as those mentioned below, and/or markers or
labels, such as fluorescent markers, isotopes, etc., as further
described hereinbelow.
[0600] The Nanobodies of the invention, the polypeptides of the
invention, and nucleic acids encoding the same, can be prepared in
a manner known per se, asd will be clear to the skilled person from
the further description herein. Some preferred, but non-limiting
methods for preparing the Nanobodies, polypeptides and nucleic
acids include the methods and techniques mentioned above and/or
further described hereinbelow.
[0601] As will be clear to the skilled person, one particularly
useful method for preparing a Nanobody and/or a polypeptide of the
invention generally comprises the steps of: [0602] the expression,
in a suitable host cell or host organism (also referred to herein
as a "host of the invention") or in another suitable expression
system of a nucleic acid that encodes said Nanobody or polypeptide
of the invention (also referred to herein as a "nucleic acid of the
invention"), optionally followed by: [0603] isolating and/or
purifying the Nanobody or polypeptide of the invention thus
obtained.
[0604] In particular, such a method may comprise the steps of:
[0605] cultivating and/or maintaining a host of the invention under
conditions that are such that said host of the invention expresses
and/or produces at least one Nanobody and/or polypeptide of the
invention; optionally followed by: [0606] isolating and/or
purifying the Nanobody or polypeptide of the invention thus
obtained.
[0607] A nucleic acid of the invention can be in the form of single
or double stranded DNA or RNA, and is preferably in the form of
double stranded DNA. For example, the nucleotide sequences of the
invention may be genomic DNA, cDNA or synthetic DNA (such as DNA
with a codon usage that has been specifically adapted for
expression in the intended host cell or host organism).
[0608] According to one embodiment of the invention, the nucleic
acid of the invention is in essentially isolated from, as defined
hereinabove.
[0609] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a vector, such as for example a
plasmid, cosmid or YAC, which again may be in essentially isolated
form.
[0610] The nucleic acids of the invention can be prepared or
obtained in a manner known per se, based on the information on the
amino acid sequences for the polypeptides of the invention given
herein, and/or can be isolated from a suitable natural source. To
provide analogs, nucleotide sequences encoding naturally occurring
V.sub.HH domains can for example be subjected to site-directed
mutagenesis, so at to provide a nucleic acid of the invention
encoding said analog. Also, as will be clear to the skilled person,
to prepare a nucleic acid of the invention, also several nucleotide
sequences, such as at least one nucleotide sequence encoding a
Nanobody and for example nucleic acids encoding one or more linkers
can be linked together in a suitable manner.
[0611] Techniques for generating the nucleic acids of the invention
will be clear to the skilled person and may for instance include,
but are not limited to, automated DNA synthesis; site-directed
mutagenesis; combining two or more naturally occurring and/or
synthetic sequences (or two or more parts thereof), introduction of
mutations that lead to the expression of a truncated expression
product; introduction of one or more restriction sites (e.g. to
create casettes and/or regions that may easily be digested and/or
ligated using suitable restriction enzymes), and/or the
introduction of mutations by means of a PCR reaction using one or
more "mismatched" primers, using for example a sequence of a
naturally occurring GPCR as a template. These and other techniques
will be clear to the skilled person, and reference is again made to
the standard handbooks, such as Sambrook et al. and Ausubel et al.,
mentioned above, as well as the Examples below.
[0612] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a genetic construct, as will be
clear to the person skilled in the art. Such genetic constructs
generally comprise at least one nucleic acid of the invention that
is optionally linked to one or more elements of genetic constructs
known per se, such as for example one or more suitable regulatory
elements (such as a suitable promoter(s), enhancer(s),
terminator(s), etc.) and the further elements of genetic constructs
referred to hereinbelow. Such genetic constructs comprising at
least one nucleic acid of the invention will also be referred to
herein as "genetic constructs of the invention".
[0613] The genetic constructs of the invention may be DNA or RNA,
and are preferably double-stranded DNA. The genetic constructs of
the invention may also be in a form suitable for transformation of
the intended host cell or host organism, in a form suitable for
integration into the genomic DNA of the intended host cell or in a
form suitable independent replication, maintenance and/or
inheritance in the intended host organism. For instance, the
genetic constructs of the invention may be in the form of a vector,
such as for example a plasmid, cosmid, YAC, a viral vector or
transposon. In particular, the vector may be an expression vector,
i.e. a vector that can provide for expression in vitro and/or in
vivo (e.g. in a suitable host cell, host organism and/or expression
system).
[0614] In a preferred but non-limiting embodiment, a genetic
construct of the invention comprises [0615] a) at least one nucleic
acid of the invention; operably connected to [0616] b) one or more
regulatory elements, such as a promoter and optionally a suitable
terminator; [0617] c) and optionally also [0618] d) one or more
further elements of genetic constructs known per se; in which the
terms "regulatory element", "promoter", "terminator" and "operably
connected" have their usual meaning in the art (as further
described below); and in which said "further elements" present in
the genetic constructs may for example be 3'- or 5'-UTR sequences,
leader sequences, selection markers, expression markers/reporter
genes, and/or elements that may facilitate or increase (the
efficiency of) transformation or integration. These and other
suitable elements for such genetic constructs will be clear to the
skilled person, and may for instance depend upon the type of
construct used, the intended host cell or host organism; the manner
in which the nucleotide sequences of the invention of interest are
to be expressed (e.g. via constitutive, transient or inducible
expression); and/or the transformation technique to be used.
[0619] Preferably, in the genetic constructs of the invention, said
at least one nucleic acid of the invention and said regulatory
elements, and optionally said one or more further elements, are
"operably linked" to each other, by which is generally meant that
they are in a functional relationship with each other. For
instance, a promoter is considered "operably linked" to a coding
sequence if said promoter is able to initiate or otherwise
control/regulate the transcription and/or the expression of a
coding sequence (in which said coding sequence should be understood
as being "under the control of" said promotor). Generally, when two
nucleotide sequences are operably linked, they will be in the same
orientation and usually also in the same reading frame. They will
usually also be essentially contiguous, although this may also not
be required.
[0620] Preferably, the regulatory and further elements of the
genetic constructs of the invention are such that they are capable
of providing their intended biological function in the intended
host cell or host organism.
[0621] For instance, a promoter, enhancer or terminator should be
"operable" in the intended host cell or host organism, by which is
meant that (for example) said promoter should be capable of
initiating or otherwise controlling/regulating the transcription
and/or the expression of a nucleotide sequence--e.g. a coding
sequence--to which it is operably linked (as defined herein).
[0622] Some particularly preferred promoters include, but are not
limited to, promoters known per se for the expression in bacterial
cells, such as those mentioned hereinbelow and/or those used in the
Examples.
[0623] A selection marker should be such that it allows--i.e. under
appropriate selection conditions--host cells and/or host organisms
that have been (succesfully) transformed with the nucleotide
sequence of the invention to be distinguished from host
cells/organisms that have not been (succesfully) transformed. Some
preferred, but non-limiting examples of such markers are genes that
provide resistance against antibiotics (such as kanamycine or
ampicilline), genes that provide for temperature resistance, or
genes that allow the host cell or host organism to be maintained in
the absence of certain factors, compounds and/or (food) components
in the medium that are essential for survival of the
non-transformed cells or organisms.
[0624] A leader sequence should be such that--in the intended host
cell or host organism--it allows for the desired post-translational
modifications and/or such that it directs the transcribed mRNA to a
desired part or organelle of a cell. A leader sequence may also
allow for secretion of the expression product from said cell. As
such, the leader sequence may be any pro-, pre-, or prepro-sequence
operable in the host cell or host organism. Leader sequences may
not be required for expression in a bacterial cell.
[0625] An expression marker or reporter gene should be such
that--in the host cell or host organism--it allows for detection of
the expression of (a gene or nucleotide sequence present on) the
genetic construct. An expression marker may optionally also allow
for the localisation of the expressed product, e.g. in a specific
part or organelle of a cell and/or in (a) specific cell(s),
tissue(s), organ(s) or part(s) of a multicellular organism. Such
reporter genes may also be expressed as a protein fusion with the
amino acid sequence of the invention. Some preferred, but
non-limiting examples include fluorescent proteins such as GFP.
[0626] Some preferred, but non-limiting examples of suitable
promoters, terminator and further elements include those used in
the Examples below. For some (further) non-limiting examples of the
promoters, selection markers, leader sequences, expression markers
and further elements that may be present/used in the genetic
constructs of the invention--such as terminators, transcriptional
and/or translational enhancers and/or integration
factors--reference is made to the general handbooks such as
Sambrook et al. and Ausubel et al. mentioned above, as well as to
the examples that are given in WO 95/07463, WO 96/23810, WO
95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO
98/21355, U.S. Pat. No. 6,207,410, U.S. Pat. No. 5,693,492 and EP 1
085 089. Other examples will be clear to the skilled person.
Reference is also made to the general background art cited above
and the further references cited hereinbelow.
[0627] The genetic constructs of the invention may generally be
provided by suitably linking the nucleotide sequence(s) of the
invention to the one or more further elements described above, for
example using the techniques described in the general handbooks
such as Sambrook et al. and Ausubel et al., mentioned above.
[0628] Often, the genetic constructs of the invention will be
obtained by inserting a nucleotide sequence of the invention in a
suitable (expression) vector known per se. Some preferred, but
non-limiting examples of suitable expression vectors are those used
in the Examples below, as well as those mentioned below.
[0629] The nucleic acids of the invention and/or the genetic
constructs of the invention may be used to transform a host cell or
host organism, i.e. for expression and/or production of the
Nanobody or polypeptide of the invention. Suitable hosts or host
cells will be clear to the skilled person, and may for example be
any suitable fungal, prokaryotic or eukaryotic cell or cell line or
any suitable fungal, prokaryotic or eukaryotic organism, for
example: [0630] a bacterial strain, including but not limited to
gram-negative strains such as strains of Escherichia coli; of
Proteus, for example of Proteus mirabilis; of Pseudomonas, for
example of Pseudomonas fluorescens; and gram-positive strains such
as strains of Bacillus, for example of Bacillus subtilis or of
Bacillus brevis; of Streptomyces, for example of Streptomyces
lividans; of Staphylococcus, for example of Staphylococcus
carnosus; and of Lactococcus, for example of Lactococcus lactis;
[0631] a fungal cell, including but not limited to cells from
species of Trichoderma, for example from Trichoderma reesei; of
Neurospora, for example from Neurospora crassa; of Sordaria, for
example from Sordaria macrospora; of Aspergillus, for example from
Aspergillus niger or from Aspergillus sojae; or from other
filamentous fungi; [0632] a yeast cell, including but not limited
to cells from species of Saccharomyces, for example of
Saccharomyces cerevisiae; of Schizosaccharomyces, for example of
Schizosaccharomyces pombe; of Pichia, for example of Pichia
pastoris or of Pichia methanolica; of Hansenula, for example of
Hansenula polymorpha; of Kluyveromyces, for example of
Kluyveromyces lactis; of Arxula, for example of Arxula
adeninivorans; of Yarrowia, for example of Yarrowia lipolytica;
[0633] an amphibian cell or cell line, such as Xenopus oocytes;
[0634] an insect-derived cell or cell line, such as cells/cell
lines derived from lepidoptera, including but not limited to
Spodoptera SF9 and Sf21 cells or cells/cell lines derived from
Drosophila, such as Schneider and Kc cells; [0635] a plant or plant
cell, for example in tobacco plants; and/or [0636] a mammalian cell
or cell line, for example derived a cell or cell line derived from
a human, from the mammals including but not limited to CHO-cells,
BHK-cells (for example BHK-21 cells) and human cells or cell lines
such as HeLa, COS (for example COS-7) and PER.C6 cells; as well as
all other hosts or host cells known per se for the expression and
production of antibodies and antibody fragments (including but not
limited to (single) domain antibodies and ScFv fragments), which
will be clear to the skilled person. Reference is also made to the
general background art cited hereinabove, as well as to for example
WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al., (1998),
supra; Riechmann and Muyldermans, (1999), supra; van der Linden,
(2000), supra; Thomassen et al., (2002), supra; Joosten et al.,
(2003), supra; Joosten et al., (2005), supra; and the further
references cited herein.
[0637] The Nanobodies and polypeptides of the invention can also be
introduced and expressed in one or more cells, tissues or organs of
a multicellular organism, for example for prophylactic and/or
therapeutic purposes (e.g. as a gene therapy). For this purpose,
the nucleotide sequences of the invention may be introduced into
the cells or tissues in any suitable way, for example as such (e.g.
using liposomes) or after they have been inserted into a suitable
gene therapy vector (for example derived from retroviruses such as
adenovirus, or parvoviruses such as adeno-associated virus). As
will also be clear to the skilled person, such gene therapy may be
performed in vivo and/or in situ in the body of a patent by
administering a nucleic acid of the invention or a suitable gene
therapy vector encoding the same to the patient or to specific
cells or a specific tissue or organ of the patient; or suitable
cells (often taken from the body of the patient to be treated, such
as explanted lymphocytes, bone marrow aspirates or tissue biopsies)
may be treated in vitro with a nucleotide sequence of the invention
and then be suitably (re-)introduced into the body of the patient.
All this can be performed using gene therapy vectors, techniques
and delivery systems which are well known to the skilled person,
for Culver, K. W., "Gene Therapy", 1994, p. xii, Mary Ann Liebert,
Inc., Publishers, New York, N.Y.). Giordano, Nature F Medicine 2
(1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson,
Science 256 (1992),808-813; Verma, Nature 389 (1994),239; Isner,
Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77
(1995),1077-1086; Onodera, Blood 91; (1998),30-36; Verma, Gene
Ther. 5 (1998),692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997),
289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang,
Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957, U.S.
Pat. No. 5,580,859; 1 U.S. Pat. No. 5,895,466; or Schaper, Current
Opinion in Biotechnology 7 (1996), 635-640. For example, in situ
expression of ScFv fragments (Afanasieva et al., Gene Ther., 10,
1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171,
1070-1077 (2003)) has been described in the art.
[0638] For expression of the Nanobodies in a cell, they may also be
expressed as so-called or as so-called "intrabodies", as for
example described in WO 94/02610, WO 95/22618 and U.S. Pat. No.
7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997)
Intracellular Antibodies: Development and Applications. Landes and
Springer-Verlag; and in Kontermann, Methods 34, (2004),
163-170.
[0639] For production, the Nanobodies and polypeptides of the
invention can for example also be produced in the milk of
transgenic mammals, for example in the milk of rabbits, cows, goats
or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No.
6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for
introducing transgenes into mammals), in plants or parts of plants
including but not limited to their leaves, flowers, fruits, seed,
roots or turbers (for example in tobacco, maize, soybean or
alfalfa) or in for example pupae of the silkworm Bombix mori.
[0640] Furthermore, the Nanobodies and polypeptides of the
invention can also be expressed and/or produced in cell-free
expression systems, and suitable examples of such systems will be
clear to the skilled person. Some preferred, but non-limiting
examples include expression in the wheat germ system; in rabbit
reticulocyte lysates; or in the E. coli Zubay system.
[0641] As mentioned above, one of the advantages of the use of
Nanobodies is that the polypeptides based thereon can be prepared
through expression in a suitable bacterial system, and suitable
bacterial expression systems, vectors, host cells, regulatory
elements, etc., will be clear to the skilled person, for example
from the references cited above. It should however be noted that
the invention in its broadest sense is not limited to expression in
bacterial systems.
[0642] Preferably, in the invention, an (in vivo or in vitro)
expression system, such as a bacterial expression system, is used
that provides the polypeptides of the invention in a form that is
suitable for pharmaceutical use, and such expression systems will
again be clear to the skilled person. As also will be clear to the
skilled person, Polypeptides of the invention suitable for
pharmaceutical use can be prepared using techniques for peptide
synthesis.
[0643] For production on industrial scale, preferred heterologous
hosts for the (industrial) production of Nanobodies or
Nanobody-containing protein therapeutics include strains of E.
coli, Pichia pastoris, S. cerevisiae that are suitable for large
scale expression/production/fermentation, and in particular for
large scale pharmaceutical expression/production/fermentation.
Suitable examples of such strains will be clear to the skilled
person. Such strains and production/expression systems are also
made available by companies such as Biovitrum (Uppsala,
Sweden).
[0644] Alternatively, mammalian cell lines, in particular Chinese
hamster ovary (CHO) cells, can be used for large scale
expression/production/fermentation, and in particular for large
scale pharmaceutical expression/production/fermentation. Again,
such expression/production systems are also made available by some
of the companies mentioned above.
[0645] The choice of the specific expression system would depend in
part on the requirement for certain post-translational
modifications, more specifically glycosylation. The production of a
Nanobody-containing recombinant protein for which glycosylation is
desired or required would necessitate the use of mammalian
expression hosts that have the ability to glycosylate the expressed
protein. In this respect, it will be clear to the skilled person
that the glycosylation pattern obtained (i.e. the kind, number and
position of residues attached) will depend on the cell or cell line
that is used for the expression. Preferably, either a human cell or
cell line is used (i.e. leading to a protein that essentially has a
human glycosylation pattern) or another mammalian cell line is used
that can provide a glycosylation pattern that is essentially and/or
functionally the same as human glycosylation or at least mimics
human glycosylation. Generally, prokaryotic hosts such as E. coli
do not have the ability to glycosylate proteins, and the use of
lower eukaryotes such as yeast are usually leads to a glycosylation
pattern that differs from human glycosylation. Nevertheless, it
should be understood that all the foregoing host cells and
expression systems can be used in the invention, depending on the
desired Nanobody or protein to be obtained.
[0646] Thus, according to one non-limiting embodiment of the
invention, the Nanobody or polypeptide of the invention is
glycosylated. According to another non-limiting embodiment of the
invention, the Nanobody or polypeptide of the invention is
non-glycosylated.
[0647] According to one preferred, but non-limiting embodiment of
the invention, the Nanobody or polypeptide of the invention is
produced in a bacterial cell, in particular a bacterial cell
suitable for large scale pharmaceutical production, such as cells
of the strains mentioned above.
[0648] According to another preferred, but non-limiting embodiment
of the invention, the Nanobody or polypeptide of the invention is
produced in a yeast cell, in particular a yeast cell suitable for
large scale pharmaceutical production, such as cells of the species
mentioned above.
[0649] According to yet another preferred, but non-limiting
embodiment of the invention, the Nanobody or polypeptide of the
invention is produced in a mammalian cell, in particular in a human
cell or in a cell of a human cell line, and more in particular in a
human cell or in a cell of a human cell line that is suitable for
large scale pharmaceutical production, such as the cell lines
mentioned hereinabove.
[0650] When expression in a host cell is used to produce the
Nanobodies and the proteins of the invention, the Nanobodies and
proteins of the invention can be produced either intracellullarly
(e.g. in the cytosol, in the periplasma or in inclusion bodies) and
then isolated from the host cells and optionally further purified;
or can be produced extracellularly (e.g. in the medium in which the
host cells are cultured) and then isolated from the culture medium
and optionally further purified. When eukaryotic hosts cells are
used, extracellular production is usually preferred since this
considerably facilitates the further isolation and downstream
processing of the Nanobodies and proteins obtained. Bacterial cells
such as the strains of E. coli mentioned above normally do not
secrete proteins extracellularly, except for a few classes of
proteins such as toxins and hemolysin, and secretory production in
E. coli refers to the translocation of proteins across the inner
membrane to the periplasmic space. Periplasmic production provides
several advantages over cytosolic production. For example, the
N-terminal amino acid sequence of the secreted product can be
identical to the natural gene product after cleavage of the
secretion signal sequence by a specific signal peptidase. Also,
there appears to be much less protease activity in the periplasm
than in the cytoplasm. In addition, protein purification is simpler
due to fewer contaminating proteins in the periplasm. Another
advantage is that correct disulfide bonds may form because the
periplasm provides a more oxidative environment than the cytoplasm.
Proteins overexpressed in E. coli are often found in insoluble
aggregates, so-called inclusion bodies. These inclusion bodies may
be located in the cytosol or in the periplasm; the recovery of
biologically active proteins from these inclusion bodies requires a
denaturation/refolding process. Many recombinant proteins,
including therapeutic proteins, are recovered from inclusion
bodies. Alternatively, as will be clear to the skilled person,
recombinant strains of bacteria that have been genetically modified
so as to secrete a desired protein, and in particular a Nanobody or
a polypeptide of the invention, can be used.
[0651] Thus, according to one non-limiting embodiment of the
invention, the Nanobody or polypeptide of the invention is a
Nanobody or polypeptide that has been produced intracellularly and
that has been isolated from the host cell, and in particular from a
bacterial cell or from an inclusion body in a bacterial cell.
According to another non-limiting embodiment of the invention, the
Nanobody or polypeptide of the invention is a Nanobody or
polypeptide that has been produced extracellularly, and that has
been isolated from the medium in which the host cell is
cultivated.
[0652] Some preferred, but non-limiting promoters for use with
these host cells include, [0653] for expression in E. coli: lac
promoter (and derivatives thereof such as the lacUV5 promoter);
arabinose promoter; left- (PL) and rightward (PR) promoter of phage
lambda; promoter of the trp operon; hybrid lac/trp promoters (tac
and trc); T7-promoter (more specifically that of T7-phage gene 10)
and other T-phage promoters; promoter of the Tn10 tetracycline
resistance gene; engineered variants of the above promoters that
include one or more copies of an extraneous regulatory operator
sequence; [0654] for expression in S. cerevisiae: constitutive:
ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c
iso-1), GAPDH (glyceraldehydes-3-phosphate dehydrogenase); PGK1
(phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated: GAL1,
10, 7 (galactose metabolic enzymes), ADH2 (alcohol dehydrogenase
2), PHOS (acid phosphatase), CUP1 (copper metallothionein);
heterologous: CaMV (cauliflower mosaic virus .sup.35S promoter);
[0655] for expression in Pichia pastoris: the AOX1 promoter
(alcohol oxidase I) [0656] for expression in mammalian cells: human
cytomegalovirus (hCMV) immediate early enhancer/promoter; human
cytomegalovirus (hCMV) immediate early promoter variant that
contains two tetracycline operator sequences such that the promoter
can be regulated by the Tet repressor; Herpes Simplex Virus
thymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal
repeat (RSV LTR) enhancer/promoter; elongation factor 1.alpha.
(hEF-1.alpha.) promoter from human, chimpanzee, mouse or rat; the
SV40 early promoter; HIV-1 long terminal repeat promoter;
.beta.-actin promoter;
[0657] Some preferred, but non-limiting vectors for use with these
host cells include: [0658] vectors for expression in mammalian
cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo
(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1
(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt
(ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag
(ATCC 37460) and 1ZD35 (ATCC 37565), as well as viral-based
expression systems, such as those based on adenovirus; [0659]
vectors for expression in bacterials cells: pET vectors (Novagen)
and pQE vectors (Qiagen); [0660] vectors for expression in yeast or
other fungal cells: pYES2 (Invitrogen) and Pichia expression
vectors (Invitrogen); [0661] vectors for expression in insect
cells: pBlueBacII (Invitrogen) and other baculovirus vectors [0662]
vectors for expression in plants or plant cells: for example
vectors based on cauliflower mosaic virus or tobacco mosaic virus,
suitable strains of Agrobacterium, or Ti-plasmid based vectors.
[0663] Some preferred, but non-limiting secretory sequences for use
with these host cells include: [0664] for use in bacterial cells
such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA,
PhoE, MalE, Lpp, LamB, and the like; TAT signal peptide, hemolysin
C-terminal secretion signal [0665] for use in yeast: .alpha.-mating
factor prepro-sequence, phosphatase (phol), invertase (Suc), etc.;
[0666] for use in mammalian cells: indigenous signal in case the
target protein is of eukaryotic origin; murine Ig .kappa.-chain
V-J2-C signal peptide; etc.
[0667] Suitable techniques for transforming a host or host cell of
the invention will be clear to the skilled person and may depend on
the intended host cell/host organism and the genetic construct to
be used. Reference is again made to the handbooks and patent
applications mentioned above.
[0668] After transformation, a step for detecting and selecting
those host cells or host organisms that have been succesfully
transformed with the nucleotide sequence/genetic construct of the
invention may be performed. This may for instance be a selection
step based on a selectable marker present in the genetic construct
of the invention or a step involving the detection of the amino
acid sequence of the invention, e.g. using specific antibodies.
[0669] The transformed host cell (which may be in the form or a
stable cell line) or host organisms (which may be in the form of a
stable mutant line or strain) form further aspects of the present
invention.
[0670] Preferably, these host cells or host organisms are such that
they express, or are (at least) capable of expressing (e.g. under
suitable conditions), an amino acid sequence of the invention (and
in case of a host organism: in at least one cell, part, tissue or
organ thereof). The invention also includes further generations,
progeny and/or offspring of the host cell or host organism of the
invention, that may for instance be obtained by cell division or by
sexual or asexual reproduction.
[0671] To produce/obtain expression of the amino acid sequences of
the invention, the transformed host cell or transformed host
organism may generally be kept, maintained and/or cultured under
conditions such that the (desired) amino acid sequence of the
invention is expressed/produced. Suitable conditions will be clear
to the skilled person and will usually depend upon the host
cell/host organism used, as well as on the regulatory elements that
control the expression of the (relevant) nucleotide sequence of the
invention. Again, reference is made to the handbooks and patent
applications mentioned above in the paragraphs on the genetic
constructs of the invention.
[0672] Generally, suitable conditions may include the use of a
suitable medium, the presence of a suitable source of food and/or
suitable nutrients, the use of a suitable temperature, and
optionally the presence of a suitable inducing factor or compound
(e.g. when the nucleotide sequences of the invention are under the
control of an inducible promoter); all of which may be selected by
the skilled person. Again, under such conditions, the amino acid
sequences of the invention may be expressed in a constitutive
manner, in a transient manner, or only when suitably induced.
[0673] It will also be clear to the skilled person that the amino
acid sequence of the invention may (first) be generated in an
immature form (as mentioned above), which may then be subjected to
post-translational modification, depending on the host cell/host
organism used. Also, the amino acid sequence of the invention may
be glycosylated, again depending on the host cell/host organism
used.
[0674] The amino acid sequence of the invention may then be
isolated from the host cell/host organism and/or from the medium in
which said host cell or host organism was cultivated, using protein
isolation and/or purification techniques known per se, such as
(preparative) chromatography and/or electrophoresis techniques,
differential precipitation techniques, affinity techniques (e.g.
using a specific, cleavable amino acid sequence fused with the
amino acid sequence of the invention) and/or preparative
immunological techniques (i.e. using antibodies against the amino
acid sequence to be isolated).
[0675] Generally, for pharmaceutical use, the polypeptides of the
invention may be formulated as a pharmaceutical preparation
comprising at least one polypeptide of the invention and at least
one pharmaceutically acceptable carrier, diluent or excipient
and/or adjuvant, and optionally one or more further
pharmaceutically active polypeptides and/or compounds. By means of
non-limiting examples, such a formulation may be in a form suitable
for oral administration, for parenteral administration (such as by
intravenous, intramuscular or subcutaneous injection or intravenous
infusion), for topical administration, for administration by
inhalation, by a skin patch, by an implant, by a suppository, etc.
Such suitable administration forms--which may be solid, semi-solid
or liquid, depending on the manner of administration--as well as
methods and carriers for use in the preparation thereof, will be
clear to the skilled person, and are further described
hereinbelow.
[0676] Thus, in a further aspect, the invention relates to a
pharmaceutical composition that contains at least one Nanobody of
the invention or at least one polypeptide of the invention and at
least one suitable carrier (i.e. a carrier suitable for veterinary
use), and optionally one or more further active substances.
[0677] One embodiment of the present invention is a polypeptide
construct comprising: at least one Nanobody of the invention, i.e.
directed against any of vWF, vWF A1 domain, A1 domain of activated
vWF, vWF A3 domain.
[0678] Another embodiment of the present invention is a polypeptide
construct as described above, wherein the Nanobody of the invention
directed against the A1 domain of activated vWF specifically
recognizes the activated vWF conformation at the site of thrombus
formation but does not bind to circulating unactivated forms of
vWF.
[0679] The Nanobodies of the invention may also be directed against
a fragment of vWF, vWF A1 domain, A1 domain of activated vWF, vWF
A3 domain, such as a fragment capable of eliciting an immune
response. A target is also a fragment of vWF, vWF A1 domain, A1
domain of activated vWF, vWF A3 domain, capable of binding to a
Nanobody of the invention raised against the `parent` full length
target.
[0680] A fragment as used herein refers to less than 100% of the
sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%
etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is
of sufficient length such that the interaction of interest is
maintained with affinity of 1.times.10-6 M or better.
[0681] A fragment as used herein also refers to optional
insertions, deletions and substitutions of one or more amino acids
which do not substantially alter the ability of the target to bind
to a Nanobody of the invention raised against the wild-type target.
The number of amino acid insertions deletions or substitutions is
preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69 or 70 amino acids.
[0682] A Nanobody of the invention directed against a target
generally means Nanobody of the invention that it is capable of
binding to its target with an affinity of better than 10.sup.-6
M.
[0683] Another embodiment of the present invention is a polypeptide
construct as described above wherein at least one Nanobody of the
invention is a humanised sequence.
[0684] Another embodiment of the present invention is a polypeptide
construct as described above wherein at least one Nanobody of the
invention is a Camelidae V.sub.HH antibody.
[0685] Another embodiment of the present invention is a polypeptide
construct as described above, wherein said Nanobody of the
invention is an homologous sequence, a functional portion, or a
functional portion of an homologous sequence of the full length
Nanobody of the invention.
[0686] Another embodiment of the present invention is a polypeptide
construct as described above, wherein said polypeptide construct is
a homologous sequence of said polypeptide construct, a functional
portion thereof, of an homologous sequence of a functional portion
thereof.
[0687] Another embodiment of the present invention is a polypeptide
construct as described above, further comprising at least one
Nanobody of the invention directed against one or more serum
proteins, in particular one or more human serum proteins.
[0688] Another embodiment of the present invention is a polypeptide
construct as described above wherein said at least one (human)
serum protein is any of (human) serum albumin, (human) serum
immunoglobulins, (human) thyroxine-binding protein, (human)
transferrine, or (human) fibrinogen or a fragment thereof.
[0689] According to a specific, but non-limiting aspect of the
invention, the polypeptides of the invention contain, besides the
one or more Nanobodies of the invention, at least one Nanobody
against human serum albumin. Although these Nanobodies against
human serum albumin may be as generally described in W04/062551 or
in the further references cited therein, according to a
particularly preferred, but non-limiting embodiment, said Nanobody
against human serum albumin consists of 4 framework regions (FR1 to
FR4 respectively) and 3 complementarity determining regions (CDR1
to CDR3 respectively), in which: [0690] i) CDR1 is an amino acid
sequence chosen from the group consisting of:
TABLE-US-00038 [0690] [SEQ ID NO: 44] SFGMS [SEQ ID NO: 45] LNLMG
[SEQ ID NO: 46] INLLG [SEQ ID NO: 47] NYWMY;
[0691] and/or from the group consisting of amino acid sequences
that have 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0692] (1) any amino acid substitution is preferably a conservative
amino acid substitution (as defined herein); and/or [0693] (2) said
amino acid sequence preferably only contains amino acid
substitutions, and no amino acid deletions or insertions, compared
to the above amino acid sequences; and in which: [0694] ii) CDR2 is
an amino acid sequence chosen from the group consisting of:
TABLE-US-00039 [0694] [SEQ ID NO: 48] SISGSGSDTLYADSVKG [SEQ ID NO:
49] TITVGDSTNYADSVKG [SEQ ID NO: 50] TITVGDSTSYADSVKG [SEQ ID NO:
51] SINGRGDDTRYADSVKG [SEQ ID NO: 52] AISADSSTKNYADSVKG [SEQ ID NO:
53] AISADSSDKRYADSVKG [SEQ ID NO: 54] RISTGGGYSYYADSVKG
[0695] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0696] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0697] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequences; [0698] and/or from the
group consisting of amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0699] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0700] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequences; and in which: [0701] iii) CDR3 is an amino acid sequence
chosen from the group consisting of:
TABLE-US-00040 [0701] [SEQ ID NO: 55] DREAQVDTLDFDY
[0702] or from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with one of the above amino acid sequences; in
which [0703] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0704] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequences; [0705] and/or from the
group consisting of amino acid sequences that have 3, 2 or only 1
"amino acid difference(s)" (as defined herein) with one of the
above amino acid sequences, in which: [0706] (1) any amino acid
substitution is preferably a conservative amino acid substitution
(as defined herein); and/or [0707] (2) said amino acid sequence
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the above amino acid
sequences; [0708] or from the group consisting of:
TABLE-US-00041 [0708] [SEQ ID NO: 56] GGSLSR [SEQ ID NO: 57]
RRTWHSEL [SEQ ID NO: 58] GRSVSRS [SEQ ID NO: 59] GRGSP
[0709] and/or from the group consisting of amino acid sequences
that have 3, 2 or only 1 "amino acid difference(s)" (as defined
herein) with one of the above amino acid sequences, in which:
[0710] (1) any amino acid substitution is preferably a conservative
amino acid substitution (as defined herein); and/or [0711] (2) said
amino acid sequence preferably only contains amino acid
substitutions, and no amino acid deletions or insertions, compared
to the above amino acid sequences.
[0712] In another aspect, the invention relates to a Nanobody
against vWF, which consist of 4 framework regions (fR1 to FR4
respectively) and 3 complementarity determining regions
[0713] (CDR1 to CDR3 respectively), which is chosen from the group
consisting of Nanobodies with the one of the following combinations
of CDR1, CDR2 and CDR3, respectively:
TABLE-US-00042 [SEQ ID NO: 44] CDR1: SFGMS; [SEQ ID NO: 48] CDR2:
SISGSGSDTLYADSVKG; [SEQ ID NO: 56] CDR3: GGSLSR; [SEQ ID NO: 45]
CDR1: LNLMG; [SEQ ID NO: 49] CDR2: TITVGDSTNYADSVKG; [SEQ ID NO:
57] CDR3: RRTWHSEL; [SEQ ID NO: 46] CDR1: INLLG; [SEQ ID NO: 50]
CDR2: TITVGDSTSYADSVKG; [SEQ ID NO: 57] CDR3: RRTWHSEL; [SEQ ID NO:
44] CDR1: SFGMS; [SEQ ID NO: 51] CDR2: SINGRGDDTRYADSVKG; [SEQ ID
NO: 58] CDR3: GRSVSRS; [SEQ ID NO: 44] CDR1: SFGMS; [SEQ ID NO: 53]
CDR2: AISADSSDKRYADSVKG; [SEQ ID NO: 59] CDR3: GRGSP; [SEQ ID NO:
44] CDR1: SFGMS; [SEQ ID NO: 53] CDR2: AISADSSDKRYADSVKG; [SEQ ID
NO: 59] CDR3: GRGSP; [SEQ ID NO: 47] CDR1: NYWMY; [SEQ ID NO: 54]
CDR2: RISTGGGYSYYADSVKG; [SEQ ID NO: 55] CDR3: DREAQVDTLDFDY.
[0714] In the Nanobodies of the invention that comprise the
combinations of CDR's mentioned above, each CDR can be replaced by
a CDR chosen from the group consisting of amino acid sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity (as
defined herein) with the mentioned CDR's; in which [0715] (1) any
amino acid substitution is preferably a conservative amino acid
substitution (as defined herein); and/or [0716] (2) said amino acid
sequence preferably only contains amino acid substitutions, and no
amino acid deletions or insertions, compared to the above amino
acid sequences; and/or chosen from the group consisting of amino
acid sequences that have 3, 2 or only 1 (as indicated in the
preceding paragraph) "amino acid difference(s)" (as defined herein)
with the mentioned CDR(s) one of the above amino acid sequences, in
which: [0717] (1) any amino acid substitution is preferably a
conservative amino acid substitution (as defined herein); and/or
[0718] (2) said amino acid sequence preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the above amino acid sequences.
[0719] However, of the Nanobodies of the invention that comprise
the combinations of CDR's mentioned above, Nanobodies comprising
one or more of the CDR's listed above are particularly preferred;
Nanobodies comprising two or more of the CDR's listed above are
more particularly preferred; and Nanobodies comprising three of the
CDR's listed above are most particularly preferred.
[0720] In these Nanobodies against human serum albumin, the
Framework regions FR1 to FR4 are preferably as defined hereinabove
for the Nanobodies of the invention.
[0721] Particularly preferred Nanobodies against human serum
albumin are chosen from the group consisting of SEQ ID NO's:
107-121. These correspond to the Nanobodies against human serum
albumin of SEQ ID NO's: 61 to 67, SEQ ID NO's 87 to 89 and SEQ ID
NO's 100-104 from applicant's co-pending US provisional application
entitled "Improved Nanobodies.TM. against Tumor Necrosis
Factor-alpha" with a filing date of May 18, 2005.
[0722] More generally, Nanobodies against serum albumin suitable
for use in the invention are described in the International
application by applicant entitled "serum albumin binding proteins"
with an international filing date of May 17, 2006.
[0723] Another embodiment of the present invention is a nucleic
acid encoding a polypeptide construct as described above.
[0724] Another embodiment of the present invention is a composition
comprising a polypeptide construct as described above and at least
one thrombolytic agent, for simultaneous, separate or sequential
administration to a subject.
[0725] Another embodiment of the present invention is a composition
as described above wherein said thrombolytic agent is any of
staphylokinase, tissue plasminogen activator, streptokinase, single
chain streptokinase, urokinase and acyl plasminogen streptokinase
complex.
[0726] Another embodiment of the present invention is a polypeptide
construct as described above, or a nucleic acid as described above,
or a composition as described above for use in the treatment,
prevention and/or alleviation of disorders relating to
platelet-mediate aggregation or dysfunction thereof.
[0727] Another embodiment of the present invention is a use of a
polypeptide construct as described above, or a nucleic acid as
described above, or a composition as described above for the
preparation of a medicament for the treatment, prevention and/or
alleviation of disorders relating to platelet-mediate aggregation
or dysfunction thereof.
[0728] Another embodiment of the present invention is a polypeptide
construct, nucleic acid or composition as described above or a use
of a polypeptide construct, nucleic acid or composition as
described above wherein said disorders are any arising from
transient cerebral ischemic attack, unstable or stable angina,
angina pectoris, cerebral infarction, myocardial infarction,
peripheral arterial occlusive disease, restenosis, coronary by-pass
graft, or coronary artery valve replacement and coronary
interventions such angioplasty, stenting, carotid endarterectomy or
atherectomy.
[0729] Another embodiment of the present invention is a polypeptide
construct, nucleic acid or composition as described above or a use
of a polypeptide construct, nucleic acid or composition as
described above wherein said disorders are any of the formation of
a non-occlusive thrombus, the formation of an occlusive thrombus,
arterial thrombus formation, acute coronary occlusion, restenosis,
restenosis after PCTA or stenting, thrombus formation in stenosed
arteries, hyperplasia after angioplasty, atherectomy or arterial
stenting, occlusive syndrome in a vascular system or lack of
patency of diseased arteries.
[0730] Another embodiment of the present invention is a polypeptide
construct, nucleic acid or composition as described above or a use
of a polypeptide construct, nucleic acid or composition as
described above wherein said disorder is plaque or thrombus
formation in high sheer environments.
[0731] Another embodiment of the present invention is a polypeptide
construct, nucleic acid or composition as described above or a use
of a polypeptide construct as described above wherein said
polypeptide construct is administered intravenously,
subcutaneously, orally, sublingually, topically, nasally,
vaginally, rectally or by inhalation.
[0732] Another embodiment of the present invention is a composition
comprising a polypeptide construct as described above or a nucleic
acid encoding said polypeptide construct, or a composition as
described above and a pharmaceutically acceptable vehicle.
[0733] Another embodiment of the present invention is a method of
producing a polypeptide as described above, comprising
(a) culturing host cells comprising nucleic acid capable of
encoding a polypeptide as described above under conditions allowing
the expression of the polypeptide, and, (b) recovering the produced
polypeptide from the culture.
[0734] Another embodiment of the present invention is a method as
described above, wherein said host cells are bacterial or
yeast.
[0735] Another embodiment of the present invention is a method for
treating invasive medical devices to prevent platelet-mediate
aggregation around the site of invasion comprising the step of
coating said device with a polypeptide construct as described
above.
[0736] Another embodiment of the present invention is an invasive
medical device for circumventing platelet-mediate aggregation
around the site of invasion, wherein said device is coated with a
polypeptide construct as described above.
[0737] Another embodiment of the present invention is a method of
identifying an agent that modulates platelet-mediated aggregation
comprising
(a) contacting a polypeptide construct as described above with a
polypeptide corresponding to its target, or a fragment thereof, in
the presence and absence of a candidate modulator under conditions
permitting binding between said polypeptides, and (b) measuring the
binding between the polypeptides of step (a), wherein a decrease in
binding in the presence of said candidate modulator, relative to
the binding in the absence of said candidate modulator identified
said candidate modulator as an agent that modulate
platelet-mediated aggregation.
[0738] Another embodiment of the present invention is a kit for
screening for agents that modulate platelet-mediated aggregation
according to the method as described above.
[0739] Another embodiment of the present invention is an unknown
agent that modulates platelet-mediated aggregation identified
according to the method as described above.
[0740] Another embodiment of the present invention is a method of
diagnosing a disease or disorder characterised by dysfunction of
platelet-mediated aggregation comprising the steps of:
(a) contacting a sample with a polypeptide construct as described
above, and (b) detecting binding of said polypeptide construct to
said sample, and (c) comparing the binding detected in step (b)
with a standard, wherein a difference in binding relative to said
sample is diagnostic of a disease or disorder characterised by
dysfunction of platelet-mediated aggregation.
[0741] Another embodiment of the present invention is a kit for
screening for diagnosing a disease or disorder characterised by
dysfunction of platelet-mediated aggregation according to the
method as described above.
[0742] Another embodiment of the present invention is a kit as
described above comprising a polypeptide construct as described
above.
[0743] In the polypeptides of the invention, the one or more
Nanobodies of the invention which are directed against a target may
be of the same sequence. Alternatively they may not all have the
same sequence. It is within the scope of the invention that a
polypeptide of the invention comprises anti-target Nanobodies of
the invention which do not all share the same sequence, but which
are directed against the same target, or fragment thereof, one or
more antigens thereof.
[0744] It is another aspect of the invention that the polypeptide
of the invention comprises two or more Nanobodies of the invention,
wherein any two Nanobodies of the invention are directed against
different epitopes/targets, i.e. against any of vWF, vWF A1 domain,
A1 domain of activated vWF, vWF A3 domain.
[0745] Another aspect of the invention is a bispecific polypeptide
of the invention comprising a Nanobody of the invention directed
against vWF A1 domain, A1 domain of activated vWF, and another
Nanobody of the invention directed against vWF A3 domain. Said
bispecific polypeptide of the invention inhibits the interaction
between vWF and collagen, and the interaction between vWF and
platelets.
[0746] According to an aspect of the present invention a
polypeptide of the invention may comprise two or more Nanobodies of
the invention which have been joined. The Nanobodies of the
invention may be identical in sequence and directed against the
same target or antigen. Depending on the number of V.sub.HHs
linked, a multivalent V.sub.HH may be bivalent (2 V.sub.HHs),
trivalent (3 V.sub.HHs), tetravalent (4 V.sub.HHs) or have a higher
valency molecules.
[0747] The present invention also relates to the finding that a
polypeptide of the invention further comprising one or more
Nanobodies of the invention each directed against a serum protein
of a subject, surprisingly has significantly prolonged half-life in
the circulation of said subject compared with the half-life of the
anti-target Nanobody of the invention(ies) when not part of said
construct. Furthermore, the said constructs were found to exhibit
the same favourable properties of V.sub.HHs such as high stability
remaining intact in mice, extreme pH resistance, high temperature
stability and high target affinity.
[0748] The serum protein may be any suitable protein found in the
serum of subject, or fragment thereof. In one aspect of the
invention, the serum protein is serum albumin, serum
immunoglobulins, thyroxine-binding protein, transferrin, or
fibrinogen. Depending on the intended use such as the required
half-life for effective treatment and/or compartimentalisation of
the target antigen, the V.sub.HH-partner can be directed to one of
the above serum proteins.
[0749] Such constructs are able to circulate in the subject's serum
for several days, reducing the frequency of treatment, the
inconvenience to the subject and resulting in a decreased cost of
treatment. Furthermore, it is an aspect of the invention that the
half-life of the polypeptide of the invention disclosed herein may
be controlled by the number of anti-serum protein Nanobodies of the
invention present in the construct. A controllable half-life is
desirable in several circumstances, for example, in the application
of a timed dose of a therapeutic polypeptide of the invention.
[0750] Another embodiment of the present invention is a polypeptide
of the invention as mentioned herein, further comprising a
thrombolytic agent.
[0751] Said thrombolytic agent may be non-covalently or covalently
attached to a Nanobody of the invention via covalent or
non-covalent means. Such covalent means are described below.
Non-covalent means include via a protein interaction such as
biotin/strepavidin, or via an immunoconjugate.
[0752] Alternatively, the thrombolytic agent may be administered
simultaneous, separate or sequential in respect of a polypeptide of
the invention.
[0753] Another aspect of the invention is a composition comprising
at least one polypeptide of the invention and at least one
thrombolytic agent, for simultaneous, separate or sequential
administration to a subject.
[0754] One aspect of the invention is a method for treating
autoimmune disease comprising administering to an individual an
effective amount of at least one polypeptide of the invention and
at least one thrombolytic agent, simultaneously, separately or
sequentially.
[0755] Another aspect of the invention is a kit containing at least
one polypeptide of the invention and at least one thrombolytic
agent for simultaneous, separate or sequential administration to a
subject. It is an aspect of the invention that the kit may be used
according to the invention. It is an aspect of the invention that
the kit may be used to treat the diseases as cited herein.
[0756] By simultaneous administration means the polypeptide and
thrombolytic agent are administered to a subject at the same time.
For example, as a mixture or a composition comprising said
components. Examples include, but are not limited to a solution
administered intraveneously, a tablet, liquid, topical cream, etc.,
wherein each preparation comprises the components of interest.
[0757] By separate administration means polypeptide and
thrombolytic agent are administered to a subject at the same time
or substantially the same time. The components are present in the
kit as separate, unmixed preparations. For example, the polypeptide
and thrombolytic agent may be present in the kit as individual
tablets. The tablets may be administered to the subject by
swallowing both tablets at the same time, or one tablet directly
following the other.
[0758] By sequential administration means the polypeptide and
thrombolytic agent are administered to a subject sequentially. The
polypeptide and thrombolytic agent are present in the kit as
separate, unmixed preparations. There is a time interval between
doses. For example, one component might be administered up to 336,
312, 288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20,
16, 12, 8, 4, 2, 1, or 0.5 hours after the other component.
[0759] In sequential administration, one component may be
administered once, or any number of times and in various doses
before and/or after administration of another component. Sequential
administration may be combined with simultaneous or sequential
administration.
[0760] The medical uses of the polypeptide of the invention
described below, also apply to the composition comprising a
polypeptide of the invention and at least one polypeptide
thrombolytic agent, for simultaneous, separate or sequential
administration to a subject as disclosed here above.
[0761] Thrombolytic agents according to the invention may include,
for example, staphylokinase, tissue plasminogen activator,
streptokinase, single chain streptokinase, urokinase and acyl
plasminogen streptokinase complex.
[0762] The Nanobodies of the invention may be joined to form any of
the polypeptide of the invention disclosed herein comprising more
than one Nanobody of the invention using methods known in the art
or any future method. For example, they may be fused by chemical
cross-linking by reacting amino acid residues with an organic
derivatisation agent such as described by Blattler et al,
Biochemistry 24, 1517-1524; EP294703. Alternatively, the Nanobody
of the invention may be fused genetically at the DNA level i.e. a
polynucleotide construct formed which encodes the complete
polypeptide of the invention comprising one or more anti-target
Nanobodies of the invention and one or more anti-serum protein
Nanobodies of the invention. A method for producing bivalent or
multivalent V.sub.HH polypeptide of the invention is disclosed in
PCT patent application WO 96/34103. One way of joining multiple
Nanobodies of the invention is via the genetic route by linking
Nanobody of the invention coding sequences either directly or via a
peptide linker. For example, the C-terminal end of the first
Nanobody of the invention may be linked to the N-terminal end of
the next Nanobody of the invention. This linking mode can be
extended in order to link additional Nanobodies of the invention
for the construction and production of tri-, tetra-, etc.
functional constructs.
[0763] The polypeptide of the invention disclosed herein may be
made by the skilled artisan according to methods known in the art
or any future method. For example, V.sub.HHs may be obtained using
methods known in the art such as by immunising a camel and
obtaining hybridoma's therefrom, or by cloning a library of
Nanobodies of the invention using molecular biology techniques
known in the art and subsequent selection by using phage
display.
[0764] Nanobodies have a unique structure that consists of a single
variable domain. V.sub.HH molecules derived from Camelidae
antibodies are among the smallest intact antigen-binding domains
known (approximately 15 kDa, or 10 times smaller than a
conventional IgG) and hence are well suited towards delivery to
dense tissues and for accessing the limited space between
macromolecules participating in or starting the process of platelet
mediated aggregation.
[0765] Despite the small size of nanobodies, and thus advantages
for penetration, it is still surprising that such a small molecule
can inhibit interactions between large polymers such as vWF (up to
60 monomers) and collagen and with such a high efficiency. It has
been described that only the large multimeric forms of vWF are
hemostatically active (Furlan, M., 1996, Ann. Hematol. 72:341-348).
Binding of multimeric vWF to collagen occurs with .about.100-fold
higher affinity than binding of monomeric vWF fragments.
[0766] The results from the high shear experiments indicate that a
lower dose may be administered to patients. Therefore, fewer side
effects are expected (such as immunogenicity or bleeding
problems).
[0767] In another embodiment of the present invention, a
polypeptide of the invention comprises one or more Nanobodies of
the invention directed to the same target, and further comprises
one or more Nanobodies of the invention directed to the same target
but to a different epitope in the same domain.
[0768] Another embodiment of the present invention is a polypeptide
of the invention wherein the number of Nanobodies of the invention
directed to the same target is two or more.
[0769] In another embodiment of the present invention, a
polypeptide of the invention comprises one or more Nanobodies of
the invention directed to one domain of the same target, and one or
more Nanobodies of the invention directed to the same target but to
another domain of the same target. Examples of different domains
might be the A1 and A3 domains of vWF
[0770] It is one non-limiting aspect of the invention that at least
one V.sub.HH directed to the A1 domain in a heterospecific
polypeptide of the invention recognizes the active conformation of
vWF. Such polypeptide of the invention may have superior
anti-thrombotic effects compared to the monomeric V.sub.HH's.
Perfusion experiment were performed in a flow chamber, to study
platelet aggregation under high shear to study the effects of these
polypeptide of the invention.
[0771] The discovery of naturally occurring Nanobodies of the
invention in llama, dromedary and camel revealed a new class of
therapeutic molecules which combine the advantages of monoclonal
antibodies for example specificity, low toxicity with the
advantages of small molecules for example tissue penetration and
stability. Unfortunately, the development of appropriate
therapeutic products based on these proteins has the drawback of
being Camelidae derived, and thus not human. Non-human proteins
contain amino acid residues that can be immunogenic when injected
into a human patient. Although studies have shown that
Camelidae-derived V.sub.HH are not immunogenic when injected in
mice, replacing Camelidae residues by human residues is preferable.
These humanized polypeptides should be substantially
non-immunogenic in humans, but retain the affinity and activity of
the wild type polypeptide.
[0772] The result of humanisation is preferably that immunogenicity
upon administration in human patients is minor or nonexistent.
Humanising a polypeptide, according to the present invention,
comprises a step of replacing one or more of the Camelidae amino
acids by their human counterpart as found in the human consensus
sequence, without that polypeptide losing its typical character,
i.e. the humanisation does not significantly affect the antigen
binding capacity of the resulting polypeptide.
[0773] WO 04/062551 and the further description herein describe
some preferred, but non-limiting examples of amino acid residues of
the antibody variable domain (V.sub.HH) which may be modified
without diminishing the native affinity of the domain for antigen
and while reducing its immunogenicity with respect to a
heterologous species; the use of V.sub.HHs having modifications at
the identified residues which are useful for administration to
heterologous species; and to the V.sub.HH so modified. More
specifically, the invention also encompasses the preparation of
modified V.sub.HHs, which are modified for administration to
humans, the resulting V.sub.HH themselves, and the use of such
"humanized" V.sub.HHs in the treatment of diseases in humans.
[0774] As mentioned in WO 04/062551 and in the further description
herein, humanization of V.sub.HH polypeptides requires the
introduction and mutagenesis of only a limited number of amino
acids in a single polypeptide chain without dramatic loss of
binding and/or inhibition activity. This is in contrast to
humanization of scFv, Fab, (Fab).sub.2 and IgG, which requires the
introduction of amino acid changes in two chains, the light and the
heavy chain and the preservation of the assembly of both
chains.
[0775] A humanisation technique may be performed by a method
comprising the replacement of any of the following residues either
alone or in combination: FR1 positions 1, 5, 28 and 30, the
hallmark amino acid at position 37, 44, 45 and 47 in FR2, FR3
residues 74, 75, 76, 83, 84, 93 and 94 and positions 103, 104, 108
and 111 in FR4; numbering according to the Kabat numbering.
[0776] The Nanobodies of the invention have a high degree of
homology to human germline VH DP-47. Further humanization may also
involve the introduction and mutagenesis of a limited amount of
amino acids in a single polypeptide chain. This is in contrast to
humanization of scFv, Fab, (Fab).sub.2 and IgG, which requires the
introduction of amino acid changes in two chains, the light and the
heavy chain and the preservation of the assembly of both
chains.
[0777] The polypeptides contain human-like residues in FR2.
Humanization may also involve mutagenesis of residues in FR1 at
position 1 and 5 which were introduced by the primer used for
repertoire cloning and do not occur naturally in the llama
sequence. Mutagenesis of those residues did not result in loss of
binding and/or inhibition activity. Humanization of FR1 also
required mutagenesis of position 28 and 30. Mutagenesis of those
residues also did not result in loss of binding and/or inhibition
activity.
[0778] Humanization may also involve mutagenesis of residues in FR3
at position 74, 75, 76, 83, 84, 93, 94. Mutagenesis of those
residues did not result in loss of binding and/or inhibition
activity.
[0779] Humanization may also involve mutagenesis of residues in FR4
at position 104, 108 and 111. Mutagenesis of Q108L resulted in
lower production level in Escherichia coli. Position 108 is solvent
exposed in camelid V.sub.HH, while in human antibodies this
position is buried at the VH-VL interface (Spinelli, 1996; Nieba,
1997). In isolated V.sub.Hs position 108 is solvent exposed. The
introduction of a non-polar hydrophobic Leu instead of polar
uncharged Gln can have a drastic effect on the intrinsic
foldability/stability of the molecule.
[0780] One embodiment of the present invention is a method for
humanizing a V.sub.HH comprising the steps of:
(a) replacing of any of the following residues either alone or in
combination: FR1 positions 1, 5, 28 and 30, the hallmark amino acid
at position 37, 44, 45 and 47 in FR2, FR3 residues 74, 75, 76, 83,
84, 93 and 94, and positions 103, 104, 108 and 111 in FR4;
numbering according to the Kabat numbering.
[0781] Examples of such humanized sequences are given below and in
the appended sequence listing.
[0782] The use of antibodies derived from sources such as mouse,
sheep, goat, rabbit etc., and humanised derivatives thereof as a
treatment for conditions which require a modulation of
platelet-associated aggregation, is problematic for several
reasons. Traditional antibodies are not stable at room temperature,
and have to be refrigerated for preparation and storage, requiring
necessary refrigerated laboratory equipment, storage and transport,
which contribute towards time and expense. Refrigeration is
sometimes not feasible in developing countries. The yields of
expression of said Fab molecules are very low and the method of
production is very labor intensive. Furthermore, the manufacture or
small-scale production of said antibodies is expensive because the
mammalian cellular systems necessary for the expression of intact
and active antibodies require high levels of support in terms of
time and equipment, and yields are very low. Furthermore,
traditional antibodies have a binding activity which depends upon
pH, and hence are unsuitable for use in environments outside the
usual physiological pH range such as, for example, in treating
gastric bleeding, gastric surgery. Furthermore, traditional
antibodies are unstable at low or high pH and hence are not
suitable for oral administration. However, it has been demonstrated
that camelid antibodies resist harsh conditions, such as extreme
pH, denaturing reagents and high temperatures (Ewert S et al,
Biochemistry 2002 Mar. 19; 41(11):3628-36), so making them suitable
for delivery by oral administration. Furthermore, traditional
antibodies have a binding activity which depends upon temperature,
and hence are unsuitable for use in assays or kits performed at
temperatures outside biologically active-temperature ranges (e.g.
37.+-.20.degree. C.).
[0783] The Nanobodies and polypeptides of the invention not only
possess the advantageous characteristics of conventional
antibodies, such as low toxicity and high selectivity, but they
also exhibit additional properties. They are more soluble, meaning
they may be stored and/or administered in higher concentrations
compared with conventional antibodies. They are stable at room
temperature meaning they may be prepared, stored and/or transported
without the use of refrigeration equipment, conveying a cost, time
and environmental savings. Other advantageous characteristics as
compared to conventional antibodies include short half-life in the
circulation which may be modulated according to the invention by,
for example, albumin-coupling, a bispecific nanobody with one
specificity against albumin and the other against the target, Fc
coupling, V.sub.HH coupling (bivalent V.sub.HHs) or by pegylation.
A short and controllable half-life is desirable for surgical
procedures, for example, which require an inhibition of
platelet-mediated aggregation for a limited time period. Also, when
bleeding problems occur or other complications, dosage can be
lowered immediately. The polypeptides of the present invention also
retain binding activity at a pH and temperature outside those of
usual physiological ranges, which means they may be useful in
situations of extreme pH and temperature which require a modulation
of platelet-mediated aggregation, such as in gastric surgery,
control of gastric bleeding, assays performed at room temperature
etc. The polypeptides of the present invention also exhibit a
prolonged stability at extremes of pH, meaning they would be
suitable for delivery by oral administration. The polypeptides of
the present invention may be cost-effectively produced through
fermentation in convenient recombinant host organisms such as
Escherichia coli and yeast; unlike conventional antibodies which
also require expensive mammalian cell culture facilities,
achievable levels of expression are high. Examples of yields of the
polypeptides of the present invention are 1 to 10 mg/ml (E. coli)
and up to 1 g/l (yeast). The polypeptides of the present invention
also exhibit high binding affinity for a broad range of different
antigen types, and ability to bind to epitopes not recognised by
conventional antibodies; for example they display long CDR-based
loop structures with the potential to penetrate into cavities and
exhibit enzyme function inhibition. Furthermore, since binding
often occurs through the CDR3 loop only, it is envisaged that
peptides derived from CDR3 could be used therapeutically (Desmyter
et al., J Biol Chem, 2001, 276: 26285-90). The polypeptides of the
invention are also able to retain full binding capacity as fusion
protein with an enzyme or toxin. Furthermore, it might be expected
that the undesirable thrombocytopenia caused by Fc:Fc receptor
mediated activation of platelet aggregation and/or
F(ab')(2)-mediated crosslinking of platelets which has been
observed when using intact IgG or F(ab')(2) therapeutically in vivo
(see Cauwenberghs N. et al, Arteriosclerosis, Thrombosis and
Vascular biology, 2000, 20: 1347), will be avoided in the use of
V.sub.HH, since V.sub.HH contains no Fc and it is not bivalent.
Thus the polypeptides of the invention, homologues or functional
portions thereof provide a considerable cost and time saving in the
treatment and diagnosis of conditions related to platelet-mediated
aggregation, and the patient in need of said polypeptides would
encounter fewer of the problems associated with conventional
agents.
[0784] Platelet-mediated aggregation is the process wherein
vWF-bound collagen adheres to platelets and/or platelet receptors,
ultimately resulting in platelet activation. Platelet activation
leads to fibrinogen binding, and finally to platelet aggregation.
It is within the scope of the present invention to provide
polypeptides which modulate the processes which comprise
platelet-mediated aggregation such as vWF-collagen binding,
vWF-platelet receptor adhesion, collagen-platelet receptor
adhesion, platelet activation, fibrinogen binding and/or platelet
aggregation.
[0785] According to an aspect of the invention a polypeptide of the
invention may be a homologous sequence of a full-length polypeptide
of the invention. According to another aspect of the invention, a
polypeptide of the invention may be a functional portion of a
full-length polypeptide of the invention. According to another
aspect of the invention, a polypeptide of the invention may be a
homologous sequence of a full length polypeptide of the invention.
According to another aspect of the invention, a polypeptide of the
invention may be a functional portion of a homologous sequence of a
full length polypeptide of the invention. According to an aspect of
the invention a polypeptide of the invention may comprise a
sequence of a polypeptide of the invention.
[0786] According to an aspect of the invention a Nanobody of the
invention used to form a polypeptide of the invention may be a
complete Nanobody of the invention (e.g. a V.sub.HH) or a
homologous sequence thereof. According to another aspect of the
invention, a Nanobody of the invention used to form the polypeptide
of the invention may be a functional portion of a complete Nanobody
of the invention. According to another aspect of the invention, a
Nanobody of the invention used to form the polypeptide of the
invention may be a homologous sequence of a complete Nanobody of
the invention. According to another aspect of the invention, a
Nanobody of the invention used to form the polypeptide of the
invention may be a functional portion of a homologous sequence of a
complete Nanobody of the invention.
[0787] According to another aspect of the invention a polypeptide
of the invention may be an homologous sequence of the parent
sequence. According to another aspect of the invention, a
polypeptide of the invention may be a functional portion parent
sequence. According to another aspect of the invention, a
polypeptide of the invention may be a functional portion of a
homologous sequence of the parent sequence.
[0788] As used herein, an homologous sequence may comprise
additions, deletions or substitutions of one or more amino acids,
which do not substantially alter the functional characteristics of
the polypeptide. The number of amino acid deletions or
substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
[0789] A homologous sequence according to the present invention
includes polypeptides extended by the addition of amino acids to
form human heavy chain antibody or human single domain heavy chain
antibody, which do not substantially alter the functional
characteristics of the unmodified polypeptide.
[0790] Where homologous sequence indicates sequence identity, it
means a sequence which presents a high sequence identity (more than
70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with the
parent sequence, and is preferably characterised by similar
properties of the parent sequence, namely affinity, said identity
calculated using known methods.
[0791] Alternatively, an homologous sequence may also be any amino
acid sequence resulting from allowed substitutions at any number of
positions of the parent sequence according to the formula
below:
Ser substituted by Ser, Thr, Gly, and Asn; Arg substituted by one
of Arg, His, Gln, Lys, and Glu; Leu substituted by one of Leu, Ile,
Phe, Tyr, Met, and Val; Pro substituted by one of Pro, Gly, Ala,
and Thr; Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His,
and Gln; Ala substituted by one of Ala, Gly, Thr, and Pro; Val
substituted by one of Val, Met, Tyr, Phe, Ile, and Leu; Gly
substituted by one of Gly, Ala, Thr, Pro, and Ser; Ile substituted
by one of Ile, Met, Tyr, Phe, Val, and Leu; Phe substituted by one
of Phe, Trp, Met, Tyr, Ile, Val, and Leu; Tyr substituted by one of
Tyr, Trp, Met, Phe, Ile, Val, and Leu; His substituted by one of
His, Glu, Lys, Gln, Thr, and Arg; Gln substituted by one of Gln,
Glu, Lys, Asn, His, Thr, and Arg; Asn substituted by one of Asn,
Glu, Asp, Gln, and Ser; Lys substituted by one of Lys, Glu, Gln,
His, and Arg; Asp substituted by one of Asp, Glu, and Asn; Glu
substituted by one of Glu, Asp, Lys, Asn, Gln, His, and Arg; Met
substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.
[0792] A homologous according to the present invention may refer to
nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600,
800 or 1000 nucleotides able to hybridize to the reverse-complement
of the nucleotide sequence capable of encoding a polypeptide under
stringent hybridisation conditions (such as the ones described by
SAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring,
Harbor Laboratory press, New York).
[0793] As used herein, a functional portion refers to a Nanobody of
the invention of sufficient length such that the interaction of
interest is maintained with affinity of 1.times.10-6 M or
better.
[0794] Alternatively a functional portion of a Nanobody of the
invention comprises a partial deletion of the complete amino acid
sequence and still maintains the binding site(s) and protein
domain(s) necessary for the binding of and interaction with the
target.
[0795] Alternatively a functional portion of any of Nanobody of the
invention is a polypeptide which comprises a partial deletion of
the complete amino acid sequence and which still maintains the
binding site(s) and protein domain(s) necessary for the inhibition
of binding of vWF to collagen.
[0796] Alternatively a functional portion of any Nanobody of the
invention is a polypeptide which comprises a partial deletion of
the complete amino acid sequence and which still maintains the
binding site(s) and protein domain(s) necessary for the binding of
and interaction with the A1 domain of vWF.
[0797] Alternatively a functional portion of any Nanobody of the
invention is a polypeptide which comprises a partial deletion of
the complete amino acid sequence and which still maintains the
binding site(s) and protein domain(s) necessary for the binding of
and interaction with collagen.
[0798] Alternatively a functional portion comprises a partial
deletion of the complete amino acid sequence of a polypeptide and
which still maintains the binding site(s) and protein domain(s)
necessary for the binding of and interaction with the antigen
against which it was raised. It includes, but is not limited to
V.sub.HH domains.
[0799] As used herein, a functional portion as it refers to a
polypeptide sequence refers to less than 100% of the sequence
(e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 5 or more
amino acids.
[0800] A portion as it refers to a nucleotide sequence encoding a
polypeptide sequence refers to less than 100% of the sequence
(e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 15 or more
nucleotides.
[0801] An aspect of the present invention is the administration of
a polypeptide of the invention according to the invention can avoid
the need for injection. Conventional antibody-based therapeutics
have significant potential as drugs because they have exquisite
specificity to their target and a low inherent toxicity, however,
they have one important drawback: they are relatively unstable, and
are sensitive to breakdown by proteases. This means that
conventional antibody drugs cannot be administered orally,
sublingually, topically, nasally, vaginally, rectally or by
inhalation because they are not resistant to the low pH at these
sites, the action of proteases at these sites and in the blood
and/or because of their large size. They have to be administered by
injection (intravenously, subcutaneously, etc.) to overcome some of
these problems. Administration by injection requires specialist
training in order to use a hypodermic syringe or needle correctly
and safely. It further requires sterile equipment, a liquid
formulation of the therapeutic polypeptide, vial packing of said
polypeptide in a sterile and stable form and, of the subject, a
suitable site for entry of the needle. Furthermore, subjects
commonly experience physical and psychological stress prior to and
upon receiving an injection.
[0802] An aspect of the present invention overcomes these problems
of the prior art, by providing the polypeptides constructs of the
present invention. Said constructs are sufficiently small,
resistant and stable to be delivered orally, sublingually,
topically, nasally, vaginally, rectally or by inhalation
substantial without loss of activity. The polypeptides constructs
of the present invention avoid the need for injections, are not
only cost/time savings, but are also more convenient and more
comfortable for the subject.
[0803] One embodiment of the present invention is a polypeptide of
the invention for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by a substance
that controls platelet mediated aggregation which is able pass
through the gastric environment without the substance being
inactivated.
[0804] As known by persons skilled in the art, once in possession
of said polypeptide of the invention, formulation technology may be
applied to release a maximum amount of polypeptide in the right
location (in the stomach, in the colon, etc.). This method of
delivery is important for treating, prevent and/or alleviate the
symptoms of disorders whose targets are located in the gut
system.
[0805] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of a disorder
susceptible to modulation by a substance that controls platelet
mediated aggregation which is able pass through the gastric
environment without being inactivated, by orally administering to a
subject a polypeptide of the invention.
[0806] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation which is able pass through the
gastric environment without being inactivated.
[0807] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the gut
system without said substance being inactivated, by orally
administering to a subject a Nanobody or polypeptide of the
invention.
[0808] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject without the substance being inactivated,
by orally administering to a subject a Nanobody or polypeptide of
the invention.
[0809] Another embodiment of the present invention is a Nanobody or
polypeptide of the invention for use in treating, preventing and/or
alleviating the symptoms or disorders susceptible to modulation by
a substance that controls platelet mediated aggregation delivered
to the vaginal and/or rectal tract.
[0810] In a non-limiting example, a formulation according to the
invention comprises a Nanobody or polypeptide of the invention, in
the form of a gel, cream, suppository, film, or in the form of a
sponge or as a vaginal ring that slowly releases the active
ingredient over time (such formulations are described in EP 707473,
EP 684814, U.S. Pat. No. 5,629,001).
[0811] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a substance that controls platelet mediated
aggregation delivered to the vaginal and/or rectal tract, by
vaginally and/or rectally administering to a subject a Nanobody or
polypeptide of the invention.
[0812] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation delivered to the vaginal and/or
rectal tract.
[0813] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
vaginal and/or rectal tract without being said substance being
inactivated, by administering to the vaginal and/or rectal tract of
a subject a Nanobody or polypeptide of the invention.
[0814] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject without said substance being inactivated,
by administering to the vaginal and/or rectal tract of a subject a
Nanobody or polypeptide of the invention.
[0815] Another embodiment of the present invention is a Nanobody or
polypeptide of the invention, for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by a substance that controls platelet mediated
aggregation delivered to the nose, upper respiratory tract and/or
lung.
[0816] In a non-limiting example, a formulation according to the
invention, comprises a Nanobody or polypeptide of the invention in
the form of a nasal spray (e.g. an aerosol) or inhaler. Since the
Nanobody or polypeptide of the invention is small, it can reach its
target much more effectively than therapeutic IgG molecules.
[0817] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a substance that controls platelet mediated
aggregation delivered to the upper respiratory tract and lung, by
administering to a subject a Nanobody or polypeptide of the
invention, by inhalation through the mouth or nose.
[0818] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation delivered to the nose, upper
respiratory tract and/or lung, without said polypeptide being
inactivated.
[0819] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the nose,
upper respiratory tract and lung without inactivation, by
administering to the nose, upper respiratory tract and/or lung of a
subject a Nanobody or polypeptide of the invention.
[0820] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject without inactivation by administering to
the nose, upper respiratory tract and/or lung of a subject a
Nanobody or polypeptide of the invention.
[0821] One embodiment of the present invention is a Nanobody or
polypeptide of the invention for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
a substance that controls platelet mediated aggregation delivered
to the intestinal mucosa, wherein said disorder increases the
permeability of the intestinal mucosa. Because of their small size,
a Nanobody or polypeptide of the invention can pass through the
intestinal mucosa and reach the bloodstream more efficiently in
subjects suffering from disorders which cause an increase in the
permeability of the intestinal mucosa.
[0822] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a substance that controls platelet mediated
aggregation delivered to the intestinal mucosa, wherein said
disorder increases the permeability of the intestinal mucosa, by
orally administering to a subject a Nanobody or polypeptide of the
invention.
[0823] This process can be even further enhanced by an additional
aspect of the present invention--the use of active transport
carriers. In this aspect of the invention, V.sub.HH is fused to a
carrier that enhances the transfer through the intestinal wall into
the bloodstream. In a non-limiting example, this "carrier" is a
second V.sub.HH which is fused to the therapeutic V.sub.HH. Such
fusion constructs are made using methods known in the art. The
"carrier" V.sub.HH binds specifically to a receptor on the
intestinal wall which induces an active transfer through the
wall.
[0824] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation delivered to the intestinal mucosa,
wherein said disorder increases the permeability of the intestinal
mucosa.
[0825] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
intestinal mucosa without being inactivated, by administering
orally to a subject a Nanobody or polypeptide of the invention.
[0826] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject without being inactivated, by
administering orally to a subject a Nanobody or polypeptide of the
invention.
[0827] This process can be even further enhanced by an additional
aspect of the present invention--the use of active transport
carriers. In this aspect of the invention, a Nanobody or
polypeptide of the invention as described herein is fused to a
carrier that enhances the transfer through the intestinal wall into
the bloodstream. In a non-limiting example, this "carrier" is a
V.sub.HH which is fused to said polypeptide. Such fusion constructs
made using methods known in the art. The "carrier" V.sub.HH binds
specifically to a receptor on the intestinal wall which induces an
active transfer through the wall.
[0828] One embodiment of the present invention is a Nanobody or
polypeptide of the invention for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
a substance that controls platelet mediated aggregation which is
able pass through the tissues beneath the tongue effectively. A
formulation of said Nanobody or polypeptide of the invention, for
example, a tablet, spray, drop is placed under the tongue and
adsorbed through the mucus membranes into the capillary network
under the tongue.
[0829] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a substance that controls platelet mediated
aggregation which is able pass through the tissues beneath the
tongue effectively, by sublingually administering to a subject a
Nanobody or polypeptide of the invention.
[0830] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation which is able to pass through the
tissues beneath the tongue.
[0831] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
tissues beneath the tongue without being inactivated, by
administering sublingually to a subject a Nanobody or polypeptide
of the invention.
[0832] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject without being inactivated, by
administering orally to a subject a Nanobody or polypeptide of the
invention.
[0833] One embodiment of the present invention is a Nanobody or
polypeptide of the invention for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
a substance that controls platelet mediated aggregation which is
able pass through the skin effectively.
[0834] A formulation of said Nanobody or polypeptide of the
invention, for example, a cream, film, spray, drop, patch, is
placed on the skin and passes through.
[0835] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a substance that controls platelet mediated
aggregation which is able pass through the skin effectively, by
topically administering to a subject a Nanobody or polypeptide of
the invention.
[0836] Another embodiment of the present invention is a use of a
Nanobody or polypeptide of the invention for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by a substance that controls
platelet mediated aggregation which is able pass through the skin
effectively.
[0837] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the skin
without being inactivated, by administering topically to a subject
a Nanobody or polypeptide of the invention.
[0838] An aspect of the invention is a method for delivering a
substance that controls platelet mediated aggregation to the
bloodstream of a subject, by administering topically to a subject a
Nanobody or polypeptide of the invention.
[0839] In another embodiment of the present invention, a Nanobody
or polypeptide of the invention further comprises a carrier
Nanobody of the invention (e.g. V.sub.HH) which acts as an active
transport carrier for transport of said Nanobody or polypeptide of
the invention via the lung lumen to the blood.
[0840] A Nanobody or polypeptide of the invention further
comprising a carrier that binds specifically to a receptor present
on the mucosal surface (bronchial epithelial cells) resulting in
the active transport of the polypeptide from the lung lumen to the
blood. The carrier Nanobody of the invention may be fused to the
Nanobody or polypeptide of the invention. Such fusion constructs
made using methods known in the art and are describe herein. The
"carrier" Nanobody of the invention binds specifically to a
receptor on the mucosal surface which induces an active transfer
through the surface.
[0841] Another aspect of the present invention is a method to
determine which Nanobodies of the invention (e.g. V.sub.HHs) are
actively transported into the bloodstream upon nasal
administration. Similarly, a naive or immune V.sub.HH phage library
can be administered nasally, and after different time points after
administration, blood or organs can be isolated to rescue phages
that have been actively transported to the bloodstream. A
non-limiting example of a receptor for active transport from the
lung lumen to the bloodstream is the Fc receptor N (FcRn). One
aspect of the invention includes the V.sub.HH molecules identified
by the method. Such V.sub.HH can then be used as a carrier V.sub.HH
for the delivery of a therapeutic V.sub.HH to the corresponding
target in the bloodstream upon nasal administration.
[0842] One embodiment of the present invention is a Nanobody or
polypeptide of the invention for use in treating, preventing and/or
alleviating the symptoms of disorders relating to platelet-mediated
aggregation or dysfunction thereof. Said disorders include,
thrombotic thrombocytopenic purpura (TTP), transient cerebral
ischemic attack, unstable or stable angina pectoris, cerebral
infarction, myocardial infarction, peripheral arterial occlusive
disease, restenosis. Said disorders further include those arising
from coronary by-pass graft, coronary artery valve replacement and
coronary interventions such angioplasty, stenting, or
atherectomy.
[0843] Other disorders are any of the formation of a non-occlusive
thrombus, the formation of an occlusive thrombus, arterial thrombus
formation, acute coronary occlusion, restenosis, restenosis after
PCTA or stenting, thrombus formation in stenosed arteries,
hyperplasia after angioplasty, atherectomy or arterial stenting,
occlusive syndrome in a vascular system or lack of patency of
diseased arteries.
[0844] One aspect of the invention is a Nanobody or polypeptide of
the invention for use in the treatment, prevention and/or
alleviation of disorders or conditions relating to
platelet-mediated aggregation or dysfunction thereof, wherein said
Nanobody or polypeptide of the invention is administered
intravenously, subcutaneously, orally, sublingually, topically,
nasally, vaginally, rectally or by inhalation.
[0845] Another aspect of the invention is the use of a Nanobody or
polypeptide of the invention for the preparation of a medicament
for the treatment, prevention and/or alleviation of disorders or
conditions relating to platelet-mediated aggregation or dysfunction
thereof, wherein said Nanobody or polypeptide of the invention is
administered intravenously, subcutaneously, orally, sublingually,
topically, nasally, vaginally, rectally or by inhalation.
[0846] Another aspect of the invention is a method of treating,
preventing and/or alleviating disorders or conditions relating to
relating to platelet-mediated aggregation or dysfunction thereof,
comprising administering to a subject a Nanobody or polypeptide of
the invention, wherein said heterospecific Nanobody or polypeptide
of the invention is administered intravenously, subcutaneously,
orally, sublingually, topically, nasally, vaginally, rectally or by
inhalation.
[0847] Another aspect of the invention is a Nanobody or polypeptide
of the invention for use in the treatment, prevention and/or
alleviation of disorders or conditions relating to
platelet-mediated aggregation or dysfunction thereof.
[0848] Another aspect of the invention is a use of a polypeptide of
the invention for the preparation of a medicament for the
treatment, prevention and/or alleviation of disorders or conditions
relating to platelet-mediated aggregation or dysfunction
thereof.
[0849] One can use a Nanobody or polypeptide of the invention of
the present invention in order to screen for agents that modulate
the binding of the polypeptide to a vWF. When identified in an
assay that measures binding or said polypeptide displacement alone,
agents will have to be subjected to functional testing to determine
whether they act as modulators of platelet-mediated aggregation.
Some examples of suitable screening methods are discussed in WO
04/062551. Of course, these methods can easily be applied to
screening for candidate modulators which alter the binding between
the Nanobody or polypeptide of the invention disclosed herein and
vWF.
[0850] A cell that is useful according to the invention is
preferably selected from the group consisting of bacterial cells
such as, for example, E. coli, yeast cells such as, for example, S.
cerevisiae, P. pastoris, insect cells or mammalian cells, e.g. as
mentioned above.
[0851] A cell that is useful according to the invention can be any
cell into which a nucleic acid sequence encoding a Nanobody or
polypeptide of the invention can be or has been introduced such
that the polypeptide is expressed at natural levels or above
natural levels, as defined herein. Preferably a polypeptide of the
invention that is expressed in a cell exhibits normal or near
normal pharmacology, as defined herein.
[0852] According to a preferred embodiment of the present
invention, a cell is selected from the group consisting of
COST-cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293
cell, K-562 cell or a 1321N1 astrocytoma cell but also other
transfectable cell lines.
[0853] In general, "therapeutically effective amount",
"therapeutically effective dose" and "effective amount" means the
amount needed to achieve the desired result or results (treating or
preventing platelet aggregation). One of ordinary skill in the art
will recognize that the potency and, therefore, an "effective
amount" can vary for the various Nanobodies or polypeptides that
inhibit platelet-mediated aggregation used in the invention. One
skilled in the art can readily assess the potency of the Nanobody
or polypeptide.
[0854] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to an individual along with the Nanobody or
polypeptide without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained.
[0855] The invention disclosed herein is useful for treating or
preventing a condition of platelet-mediated aggregation, in a
subject and comprising administering a pharmaceutically effective
amount of a Nanobody or polypeptide or composition that inhibits
BTK and that inhibits platelet-mediated aggregation.
[0856] The invention disclosed herein is useful for treating or
preventing the first steps of thrombus formation, in a subject and
comprising administering a pharmaceutically effective amount of a
Nanobody or polypeptide or composition according to the
invention.
[0857] The invention disclosed herein is useful for treating or
preventing restenosis, in a subject and comprising administering a
pharmaceutically effective amount of a Nanobody or polypeptide or
composition according to the invention.
[0858] One aspect of the present invention is the use of Nanobodies
or polypeptides of the invention for treating or preventing a
condition of platelet-mediated aggregation, in a subject and
comprising administering a pharmaceutically effective amount of a
Nanobody or polypeptide in combination with another, such as, for
example, aspirin.
[0859] One aspect of the present invention is the use of Nanobodies
or polypeptides of the invention for treating or preventing a
condition of platelet-mediated aggregation, in a subject and
comprising administering a pharmaceutically effective amount of a
Nanobody or polypeptide in combination with another, such as, for
example, a thrombolytic agent.
[0860] Another aspect of the present invention is a use of a
Nanobody or polypeptide of the invention for treating or preventing
plaque or thrombus in an individual. Said plaque or thrombus
formation may be under conditions of high sheer. In both thrombosis
and reocclusion, the reversible adhesion or tethering of the
platelets at high shear rate is followed by a firm adhesion through
the collagen receptor on platelets resulting in platelet
activation; the tethering of platelets by vWF to collagen exposed
in the damaged vessel wall is especially important under high shear
conditions. The inventors have found that Nanobody or polypeptide
of the invention of the present invention unexpected performed well
under high sheer conditions.
[0861] The present invention is not limited to the administration
of formulations comprising a single Nanobody or polypeptide of the
invention. It is within the scope of the invention to provide
combination treatments wherein a formulation is administered to a
patient in need thereof that comprises more than one Nanobody or
polypeptide of the invention.
[0862] Conditions of platelet-mediated aggregation include, but are
not limited to, unstable angina, stable angina, angina pectoris,
embolus formation, deep vain thrombosis, hemolytic uremic syndrome,
hemolytic anemia, acute renal failure, thrombolytic complications,
thrombotic thrombocytopenic purpura, disseminated intravascular
comgelopathy, thrombosis, coronary heart disease, thromboembolic
complications, myocardial infarction, restenosis, and atrial
thrombosis formation in atrial fibrillation, chronic unstable
angina, transient ischemic attacks and strokes, peripheral vascular
disease, arterial thrombosis, pre-eclampsia, embolism, restenosis
and/or thrombosis following angioplasty, carotid endarterectomy,
anastomosis of vascular grafts, and chronic exposure to
cardiovascular devices. Such conditions may also result from
thromboembolism and reocculsion during and after thrombolytic
therapy, after angioplasty, and after coronary artery bypass.
[0863] It is well known in the art how to determine the inhibition
of platelet-mediated aggregation using the standard tests described
herein, or using other similar tests. Preferably, the method would
result in at least a 10% reduction in platelet-mediated
aggregation, including, for example, 15%, 20%, 25%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably
by 90%.
[0864] Similarly, the method would result in at least a 10%
reduction in intracellular calcium mobilisation including, for
example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.
Similarly, the method would result in at least a 10% reduction in
the level of phosphorylated PLCg 2 including, for example, 15%,
20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.
[0865] The reduction can be measured, for example, by comparing the
optical impedence in a chronology platelet aggregometer. Any other
known measurement method may also be used. For example, (1) upon
collagen stimulation, the level of collagen-induced intracellular
calcium mobilization increases over time and so the measurement may
include measuring the level of collagen-induced intracellular
calcium or (2) upon collagen stimulation, the level of
phosphorylated PLCg 2 increases over time and so the measurement
may include measuring the level of phosphorylated PLCg 2.
[0866] The cells can be contacted in vitro, for example, by adding
a Nanobody or polypeptide of the invention to the culture medium
(by continuous infusion, by bolus delivery, or by changing the
medium to a medium that contains the Nanobody or polypeptide) or by
adding the Nanobody or polypeptide to the extracellular fluid in
vivo (by local delivery, systemic delivery, inhalation, intravenous
injection, bolus delivery, or continuous infusion). The duration of
"contact" with a cell or population of cells is determined by the
time the Nanobody or polypeptide is present at physiologically
effective levels or at presumed physiologically effective levels in
the medium or extracellular fluid bathing the cell or cells.
Preferably, the duration of contact is 1-96 hours, and more
preferably, for 24 hours, but such time would vary based on the
half life of the Nanobody or polypeptide and could be optimized by
one skilled in the art using routine experimentation.
[0867] The Nanobody or polypeptide useful in the present invention
can be formulated as pharmaceutical compositions and administered
to a mammalian host, such as a human patient or a domestic animal
in a variety of forms adapted to the chosen route of
administration, i.e., orally or parenterally, by intra-nasally by
inhalation, intravenous, intramuscular, topical or subcutaneous
routes.
[0868] The Nanobody or polypeptide of the present invention can
also be administered using gene therapy methods of delivery. See,
e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference
in its entirety. Using a gene therapy method of delivery, primary
cells transfected with the gene for the Nanobody or polypeptide of
the present invention can additionally be transfected with tissue
specific promoters to target specific organs, tissue, grafts,
tumors, or cells.
[0869] Thus, the present Nanobody or polypeptide may be
systemically administered, e.g., orally, in combination with a
pharmaceutically acceptable vehicle such as an inert diluent or an
assimilable edible carrier. They may be enclosed in hard or soft
shell gelatin capsules, may be compressed into tablets, or may be
incorporated directly with the food of the patient's diet. For oral
therapeutic administration, the Nanobody or polypeptide may be
combined with one or more excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and
preparations should contain at least 0.1% of the Nanobody or
polypeptide. The percentage of the compositions and preparations
may, of course, be varied and may conveniently be between about 2
to about 60% of the weight of a given unit dosage form. The amount
of the Nanobody or polypeptide in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0870] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol.
[0871] Various other materials may be present as coatings or to
otherwise modify the physical form of the solid unit dosage form.
For instance, tablets, pills, or capsules may be coated with
gelatin, wax, shellac or sugar and the like. A syrup or elixir may
contain the Nanobody or polypeptide, sucrose or fructose as a
sweetening agent, methyl and propylparabens as preservatives, a dye
and flavoring such as cherry or orange flavor. Of course, any
material used in preparing any unit dosage form should be
pharmaceutically acceptable and substantially non-toxic in the
amounts employed. In addition, the Nanobody or polypeptide may be
incorporated into sustained-release preparations and devices.
[0872] The Nanobody or polypeptide may also be administered
intravenously or intraperitoneally by infusion or injection.
Solutions of the Nanobody or polypeptide can be prepared in water,
optionally mixed with a nontoxic surfactant. Dispersions can also
be prepared in glycerol, liquid polyethylene glycols, triacetin,
and mixtures thereof and in oils. Under ordinary conditions of
storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
[0873] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form must be sterile,
fluid and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion
medium comprising, for example, water, ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols,
and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the formation of liposomes, by the maintenance of
the required particle size in the case of dispersions or by the use
of surfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, buffers or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[0874] Sterile injectable solutions are prepared by incorporating
the Nanobody or polypeptide in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filter sterilization. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and the freeze drying techniques, which yield a
powder of the active ingredient plus any additional desired
ingredient present in the previously sterile-filtered
solutions.
[0875] For topical administration, the present Nanobody or
polypeptide may be applied in pure form, i.e., when they are
liquids. However, it will generally be desirable to administer them
to the skin as compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0876] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, hydroxyalkyls or
glycols or water-alcohol/glycol blends, in which the present
Nanobody or polypeptide can be dissolved or dispersed at effective
levels, optionally with the aid of non-toxic surfactants. Adjuvants
such as fragrances and additional antimicrobial agents can be added
to optimize the properties for a given use. The resultant liquid
compositions can be applied from absorbent pads, used to impregnate
bandages and other dressings, or sprayed onto the affected area
using pump-type or aerosol sprayers.
[0877] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0878] Examples of useful dermatological compositions which can be
used to deliver the Nanobody or polypeptide to the skin are known
to the art; for example, see Jacquet et al. (U.S. Pat. No.
4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S.
Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0879] Useful dosages of the Nanobody or polypeptide can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0880] Generally, the concentration of the Nanobody or polypeptide
in a liquid composition, such as a lotion, will be from about
0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration
in a semi-solid or solid composition such as a gel or a powder will
be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0881] The amount of the Nanobody or polypeptide required for use
in treatment will vary with the route of administration, the nature
of the condition being treated and the age and condition of the
patient and will be ultimately at the discretion of the attendant
physician or clinician. Also the dosage of the Nanobody or
polypeptide varies depending on the target cell, tumor, tissue,
graft, or organ.
[0882] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0883] An administration regimen could include long-term, daily
treatment. By "long-term" is meant at least two weeks and
preferably, several weeks, months, or years of duration. Necessary
modifications in this dosage range may be determined by one of
ordinary skill in the art using only routine experimentation given
the teachings herein. See Remington's Pharmaceutical Sciences
(Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage
can also be adjusted by the individual physician in the event of
any complication.
[0884] The invention provides for an agent that is a modulator of
platelet-mediated aggregation.
[0885] The candidate agent may be a synthetic agent, or a mixture
of agents, or may be a natural product (e.g. a plant extract or
culture supernatant). A candidate agent according to the invention
includes a small molecule that can be synthesized, a natural
extract, peptides, proteins, carbohydrates, lipids etc.
[0886] Candidate modulator agents from large libraries of synthetic
or natural agents can be screened. Numerous means are currently
used for random and directed synthesis of saccharide, peptide, and
nucleic acid based agents. Synthetic agent libraries are
commercially available from a number of companies including
Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex
(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and
Microsource (New Milford, Conn.). A rare chemical library is
available from Aldrich (Milwaukee, Wis.). Combinatorial libraries
are available and can be prepared. Alternatively, libraries of
natural agents in the form of bacterial, fungal, plant and animal
extracts are available from e.g., Pan Laboratories (Bothell, Wash.)
or MycoSearch (NC), or are readily producible by methods well known
in the art. Additionally, natural and synthetically produced
libraries and agents are readily modified through conventional
chemical, physical, and biochemical means.
[0887] Useful agents may be found within numerous chemical classes.
Useful agents may be organic agents, or small organic agents. Small
organic agents have a molecular weight of more than 50 yet less
than about 2,500 daltons, preferably less than about 750, more
preferably less than about 350 daltons. Exemplary classes include
heterocycles, peptides, saccharides, steroids, and the like. The
agents may be modified to enhance efficacy, stability,
pharmaceutical compatibility, and the like. Structural
identification of an agent may be used to identify, generate, or
screen additional agents. For example, where peptide agents are
identified, they may be modified in a variety of ways to enhance
their stability, such as using an unnatural amino acid, such as a
D-amino acid, particularly D-alanine, by functionalizing the amino
or carboxylic terminus, e.g. for the amino group, acylation or
alkylation, and for the carboxyl group, esterification or
amidification, or the like.
[0888] For primary screening, a useful concentration of a candidate
agent according to the invention is from about 10 mM to about 100
.mu.M or more (i.e. 1 mM, 10 mM, 100 mM, 1 M etc.). The primary
screening concentration will be used as an upper limit, along with
nine additional concentrations, wherein the additional
concentrations are determined by reducing the primary screening
concentration at half-log intervals (e.g. for 9 more
concentrations) for secondary screens or for generating
concentration curves.
[0889] A high throughput screening kit according to the invention
comprises all the necessary means and media for performing the
detection of an agent that modulates platelet-mediated aggregation
by interacting with a target of the invention, such as for example
vWF, or fragment thereof in the presence of a polypeptide,
preferably at a concentration in the range of 1 .mu.M to 1 mM. The
kit comprises the following. Recombinant cells of the invention,
comprising and expressing the nucleotide sequence encoding vWF, or
fragment thereof, which are grown according to the kit on a solid
support, such as a microtiter plate, more preferably a 96 well
microtiter plate, according to methods well known to the person
skilled in the art especially as described in WO 00/02045.
Alternatively vWF, or fragment thereof is supplied in a purified
form to be immobilized on, for example, a 96 well microtiter plate
by the person skilled in the art. Alternatively vWF, or fragment
thereof is supplied in the kit pre-immobilized on, for example, a
96 well microtiter plate. Modulator agents according to the
invention, at concentrations from about 1 .mu.M to 1 mM or more,
are added to defined wells in the presence of an appropriate
concentration of Nanobody or polypeptide of the invention said
concentration of said polypeptide preferably in the range of 1
.mu.M to 1 mM. Kits may contain more than one polypeptide
[0890] Binding assays are performed as according to the methods
already disclosed herein and the results are compared to the
baseline level of, for example vWF, or fragment thereof binding to
a polypeptide of the invention, but in the absence of added
modulator agent. Wells showing at least 2 fold, preferably 5 fold,
more preferably 10 fold and most preferably a 100 fold or more
increase or decrease in vWF-polypeptide binding (for example) as
compared to the level of activity in the absence of modulator, are
selected for further analysis.
[0891] The invention provides for kits useful for screening for
modulators of platelet-mediated aggregation, as well as kits useful
for diagnosis of diseases or disorders characterised by
dysregulation platelet-mediated aggregation. Kits useful according
to the invention can include an isolated vWF, or fragment thereof.
Alternatively, or in addition, a kit can comprise cells transformed
to express vWF, or fragment thereof. In a further embodiment, a kit
according to the invention can comprise a polynucleotide encoding
vWF, or fragment thereof. In a still further embodiment, a kit
according to the invention may comprise the specific primers useful
for amplification of vWF, or fragment thereof. Kits useful
according to the invention can comprise a Nanobody or polypeptide
of the invention. A kit according to the invention can comprise
cells transformed to express said polypeptide. Kits may contain
more than one polypeptide. In a further embodiment, a kit according
to the invention can comprise a polynucleotide encoding a
macromolecule, for example, vWF, or fragment thereof. In a still
further embodiment, a kit according to the invention may comprise
the specific primers useful for amplification of a macromolecule
such as, for example, vWF, or fragment thereof. All kits according
to the invention will comprise the stated items or combinations of
items and packaging materials therefore. Kits will also include
instructions for use.
[0892] The invention also provides for invasive medical devices
coated with a Nanobody or polypeptide of the invention or an agent
resulting from a screening method of the invention for use in
devices requiring the same. Non-limiting examples of devices
include surgical tubing, occlusion devices, prosthetic devices.
Application for said devices include surgical procedures which
require a modulation of platelet-mediated aggregation around the
site of invasion.
[0893] One embodiment of the present is a method for treating
invasive medical devices to prevent platelet-mediate aggregation
around the site of invasion comprising the step of coating said
device with a Nanobody or polypeptide of the invention or agent
according to the invention.
[0894] Another embodiment of the present is a invasive medical
devices that circumvents platelet-mediate aggregation around the
site of invasion, wherein said device is coated with a Nanobody or
polypeptide of the invention or agent according to the
invention.
[0895] In another aspect, the invention relates to a method for the
prevention and/or treatment of at least one aggregation-mediated
disorder (as described herein), said method comprising
administering, to a subject in need thereof, a pharmaceutically
active amount of a Nanobody of the invention, of a polypeptide of
the invention, and/or of a pharmaceutical composition comprising
the same.
[0896] In the context of the present invention, the term
"prevention and/or treatment" not only comprises preventing and/or
treating the disease, but also generally comprises preventing the
onset of the disease, slowing or reversing the progress of disease,
preventing or slowing the onset of one or more symptoms associated
with the disease, reducing and/or alleviating one or more symptoms
associated with the disease, reducing the severity and/or the
duration of the disease and/or of any symptoms associated therewith
and/or preventing a further increase in the severity of the disease
and/or of any symptoms associated therewith, preventing, reducing
or reversing any physiological damage caused by the disease, and
generally any pharmacological action that is beneficial to the
patient being treated.
[0897] The subject to be treated may be any warm-blooded animal,
but is in particular a mammal, and more in particular a human
being. As will be clear to the skilled person, the subject to be
treated will in particular be a person suffering from, or at risk
from, the diseases and disorders mentioned herein.
[0898] The invention also relates to a method for the prevention
and/or treatment of at least one disease or disorder that can be
prevented and/or treated by administering a Nanobody or polypeptide
of the invention to a patient, said method comprising
administering, to a subject in need thereof, a pharmaceutically
active amount of a Nanobody of the invention, of a polypeptide of
the invention, and/or of a pharmaceutical composition comprising
the same.
[0899] More in particular, the invention relates to a method for
the prevention and/or treatment of at least one disease or disorder
chosen from the group consisting of the diseases and disorders
listed herein, said method comprising administering, to a subject
in need thereof, a pharmaceutically active amount of a Nanobody of
the invention, of a polypeptide of the invention, and/or of a
pharmaceutical composition comprising the same.
[0900] In another embodiment, the invention relates to a method for
immunotherapy, and in particular for passive immunotherapy, which
method comprises administering, to a subject suffering from or at
risk of the diseases and disorders mentioned herein, a
pharmaceutically active amount of a Nanobody of the invention, of a
polypeptide of the invention, and/or of a pharmaceutical
composition comprising the same.
[0901] In the above methods, the Nanobodies and/or polypeptides of
the invention and/or the compositions comprising the same can be
administered in any suitable manner, depending on the specific
pharmaceutical formulation or composition to be used. Thus, the
Nanobodies and/or polypeptides of the invention and/or the
compositions comprising the same can for example be administered
orally, intraperitoneally (e.g. intravenously, subcutaneously,
intramuscularly, or via any other route of administration that
circumvents the gastrointestinal tract), intranasally,
transdermally, topically, by means of a suppository, by inhalation,
again depending on the specific pharmaceutical formulation or
composition to be used. The clinician will be able to select a
suitable route of administration and a suitable pharmaceutical
formulation or composition to be used in such administration,
depending on the disease or disorder to be prevented or treated and
other factorse well known to the clinician.
[0902] As mentioned herein and as will be clear to the skilled
person, for acute conditions and complications (i.e. as may occur
with some of the aggregation-mediated disorders mentioned herein),
usually administration directly into the blood stream such by
infusion or injection or any other suitable means will be
preferred.
[0903] The Nanobodies and/or polypeptides of the invention and/or
the compositions comprising the same are administered according to
a regime of treatment that is suitable for preventing and/or
treating the disease or disorder to be prevented or treated. The
clinician will generally be able to determine a suitable treatment
regimen, depending on factors such as the disease or disorder to be
prevented or treated, the severity of the disease to be treated
and/or the severity of the symptoms thereof, the specific Nanobody
or polypeptide of the invention to be used, the specific route of
administration and farmaceutical formulation or composition to be
used, the age, gender, weight, diet, general condition of the
patient, and similar factors well known to the clinician.
[0904] Generally, the treatment regimen will comprise the
administration of one or more Nanobodies and/or polypeptides of the
invention, or of one or more compositions comprising the same, in
one or more pharmaceutically effective amounts or doses. The
specific amount(s) or doses to administered can be determined by
the clinician, again based on the factors cited above.
[0905] Generally, for the prevention and/or treatment of the
diseases and disorders mentioned herein and depending on the
specific disease or disorder to be treated, the potency of the
specific Nanobody and polypeptide of the invention to be used, the
specific route of administration and the specific pharmaceutical
formulation or composition used, the Nanobodies and polypeptides of
the invention will generally be administered in an amount between 1
gram and 0.01 microgram per kg body weight per day, preferably
between 0.1 gram and 0.1 microgram per kg body weight per day, such
as about 1, 10, 100 or 1000 microgram per kg body weight per day,
either continuously (e.g. by infusion), as a single daily dose or
as multiple divided doses during the day. The clinician will
generally be able to determine a suitable daily dose, depending on
the factors mentioned herein. It will also be clear that in
specific cases, the clinician may choose to deviate from these
amounts, for example on the basis of the factors cited above and
his expert judgment. Generally, some guidance on the amounts to be
administered can be obtained from the amounts usually administered
for comparable conventional antibodies or antibody fragments
against the same target administered via essentially the same
route, taking into account however differences in affinity/avidity,
efficacy, biodistribution, half-life and similar factors well known
to the skilled person.
[0906] Usually, in the above method, a single Nanobody or
polypeptide of the invention will be used. It is however within the
scope of the invention to use two or more Nanobodies and/or
polypeptides of the invention in combination.
[0907] The Nanobodies and polypeptides of the invention may also be
used in combination with one or more further pharmaceutically
active compounds or principles, i.e. as a combined treatment
regimen, which may or may not lead to a synergistic effect. Again,
the clinician will be able to select such further compounds or
principles, as well as a suitable combined treatment regimen, based
on the factors cited above and his expert judgement.
[0908] In particular, the Nanobodies and polypeptides of the
invention may be used in combination with other pharmaceutically
active compounds or principles that are or can be used for the
prevention and/or treatment of the diseases and disorders cited
herein, as a result of which a synergistic effect may or may not be
obtained. Examples of such compounds and principles, as well as
routes, methods and pharmaceutical formulations or compositions for
administering them will be clear to the clinician, and for example
include, but are not limited to heparin, aspirin (e.g.
Aspegic.RTM.), Plavix and/or Reopro.
[0909] When two or more substances or principles are to be used as
part of a combined treatment regimen, they can be administered via
the same route of administration or via different routes of
administration, at essentially the same time or at different times
(e.g. essentially simultaneously, consecutively, or according to an
alternating regime). When the substances or principles are
administered to be simultaneously via the same route of
administration, they may be administered as different
pharmaceutical formulations or compositions or part of a combined
pharmaceutical formulation or composition, as will be clear to the
skilled person.
[0910] Also, when two or more active substances or principles are
to be used as part of a combined treatment regimen, each of the
substances or principles may be administered in the same amount and
according to the same regimen as used when the compound or
principle is used on its own, and such combined use may or may not
lead to a synergistic effect. However, when the combined use of the
two or more active substances or principles leads to a synergistic
effect, it may also be possible to reduce the amount of one, more
or all of the substances or principles to be administered, while
still achieving the desired therapeutic action. This may for
example be useful for avoiding, limiting or reducing any unwanted
side-effects that are associated with the use of one or more of the
substances or principles when they are used in their usual amounts,
while still obtaining the desired pharmaceutical or therapeutic
effect.
[0911] The effectiveness of the treatment regimen used according to
the invention may be determined and/or followed in any manner known
per se for the disease or disorder involved, as will be clear to
the clinician. The clinician will also be able, where appropriate
and or a case-by-case basis, to change or modify a particular
treatment regimen, so as to achieve the desired therapeutic effect,
to avoid, limit or reduce unwanted side-effects, and/or to achieve
an appropriate balance between achieving the desired therapeutic
effect on the one hand and avoiding, limiting or reducing undesired
side effects on the other hand.
[0912] The subject to be treated may be any warm-blooded animal,
but is in particular a mammal, and more in particular a human
being. As will be clear to the skilled person, the subject to be
treated will in particular be a person suffering from, or at risk
from, the diseases and disorders mentioned herein.
[0913] The invention also relates to the use of a Nanobody or
polypeptide of the invention in the preparation of a pharmaceutical
composition for the prevention and/or treatment of at least one
disease or disorder (e.g. an aggregation disorder as mentioned
herein) that can be prevented and/or treated by administering a
Nanobody or polypeptide of the invention to a patient.
[0914] Generally, the treatment regimen will be followed until the
desired therapeutic effect is achieved and/or for as long as the
desired therapeutic effect is to be maintained. Again, this can be
determined by the clinician.
[0915] Finally, it will also be clear to the skilled person that it
may be possible to "graft" one or more of the CDR's mentioned above
for the Nanobodies of the invention onto other "scaffolds",
including but not limited to human scaffolds or non-immunoglobulin
scaffolds. Suitable scaffolds and techniques for such CDR grafting
will be clear to the skilled person and are well known in the art,
see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605 522,
EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., Protein
Science (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;
34(2):184-199; Kettleborough et al., Protein Eng. 1991 October;
4(7): 773-783; O'Brien and Jones, Methods Mol. Biol.
2003:207:81-100; and Skerra, J. Mol. Recognit. 2000:13:167-187, and
Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the
further references cited therein; and also include for example the
framework regions of other (single) domain antibodies. For example,
techniques known per se for grafting mouse or rat CDR's onto human
frameworks and scaffolds can be used in an analogous manner to
provide chimeric proteins comprising one or more of the CDR's of
the Nanobodies of the invention and one or human framework regions
or sequences.
[0916] Thus, in another embodiment, the invention comprises a
chimeric polypeptide comprising at least one CDR sequence chosen
from the group consisting of CDR1 sequences, CDR2 sequences and
CDR3 sequences mentioned herein for the Nanobodies of the
invention. Preferably, such a chimeric polypeptide comprises at
least one CDR sequence chosen from the group consisting of the CDR3
sequences mentioned herein for the Nanobodies of the invention, and
optionally also at least one CDR sequence chosen from the group
consisting of the CDR1 sequences and CDR2 sequences mentioned
herein for the Nanobodies of the invention. For example, such a
chimeric polypeptide may comprise one CDR sequence chosen from the
group consisting of the CDR3 sequences mentioned herein for the
Nanobodies of the invention, one CDR sequence chosen from the group
consisting of the CDR1 sequences mentioned herein for the
Nanobodies of the invention and one CDR sequence chosen from the
group consisting of the CDR1 sequences and CDR2 sequences mentioned
herein for the Nanobodies of the invention. The combinations of
CDR's that are mentioned herein as being preferred for the
Nanobodies of the invention will usually also be preferred for
these chimeric polypeptides.
[0917] In said chimeric polypeptides, the CDR's may be linked to
further amino acid sequences sequences and/or may be linked to each
other via amino acid sequences, in which said amino acid sequences
are preferably framework sequences or are amino acid sequences that
act as framework sequences, or together form a scaffold for
presenting the CDR's. Reference is again made to the prior art
mentioned in the last paragraph. According to one preferred
embodiment, the amino acid sequences are human framework sequences,
for example V.sub.H3 framework sequences. However, non-human,
synthetic, semi-synthetic or non-immunoglobulin framework sequences
may also be used. Preferably, the framework sequences used are such
that (1) the chimeric polypeptide is capable of binding xxxx, i.e.
with an affinity that is at least 1%, preferably at least 5%, more
preferably at least 10%, such as at least 25% and up to 50% or 90%
or more of the affinity of the corresponding Nanobody of the
invention; (2) the chimeric polypeptide is suitable for
pharmaceutical use; and (3) the chimeric polypeptide is preferably
essentially non-immunogenic under the intended conditions for
pharmaceutical use (i.e. indication, mode of administration, dosis
and treatment regimen) thereof (which may be essentially analogous
to the conditions described herein for the use of the Nanobodies of
the invention).
[0918] According to one non-limiting embodiment, the chimeric
polypeptide comprises at least two CDR sequences (as mentioned
above) linked via at least one framework sequence, in which
preferably at least one of the two CDR sequences is a CDR3
sequence, with the other CDR sequence being a CDR1 or CDR2
sequence. According to a preferred, but non-limiting embodiment,
the chimeric polypeptide comprises at least two CDR sequences (as
mentioned above) linked at least two framework sequences, in which
preferably at least one of the three CDR sequences is a CDR3
sequence, with the other two CDR sequences being CDR1 or CDR2
sequences, and preferably being one CDR1 sequence and one CDR2
sequence. According to one specifically preferred, but non-limiting
embodiment, the chimeric polypeptides have the structure
FR1'-CDR1-FR2'-CDR2-FR3'-CDR3-FR4', in which CDR1, CDR2 and CDR3
are as defined herein for the CDR's of the Nanobodies of the
invention, and FR1', FR2', FR3' and FR4' are framework sequences.
FR1', FR2', FR3' and FR4' may in particular be Framework 1,
Framework 2, Framework 3 and Framework 4 sequences, respectively,
of a human antibody (such as V.sub.H3 sequences) and/or parts or
fragments of such Framework sequences. It is also possible to use
parts or fragments of a chimeric polypeptide with the structure
FR1'-CDR1-FR2'-CDR2-FR3'-CDR3-FR4. Preferably, such parts or
fragments are such that they meet the criteria set out in the
preceding paragraph.
[0919] The invention will now be further described by means of the
following non-limiting examples and figures, in which the Figures
show:
[0920] FIG. 1: Binding of nanobodies to vWF in ELISA
[0921] FIG. 2: Alignment of 12A5 homologue nanobody sequences
(12A5, SEQ ID NO:60; 12B4, SEQ ID NO:67; 12E8, SEQ ID NO:68; 12A6,
SEQ ID NO:69; 12D8, SEQ ID NO:70)
[0922] FIG. 3: Alignment of 12B6 homologue nanobody sequences
(12B6, SEQ ID NO:62; 12A2, SEQ ID NO:71; 12F2, SEQ ID NO:72; 14H10,
SEQ ID NO:73)
[0923] FIG. 4: Binding of 12A5 homologue nanobodies to vWF in
BIACORE
[0924] FIG. 5: Binding of 12B6 homologue nanobodies to vWF in
BIACORE
[0925] FIG. 6: Platelet adhesion at different concentrations of
12B6, 12A2 and 12A5 nanobodies
[0926] FIG. 7a: Binding in ELISA to vWF for 12B6 nanobody after
heating at increasing temperatures
[0927] FIG. 7b: Binding in ELISA to vWF for 12A2 nanobody after
heating at increasing temperatures
[0928] FIG. 7c: Binding in ELISA to vWF for 12A5 nanobody after
heating at increasing temperatures
[0929] FIG. 8a: Binding of vWF from different species to 12B6
nanobody in ELISA
[0930] FIG. 8b: Binding of vWF from different species to 12A2
nanobody in ELISA
[0931] FIG. 8c: Binding of vWF from different species to 12A5
nanobody in ELISA
[0932] FIG. 9: Binding of bivalent 12B6 nanobodies to vWF in
BIACORE
[0933] FIG. 10: Binding of bivalent 12A2 nanobodies to vWF in
BIACORE
[0934] FIG. 11: Binding of bivalent 12A5 nanobodies to vWF in
BIACORE
[0935] FIG. 12: Binding in ELISA to vWF of bivalent 12B6 nanobodies
after heating at increasing temperatures
[0936] FIG. 13: Binding in ELISA to vWF of bivalent 12A2 nanobodies
after heating at increasing temperatures
[0937] FIG. 14: Binding in ELISA to vWF of bivalent 12A5 nanobodies
after heating at increasing temperatures
[0938] FIG. 15: Alignment of humanised 12B6 nanobody sequences
(12B6, SEQ ID NO:62; 12B6H1, SEQ ID NO:86; 12B6H2, SEQ ID NO:87;
12B6H3, SEQ ID NO:88; 12B6H4, SEQ ID NO:89)
[0939] FIG. 16: Binding in ELISA to vWF of wild type and humanised
12B6 nanobody
[0940] FIG. 17: Alignment of humanised 12A2 nanobody sequences
(12A2, SEQ ID NO:71, 12A2H1, SEQ ID NO:90, 12A2H3, SEQ ID NO:91;
12A2H4, SEQ ID NO:92; 12A2H11, SEQ ID NO:93; 12A2H13, SEQ ID
NO:94)
[0941] FIG. 18: Binding in ELISA to vWF of humanised 12A2
nanobodies, after heating at increasing temperatures
[0942] FIG. 19: Binding in ELISA to vWF of humanised 12A2
nanobodies
[0943] FIG. 20: Alignment of humanised 12A5 nanobody sequences
(12A5, SEQ ID NO:60; 12A5H1, SEQ ID NO:95; 12A5H2, SEQ ID NO:96;
12A5H3, SEQ ID NO:97)
[0944] FIG. 21: Binding in ELISA to vWF of wild type and humanised
12A5 nanobody
[0945] FIG. 22: Alignment of nanobodies selected for bivalent form
(12A2H1, SEQ ID NO:90; 12A2H4, SEQ ID NO:92; 12B6H2, SEQ ID
NO:87)
[0946] FIG. 23: Platelet adhesion at different concentrations of
bivalent (humanised) nanobodies
[0947] FIG. 24: Blood flow pattern for Folts model in baboons
[0948] FIG. 25: Experimental setup for Folts model in baboons
[0949] FIG. 26: Folts study of baboon control group. The blood flow
in function of time is shown, indicating the CFRs (representative
of 2 independent experiments)
[0950] FIG. 27: Folts study of baboon group treated with Aspegic.
The blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0951] FIG. 28: study of baboon group treated with Heparin. The
blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0952] FIG. 29: Folts study of baboon group treated with Plavix.
The blood flow in function of time is shown, indicating the CFRs
(representative of 4 independent experiments)
[0953] FIG. 30: Folts study of baboon group treated with Reopro.
The blood flow in function of time is shown, indicating the CFRs
(representative of 3 independent experiments)
[0954] FIG. 31: Folts study of baboon group treated with ALX-0081
(SEQ ID NO:98). The blood flow in function of time is shown,
indicating the CFRs (representative of 8 independent
experiments)
[0955] FIG. 32: Flow read out from baboon ID 6 treated with a
combination of Aspegic, Heparin, Plavix and ALX-0081
[0956] FIG. 33: Averages of relative blood loss in function of
different doses of Plavix, Reopro and ALX-0081
[0957] FIG. 34: Average length of CFRs and average relative amount
of blood loss for animals treated with Plavix in function of
increasing drug dose.
[0958] FIG. 35: Average length of CFRs and average relative amount
of blood loss for animals treated with Reopro in function of
increasing drug dose
[0959] FIG. 36: Average length of CFRs and average relative amount
of blood loss for animals treated with ALX-0081 in function of
increasing drug dose
[0960] FIG. 37: ristocetin-induced aggregation (%, .box-solid.) and
length of CFRs (s, .diamond-solid.) for each baboon treated with
ALX-0081 in function of all doses
[0961] FIG. 38: Concentration of ALX-0081 in plasma versus the
length of CFRs for all baboons treated with ALX-0081
[0962] FIG. 39: Concentration of ALX-0081 in plasma versus relative
amount of blood loss from the gauzes
[0963] FIG. 40: Folts study of baboon 1 treated with ALX-0081 and
vWF. The blood flow in function of time is shown, indicating the
CFRs
[0964] FIG. 41: strings (arrows) of adhered platelets on ULvWF
secreted from stimulated endothelial cells
[0965] FIG. 42: Absence of strings when platelets are perfused over
ULvWF in the presence of ALX-0081
[0966] FIG. 43: Control perfusion experiment: ULvWF strings before
(panel A, indicated with red arrows) and during (panel B) perfusion
with normal plasma. In panel B, ULvWF strings being cleaved by
ADAMTS-13 are indicated with a blue and red arrow for a piece of an
ULvWF string moving away or for largely cleaved ULvWF strings
respectively
[0967] FIG. 44: Perfusion experiment in presence of ALX-0081.
Microscopic image of a field before (panel A) and of the same field
after (panel B) perfusion with normal plasma. An ULvWF string is
indicated in panel A with a red arrow which is absent in panel B
due to cleavage of the ULvWF by ADAMTS-13.
[0968] FIG. 45: cleavage of A1-A2-A3 by ADAMTS-13 present in normal
pool plasma (NPP) in the absence and presence of ALX-0081.
and in which the Tables, which form an integral part of the present
description, are as follows: Table 8: Sequence listing of anti-vWF
nanobodies Table 9: Expression yields of anti-vWF nanobodies Table
10: Platelet adhesion in perfusion chamber of anti-vWF nanobodies
Table 11: Sequence listing of 12B6 and 12A5 homologue nanobodies
Table 12: Estimated K-on, K-off and KD values for 12A5 homologue
nanobodies Table 13: Estimated K-on, K-off and KD values for 12B6
homologue nanobodies Table 14: Real KD value of 12B6, 12A2 and 12A5
nanobodies Table 15: Platelet adhesion in perfusion chamber of
12B6, 12A2 and 12A5 nanobodies Table 16: Concentration of 12B6,
12A2 and 12A5 nanobodies after heating at increasing temperatures
Table 17: Sequence listing of bivalent nanobodies Table 18:
Sequence listing of linker sequences Table 19: Expression yields of
bivalent 12B6, 12A2 and 12A5 nanobodies Table 20: Concentration of
12B6 bivalent nanobodies after heating at increasing temperatures
Table 21: Concentration of 12A2 bivalent nanobodies after heating
at increasing temperatures Table 22: Concentration of 12A5 bivalent
nanobodies after heating at increasing temperatures Table 23:
Platelet adhesion in perfusion chamber of 12A2 bivalent nanobodies
Table 24: Sequence listing of humanised 12B6 nanobodies Table 25:
Expression yields of wild type and humanised 12B6 nanobodies Table
26: Concentration of wild type and humanised 12B6 nanobodies after
heating at increasing temperatures Table 27: KD values for wild
type and humanised 12B6 nanobodies Table 28: Sequence listing of
humanised 12A2 nanobodies Table 29: Expression yields of wild type
and humanised 12A2 nanobodies Table 30: Concentration of wild type
and humanised 12A2 nanobodies after heating at increasing
temperatures Table 31: Platelet adhesion of wild type and humanised
12A2 nanobodies in perfusion chamber at 0.7 and 1.5 ug/ml Table 32:
Platelet adhesion of wild type and humanised 12A2 nanobodies in
perfusion chamber at 0.5, 1 and 2 ug/ml Table 33: KD values for
wild type and humanised 12A2 nanobodies Table 34: Sequence listing
of humanised 12A5 nanobodies Table 35: Expression yields of wild
type and humanised 12A5 nanobodies Table 36: Concentration of wild
type and humanised 12A5 nanobodies after heating at increasing
temperatures Table 37: KD values for wild type and humanised 12A5
nanobodies Table 38: Sequence listing of humanised bivalent
nanobodies Table 39: Expression yields of humanised bivalent
nanobodies Table 40: Concentration of humanised bivalent nanobody
after heating at increasing temperatures Table 41: Platelet
adhesion of wild type and humanised bivalent nanobodies Table 42:
baboons used with the different test compounds in the Folts study
Table 43: Length of CFRs (s) for control animals (ND=not done)
Table 44: Length of CFRs (s) for animals treated with Aspegic.TM.
(ND=not done) Table 45: Length of CFRs (s) for animals treated with
Heparin.TM. (ND=not done) Table 46: Length of CFRs (s) for animals
treated with Plavix.TM. (ND=not done) Table 47: Length of CFRs (s)
for animals treated with Reopro.TM. (ND=not done) Table 48: Length
of CFRs (s) for animals treated with ALX-0081 (ND=not done) Table
49: baboons used with the different test compounds in the Folts
study Table 50: Inhibition of CFRs in the Folts model for the
different drugs tested. The number of experiments in which an
inhibition of CFRs was observed in the mentioned different
conditions is shown as a function of the total number of
independent repeats of that condition. Table 51: Length of CFRs
(seconds) for each baboon and each dose of Aspegic, Heparin, Plavix
and ALX-0081. The effective dose is indicated in yellow Table 52:
Blood loss relative to the second control gauze for animals treated
with Plavix.TM. in function of final dose (STD=standard deviation)
Table 53: Blood loss relative to the second control gauze for
animals treated with Reopro.TM. in function of final dose
(STD=standard deviation) Table 54: Blood loss relative to the
second control gauze for animals treated with ALX-0081M function of
final dose (STD=standard deviation) Table 55: The average of the
total amount of blood loss (=sum of blood loss from the first five
doses of test compound) as relative to the second control gauze
Table 56: Blood loss in gauzes relative to the second control gauze
for each baboon treated with Aspegic, Heparin, Plavix and ALX-0081
in function of drug dose. The effective drug dose in which a
complete inhibition of CFRs was observed, is indicated in yellow
Table 57: % ristocetin-induced platelet aggregation for each baboon
treated with Aspegic, Heparin, Plavix and ALX-0081 in function of
drug dose Table 58: concentration of ALX-0081 [.mu.g/ml] in blood
samples obtained at 10 minutes after administration Table 59:
Length of CFRs [seconds] for baboons treated with ALX-0081 and with
vWF Table 60: Volumes [.mu.l] to prepare the different mixtures for
study of cleavage of A1A2A3 by ADAMTS13.
EXAMPLES
A. Selection and Screening of Nanobodies Specific for vWF and
Inhibiting Platelet Adhesion
Example 1
Antigen Specific Monovalent Nanobodies
[0969] The nanobodies represented in Table 8 SEQ ID Nos: 60 to 66
are obtained from llamas immunized with human vWF or with
recombinant A1 domain of vWF. The nanobodies bind to the A1 domain
of vWF and inhibit the interaction between vWF and gplb on the
platelets.
Example 2
Expression and Purification of Nanobodies
[0970] Plasmid was prepared (QIAGEN, according to the manufacturers
instructions) and was transformed into WK6 or TG1 electro-competent
cells. A single colony was used to start an overnight culture in LB
containing 2% glucose and 100 .mu.g/ml ampicillin This overnight
culture was diluted 100-fold in 2.times.300 ml TB medium containing
100 .mu.g/ml ampicillin, and incubated at 37.degree. C. until OD600
nm=0.5. 1 mM IPTG was added and the culture was incubated for 3
more hours at 37.degree. C. or overnight at 28.degree. C.
[0971] Cultures were centrifuged for 20 minutes at 10000 rpm at
4.degree. C. The pellet was frozen overnight or for 1 hour at
-20.degree. C. Next, the pellet was thawed at room temperature for
40 minutes, resuspended in 20 ml peri buffer (50 mM
NaH.sub.2PO.sub.4 and 300 mM NaCl) and shaken at room temperature
for 1 hour. Periplasmic fraction was isolated by centrifugation for
20 minutes at 4.degree. C. at 20000 rpm. The nanobodies were
purified on a Nickel column (TALON, Clonetech) as described by the
manufacturer and expression yields were calculated as represented
in Table 9.
Example 3
Binding of Nanobodies to vWF in ELISA
[0972] The nanobodies of Example 1 were tested for binding to vWF
in ELISA. Therefore, a microtiterplate (Nunc, Maxisorb) was coated
with vWF (Red Cross) at a 200-fold dilution and pre-warmed for 15
minutes at 37.degree. C. The plate was coated overnight at
4.degree. C. The plate was then washed with PBS-Tween and blocked
for two hours at room temperature with PBS-1% casein. After
washing, the samples were applied starting at a concentration of 10
.mu.g/ml and 3-fold dilutions were made in PBS. After a two hours
incubation period, the plates were washed and mouse monoclonal
anti-myc antibody at a 1000-fold dilution was applied for 1 hour at
room temperature. The plates were washed and polyclonal
anti-mouse-HRP (DAKO) was applied at a 1000-fold dilution for one
hour at room temperature. The plates were washed and
ABTS/H.sub.2O.sub.2 substrate was applied. The OD 405 nm was
measured. Results are shown in FIG. 1.
Example 4
Inhibition of Platelet Adhesion by Nanobodies in a Flow Chamber
[0973] The protein samples were analysed in a perfusion chamber.
Thermanox coverslips (Nunc) were soaked overnight in 80% ethanol,
rinsed thoroughly with distilled water and air-dried. Human
placental collagen type III (Sigma) was solubilized in 0.05 mol/l
acetic acid and sprayed on the coverslips at a final density of 30
.mu.g/cm.sup.2 with a retouching airbrush. After spraying the
coverslips were blocked with 1% human albumin solution in PBS for
at least 1 hour at RT. Perfusions were performed with a single-pass
perfusion chamber under non-pulsatile flow conditions using a
modified small perfusion chamber with a slit height of 0.1 mm and a
slit width of 2 mm Blood was obtained by venipuncture from healthy
volunteers and anti-coagulated with Penta/PPACK. Triplicate
coverslips were inserted into the chamber. Five milliliters of
blood was pre-warmed at 37.degree. C. for 5 minutes with or without
addition of 2 microgram/ml nanobody and then circulated through the
chamber for 5 minutes at a wall shear rate of 1600 s.sup.-1 using
an infusion pump. After a perfusion run, the coverslip was taken
from the chamber, rinsed in Hepes buffered saline (10 mM Hepes, 150
mM NaCL, ph 7.4), fixed in 0.5% glutaraldehyde in PBS, dehydrated
in methanol and stained with May-Grunwald and Giemsa (Riedel de
Haen). Platelet deposition was evaluated as platelet surface
coverage using light microscopy and computer-assisted analysis.
Results are shown in Table 10. Nanobodies 12B6 and 12A5 clearly
inhibit platelet adhesion to collagen type III in the perfusion
chamber at high shear rate.
Example 5
Analysis in BIACORE for Binding to vWF for Homologues
Nanobodies
[0974] Nanobodies 12B6 and 12A5 inhibit platelet adhesion in the
perfusion chamber. Homologue sequences were obtained from the llama
comprising the amino acid differences as shown in Table 11 SEQ ID
Nos 67 to 73. FIGS. 2 and 3 represent the alignment of the 12A5 and
12B6 homologue nanobody sequences.
[0975] vWF was covalently bound to the sensor chip surface via
amine coupling. The CM5 surface of the chip was activated by the
injection of EDC/NHS (1:1 mix of 0.4 M
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.1 M
N-hydroxysuccinimide in water) for 7 minutes. Upon activation vWF
was injected until an increase of 6000 response units was detected.
The excess of reactive groups was deactivated with 1 M
Ethanolamine-HCl (pH 8.5) for 7 minutes. The flowrate was kept
constant during the immobilization procedure at 5 ul/min The eluent
buffer was 0.01 M HEPES (pH 7.4) with 0.15 M NaCl, 3 mM EDTA and
0.005% Surfactant P20.
[0976] The nanobodies 12B6 and 12A5 and their homologue proteins
were analysed in BIACORE on vWF at a concentration of the nanobody
of 5 .mu.g/ml as shown in FIGS. 4 and 5. Estimated kon, K-off and
KD values are represented in Table 12 and 13. Nanobodies 12A5 and
12B5 have the best K-on and K-off rates. Nanobodies 12A2 and 12B6
have the best K-on rate, the K-off rates are very comparable for
all nanobodies tested.
[0977] Table 14 shows the real KD value for vWF on BIACORE using a
range of concentrations of the nanobodies. From the set of curves
that were generated for each nanobody, only those curves where
equilibrium was reached, were used to derive the KD value via
steady state affinity.
[0978] For the treatment of acute events, the fast inhibition of
vWF is very important, and thus a fast K-on rate is preferred. The
K-on rate determines how fast a nanobody binds its target (vWF)
when injected into human or animals
Example 6
Compare Inhibiting Nanobodies for Potency in the Perfusion
Chamber
[0979] To compare the potency for inhibition of platelet adhesion,
the nanobodies 12A2, 12B6 and 12A5 were tested in the perfusion
chamber at 0.2, 0.4 and 0.6 .mu.g/ml. The experiment was performed
using the same donor for all nanobodies. Results are shown in Table
15 and FIG. 6. The nanobodies show a very comparable inhibiting
capacity in the perfusion chamber, with full inhibition of platelet
adhesion at a concentration of 0.6 .mu.g/ml nanobody.
Example 7
Stability of Nanobodies at Elevated Temperatures
[0980] A stock solution of nanobodies at a concentration of 200
.mu.g/ml in PBS was prepared and divided into several tubes. Each
tube containing nanobody was incubated at different temperatures
for 1 hour, then cooled at room temperature for 2 hours and put at
4.degree. C. overnight. The next day, the samples were centrifuged
for 30 minutes at 13000 rpm, and the supernatant was tested for
OD280 nm. The concentration of supernatants were measured
spectophotometrically and expressed as a percentage of the
concentration at room temperature. The results are summarized in
Table 16.
[0981] The supernatants were also tested in ELISA for binding to
vWF as described above in Example 3. As shown in FIGS. 7a (12B6),
7b (12A2) and 7c (12A5), the nanobodies are very stable at elevated
temperatures.
Example 8
Cross-Reactivity of the Nanobodies with vWF from Other Species
[0982] A microtiterplate was coated with mouse anti-myc at 1/1000
overnight at 4.degree. C. The plate was washed with PBS-Tween and
blocked for two hours at room temperature with PBS-1% casein. After
washing, the nanobodies were applied at a concentration of 10
.mu.g/ml in PBS. After a one hour incubation period, the plates
were washed and plasma (dog, pig, human, baboon and cynomologues
monkey) was applied starting at a five-fold dilution and making
further two-fold dilutions in PBS. The plates were incubated for 1
hour at room temperature. The plates were washed and polyclonal
anti-vWF-HRP (DAKO) was applied at a 2000-fold dilution for one
hour at room temperature. The plates were washed and
ABTS/H.sub.2O.sub.2 substrate was applied. The OD 405 nm was
measured.
[0983] As shown in FIGS. 8a (12B6), 8b (12A2) and 8c (12A5),
nanobodies 12A5, 12A2 and 12B6 are cross-reactive with human,
baboon and cynomologues monkey vWF. The nanobodies 12A2 and 12B6
are also cross-reactive with pig vWF. These nanobodies can
therefore be tested for efficacy and safety in pigs. None of the
nanobodies is cross-reactive with dog vWF.
B. Construction of Bivalent Nanobodies Specific for vWF and
Inhibiting Platelet Adhesion
Example 9
Amino Acid Sequences of the Bivalent Nanobodies
[0984] Table 17 SEQ ID Nos 74 to 82 represents bivalent nanobodies
constructed for 12B6, 12A2 and 12A5. The nanobodies were linked
with the linkers represented in Table 18 SEQ ID Nos 83 to 85.
Example 10
Expression and Purification of the Bivalent Nanobodies
[0985] Expressions were performed as described above in Example 2.
Expression yields are summarized in Table 19.
Example 11
Analysis of the Bivalent Nanobodies in BIACORE
[0986] The bivalent nanobodies of Example 9 were analyzed in
BIACORE at 1.3 nM as described above in Example 5 to compare the
affinities for vWF versus the monovalent nanobody. The bivalent
nanobodies 12B6 (FIG. 9), 12A2 (FIG. 10) and 12A5 (FIG. 11) have an
improved affinity for vWF when compared to the monovalent
nanobody.
Example 12
Stability of the Bivalent Nanobodies at Elevated Temperatures
[0987] Stability of the bivalent nanobodies was measured as
described above in Example 7. The concentration (.mu.g/ml) of the
supernatants was measured and expressed as a percentage of the
concentration at room temperature. The results are summarized in
Table 20 (bivalent 12B6), Table 21 (bivalent 12A2) and Table 22
(bivalent 12A5).
[0988] The supernatants were tested in ELISA for binding to vWF as
described above in Example 3. The supernatants were applied
starting at a 1/100 dilution and 1/5 dilutions were made in PBS.
Results are shown in FIG. 12 (12B6), FIG. 13 (12A2) and FIG. 14
(12A5).
Example 13
Analysis of Monovalent and Bivalent 12A2 in the Flow Chamber
[0989] Nanobody 12A2 (monovalent and bivalent forms) was tested in
the perfusion chamber as described above in Example 4. The
experiment was performed using the same donor for all nanobodies.
Results are summarized in Table 23. The bivalent nanobodies inhibit
platelet adhesion more efficiently then the monovalent form.
C. Humanisation of Nanobodies Specific for vWF and Inhibiting
Platelet Adhesion
Example 14
Humanisation of 12B6 Nanobody
[0990] Table 24 SEQ ID Nos: 86 to 89 represents four humanised 12B6
nanobodies. Table II lists the amino acid changes that were
performed to achieve these sequences. FIG. 15 represents the
alignment of the humanised sequences for 12B6.
TABLE-US-00043 TABLE II non-limiting humanizing substitutions Q1E
A14P A17S E44G D46E R76N M77S A82bS K83R P84A 12B6H1 X X X X X X X
12B6H2 X X X X X X X X 12B6H3 X X X X X X X X 12B6H4 X X X X X X X
X
[0991] Expressions were performed as described in Example 2.
Expression yields are summarized in Table 25.
[0992] The stability of the humanised nanobodies was measured as
described in Example 7. Table 26 summarizes the OD280 nm
concentrations (.mu.g/ml) of the supernatants expressed as a
percentage of the concentration at room temperature.
[0993] FIG. 16 shows the binding of humanised 12B6 nanobodies to
vWF in ELISA performed as described in Example 3.
[0994] The affinity of humanised 12B6 nanobodies for vWF was
determined in BIACORE. KD values are summarized in Table 27.
Example 15
Humanisation of 12A2 Nanobody
[0995] Table 28 SEQ ID Nos: 90 to 94 represents five humanised 12A2
nanobodies. Tables III and IV list the following amino acid changes
that were performed to achieve these sequences. FIG. 17 represents
the alignment of the humanised sequences for 12A2.
TABLE-US-00044 TABLE III non-limiting humanizing substitutions: Q1E
K3Q E5V A14P A17S R27F E44G D46E 12A2H1 X X X X X X X 12A2H3 X X X
X X X X 12A2H4 X X X X X X X 12A2H11 X X X X X X X X 12A2H13 X X X
X X X X X
TABLE-US-00045 TABLE IV R76N M77S N82bS K83R P84A Q108L 12A2H1 X X
X 12A2H3 X X X X 12A2H4 X X X X 12A2H11 X X X 12A2H13 X X X X X
X
[0996] Expressions were performed as described in Example 2.
Expression yields are summarized in Table 29.
[0997] Stability of the humanised nanobodies was measured as
described in Example 7. Table 30 summarizes the OD280 nm
concentrations (.mu.g/ml) of the supernatants expressed as a
percentage of the concentration at room temperature. All humanised
12A2 nanobodies are very stable upon heating at increasing
temperatures.
[0998] The ELISA of FIGS. 18 and 19 was performed as described in
Example 3. 12A2H1 and 12A2H4 bind very well to vWF in ELISA.
[0999] The nanobodies were tested in the flow chamber at a
concentration of 0.7 .mu.g/ml and 1.5 .mu.g/ml. The same donor was
used for all the experiments. The experiment was performed as
described in Example 4. Results are summarized in Table 31 and
32.
[1000] The affinity of humanised 12A2 nanobodies for vWF was
determined in BIACORE. KD values are summarized in Table 33.
Example 16
Humanisation of 12A5 Nanobody
[1001] Table 34 SEQ ID Nos: 95 to 97 represents three humanised
12A5 nanobodies. Table V lists the amino acid changes that were
performed to achieve these sequences. FIG. 20 represents the
alignment of the humanised sequences for 12A5.
TABLE-US-00046 TABLE V non-limiting humanizing substitutions: A1E
L23A Q44G G73N P74A K83R P84A 12A5H1 X X X X X 12A5H2 X X X X X X
12A5H3 X X X X X X X
[1002] Expressions were performed as described above in Example 2.
Expression yields after TALON purification are summarized in Table
35 for each humanised 12A5 nanobody.
[1003] Stability of the humanised 12A5 nanobodies was measured as
described above in Example 7. The OD280 of the supernatants was
measured and expressed as percentage of the OD280 at room
temperature. The results are summarized in Table 36. All humanised
12A5 nanobodies are comparably stable upon heating at increasing
temperatures to the wild type.
[1004] ELISA was performed as described in Example 3. FIG. 21
illustrates the binding activity for vWF in ELISA.
[1005] The affinity of humanised 12A5 nanobodies for vWF was
determined in BIACORE. KD values are summarized in Table 37.
Example 17
Bivalent Humanised Nanobodies
[1006] Three humanised nanobodies 12A2H1, 12A2H4 and 12B6H2 were
selected for bivalent form with the 3a linker. The sequences of
these 3 nanobodies differ only by a few amino acids as shown in
FIG. 22. Table 38 SEQ ID Nos 98 to 100 lists the sequences of the
bivalent nanobodies. Table 38 SEQ ID Nos 101- to 106 lists the
sequences of humanised bivalent nanobodies linked with the GS9 and
GS30 linker, respectively.
[1007] Expressions were performed as described in Example 2. The
nanobodies containing a (His).sub.6-tag were purified on a Nickel
column (TALON, Clonetech) as described by the manufacturer. The
tag-sequence is EQKLISEEDLNGAA.sub.HHHHHH. Nanobodies without tags
were purified on protein A. Expression yields were calculated and
are summarized in Table 39.
[1008] Stability of the humanised bivalent nanobodies was measured
as described in Example 7. OD280 of the supernatants was measured
and expressed as percentage of the OD280 at room temperature. The
results are summarized in Table 40.
[1009] The nanobodies (humanised but also wild type) were tested in
the flow chamber at a concentration of 0.15 .mu.g/ml, 0.3 .mu.g/ml
and 0.6 .mu.g/ml. The same donor was used for all the experiments.
The experiment was performed as described in Example 4. FIG. 23
shows the platelet adhesion at different concentrations of bivalent
nanobodies. Table 41 lists platelet adhesion of wild type and
humanised bivalent nanobodies.
D. Effect of (Bivalent) Nanobody on Arterial Thrombosis in a Baboon
FOLTS Model
Example 18
Baboon FOLTS Model with ALX-0081
[1010] In this study the efficacy and safety of ALX-0081 was
evaluated in a Folts thrombosis model in baboons.
[1011] Also, the efficacy and safety of ALX-0081 in a Folts
thrombosis model in baboons was compared to other drugs currently
used in the clinic, such as Reopro, Plavix, Aspegic, Heparin and
Epinephrin. All these were diluted in 0.9% sodium chloride and
administered as intravenous bolus injections. This study was as
well designed to determine the effective dose for each of these
compounds.
[1012] Finally, the efficacy in a Folts thrombosis model in baboons
of a combination of drugs that is currently used in the clinic in a
percutaneous coronary intervention (PCI) setting was tested:
Aspegic, Heparin, and Plavix. We furthermore evaluated if ALX-0081
can improve the efficacy of this combination when added on top.
[1013] We looked at safety parameters such as induction of
bleeding, vWF and factor VIII levels, and platelet count, PT, and
aPTT.
Study Protocol
[1014] The study protocol that was applied is the original Folts
model and some modifications described below (Folts J D, et al,
Circulation. 1976; 54:365-370).
[1015] Healthy male or female baboons (Papio ursinus) were used.
The animals were 8-17 kg of weight and were disease-free for at
least 2 weeks prior to use. The baboons were fed with dry standard
food only. The baboons were used at different time points. The
weight of the baboons are summarized in table 42 (efficacy study
ALX-0081 and comparison with individual drugs) and table 50
(efficacy of a combination of drugs and ALX-0081 on top of this
combination).
[1016] Animals were anaesthetized and body temperature is
maintained at 37.degree. C. with a heating table. A segment of a
femoral artery was dissected free from surrounding tissue. A shunt
was placed between the femoral vein and femoral artery to obtain
high shear rates. The mean and phasic blood flow was recorded
continuously throughout the experiment. Baseline flow was recorded
for 20 minutes. The proximal dissection site of the femoral artery
was then injured by applying two overlapping occlusions of the
artery for 1 second using a forceps. A clamp was placed over the
injured site to create an external stenosis.
[1017] A gradual decline in blood flow due to platelet adhesion and
aggregation was observed. When flow was reduced to zero, blood flow
was restored by opening the clamp to dislodge the platelet-rich
thrombus. This repetitive pattern of decreasing blood flow
following mechanical restoration is referred to as cyclic flow
reductions (CFRs). Additional endothelial injury was repeated if
needed to finally obtain stable CFRs in these baboons. The number
of times the thrombus needed to be dislodged determines the number
of CFRs. FIG. 24 illustrates blood flow pattern during the Folts
model in baboons.
[1018] After a 30-minute control period of reproducible CFRs, the
vehicle was administered as an internal control and CFRs were
followed up for 30 more minutes. After this period, test agents
(saline (n=2), Reopro (n=3), Aspegic (n=3), Plavix (n=4), Heparin
(n=3) or ALX-0081 Nanobody.TM. (n=9)) were provided via an
intravenous bolus injection (followed by a continuous infusion for
ALX-0081) and monitoring was continued up to 30 minutes after drug
administration. This procedure was repeated for several times with
escalating doses of the test substance. The anti-thrombotic effect
was quantified by comparing the length of CFRs before and after
drug administration. When full inhibition of CFRs was observed, a
new injury was applied in order to confirm that the inhibition was
an effect of the treatment but not of a natural healing phenomenon.
At the end of the experiments, Epinephrin (2.2 .mu.g/kg/min) was
injected in order to distinguish between a weak and a strong
inhibition of the CFRs. Indeed, it has been demonstrated before
that CFRs reappear in the presence of Epinephrin when aspirin (a
weak anti-platelet drug) is used in the same model.
The setup of the experiment is illustrated FIG. 25.
[1019] The length of the CFRs, after each dose of test compound are
summarized in tables 43-48. Doses at which full inhibition of CFRs
is obtained are shaded.
[1020] A representative read out of the blood flow during the Folts
model experiments is shown in FIGS. 26-31.
[1021] The results demonstrate that CFRs can be obtained in the
control animals for at least 3 hours, without the need for a new
injury in between. The mean length of the CFRs is 2-5 minutes and
there is no effect on the length of the CFRs by injection of saline
(FIG. 26, table 43).
Aspegic
[1022] Three animals were injected with Aspegic (injectable
Aspirin) and looked for inhibition of CFRs. In the clinic, a bolus
injection of 250 mg (.+-.3-5 mg/kg) is administered to the patient,
just before the start of a percutaneous coronary intervention (PCI)
procedure. In two animals (baboon 3 and 5) no inhibition of CFRs
could be obtained at doses as high as 80 and 40 mg/kg Aspegic
respectively (FIG. 27, table 44). In baboon 4, it was very
difficult to establish a stable repetitive pattern of CFRs in the
control phase. After several new injuries were made (this is at the
time we injected saline), stable CFRs were obtained. Full
inhibition of CFRs was obtained at the dose of 5 mg/kg Aspegic, but
at higher doses and upon new injury, the CFRs returned, although
the mean length of the CFRs was 3-4 times longer than before
administration of Aspegic. After infusion of Epinephrin, the CFRs
returned immediately and completely (table 44).
Heparin
[1023] Three animals were injected with unfractionated Heparin and
looked for inhibition of CFRs. In the clinic, a bolus injection of
60-70 IU/kg is administered to the patient, and the aPTT (activated
partial thromboplastin time) is monitored every 30 minutes. Extra
Heparin is administered if the aPTT is <250 seconds. In baboons
7 and 8, no inhibition of the CFRs could be obtained even not at
doses as high as 240 IU/kg (FIG. 28, table 45). In baboon 6, full
inhibition of the CFRs was obtained at the first dose of 15 IU/kg
and at higher doses, but when we made a new injury the CFRs
returned each time. At the highest dose of 240 IU/kg, CFRs were
inhibited even after a new injury, but the flow was decreasing and
upon infusion of Epinephrine, the CFRs returned immediately.
Plavix
[1024] Four baboons were treated with Plavix and used for the Folts
study. We used Plavix as injectable drug by re-suspending tablets
in methanol. Therefore, we were able to perform a dose escalation
experiment as for the other drugs. In patients, 300-600 mg Plavix
is administered orally, inhibition of platelet aggregation can be
seen 2 hours after single oral doses of Plavix. Already at the 2.5
mg/kg final dose in the baboons, an effect on the length of the
CFRs could be demonstrated, but this inhibitory effect started only
10 minutes after injection (FIG. 29, table 46). In baboon 12, full
inhibition of the CFRs was obtained at this dose of 2.5 mg/kg. In
the other three baboons full inhibition of CFRs was obtained at the
5 mg/kg final dose. CFRs remained inhibited when a new injury was
made, but returned after infusion of Epinephrin. When Epinephrin
infusion was stopped, the CFRs remained for another 5 minutes, but
then again full inhibition was obtained (FIG. 29).
Reopro
[1025] Reopro was tested for efficacy in the Folts model in three
baboons. In the clinic, patients receive a dose of 250 .mu.g/kg
followed by a continuous infusion of 7.5 .mu.g/kg/hour. This is
also the dose which we needed in baboons 13, 14 and 15 to obtain
full inhibition of the CFRs (final dose of 170-420 .mu.g/kg (FIG.
30, table 47). We administered to the baboons a bolus injection
only. When a new injury was applied, complete inhibition of the
CFRs was retained and infusion of Epinephrin could not reverse this
inhibition (FIG. 30).
ALX-0081
[1026] Nine baboons received ALX-0081 and were used in the Folts
model. In all baboons full inhibition of CFRs was obtained at the
dose of 30 .mu.g/kg+45 .mu.g/kg/hour (final dose of 43 .mu.g/kg).
In 2 baboons, full inhibition was already obtained at the 10
.mu.g/kg+15 .mu.g/kg/hour (baboons 17 and 22) Inhibition was
retained upon a new injury and after infusion of Epinephrin in all
nine baboons (FIG. 31, table 48).
Aspegic-Heparin-Plavix-ALX-0081 (Asp/Hep/Plav/ALX) Combinations
[1027] Seven baboons received a bolus injection of 5 mg/kg Aspirin,
60 IU/kg Heparin and increasing doses of Plavix. Extra Heparin was
administered at different time points to sustain a certain level
(aPTT should be at least doubled versus control). CFRs were
monitored for 30 minutes after each dose of test compounds. We
started at a dose of 1 mg/kg Plavix and added 1 mg/kg after 30
minutes. The anti-thrombotic effect was quantified by comparing the
length of CFRs before and after drug administration. When full
inhibition of CFRs was observed, a new injury was applied.
Epinephrin was injected and continued till the end of the
experiment. If CFRs did not return, a new injury was applied. After
2-3 CFRs increasing doses of ALX-0081 were added: (1), 3, 10 or 30
.mu.g/kg. We waited after each dose of ALX-0081 for 2 CFRs and
increased the dose until full inhibition of CFRs was obtained. At
full inhibition of the CFRs, continuous infusion was started of 1.5
times dose ALX-0081/kg/hour for 30 minutes and epinephrine infusion
was continued. A new injury was applied after 10-15 minutes. The
specifications of the baboons that were used in this study are
represented in table 49.
[1028] The results from these studies for each test compound
individually are summarized in table 50. A clear superior
anti-thrombotic effect in the Folts thrombosis baboon model is
observed for ALX-0081 and Reopro when compared to Aspirin, Heparin
or Plavix: upon new injury and after infusion of Epinephrin, the
CFRs do not return in the Folts model in the ALX-0081 and Reopro
treated baboons in contrast to the model in Aspegic, Heparin or
Plavix treated animals The dose of ALX-0081 required for full
inhibition of CFRs is approximately 10-fold lower than the dose
needed for Reopro. Therefore, it is concluded that ALX-0081 is more
potent than Reopro.
[1029] After administration of a combination of 5 mg/kg Aspirin, 60
IU/kg Heparin and increasing doses of Plavix, in all seven baboon
full inhibition of CFRs was obtained at this final dose. The dose
of Plavix required for full inhibition is 2.5-fold lower than the
dose needed when Plavix alone is administered. For all baboons
tested, CFRs did not return when a new injury was made (FIG. 32).
However, upon injection of Epinephrin, CFRs returned spontaneously
in baboons 5, 8, 9 and 10 and upon a new injury in baboons 4, 6 and
7. Extra Heparin was injected at the same time as epinephrin. After
2 CFRs, increasing doses of ALX-0081 were administered while
continuing the infusion of Epinephrin. The dose of ALX-0081 was
increased from 1 over 3-10 to 30 .mu.g/kg. When full inhibition of
CFRs was obtained, a continuous infusion of ALX-0081 was started at
1.5 times the effective dose/kg/hour. In all seven baboons, full
inhibition of the CFRs was obtained at the 30 .mu.g/kg dose. This
effective dose of ALX-0081 is the same as required for complete
inhibition of CFRs in the Folts model when ALX-0081 is administered
alone.
[1030] Therefore, we can conclude that the efficacy of ALX-0081 is
not increased by simultaneous infusion of Plavix, Heparin and
Aspegic. This observation is completely in line with our
hypothesis: ALX-0081 inhibits the very first interaction between
platelets and the exposed collagen in the damaged arterial wall.
Plavix and Aspegic on the other hand inhibit further downstream in
the cascade leading to the development of a thrombus. Therefore,
Plavix and Aspirin do not contribute to better efficacy as ALX-0081
interferes already with the first step in thrombus formation.
Moreover, when a new injury was applied at the effective dose of
ALX-0081, the CFRs did not return, demonstrating a potent
antithrombotic effect of this Nanobody.TM.. The results are
summarized in table 51.
Measurements
[1031] The following parameters were measured: a) bleeding
analysis, b) vWF concentration, Factor VIII levels, and platelet
count, PT and aPTT c) ristocetin-induced platelet aggregation d)
ALX-0081 concentration, and e) analysis of arterial sections for
restenosis, f) immunogenicity of ALX-0081
a) Bleeding Analysis
[1032] To analyze bleeding, an incision was made with a scalpel in
the groin. This was done at 15 minutes after recording baseline
flow, when the injury was made in the artery. Gauzes were inserted
in the wound and replaced every 30 minutes just before each new
dose of test compound. The amount of blood loss after each dose of
test compound was determined by weighing the gauzes. Blood loss is
expressed relative to the amount of blood loss in the second
control gauze (during the saline injection) (tables 52-54).
[1033] For all baboons treated with Plavix and Reopro, blood loss
is high (up to 9-40 fold respectively at the highest dose),
starting from the effective dose on. For animals treated with
ALX-0081 bleeding is lower than in the Plavix treated animals, and
much lower when compared to Reopro treated animals.
[1034] In order to determine the safety versus efficacy level of
Plavix, Reopro and ALX-0081 as antithrombotic drugs, the averages
of blood loss relative to the second control gauze are shown for
these drugs in function of the drug dose as multiple of the
effective dose (FIG. 33). The effective dose for Plavix is 5 mg/kg,
for Reopro 250 .mu.g/kg and for ALX-0081 30 .mu.g/kg (table 46-48).
These results nicely demonstrate the superior safety of ALX-0081
when compared to Reopro and Plavix: the window in which ALX-0081
could be administered without a major increase in bleeding is much
wider compared to Plavix and Reopro.
[1035] The results from the averages of blood loss in the gauzes
(if available) were combined with the averages of the lengths of
the CFRs in the FIGS. 34-36.
[1036] A broad therapeutic window was observed for ALX-0081 in the
Folts model: a strong antithrombotic effect could be demonstrated
without any major bleedings for cumulative doses ranging from 43
.mu.g/kg up to 403 .mu.g/kg (FIG. 36). In contrast however, the
therapeutic window for Reopro and Plavix in the same model was much
more narrow compared to ALX-0081, combining an effective
antithrombotic effect with a high blood loss (FIG. 34-35).
[1037] The average of the total amount of blood loss (=sum of blood
loss from the gauzes of the first five doses of test compound) as
relative to the second control gauze are summarized in table 55. In
this table we also indicate the final dose as multiple of the
effective dose (=sum of the five doses divided by the effective
dose). As mentioned before, the effective dose for Plavix is 5
mg/kg, for Reopro 250 .mu.g/kg and for ALX-0081 30 .mu.g/kg.
[1038] The results shown in table 55 clearly show that total blood
loss is significantly increased in the animals treated with Plavix
and to an even higher extent in the animals treated with Reopro.
Blood loss in animals receiving ALX-0081 is 2-fold and 4 fold less
than in Plavix or Reopro treated animals, respectively. This again
clearly demonstrates that ALX-0081 is safer than Plavix and Reopro
in terms of bleeding risk, although doses of more than 10-fold the
effective dose were used.
[1039] The effective combination of Aspegic, Heparin and Plavix
results in an increase in blood loss of up to 14 fold when compared
to the control gauze (table 56). Addition of ALX-0081 on top of the
combination of Aspegic, Heparin and Plavix does not result in
increased bleeding except for baboons 4 and 7. In baboon 7,
bleeding was much increased after administration of epinephrine,
extra heparin and non effective doses of ALX-0081, but was lower
again after administration of the effective dose of ALX-0081. These
results demonstrated that ALX-0081 is safe when added on top of the
combination of drugs that is currently used in a clinical
setting.
b) vWF Concentration, Factor VIII Level, Platelet Count, PT and
aPTT vWF
[1040] The vWF levels in the platelet rich plasma (PRP) of blood
samples taken after administration of the different doses of the
drugs in the Folts model were determined using an immunosorbent
assay and expressed as a percentage of the human standard (WHO
5.sup.th International Standard for factor VIII and VWF)
[1041] The results clearly demonstrate that the different drugs
used in the model have no major effect on the vWF level.
Factor VIII
[1042] The factor VIII levels in the PRP of blood samples taken
after administration of the different doses of the drugs in the
Folts model were determined using the aPTT test. We did not test
the plasma samples of the baboons treated with Heparin, as we
demonstrated that Heparin prolongs the aPTT time. The FVIII levels
were expressed as percentage of the first control sample, taken 10
minutes after injury of the femoral artery. We do not see any
effect of the treatments on the aPTT test.
Platelet Count, PT and aPTT
[1043] The platelet count measurements during the Folts model
experiments were performed. The data showed that baboon 15 has a
very low platelet count when compared to the other animals The
platelet counts for all kind of treatments, except for the Plavix
treatment, are very comparable to what we see in the control
animals and are fairly constant over time.
[1044] The PT values demonstrate no effect of the test compounds on
the PT time, except for baboons treated with the 240 IU/kg dose of
Heparin where a minor increase in PT was observed.
[1045] The aPTT values observed during the Folts model studies are
summarized. These results indicate that the test compounds have no
effect on the aPTT values, except for the baboons treated with
Heparin. In these animals, aPTT values are prolonged from the 30-60
IU/kg dose on, as is also observed in patients. Heparin acts as an
anticoagulant by forming a complex with antithrombin and catalyzing
the inhibition of activated blood coagulation factors such as XIa,
IXa, Xa and thrombin (factor IIa). These factors are all involved
in the intrinsic coagulation cascade of which its functionality is
measured in the aPTT test.
c) Measurement of Ristocetin-Induced Platelet Aggregation in Blood
Obtained from Baboons Treated with ALX-0081
[1046] Blood obtained from baboons treated with ALX-0081 was
analyzed for inhibition of platelet aggregation. Platelet
aggregations were performed on a Chronolog whole blood and optical
Aggregometer (Model 560CA, Chronolog, USA). PRP was prepared
(collected on 0.38 mol/L citrate), by centrifuging the whole blood
at 1200 rpm for 5 minutes. The upper fraction containing the PRP
was carefully removed. The lower fraction was further centrifuged
at 3000 rpm for 10 minutes to prepare platelet poor plasma (PPP).
Platelets were counted in PRP and diluted in PPP to a final
concentration of 200.000 platelets per microliter. 3 mg/ml
ristocetin (DAKO) was added and aggregation was measured.
[1047] The ex vivo platelet aggregation is measured in the blood
samples taken during the Folts experiment in the baboons treated
with ALX-0081. The GPIb-IX-V dependent platelet aggregation through
vWF is measured using ristocetin as a modulator. The % aggregation
is measured at each time point and at each dose. The control sample
is taken at 10 minutes after arterial injury.
[1048] Results from the RIPA test are compared to the inhibition of
the CFRs for each baboon treated with ALX-0081 (FIG. 37). As shown
in FIG. 37, an inverse relation between the RIPA and the length of
the CFRs is observed. Moreover, these results demonstrate that full
inhibition in the RIPA test is obtained at lower doses than full
inhibition of CFRs in baboons 16, 18, 19 and 23. For baboons 20,
21, 22 and 24 the results with RIPA compare very well with the
results for efficacy in the Folts model.
d) Concentration of ALX-0081
[1049] Microtiterplates are coated with mouse polyclonal anti-myc
overnight at 4.degree. C. at a 1000-fold dilution. The plates are
washed with PBS-Tween and blocked for 2 hours at RT with PBS-1%
casein. Samples are diluted in a non-coated microtiterplate in 25%
reference baboon plasma. The standard curve is prepared by diluting
the nanobody in the same reference baboon plasma sample. Samples
are applied on the anti-myc coated plates and allowed to bind for 2
hours at RT. The plates are washed 5 times with PBS-Tween. Rabbit
anti-vWF-HRP (DAKO) is applied at a 3000-fold dilution for one hour
at RT. For measuring OD405 nm, samples are washed 5 times with
PBS-Tween and ABTS/H.sub.2O.sub.2 substrate is added.
[1050] We determined the concentration of ALX-0081 in plasma
samples taken at 10 minutes after each bolus injection. The bolus
injection was immediately followed by a continuous infusion. The
concentrations (.mu.g/ml) are summarized in table 58.
[1051] For all baboons an increasing level of ALX-0081 in the
plasma samples was measured by ELISA after dose-escalation of
ALX-0081. For baboon 16, consistently higher amounts of ALX-0081
were determined in the plasma sample taken after the 10 .mu.g/kg
dose compared to the sample taken after 30 .mu.g/kg. The ALX-0081
level in that sample is also substantially higher than what is
noted for all other baboons given the same dosing schedule of
ALX-0081. As the levels of ALX-0081 for baboon 16 after the higher
doses are in line of the expectations, we assume that an unknown
abnormality during blood sampling accounts for this outlier.
[1052] The concentration of ALX-0081 for each dose in the different
baboons is variable. For the 3 .mu.g/kg dose, the concentration
ranges between 0.03 and 0.14 .mu.g/ml for 10 .mu.g/kg between 0.18
and 1.23 .mu.g/ml, for 30 .mu.g/kg between 0.51 and 1.14 .mu.g/ml,
for 90 .mu.g/kg between 1.38 and 6.77 .mu.g/ml and for 270 .mu.g/kg
between 4.03 and 35.14 .mu.g/ml.
In FIG. 38, we plot the concentration of ALX-0081 in plasma versus
the length of the CFRs.
[1053] The concentration of ALX-0081 required for full inhibition
of CFRs is between 0.3 and 0.5 .mu.g/ml, which is in full agreement
with the concentration required to inhibit platelet adhesion to
collagen in the flow chamber at high shear rate, when ALX-0081 is
spiked in human blood. In blue (FIG. 38, panel B) we indicated the
concentration range of ALX-0081 where inhibition starts (leaving
out the 10 .mu.g/kg dose in baboon 16).
[1054] When we plot the concentration of ALX-0081 versus the
relative amount of blood loss from the gauzes, we observe a more
than 2-fold increase in bleeding at doses above 1 .mu.g/ml (FIG.
39). A 10-fold increase in blood loss was observed when the
ALX-0081 concentration is 19 .mu.g/ml, which is 40-60-fold the
effective concentration.
e) Analysis of Arterial Sections for Restenosis
[1055] Four weeks after treatment of the second artery, the
arteries are dissected free from surrounding tissue. The artery is
tied at the upper and low site of the endothelial injury including
the site where the shunt was placed. A section is removed of 2
centimetre, cut in a lower (shunt site) and upper part (stenosed
and injured site) and stored in 10% formaldehyde. The baboons are
then sacrificed by euthanase injection. The arteries are marked
according to origin and cut into rings of 2 mm each. The rings are
placed into marked cassettes suitable for histology processing. The
cassettes are then placed overnight in an automated VIP Tissue Tek
processor following the overnight processing schedule as described
in Bancroft (Bancroft, John D., Stevens Alan (1990). Theory and
Practice of Histological Techniques. Third Edition).
[1056] After processing, the arteries are embedded in marked
paraffin wax blocks and cooled on a freeze plate. The wax blocks
are cut in series sections of 4 micron each on a rotary microtome.
Sections are picked up on glass slides and stained for histological
evaluation. Haematoxylin and Eosin as well as Verhoeff's method for
elastic fibers stains are performed on each of the arteries
(Bancroft, John D., Stevens Alan (1990). Theory and Practice of
Histological Techniques. Third Edition). After staining, the slides
are dehydrated, cleared, mounted and labelled. Blind analysis of
the sections is performed.
f) Immunogenicity Analysis
[1057] The presence of ALX-0081 immunoglobulins in plasma of three
baboons was evaluated by two methods, respectively an ELISA method
and a SPR-based method on Biacore. The baboons were treated for 8
weeks with increasing doses of ALX-0081 (starting from 10
.mu.g/kg). During said 8 weeks, no immunogenic response could be
observed upon injection of ALX-0081. The half-life of ALX-0081
ranged between 7 and 9 hours.
Example 20
Use of vWF as an Antidote for ALX-0081
[1058] Despite the proven safety of ALX-0081 in baboons, and the
rapid clearance of the Nanobody.TM., we decided to evaluate the use
of vWF as an antidote for ALX-0081. This was tested in a Folts
model in baboons where we evaluated if the inhibitory effect of
ALX-0081 on arterial thrombus formation can be reversed by
injection of vWF.
[1059] The experimental procedure followed the original Folts model
with the modifications as described in the previous example 18.
[1060] Healthy male baboons (Papio ursinus) were used in this
study. The animals were 9-12 kg of weight and were disease-free for
at least 2 weeks prior to use. The baboons were fed with dry
standard food only.
[1061] Three baboons were used in this study, the length of the
CFRs during the control phase, after administration saline,
ALX-0081 and vWF is summarized in table 59.
[1062] The blood flow as a function of time for each baboon and
experiment is shown in FIG. 40.
[1063] In all three baboons, full inhibition of CFRs was obtained
at the 30 .mu.g/kg+45 .mu.g/kg/hour dose of ALX-0081, even when a
new injury was applied, the CFRs did not return. Upon injection of
the first dose of vWF (250 IU), the flow gradually decreased, but
CFRs did not return until an extra dose of 250 IU of vWF was
administered. This result demonstrated nicely that the activity of
ALX-0081 can be reversed by administration of vWF, and that
therefore, vWF would be a good antidote for this Nanobody.TM..
[1064] Thus, another aspect of the invention relates to the use of
vWF, of a suitable fragment thereof, of DDAVP (desmopressinor) or a
suitable fragment thereof, or of a pharmaceutical composition
comprising any of the foregoing, as an antidote for complications
or undesired side effects associated with the use of a Nanobody,
protein or polypeptide against vWF, in particular a Nanobody,
protein or polypeptide as described herein.
Example 21
Effects of ALX-0081 on Platelet Adhesion to Endothelial
Cell-Derived ULvWF and on the Activity of ADAMTS-13
[1065] This study serves as a proof of concept for the use of
ALX-0081 as a drug in TTP patients. Perfusions of platelets
reconstituted in TTP plasma (no ADAMTS13) are performed on
endothelial cells secreting ULvWF, in the absence and presence of
ALX-0081. In a separate experiment we test if ALX-0081, which binds
to the A1 domain of vWF, interferes with the ADAMTS-13 activity.
ADAMTS-13 binds to and cleaves the A2 domain of vWF.
[1066] Endothelial cells were obtained from human umbilical cord
veins by the method of Maruyama (Z.Zellforsch. Mikrosk. A4nat.
60:69; 1963). Endothelial cells were activated with 100 .mu.M
histamine (Sigma-Aldrich, St Louis, Mo.) for 15 minutes at room
temperature before the perfusion experiments.
[1067] Blood was drawn from healthy volunteers who denied ingestion
of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs)
for the preceding 10 days into one-tenth volume 3.4% sodium
citrate. Platelet-rich plasma (PRP) was prepared from whole blood
by centrifugation (10 minutes at 200 g at room temperature). The
PRP was acidified by addition of one-tenth volume of ACD (2.5%
trisodium citrate, 1.5% citric acid, and 2% D-glucose), and the
platelets were spun down (500 g, 15 minutes). The platelet pellet
was resuspended in HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-Tyrode buffer
(10 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH.sub.2PO.sub.4,
1.7 mM MgCl.sub.2, 5 mM D-glucose, pH 6.5). Prostacyclin
(PGI.sub.2, 10 ng/mL) was added to prevent platelet activation
during the subsequent washing step. Platelets were spun down and
resuspended in a small volume of HEPES-Tyrode buffer. This platelet
suspension was diluted in HEPES buffer at pH 7.4, or in TTP
plasma.
[1068] Perfusions were performed in a single-pass perfusion chamber
as described previously. The experiment was followed by real-time
videomicroscopy.
[1069] In a second type of experiment, different reaction mixtures
were prepared as summarized in table 60, however, without the
addition of the A1A2A3 construct. The A1A2A3 construct is a
recombinant fragment consisting out of the A1, A2 and A3 domain of
vWF. Mixtures were pre-incubated for 5 minutes at 37.degree. C.
after which the A1A2A3 fragment is added and the mixture is
incubated in a waterbath overnight at 37.degree. C. The next day a
reducing SDS-PAGE is run on the samples (12%, and using as marker
the Precision Plus Protein Standards from BioRad) and blotted on
Immobilon-FL (Millipore). The blot was blocked for 2 hours at room
temperature with blockbuffer (1:1 Odyssey blockbuffer in
1.times.TBS pH=7.4) and incubated with Rabbit polyclonal anti-vWF
(DAKO). Alexa Fluor 680 goat anti-rabbit was used for detection.
Scanning was done on the ODYSSEY to detect the degradation
products.
Control Experiment: Binding of Platelets to ULvWF
[1070] Endothelial cells were isolated from freshly obtained human
umbilical cords by collagenase digestion of the interior of the
umbilical vein. The cells were grown in tissue culture as a
homogeneous population. Cultured human endothelial cells grow as
monolayers of closely opposed, polygonal large cells and they
contain cytoplasmic inclusions (Weibel-Palade bodies).
Histamine-stimulated endothelial cells isolated from human
umbilical cords express ultra-large von Willebrand factor (ULvWF)
on their surface. Perfusion of these stimulated cells with isolated
blood platelets, which are suspended into either buffer or plasma
from a patient with acquired TTP, results in deposition of
platelets onto the ULvWF (8). These ULvWF-adhered platelets appear
as so-called `strings`, which are visible when the perfusion
experiment is monitored by real-time video microscopy (FIG.
41).
Inhibition by ALX-0081 for the Generation of Platelet Strings
[1071] Platelets were resuspended in buffer or in TTP plasma and
the concentrations of ALX-0081 used in this experiment were 0.2, 2
and 10 .mu.g/ml. Histamine-stimulated endothelial cells isolated
from human umbilical cords are perfused with these platelet
suspensions as described above.
[1072] Addition of ALX-0081 to platelets resuspended in buffer or
in plasma from a TTP patient results in a complete inhibition of
string formation under all conditions tested (FIG. 42). Perfusion
experiments were performed at a shear stress of 2.5 dyn/cm2 for 4
minutes. During this 4 minute perfusion at least 20 microscopic
fields were examined, and in the presence of the Nanobody.TM., no
strings could be demonstrated at all conditions tested (FIG.
42).
Cleavage of ULvWF by ADAMTS-13
[1073] ADAMTS-13 reduces the size of large and ultralarge VWF
multimers to smaller forms by specifically cleaving the Y842/M843
peptide bond in the VWF A2 domain. Two types of assays were used to
evaluate the effect of ALX-0081 on the cleavage of ULvWF by
ADAMTS-13: i.e. a perfusion assay and an assay observing the
cleavage of a recombinant vWF fragment.
[1074] In a first experiment, strings were generated by a 4 minute
perfusion of washed platelets resuspended in buffer over
histamine-stimulated endothelial cells. Subsequently, the
non-adhered platelets were washed away by a 4 minute perfusion of
buffer. After that, buffer was perfused for an other 4 minutes,
followed by a 4 minute perfusion of pooled normal plasma containing
ADAMTS-13. Detachment of the platelet strings was observed after
perfusion of pooled normal plasma (FIG. 43). More than 95% of the
strings were cleaved after the 4 minute perfusion.
[1075] In a next experiment, strings were generated by a 4 minute
perfusion of washed platelets resuspended in buffer over
histamine-stimulated endothelial cells and the non-adhered
platelets were washed away by a 4 minute perfusion of buffer as
above. After that, ALX-0081 (10 .mu.g/ml in buffer) was perfused
for 4 minutes, followed by a 4 minute perfusion of pooled normal
plasma containing ALX-0081 (10 .mu.g/ml=10-fold molar excess over
vWF) and ADAMTS-13. We could clearly demonstrate detachment of the
platelet strings and 95% of the strings were cleaved after the 4
minute perfusion (FIG. 44)
[1076] These results clearly demonstrate that ALX-0081 does not
have an effect on the cleavage of ULvWF strings by ADAMTS-13.
[1077] In a second assay, a recombinant fragment containing the
A1-A2-A3 domain of vWF was mixed with normal pool plasma (NPP)
containing ADAMTS-13, resulting in proteolytic cleavage of the
fragment which was observed by a Western Blot analysis. ADAMTS-13
activity was tested in the absence and presence of 10 .mu.g/ml
ALX-0081. As indicated in FIG. 45 ALX-0081 has no effect on the
cleavage of the vWF fragment (lanes 6-7-8).
[1078] In order to demonstrate that the observed cleavage is
specific for ADAMTS-13, a control experiment in the presence of
EDTA was performed as EDTA inhibits the activity of ADAMTS-13. As
expected, the presence of EDTA in NPP resulted in inhibition of
cleavage of the fragment (FIG. 45 lane 4).
[1079] This experiment again proves that ALX-0081 has no effect on
the ADAMTS-13 activity.
TABLE-US-00047 TABLE 8 Sequence listing of anti-vWF nanobodies SEQ
Name ID NO Sequence 12A5 60
AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTN
YADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGT QVTVSS
12B1 61 QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYGMGWFRQAPGKEREFVTSISWSGTYT
AYSDNVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAAQSRYRSNYYDHDDKYAYW
GQGTQVTVSS 12B6 62
QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGST
YYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYN
FWGQGTQVTVSS 12D11 63
AVQLVDSGGGLVQAGGSLRLSCTASERTTFSSYTLGWFRQAPGKEREFVGGISWSGVS
TDYAEFAKGRFTISRDHAANTVYLEMNSLKPEDTAVYYCAALGRYRSNWRNIGQYDYW
GQGTQVTVSS 12E3 64
EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYGMGWFRQAPGKEREFVTSISWSGSYT
AYADNVKGRFTISRDNAKNTVYLQMDSLKPGDTAVYYCAAQSRYSSNYYDHDDKYAYW
GQGTQVTVSS 12C9 65
AVQLVESGGGLVQPGGSLKLSCATSGSIFSSSAMAWYRQASGKQRELVATITSGGRTS
YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYDCNFVVDGKRAPWGQGTQVTVSS 14F8 66
AVQLVESGGGLVQAGESLRLSCTSSGRAFSYYNTGWFRQAPGKEREFVAAISWSGGLT
YYADSVKGRFTISRDNAKDMVYLQMASLKPEDTAVYYCAANRRQKTVQMGERAYDYWG
QGTQVTVSS
TABLE-US-00048 TABLE 9 Expression yields of anti-vWF nanobodies
Nanobody Yield (mg/l) after TALON 12A5 13 12B1 6 12B6 16 12D11 8
12E3 4 12C9 25 14F8 48
TABLE-US-00049 TABLE 10 Platelet adhesion in perfusion chamber of
anti-vWF nanobodies Nanobody Control % Platelet adhesion at 2
.mu.g/ml 12A5 60 .+-. 7 10 .+-. 3 12B1 60 .+-. 7 56 .+-. 3 12B6 60
.+-. 7 19 .+-. 5 12D11 60 .+-. 7 61 .+-. 7 12E3 60 .+-. 7 54 .+-. 1
12C9 71 .+-. 3 68 .+-. 8 14F8 71 .+-. 3 51 .+-. 10
TABLE-US-00050 TABLE 11 Sequence listing of 12B6 and 12A5 homologue
nanobodies SEQ Name ID NO Sequence 12A5 homologue sequences 12B4 67
QVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGLYRQAPGKQRELVATITSGGSTN
YADSVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGT QVTVSS
12E8 68 AVQLEESGGGLVQPGGSLRLSCLASGRIFSIGAMGLYRQAPGKQRELVATITSGGSTN
YADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCYANLKQGDYGYRFNDYWGQGT QVTVSS
12A6 69 QVQLVESGGGLVQPGGSLRLSCLASGRIFSIGTMGLYRQAPGKQRELVATITSGGSTN
YADSVKGRFTISRDGAKNTVYLQMNSLRPEDTAVYYCYANLKQGDYGYRFNDYWGQGT QVTVSS
12D8 70 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGTMGLYRQAPGKQRELVATITSGGSTN
YADSVKGRFTISRDGAKNTVYLQMNSLRPEDTAVYYCYANLKQGDYGYRFNDYWGQGT QVTVSS
12B6 homologue sequences 12A2 71
QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGST
YYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYT
FWGQGTQVTVSS 12F2 72
QVKLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGRERDVVAAISRTGGST
YYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRSLPSEYT
FWGQGTQVTVSS 14H10 73
QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGST
YYPDSVEGRFTISRDNAKRMVYLEMNNLKPDDTAVYYCAAAGVRAEDGRVRTLPSEYT
FWGQGTQVTVSS
TABLE-US-00051 TABLE 12 Estimated K-on, K-off and KD values for
12A5 homologue nanobodies Nanobody Koff (.times.10.sup.-3/s) Kon
(.times.10.sup.6 1/Ms) KD (nM) 12A5 2.51 0.629 3.98 12B4 2.2 0.544
4.05 12E8 2.93 0.171 17.1 12A6 4.72 0.188 25.1 12D8 5.84 0.139
41.9
TABLE-US-00052 TABLE 13 Estimated K-on, K-off and KD values for
12B6 homologue nanobodies Nanobody Koff (.times.10.sup.-3/s) Kon
(.times.10.sup.6 1/Ms) KD (nM) 12B6 5.97 2.55 2.33 12A2 3.49 1.11
3.13 12F2 4.04 6.41 6.3 14H10 3.97 6.84 5.81
TABLE-US-00053 TABLE 14 Real KD value of 12B6, 12A2 and 12A5
nanobodies Nanobody Koff (.times.10.sup.-3/s) Kon (.times.10.sup.6
1/Ms) KD (nM) 12B6 10.03 2.28 4.5 12A2 9.9 2.24 4.4 12A5 3.3 1.22
2.7
TABLE-US-00054 TABLE 15 Platelet adhesion in perfusion chamber of
12B6, 12A2 and 12A5 nanobodies % Platelet adhesion Nanobody 0
.mu.g/ml 0.2 .mu.g/ml 0.4 .mu.g/ml 0.6 .mu.g/ml Control 71 .+-. 3
-- -- -- 12B6 -- 59 .+-. 6 43 .+-. 7 27 .+-. 8 12A2 -- 58 .+-. 8 40
.+-. 10 11 .+-. 8 12A5 -- 50 .+-. 7 27 .+-. 10 2 .+-. 2
TABLE-US-00055 TABLE 16 Concentration of 12B6, 12A2 and 12A5
nanobodies after heating at increasing temperatures 37.degree.
50.degree. 60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT
C. C. C. C. C. C. 12B6 100 97 100 104 94 91 68 12A2 100 106 104 100
93 87 90 12A5 100 108 107 98 83 75 66
TABLE-US-00056 TABLE 17 Sequence listing of bivalent nanobodies SEQ
ID Name NO Sequence 12A2-3a-12A2 74
QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQAGG
ALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVE
GRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSE YTFWGQGTQVTVSS
12A2-GS9-12A2 75 QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTY
YPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRV
RTLPSEYTFWGQGTQVTVSS 12A2-GS30-12A2 76
QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFR
QAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLK
PEGTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A5-3a-12A5 77
AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVA
TITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYAN
LKQGSYGYRFNDYWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSC
LASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISR
DGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTV SS 12A5-GS9-12A5
78 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVA
TITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYAN
LKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGG
SLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKG
RFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRENDYWGQ GTQVTVSS
12A5-GS30-12A5 79 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVA
TITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYAN
LKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS
GGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQ
RELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVY
YCYANLKQGSYGYRFNDYWGQGTQVTVSS 12B6-3a-12B6 80
QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQAGG
ALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVE
GRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSE YNFWGQGTQVTVSS
12B6-GS9-12B6 81 QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTY
YARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRV
RTLPSEYNFWGQGTQVTVSS 12B6-GS30-12B6 82
QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFR
QAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALK
PEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS
TABLE-US-00057 TABLE 18 Sequence listing of linker sequences SEQ
Name ID NO Sequence 3a 83 AAA GS9 84 GGGGSGGGS GS30 85
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
TABLE-US-00058 TABLE 19 Expression yields of bivalent 12B6, 12A2
and 12A5 nanobodies Yield (mg/l) after TALON or Nanobody other
purification 12B6 16 12B6-3a-12B6 9 12B6-GS9-12B6 16 12B6-GS30-12B6
17 12A2 18 12A2-3a-12A2 45 12A2-GS9-12A2 22 12A2-GS30-12A2 11 12A5
13 12A5-3a-12A5 10 12A5-GS9-12A5 11 12A5-GS30-12A5 18
TABLE-US-00059 TABLE 20 Concentration of 12B6 bivalent nanobodies
after heating at increasing temperatures 37.degree. 50.degree.
60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT C. C. C. C.
C. C. 12B6 100 97 100 104 94 91 68 12B6-3a-12B6 100 103 96 87 9 8 6
12B6-GS9-12B6 100 103 94 88 19 8 7 12B6-GS30-12B6 100 100 100 98 46
14 11
TABLE-US-00060 TABLE 21 Concentration of 12A2 bivalent nanobodies
after heating at increasing temperatures 37.degree. 50.degree.
60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT C. C. C. C.
C. C. 12A2 100 106 104 100 93 87 90 12A2-3a-12A2 100 87 88 91 55 50
43 12A2-GS9-12A2 100 102 113 138 91 13 15 12A2-GS30-12A2 100 115 93
116 81 49 34
TABLE-US-00061 TABLE 22 Concentration of 12A5 bivalent nanobodies
after heating at increasing temperatures 37.degree. 50.degree.
60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT C. C. C. C.
C. C. 12A5 100 108 107 98 83 75 66 12A5-3a-12A5 100 101 114 29 6 4
6 12A5-GS9-12A5 100 104 115 32 13 14 10 12A5-GS30-12A5 100 104 87 7
6 35 21
TABLE-US-00062 TABLE 23 Platelet adhesion in perfusion chamber of
12A2 bivalent nanobodies % Platelet adhesion Nanobody 0 .mu.g/ml
0.1 .mu.g/ml 0.2 .mu.g/ml 0.4 .mu.g/ml Control 81 .+-. 5 -- -- --
12A2 -- 78 .+-. 2 72 .+-. 6 61 .+-. 8 12A2-3a-12A2 -- 74 .+-. 5 50
.+-. 3 33 .+-. 0 12A2-GS9-12A2 -- 81 .+-. 1 73 .+-. 1 40 .+-. 2
12A2-GS30-12A2 -- 81 .+-. 3 73 .+-. 3 37 .+-. 1
TABLE-US-00063 TABLE 24 Sequence listing of humanised 12B6
nanobodies SEQ ID Name NO Sequence 12B6H1 86
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAAISRTGGST
YYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN
FWGQGTQVTVSS 12B6H2 87
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGST
YYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN
FWGQGTQVTVSS 12B6H3 88
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAAISRTGGST
YYARSVEGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN
FWGQGTQVTVSS 12B6H4 89
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAAISRTGGST
YYARSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN
FWGQGTQVTVSS
TABLE-US-00064 TABLE 25 Expression yields of wild type and
humanised 12B6 nanobodies Yield (mg/l) after TALON or Nanobody
other purification 12B6 16 12B6H1 3 12B6H2 9 12B6H3 8 12B6H4 3
TABLE-US-00065 TABLE 26 Concentration of wild type and humanised
12B6 nanobodies after heating at increasing temperatures 37.degree.
50.degree. 60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT
C. C. C. C. C. C. 12B6 100 97 100 104 94 91 68 12B6H1 100 101 100
97 45 58 54 12B6H2 100 97 96 96 83 46 53 12B6H3 100 101 98 97 74 73
65 12B6H4 100 101 100 93 41 66 54
TABLE-US-00066 TABLE 27 KD values for wild type and humanised 12B6
nanobodies Nanobody KD (nM) 12B6 4.4 12B6H1 4.4 12B6H2 3.5 12B6H3 9
12B6H4 7.3
TABLE-US-00067 TABLE 28 Sequence listing of humanised 12A2
nanobodies SEQ ID Name NO Sequence 12A2H1 90
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGG
STYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLP
SEYTFWGQGTQVTVSS 12A2H3 91
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGG
STYYPDSVEGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLP
SEYTFWGQGTQVTVSS 12A2H4 92
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGG
STYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLP
SEYTFWGQGTQVTVSS 12A2H11 93
EVQLVESGGGLVQPGGSLRLSCAASGFTFSYNPMGWFRQAPGKGRELVAAISRTGG
STYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLP
SEYTFWGQGTQVTVSS 12A2H13 94
EVQLVESGGGLVQPGGSLRLSCAASGFTFSYNPMGWFRQAPGKGRELVAAISRTGG
STYYPDSVEGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLP
SEYTFWGQGTLVTVSS
TABLE-US-00068 TABLE 29 Expression yields of wild type and
humanised 12A2 nanobodies Yield (mg/l) after TALON or Nanobody
other purification 12A2 18 12A2H1 11 12A2H3 11 12A2H4 11 12A2H11 15
12A2H13 11
TABLE-US-00069 TABLE 30 Concentration of wild type and humanised
12A2 nanobodies after heating at increasing temperatures 37.degree.
50.degree. 60.degree. 70.degree. 80.degree. 90.degree. Nanobody RT
C. C. C. C. C. C. 12A2 100 106 104 100 93 87 90 12A2H1 100 99 99 99
100 89 80 12A2H3 100 102 101 102 102 90 89 12A2H4 100 100 101 100
99 90 83 12A2H11 100 111 113 107 103 85 67 12A2H13 100 104 103 103
100 90 81
TABLE-US-00070 TABLE 31 Platelet adhesion of wild type and
humanised 12A2 nanobodies in perfusion chamber at 0.7 and 1.5 ug/ml
% Platelet adhesion Nanobody 0 .mu.g/ml 0.7 .mu.g/ml 1.5 .mu.g/ml
Control 73 .+-. 4 -- -- 12A2 -- 36 .+-. 3 34 .+-. 5 12A2H1 -- 49
.+-. 1 47 .+-. 3 12A2H3 -- 62 .+-. 4 63 .+-. 1 12A2H4 -- 55 .+-. 1
54 .+-. 1 12A2H11 -- 57 .+-. 1 52 .+-. 1 12A2H13 -- 67 .+-. 4 67
.+-. 4
TABLE-US-00071 TABLE 32 Platelet adhesion of wild type and
humanised 12A2 nanobodies in perfusion chamber at 0.5, 1 and 2
ug/ml % Platelet adhesion Nanobody 0 .mu.g/ml 0.5 .mu.g/ml 1
.mu.g/ml 2 .mu.g/ml Control 72 .+-. 1 -- -- -- 12A2 -- 33 .+-. 10
35 .+-. 11 10 .+-. 10 12A2H1 -- 40 .+-. 9 43 .+-. 3 38 .+-. 5
12A2H4 -- 61 .+-. 1 57 .+-. 1 46 .+-. 5
TABLE-US-00072 TABLE 33 KD values for wild type and humanised 12A2
nanobodies Nanobody KD (nM) 12A2 3.1 12A2H3 14.6 12A2H11 10.6
12A2H13 38.8
TABLE-US-00073 TABLE 34 Sequence listing of humanised 12A5
nanobodies SEQ ID Name NO Sequence 12A5H1 95
EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVATITSGGS
TNYADPVKGRFTISRDGPKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRFNDYW GQGTQVTVSS
12A5H2 96 EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVATITSGGS
TNYADPVKGRFTISRDGAKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRFNDYW GQGTQVTVSS
12A5H3 97 EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVATITSGGS
TNYADPVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRENDYW
GQGTQVTVSS
TABLE-US-00074 TABLE 35 Expression yields of wild type and
humanised 12A5 nanobodies Nanobody Yield (mg/l) after TALON 12A5 13
12A5H1 8 12A5H2 9 12A5H3 11
TABLE-US-00075 TABLE 36 Concentration of wild type and humanised
12A5 nanobodies after heating at increasing temperatures Nanobody
37.degree. C. 50.degree. C. 60.degree. C. 70.degree. C. 80.degree.
C. 90.degree. C. 12A5 108 107 98 83 75 66 12A5H1 99 91 86 60 69 63
12A5H2 99 108 90 58 67 60 12A5H3 101 97 97 67 73 64
TABLE-US-00076 TABLE 37 KD values for wild type and humanised 12A5
nanobodies Nanobody KD (nM) 12A5 1.6 12A5H1 1.8 12A5H2 12.8 12A5H3
ND
TABLE-US-00077 TABLE 38 Sequence listing of humanised bivalent
nanobodies SEQ ID Name NO Sequence 12A2H1-3a-12A2H1 98
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGG
SLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVE
GRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSE YTFWGQGTQVTVSS
12A2H4-3a-12A2H4 99
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGG
SLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVE
GRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSE YTFWGQGTQVTVSS
12B6H2-3a-12B6H2 100
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQPGG
SLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVE
GRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSE YNFWGQGTQVTVSS
12A2H1-GS9-12A2H1 101
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTY
YPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRV
RTLPSEYTFWGQGTQVTVSS 12A2H4-GS9-12A2H4 102
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTY
YPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRV
RTLPSEYTFWGQGTQVTVSS 12B6H2-GS9-12B6H2 103
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTY
YARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRV
RTLPSEYNFWGQGTQVTVSS 12A2H1-GS30-12A2H1 104
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFR
QAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLR
AEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A2H4-GS30-12A2H4 105
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFR
QAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLR
AEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B6H2-GS30-12B6H2 106
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVA
AISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAA
AGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFR
QAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLR
AEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS
TABLE-US-00078 TABLE 39 Expression yields of humanised bivalent
nanobodies Nanobody Tags Yield (mg/l) 12A2H1-3a-12A2H1 Yes 5
12A2H1-3a-12A2H1 No 6 12A2H4-3a-12A2H4 Yes 10 12A2H4-3a-12A2H4 No 7
12B6H2-3a-12B6H2 Yes 10 12B6H2-3a-12B6H2 No 2
TABLE-US-00079 TABLE 40 Concentration of humanised bivalent
nanobody after heating at increasing temperatures 37.degree.
50.degree. 60.degree. 70.degree. 80.degree. 90.degree. Nanobody
Tags C. C. C. C. C. C. 12A2H1-3a-12A2H1 Yes 98 96 93 63 8 9
12A2H1-3a-12A2H1 No 100 99 100 77 14 15 12A2H4-3a-12A2H4 Yes 100 99
95 9 6 9 12A2H4-3a-12A2H4 No 98 98 98 18 17 26 12B6H2-3a-12B6H2 Yes
100 91 85 7 6 7 12B6H2-3a-12B6H2 No 100 99 99 28 13 18
TABLE-US-00080 TABLE 41 Platelet adhesion of wild type and
humanised bivalent nanobodies % Platelet adhesion Nanobody 0
.mu.g/ml 0.15 .mu.g/ml 0.3 .mu.g/ml 0.6 .mu.g/ml Control 65 .+-. 8
-- -- -- 12B6-3a-12B6 -- 50 .+-. 5 15 .+-. 6 8 .+-. 6
12B6H2-3a-12B6H2 -- 53 .+-. 4 30 .+-. 16 17 .+-. 6 12A2-3a-12A2 --
36 .+-. 8 10 .+-. 8 4 .+-. 3 12A2H1-3a-12A2H1 -- 54 .+-. 4 10 .+-.
11 12 .+-. 7 12A2H4-3a-12A2H4 -- 38 .+-. 2 10 .+-. 6 8 .+-. 4
TABLE-US-00081 TABLE 42 baboons used with the different test
compounds in the Folts study Baboon ID Sex Weight [kg] Left leg
Right leg 1 male 9.8 Control 2 male 10.0 Control 13 male 12.4
Reopro 14 male 9.5 Reopro 15 male 10.8 Reopro 16 male ALX-0081 17
male 15.6 ALX-0081 18 male 17.2 ALX-0081 20/22 male 12.7 ALX-0081
ALX-0081 21 female 8.0 ALX-0081 23 male ALX-0081 24 male 9.4
ALX-0081 3/19 male 15.2 Aspegic ALX-0081 4 female 13.6 Aspegic 5
male 17.4 Aspegic 9 male 13.2 Plavix 6/10 male 10.2 Heparin Plavix
7/11 male 9.4 Heparin Plavix 8/12 male 10.5 Heparin Plavix
TABLE-US-00082 TABLE 43 Length of CFRs (s) for control animals (ND
= not done) Control Baboon ID Final dose Dose 1 2 0 control 291 249
0 saline 294 278 0 saline 427 185 0 saline 285 203 0 saline 438
175
TABLE-US-00083 TABLE 44 Length of CFRs (s) for animals treated with
Aspegic .TM. (ND = not done) Aspegic Baboon ID Final dose Dose 3 4
5 0 control 88 no CFRs 148 0 saline 147 204 184 1 mg/kg 1 mg/kg 149
164 135 2.5 mg/kg 1.5 mg/kg 102 325 115 5 mg/kg 2.5 mg/kg 102 1800
245 10 mg/kg 5 mg/kg 113 905 156 20 mg/kg 10 mg/kg 125 657 169 40
mg/kg 20 mg/kg 110 ND 145 80 mg/kg 40 mg/kg 129 ND ND Epinephrin ND
161 ND
TABLE-US-00084 TABLE 45 Length of CFRs (s) for animals treated with
Heparin .TM. (ND = not done) Heparin Baboon ID Final dose Dose 6 7
8 0 control 232 113 166 0 saline 298 131 246 15 IU/kg 15 IU/kg 630
208 255 30 IU/kg 30 IU/kg 355 241 320 60 IU/kg 60 IU/kg 432 246 332
120 IU/kg 120 IU/kg 610 160 206 240 IU/kg 240 IU/kg >1800 221
169 Epinephrin 109 65 ND
TABLE-US-00085 TABLE 46 Length of CFRs (s) for animals treated with
Plavix .TM. (ND = not done) Plavix Baboon ID Final dose Dose 9 10
11 12 0 control 215 178 84 144 0 saline 168 160 88 189 1 mg/kg 1
mg/kg 189 ND 132 179 2.5 mg/kg 2.5 mg/kg 883 400 258 >1800 5
mg/kg 2.5 mg/kg >1800 >1800 >1800 >1800 10 mg/kg 5
mg/kg >1800 >1800 >1800 >1800 20 mg/kg 10 mg/kg
>1800 >1800 >1800 >1800 Epinephrin 241 91 83 66
TABLE-US-00086 TABLE 47 Length of CFRs (s) for animals treated with
Reopro .TM. (ND = not done) Reopro Baboon ID Final dose Dose 13 14
15 0 control 144 90 308 0 saline 141 103 268 20 .mu.g/kg 20
.mu.g/kg 98 82 254 70 .mu.g/kg 50 .mu.g/kg 90 90 248 170 .mu.g/kg
100 .mu.g/kg 90 >1800 >1800 420 .mu.g/kg 250 .mu.g/kg
>1800 >1800 >1800 920 .mu.g/kg 500 g/kg.sup. >1800
>1800 >1800 Epinephrin >1200 >1200 >1200
TABLE-US-00087 TABLE 48 Length of CFRs (s) for animals treated with
ALX-0081 (ND = not done) ALX-0081 Baboon ID Final dose Dose 16 17
18 19 20 21 22 23 24 0 control 168 293 185 90 94 117 188 164 161 0
saline 151 236 242 117 98 107 233 105 178 3 .mu.g/kg 3
micro-gram/kg 193 ND ND 144 133 183 312 112 295 13 .mu.g/kg 10
micro-gram/kg 913 >1800 298 237 620 525 >1800 213 380 43
.mu.g/kg 30 micro-gram/kg >1800 >1800 >1800 >1800
>1800 >1800 >1800 >1800 >1800 133 .mu.g/kg 90
micro-gram/kg >1800 >1800 >1800 >1800 >1800 >1800
>1800 >1800 >1800 403 .mu.g/kg 270 micro-gram/kg >1800
>1800 >1800 >1800 >1800 >1800 >1800 >1800
>1800 Epinephrin >1200 >1200 >900 >900 >900
>900 >900 >900 >900
TABLE-US-00088 TABLE 49 baboons used with the different test
compounds in the Folts study Baboon ID Sex Weight [kg] Mix 1 Male
9.8 Asp/Hep/Plav/ALX 2 Female 13.6 Asp/Hep/Plav/ALX 3 Female 7.8
Asp/Hep/Plav/ALX 4 Male 12.1 Asp/Hep/Plav/ALX 5 Male 11.4
Asp/Hep/Plav/ALX 6 Male 11.4 Asp/Hep/Plav/ALX 7 Male 14.0
Asp/Hep/Plav/ALX
TABLE-US-00089 TABLE 50 Inhibition of CFRs in the Folts model for
the different drugs tested. The number of experiments in which an
inhibition of CFRs was observed in the mentioned different
conditions is shown as a function of the total number of
independent repeats of that condition. Inhibition Inhibition of
CFRs of CFRs after adminis- Test Inhibition after new tration of
Effective compound of CFRs injury Epinephrin dose Control 0/2 ND ND
-- Aspegic 0/3 0/3 0/1 -- Heparin 1/3 1/3 0/2 -- Plavix 4/4 4/4 0/4
5 mg/kg Reopro 3/3 3/3 3/3 170-420 .mu.g/kg ALX-0081 9/9 9/9 9/9
13-43 .mu.g/kg + 1.5 .times. dose/ kg/hour
TABLE-US-00090 TABLE 51 Length of CFRs (seconds) for each baboon
and each dose of Aspegic, Heparin, Plavix and ALX-0081.
##STR00001## The effective dose is indicated in yellow
TABLE-US-00091 TABLE 52 Blood loss relative to the second control
gauze for animals treated with Plavix .TM. in function of final
dose (STD = standard deviation) Plavix Baboon ID Final dose Dose 9
10 11 12 Average STD 1 mg/kg 1 mg/kg 0.6 1.3 1.6 1.2 1.4 0.2 2.5
mg/kg 1.5 mg/kg 1.5 1.4 1.1 1.0 1.2 0.2 5 mg/kg 2.5 mg/kg 5.1 4.7
1.4 6.6 4.5 2.2 10 mg/kg 5 mg/kg 6.5 4.5 5.8 13.6 7.6 4.1 20 mg/kg
10 mg/kg 3.7 4.1 9.1 2.6 4.9 2.9
TABLE-US-00092 TABLE 53 Blood loss relative to the second control
gauze for animals treated with Reopro .TM. in function of final
dose (STD = standard deviation) Reopro Baboon ID Final dose Dose 13
14 15 Average STD 20 .mu.g/kg 20 .mu.g/kg 2.7 0.6 2.1 1.8 1 70
.mu.g/kg 50 .mu.g/kg 1.2 2.1 0.4 1.2 1 170 .mu.g/kg 100 .mu.g/kg
1.2 6.0 4.7 4.0 2 420 .mu.g/kg 250 .mu.g/kg 28.3 7.1 13.9 16.4 11
920 .mu.g/kg 500 .mu.g/kg 39.8 14.5 6.3 20.2 17
TABLE-US-00093 TABLE 54 Blood loss relative to the second control
gauze for animals treated with ALX-0081in function of final dose
(STD = standard deviation) ALX-0081 Baboon ID Final dose Dose 17 18
19 20 21 22 23 24 Average STD 3 .mu.g/kg 3 .mu.g/kg ND ND 0.4 1.2
0.5 1.1 1.8 0.8 1.0 0.5 13 .mu.g/kg 10 .mu.g/kg 1.2 0.8 0.1 1.2 1.1
1.3 1.1 1.1 1.0 0.4 43 .mu.g/kg 30 .mu.g/kg 2.8 2.0 4.1 3.9 2.1 3.8
3.0 1.6 2.9 1.0 133 .mu.g/kg 90 .mu.g/kg 2.5 3.1 2.0 0.4 4.0 5.3
5.6 1.4 3.0 1.8 403 .mu.g/kg 270 .mu.g/kg 2.6 2.3 2.3 0.4 0.8 5.6
10.7 0.9 3.2 3.4
TABLE-US-00094 TABLE 55 The average of the total amount of blood
loss (=sum of blood loss from the first five doses of test
compound) as relative to the second control gauze Total dose as
Average Standard multiple of of total deviation of Test compound
effective dose blood loss blood loss Plavix 4 19.4 4.0 Reopro 3.5
44 26 ALX-0081 13 10.8 5.7
TABLE-US-00095 TABLE 56 Blood loss in gauzes relative to the second
control gauze for each baboon treated with Aspegic, Heparin, Plavix
and ALX-0081 in function of drug dose. ##STR00002## The effective
drug dose in which a complete inhibition of CFRs was observed, is
indicated in yellow
TABLE-US-00096 TABLE 57 % ristocetin-induced platelet aggregation
for each baboon treated with Aspegic, Heparin, Plavix and ALX-0081
in function of drug dose 4 5 6 7 8 9 10 control 75 78 79 70 46 70
46 saline 48 78 74 24 45 65 47 5 mg/kg Aspegic + 60 IU/kg Heparin +
62 65 89 64 66 68 55 1 mg/kg Plavix + 1 mg/kg Plavix 42 63 66 83 59
76 60 Epinephrin + effective dose ALX-0081 0 24 24 6 17 7 8
TABLE-US-00097 TABLE 58 concentration of ALX-0081 [.mu.g/ml] in
blood samples obtained at 10 minutes after administration ALX-0081
Baboon ID Final dose Dose 16 17 18 19 20 21 22 23 24 3 .mu.g/kg 3
.mu.g/kg 0.10 0.03 0.05 0.08 0.04 0.14 0.08 13 .mu.g/kg 10 .mu.g/kg
1.23 0.34 0.29 0.26 0.50 0.39 0.41 0.18 0.42 43 .mu.g/kg 30
.mu.g/kg 1.00 0.51 0.72 1.14 1.01 0.61 1.01 0.87 1.21 133 .mu.g/kg
90 .mu.g/kg 1.87 1.38 1.77 1.61 2.64 1.60 6.77 2.75 5.01 403
.mu.g/kg 270 .mu.g/kg 6.77 4.03 6.73 35.14 7.66 6.01 9.24 18.56
16.62
TABLE-US-00098 TABLE 59 Length of CFRs [seconds] for baboons
treated with ALX-0081 and with vWF Baboon ID Dose 1 2 3 Control 119
140 111 Saline ND 158 158 30 .mu.g/kg + 45 .mu.g/kg/hour >1800
>1800 >1800 ALX-0081 250 IU vWF >420 >1800 >1800 250
IU vWF 246 256 230
TABLE-US-00099 TABLE 60 Volumes [.mu.l] to prepare the different
mixtures for study of cleavage of A1A2A3 by ADAMTS13 NPP + NPP +
NPP EDTA ALX-0081 PBS Tris (100 mM) 5 5 5 5 BaCl.sub.2 (10 mM) 5 5
5 5 Pefablock (100 mM) 1.3 1.3 1.3 1.3 Plasma 3.3 3.3 3.3 3.3 ALX
0081 (2.48 mg/ml) -- -- 4 -- PBS -- -- -- 4 EDTA (0.35M) pH = 8.3
-- 2.6 -- -- H.sub.2O 81.9 79.3 77.9 77.9 A1A2A3 (460 g/ml) 3.5 3.5
3.5 3.5
Sequence CWU 1
1
303126PRTartificial sequencesynthetic peptide 1Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Xaa Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly 20 25 214PRTartificial
sequencesynthetic peptide 2Trp Xaa Arg Gln Ala Pro Gly Lys Xaa Xaa
Glu Xaa Val Ala 1 5 10 332PRTartificial sequencesynthetic peptide
3Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1
5 10 15 Met Asn Ser Leu Xaa Xaa Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Ala 20 25 30 411PRTartificial sequencesynthetic peptide 4Xaa Xaa
Gln Gly Thr Xaa Val Thr Val Ser Ser 1 5 10 526PRTartificial
sequencesynthetic peptide 5Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly 20 25 614PRTartificial sequencesynthetic peptide 6Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10
714PRTartificial sequencesynthetic peptide 7Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 813PRTartificial
sequencesynthetic peptide 8Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Gly Ala 1 5 10 914PRTartificial sequencesynthetic peptide 9Trp
Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10
1014PRTartificial sequencesynthetic peptide 10Trp Phe Arg Gln Ala
Pro Gly Lys Gln Arg Glu Phe Val Ala 1 5 10 1113PRTartificial
sequencesynthetic peptide 11Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ala 1 5 10 1232PRTartificial sequencesynthetic peptide
12Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1
5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Ala 20 25 30 1311PRTartificial sequencesynthetic peptide 13Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 1411PRTartificial
sequencesynthetic peptide 14Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 155PRTartificial sequencesynthetic peptide 15Asn Tyr Gly
Met Gly 1 5 165PRTartificial sequencesynthetic peptide 16Ser Tyr
Thr Leu Gly 1 5 175PRTartificial sequencesynthetic peptide 17Asn
Tyr Asn Met Gly 1 5 185PRTartificial sequencesynthetic peptide
18Ser Ser Ala Met Ala 1 5 195PRTartificial sequencesynthetic
peptide 19Tyr Tyr Asn Thr Gly 1 5 205PRTartificial
sequencesynthetic peptide 20Ile Gly Ala Met Gly 1 5
215PRTartificial sequencesynthetic peptide 21Ile Gly Thr Met Gly 1
5 225PRTartificial sequencesynthetic peptide 22Tyr Asn Pro Met Gly
1 5 2317PRTartificial sequencesynthetic peptide 23Ser Ile Ser Trp
Ser Gly Thr Tyr Thr Ala Tyr Ser Asp Asn Val Lys 1 5 10 15 Gly
2417PRTartificial sequencesynthetic peptide 24Gly Ile Ser Trp Ser
Gly Val Ser Thr Asp Tyr Ala Glu Phe Ala Lys 1 5 10 15 Gly
2518PRTartificial sequencesynthetic peptide 25Thr Ser Ile Ser Trp
Ser Gly Ser Tyr Thr Ala Tyr Ala Asp Asn Val 1 5 10 15 Lys Gly
2617PRTartificial sequencesynthetic peptide 26Ser Ile Ser Trp Ser
Gly Met Ser Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly
2716PRTartificial sequencesynthetic peptide 27Thr Ile Thr Ser Gly
Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
2817PRTartificial sequencesynthetic peptide 28Ala Ile Ser Trp Ser
Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
2916PRTartificial sequencesynthetic peptide 29Thr Ile Thr Ser Gly
Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys Gly 1 5 10 15
3016PRTartificial sequencesynthetic peptide 30Thr Ile Thr Ser Gly
Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
3117PRTartificial sequencesynthetic peptide 31Ala Ile Ser Arg Thr
Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly
3217PRTartificial sequencesynthetic peptide 32Ala Ile Ser Arg Thr
Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly
3317PRTartificial sequencesynthetic peptide 33Gln Ser Arg Tyr Arg
Ser Asn Tyr Tyr Asp His Asp Asp Lys Tyr Ala 1 5 10 15 Tyr
3416PRTartificial sequencesynthetic peptide 34Leu Gly Arg Tyr Arg
Ser Asn Trp Arg Asn Ile Gly Gln Tyr Asp Tyr 1 5 10 15
3517PRTartificial sequencesynthetic peptide 35Gln Ser Arg Tyr Ser
Ser Asn Tyr Tyr Asp His Asp Asp Lys Tyr Ala 1 5 10 15 Tyr
3615PRTartificial sequencesynthetic peptide 36Ser Asn Arg Tyr Arg
Thr His Thr Thr Gln Ala Met Tyr Asn Tyr 1 5 10 15 378PRTartificial
sequencesynthetic peptide 37Val Val Asp Gly Lys Arg Ala Pro 1 5
3816PRTartificial sequencesynthetic peptide 38Asn Arg Arg Gln Lys
Thr Val Gln Met Gly Glu Arg Ala Tyr Asp Tyr 1 5 10 15
3914PRTartificial sequencesynthetic peptide 39Asn Leu Lys Gln Gly
Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr 1 5 10 4014PRTartificial
sequencesynthetic peptide 40Asn Leu Lys Gln Gly Asp Tyr Gly Tyr Arg
Phe Asn Asp Tyr 1 5 10 4119PRTartificial sequencesynthetic peptide
41Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1
5 10 15 Tyr Asn Phe 4219PRTartificial sequencesynthetic peptide
42Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1
5 10 15 Tyr Thr Phe 4319PRTartificial sequencesynthetic peptide
43Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Ser Leu Pro Ser Glu 1
5 10 15 Tyr Thr Phe 445PRTartificial sequencesynthetic peptide
44Ser Phe Gly Met Ser 1 5 455PRTartificial sequencesynthetic
peptide 45Leu Asn Leu Met Gly 1 5 465PRTartificial
sequencesynthetic peptide 46Ile Asn Leu Leu Gly 1 5
475PRTartificial sequencesynthetic peptide 47Asn Tyr Trp Met Tyr 1
5 4817PRTartificial sequencesynthetic peptide 48Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
4916PRTartificial sequencesynthetic peptide 49Thr Ile Thr Val Gly
Asp Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
5016PRTartificial sequencesynthetic peptide 50Thr Ile Thr Val Gly
Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
5117PRTartificial sequencesynthetic peptide 51Ser Ile Asn Gly Arg
Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
5217PRTartificial sequencesynthetic peptide 52Ala Ile Ser Ala Asp
Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
5317PRTartificial sequencesynthetic peptide 53Ala Ile Ser Ala Asp
Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
5417PRTartificial sequencesynthetic peptide 54Arg Ile Ser Thr Gly
Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
5513PRTartificial sequencesynthetic peptide 55Asp Arg Glu Ala Gln
Val Asp Thr Leu Asp Phe Asp Tyr 1 5 10 566PRTartificial
sequencesynthetic peptide 56Gly Gly Ser Leu Ser Arg 1 5
578PRTartificial sequencesynthetic peptide 57Arg Arg Thr Trp His
Ser Glu Leu 1 5 587PRTartificial sequencesynthetic peptide 58Gly
Arg Ser Val Ser Arg Ser 1 5 595PRTartificial sequencesynthetic
peptide 59Gly Arg Gly Ser Pro 1 5 60122PRTartificial
sequencesynthetic peptide 60Ala Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala
Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Ala Met Gly Met Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85
90 95 Ala Asn Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
61126PRTartificial sequencesynthetic peptide 61Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45 Thr Ser Ile Ser Trp Ser Gly Thr Tyr Thr Ala Tyr Ser Asp Asn Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Ser Arg Tyr Arg Ser Asn Tyr
Tyr Asp His Asp Asp Lys 100 105 110 Tyr Ala Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 125 62128PRTartificial
sequencesynthetic peptide 62Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Asp Val Val 35 40 45 Ala Ala Ile Ser
Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val 50 55 60 Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80
Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu
Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 63126PRTartificial sequencesynthetic
peptide 63Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Arg Thr
Thr Phe Ser Ser 20 25 30 Tyr Thr Leu Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe 35 40 45 Val Gly Gly Ile Ser Trp Ser Gly
Val Ser Thr Asp Tyr Ala Glu Phe 50 55 60 Ala Lys Gly Arg Phe Thr
Ile Ser Arg Asp His Ala Ala Asn Thr Val 65 70 75 80 Tyr Leu Glu Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala
Ala Leu Gly Arg Tyr Arg Ser Asn Trp Arg Asn Ile Gly Gln 100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
64126PRTartificial sequencesynthetic peptide 64Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45 Thr Ser Ile Ser Trp Ser Gly Ser Tyr Thr Ala Tyr Ala Asp Asn Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Gly Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Ser Arg Tyr Ser Ser Asn Tyr
Tyr Asp His Asp Asp Lys 100 105 110 Tyr Ala Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 125 65116PRTartificial
sequencesynthetic peptide 65Ala Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr
Ser Gly Ser Ile Phe Ser Ser Ser 20 25 30 Ala Met Ala Trp Tyr Arg
Gln Ala Ser Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asp Cys Asn 85
90 95 Phe Val Val Asp Gly Lys Arg Ala Pro Trp Gly Gln Gly Thr Gln
Val 100 105 110 Thr Val Ser Ser 115 66125PRTartificial
sequencesynthetic peptide 66Ala Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ser
Ser Gly Arg Ala Phe Ser Tyr Tyr 20 25 30 Asn Thr Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser
Trp Ser Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Met Val Tyr 65 70 75 80
Leu Gln Met Ala Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Asn Arg Arg Gln Lys Thr Val Gln Met Gly Glu Arg Ala
Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125 67122PRTartificial sequencesynthetic peptide 67Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser Ile Gly 20
25 30 Ala Met Gly Leu Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
Val 35 40 45 Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Pro Lys
Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Asn Leu Lys Gln Gly Ser
Tyr Gly Tyr Arg Phe Asn Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 68122PRTartificial sequencesynthetic
peptide 68Ala Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Ile
Phe Ser Ile Gly 20 25 30 Ala Met Gly Leu Tyr Arg Gln Ala Pro Gly
Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Ser Gly Gly Ser
Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Asn
Leu
Lys Gln Gly Asp Tyr Gly Tyr Arg Phe Asn Asp Tyr Trp 100 105 110 Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 69122PRTartificial
sequencesynthetic peptide 69Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala
Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Thr Met Gly Leu Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85
90 95 Ala Asn Leu Lys Gln Gly Asp Tyr Gly Tyr Arg Phe Asn Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
70122PRTartificial sequencesynthetic peptide 70Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Thr
Met Gly Leu Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Tyr 85 90 95 Ala Asn Leu Lys Gln Gly Asp Tyr Gly Tyr
Arg Phe Asn Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 71128PRTartificial sequencesynthetic peptide 71Gln
Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10
15 Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn
20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp
Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg
Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr
Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
72128PRTartificial sequencesynthetic peptide 72Gln Val Lys Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Asp Val Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met
Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Ser Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 73128PRTartificial
sequencesynthetic peptide 73Gln Val Lys Leu Glu Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Asp Val Val 35 40 45 Ala Ala Ile Ser
Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80
Leu Glu Met Asn Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu
Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 74259PRTartificial sequencesynthetic
peptide 74Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu Arg Asp Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 Ala Ala Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 130 135 140 Ala Gly Gly Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe 145 150 155 160 Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg 165 170 175 Asp Leu Val Ala Ala Ile Ser Arg
Thr Gly Gly Ser Thr Tyr Tyr Pro 180 185 190 Asp Ser Val Glu Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg 195 200 205 Met Val Tyr Leu
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr
Cys Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg 225 230 235
240 Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr
245 250 255 Val Ser Ser 75265PRTartificial sequencesynthetic
peptide 75Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu Arg Asp Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu
Ser 130 135 140 Gly Gly Gly Leu Val Gln Ala Gly Gly Ala Leu Arg Leu
Ser Cys Ala 145 150 155 160 Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro
Met Gly Trp Phe Arg Gln 165 170 175 Ala Pro Gly Lys Glu Arg Asp Leu
Val Ala Ala Ile Ser Arg Thr Gly 180 185 190 Gly Ser Thr Tyr Tyr Pro
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser 195 200 205 Arg Asp Asn Ala
Lys Arg Met Val Tyr Leu Gln Met Asn Asn Leu Lys 210 215 220 Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala 225 230 235
240 Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly
245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265
76286PRTartificial sequencesynthetic peptide 76Gln Val Lys Leu Glu
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Leu Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met
Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ala Leu 165 170
175 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro Met
180 185 190 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Leu Val
Ala Ala 195 200 205 Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp
Ser Val Glu Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Arg Met Val Tyr Leu Gln 225 230 235 240 Met Asn Asn Leu Lys Pro Glu
Gly Thr Ala Val Tyr Tyr Cys Ala Ala 245 250 255 Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 260 265 270 Tyr Thr Phe
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 275 280 285
77247PRTartificial sequencesynthetic peptide 77Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Ala
Met Gly Met Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Pro Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Tyr 85 90 95 Ala Asn Leu Lys Gln Gly Ser Tyr Gly Tyr
Arg Phe Asn Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val
Ser Ser Ala Ala Ala Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Leu
Ala Ser Gly Arg Ile Phe Ser Ile Gly Ala Met Gly 145 150 155 160 Met
Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile 165 170
175 Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys Gly Arg Phe
180 185 190 Thr Ile Ser Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu Gln
Met Asn 195 200 205 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Tyr Ala Asn Leu 210 215 220 Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn
Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Gln Val Thr Val Ser Ser
245 78253PRTartificial sequencesynthetic peptide 78Ala Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30
Ala Met Gly Met Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35
40 45 Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Pro Lys Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Tyr 85 90 95 Ala Asn Leu Lys Gln Gly Ser Tyr Gly
Tyr Arg Phe Asn Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly
Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe 145 150 155 160
Ser Ile Gly Ala Met Gly Met Tyr Arg Gln Ala Pro Gly Lys Gln Arg 165
170 175 Glu Leu Val Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala
Asp 180 185 190 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Pro
Lys Asn Thr 195 200 205 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Tyr Ala Asn Leu Lys Gln Gly
Ser Tyr Gly Tyr Arg Phe Asn 225 230 235 240 Asp Tyr Trp Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 245 250 79274PRTartificial
sequencesynthetic peptide 79Ala Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala
Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Ala Met Gly Met Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85
90 95 Ala Asn Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly 145 150 155 160 Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Leu Ala 165 170 175 Ser Gly Arg Ile
Phe Ser Ile Gly Ala Met Gly Met Tyr Arg Gln Ala 180 185 190 Pro Gly
Lys Gln Arg Glu Leu Val Ala Thr Ile Thr Ser Gly Gly Ser 195 200 205
Thr Asn Tyr Ala Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 210
215 220 Gly Pro Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Tyr Ala Asn Leu Lys
Gln Gly Ser Tyr 245 250 255 Gly Tyr Arg Phe Asn Asp Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val 260 265
270 Ser Ser 80259PRTartificial sequencesynthetic peptide 80Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20
25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Val
Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala
Arg Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ala Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Ala
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Ala
Gly Gly Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 145 150
155 160 Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg 165 170 175 Asp Val Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala 180 185 190 Arg Ser Val Glu Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Arg 195 200 205 Met Val Tyr Leu Gln Met Asn Ala Leu
Lys Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Ala Gly
Val Arg Ala Glu Asp Gly Arg Val Arg 225 230 235 240 Thr Leu Pro Ser
Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser
Ser 81265PRTartificial sequencesynthetic peptide 81Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Val Val 35
40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser
Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp
Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 130 135 140 Gly Gly Gly
Leu Val Gln Ala Gly Gly Ala Leu Arg Leu Ser Cys Ala 145 150 155 160
Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln 165
170 175 Ala Pro Gly Lys Glu Arg Asp Val Val Ala Ala Ile Ser Arg Thr
Gly 180 185 190 Gly Ser Thr Tyr Tyr Ala Arg Ser Val Glu Gly Arg Phe
Thr Ile Ser 195 200 205 Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln
Met Asn Ala Leu Lys 210 215 220 Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Ala Gly Val Arg Ala 225 230 235 240 Glu Asp Gly Arg Val Arg
Thr Leu Pro Ser Glu Tyr Asn Phe Trp Gly 245 250 255 Gln Gly Thr Gln
Val Thr Val Ser Ser 260 265 82286PRTartificial sequencesynthetic
peptide 82Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu Arg Asp Val Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Ala Arg Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Ala Gly Gly Ala Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Tyr Asn Pro Met 180 185 190 Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Asp Val Val Ala Ala 195 200 205 Ile Ser Arg Thr
Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val Glu Gly 210 215 220 Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 225 230 235
240 Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
245 250 255 Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro
Ser Glu 260 265 270 Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 275 280 285 833PRTartificial sequencesynthetic peptide
83Ala Ala Ala 1 849PRTartificial sequencesynthetic peptide 84Gly
Gly Gly Gly Ser Gly Gly Gly Ser 1 5 8530PRTartificial
sequencesynthetic peptide 85Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 20 25 30 86128PRTartificial
sequencesynthetic peptide 86Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Asp Val Val 35 40 45 Ala Ala Ile Ser
Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val 50 55 60 Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu
Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 87128PRTartificial sequencesynthetic
peptide 87Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Gly Arg Glu Val Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Ala Arg Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 88128PRTartificial sequencesynthetic peptide 88Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25
30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Asp Val Val
35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg
Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu
Asp Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
89128PRTartificial sequencesynthetic peptide 89Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Asp Val Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 90128PRTartificial
sequencesynthetic peptide 90Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser
Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu
Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 91128PRTartificial sequencesynthetic
peptide 91Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 92128PRTartificial sequencesynthetic peptide 92Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25
30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp
Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Arg Ser Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu
Asp Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
93128PRTartificial sequencesynthetic peptide 93Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 94128PRTartificial
sequencesynthetic peptide 94Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser
Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu
Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 125 95122PRTartificial sequencesynthetic
peptide 95Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile
Phe Ser Ile Gly 20 25 30 Ala Met Gly Met Tyr Arg Gln Ala Pro Gly
Lys Gly Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Ser Gly Gly Ser
Thr Asn Tyr Ala Asp Pro Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Asn
Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 96122PRTartificial
sequencesynthetic peptide 96Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Ala Met Gly Met Tyr Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Asn
Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 97122PRTartificial
sequencesynthetic peptide 97Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Ile Phe Ser Ile Gly 20 25 30 Ala Met Gly Met Tyr Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr
Ser Gly Gly Ser Thr Asn Tyr Ala Asp Pro Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85
90 95 Ala Asn Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
98259PRTartificial sequencesynthetic peptide 98Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Ala Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 145 150 155 160 Ser
Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg 165 170
175 Glu Leu Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro
180 185 190 Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Arg 195 200 205 Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg 225 230 235 240 Thr Leu Pro Ser Glu Tyr Thr
Phe Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser
99259PRTartificial sequencesynthetic peptide 99Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40
45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Ala Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 145 150 155 160 Ser
Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg 165 170
175 Glu Leu Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro
180 185 190 Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Arg 195 200 205 Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg 225 230 235 240 Thr Leu Pro Ser Glu Tyr Thr
Phe Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser
100259PRTartificial sequencesynthetic peptide 100Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Val Val 35
40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser
Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp
Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Ala Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 145 150 155 160
Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg 165
170 175 Glu Val Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr
Ala 180 185 190 Arg Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Arg 195 200 205 Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Ala Gly Val Arg
Ala Glu Asp Gly Arg Val Arg 225 230 235 240 Thr Leu Pro Ser Glu Tyr
Asn Phe Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser
101265PRTartificial sequencesynthetic peptide 101Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35
40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser
Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp
Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Thr Phe Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 130 135 140 Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 145 150 155 160
Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln 165
170 175 Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Ala Ile Ser Arg Thr
Gly 180 185 190 Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu Gly Arg Phe
Thr Ile Ser 195 200 205 Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln
Met Asn Ser Leu Arg 210 215 220 Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Ala Gly Val Arg Ala 225 230 235 240 Glu Asp Gly Arg Val Arg
Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly 245 250 255 Gln Gly Thr Gln
Val Thr Val Ser Ser 260 265 102265PRTartificial sequencesynthetic
peptide 102Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu
Ser 130 135 140 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala 145 150 155 160 Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro
Met Gly Trp Phe Arg Gln 165 170 175 Ala Pro Gly Lys Gly Arg Glu Leu
Val Ala Ala Ile Ser Arg Thr Gly 180 185 190 Gly Ser Thr Tyr Tyr Pro
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser 195 200 205 Arg Asp Asn Ala
Lys Arg Ser Val Tyr Leu Gln Met Asn Ser Leu Arg 210 215 220 Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala 225 230 235
240 Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly
245 250 255 Gln Gly Thr Gln Val Thr Val Ser Ser 260 265
103265PRTartificial sequencesynthetic peptide 103Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Val Val 35
40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser
Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp
Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 130 135 140 Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 145 150 155 160
Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln 165
170 175 Ala Pro Gly Lys Gly Arg Glu Val Val Ala Ala Ile Ser Arg Thr
Gly 180 185 190 Gly Ser Thr Tyr Tyr Ala Arg Ser Val Glu Gly Arg Phe
Thr Ile Ser 195 200 205 Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln
Met Asn Ser Leu Arg 210 215 220 Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Ala Gly Val Arg Ala 225 230 235 240 Glu Asp Gly Arg Val Arg
Thr Leu Pro Ser Glu Tyr Asn Phe Trp Gly 245 250 255 Gln Gly Thr Gln
Val Thr Val Ser Ser 260 265 104286PRTartificial sequencesynthetic
peptide 104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110
Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Tyr Asn Pro Met 180 185 190 Gly Trp Phe Arg Gln Ala
Pro Gly Lys Gly Arg Glu Leu Val Ala Ala 195 200 205 Ile Ser Arg Thr
Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu Gly 210 215 220 Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 225 230 235
240 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
245 250 255 Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro
Ser Glu 260 265 270 Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 275 280 285 105286PRTartificial sequencesynthetic peptide
105Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
Tyr Asn 20 25 30 Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly
Arg Glu Leu Val 35 40 45 Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Arg Ser Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly
Val Arg Ala Glu Asp Gly Arg Val Arg Thr Leu Pro 100 105 110 Ser Glu
Tyr Thr Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130
135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
Phe Ser Tyr Asn Pro Met 180 185 190 Gly Trp Phe Arg Gln Ala Pro Gly
Lys Gly Arg Glu Leu Val Ala Ala 195 200 205 Ile Ser Arg Thr Gly Gly
Ser Thr Tyr Tyr Pro Asp Ser Val Glu Gly 210
215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu
Gln 225 230 235 240 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Ala 245 250 255 Ala Gly Val Arg Ala Glu Asp Gly Arg Val
Arg Thr Leu Pro Ser Glu 260 265 270 Tyr Thr Phe Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 275 280 285 106286PRTartificial
sequencesynthetic peptide 106Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Tyr Asn 20 25 30 Pro Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Gly Arg Glu Val Val 35 40 45 Ala Ala Ile
Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val 50 55 60 Glu
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg
Thr Leu Pro 100 105 110 Ser Glu Tyr Asn Phe Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn Pro Met 180 185 190
Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Val Val Ala Ala 195
200 205 Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Ala Arg Ser Val Glu
Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val
Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Ala 245 250 255 Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro Ser Glu 260 265 270 Tyr Asn Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 275 280 285 107117PRTartificial
sequencesynthetic peptide 107Ala Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Gly Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala
Ala Ser Glu Arg Ile Phe Asp Leu Asn 20 25 30 Leu Met Gly Trp Tyr
Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Cys
Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Met Asp Tyr Thr Lys Gln Thr Val Tyr 65 70
75 80 Leu His Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr
Cys 85 90 95 Lys Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln
Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 108116PRTartificial
sequencesynthetic peptide 108Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Glu Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala
Ala Ser Glu Arg Ile Trp Asp Ile Asn 20 25 30 Leu Leu Gly Trp Tyr
Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile
Thr Val Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Tyr Asp Lys Asn Thr Leu Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys
Lys 85 90 95 Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly
Thr Gln Val 100 105 110 Thr Val Ser Ser 115 109115PRTartificial
sequencesynthetic peptide 109Ala Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile
Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr
Gln Val Thr 100 105 110 Val Ser Ser 115 110116PRTartificial
sequencesynthetic peptide 110Ala Val Gln Leu Val Asp Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Ser Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val
Arg Gln Tyr Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile
Asn Gly Arg Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Glu Tyr Tyr
Cys 85 90 95 Thr Ile Gly Arg Ser Val Ser Arg Ser Arg Thr Gln Gly
Thr Gln Val 100 105 110 Thr Val Ser Ser 115 111114PRTartificial
sequencesynthetic peptide 111Ala Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr
Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Asp Gln Glu Trp Val 35 40 45 Ser Ala Ile
Ser Ala Asp Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70
75 80 Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Pro Gly Thr Gln
Val Thr Val 100 105 110 Ser Ser 112114PRTartificial
sequencesynthetic peptide 112Gln Val Gln Leu Ala Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr
Ala Ser Gly Phe Thr Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Ser Ala Asp Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70
75 80 Leu Glu Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln
Val Thr Val 100 105 110 Ser Ser 113123PRTartificial
sequencesynthetic peptide 113Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Tyr Trp Val
Arg Val Ala Pro Gly Lys Gly Leu Glu Arg Ile 35 40 45 Ser Arg Asp
Ile Ser Thr Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser 50 55 60 Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70
75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr
Tyr 85 90 95 Cys Ala Lys Asp Arg Glu Ala Gln Val Asp Thr Leu Asp
Phe Asp Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 114115PRTartificial sequencesynthetic peptide 114Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20
25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp
Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser
Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
115115PRTartificial sequencesynthetic peptide 115Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
116115PRTartificial sequencesynthetic peptide 116Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
117115PRTartificial sequencesynthetic peptide 117Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
118115PRTartificial sequencesynthetic peptide 118Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
119115PRTartificial sequencesynthetic peptide 119Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
120115PRTartificial sequencesynthetic peptide 120Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
121115PRTartificial sequencesynthetic peptide 121Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
12230PRTartificial sequencesynthetic peptide 122Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser 20 25 30
12330PRTartificial sequencesynthetic peptide 123Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
12430PRTartificial sequencesynthetic peptide 124Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20
25 30 12530PRTartificial sequencesynthetic peptide 125Ala Val Gln
Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Thr Ala Ser Glu Arg Thr Thr Phe 20 25 30
12630PRTartificial sequencesynthetic peptide 126Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn 20 25 30
12730PRTartificial sequencesynthetic peptide 127Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys
Leu Ser Cys Ala Thr Ser Gly Ser Ile Phe Ser 20 25 30
12830PRTartificial sequencesynthetic peptide 128Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg
Leu Ser Cys Thr Ser Ser Gly Arg Ala Phe Ser 20 25 30
12930PRTartificial sequencesynthetic peptide 129Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser 20 25 30
13030PRTartificial sequencesynthetic peptide 130Ala Val Gln Leu Glu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser 20 25 30
13130PRTartificial sequencesynthetic peptide 131Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser 20 25 30
13230PRTartificial sequencesynthetic peptide 132Ala Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Leu Ala Ser Gly Arg Ile Phe Ser 20 25 30
13330PRTartificial sequencesynthetic peptide 133Gln Val Lys Leu Glu
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13430PRTartificial sequencesynthetic peptide 134Gln Val Lys Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13530PRTartificial sequencesynthetic peptide 135Gln Val Lys Leu Glu
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ala Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13630PRTartificial sequencesynthetic peptide 136Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13730PRTartificial sequencesynthetic peptide 137Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13830PRTartificial sequencesynthetic peptide 138Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
13930PRTartificial sequencesynthetic peptide 139Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
14030PRTartificial sequencesynthetic peptide 140Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
14130PRTartificial sequencesynthetic peptide 141Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
14230PRTartificial sequencesynthetic peptide 142Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30
14330PRTartificial sequencesynthetic peptide 143Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30
14430PRTartificial sequencesynthetic peptide 144Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30
14530PRTartificial sequencesynthetic peptide 145Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser 20 25 30
14630PRTartificial sequencesynthetic peptide 146Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser 20 25 30
14730PRTartificial sequencesynthetic peptide 147Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser 20 25 30
1485PRTartificial sequencesynthetic peptide 148Ile Gly Ala Met Gly
1 5 1495PRTartificial sequencesynthetic peptide 149Asn Tyr Gly Met
Gly 1 5 1505PRTartificial sequencesynthetic peptide 150Tyr Asn Pro
Met Gly 1 5 1516PRTartificial sequencesynthetic peptide 151Ser Ser
Tyr Thr Leu Gly 1 5 1525PRTartificial sequencesynthetic peptide
152Asn Tyr Gly Met Gly 1 5 1535PRTartificial sequencesynthetic
peptide 153Ser Ser Ala Met Ala 1 5 1545PRTartificial
sequencesynthetic peptide 154Tyr Tyr Asn Thr Gly 1 5
1555PRTartificial sequencesynthetic peptide 155Ile Gly Ala Met Gly
1 5 1565PRTartificial sequencesynthetic peptide 156Ile Gly Ala Met
Gly 1 5 1575PRTartificial sequencesynthetic peptide 157Ile Gly Thr
Met Gly 1 5 1585PRTartificial sequencesynthetic peptide 158Ile Gly
Thr Met Gly 1 5 1595PRTartificial sequencesynthetic peptide 159Tyr
Asn Pro Met Gly 1 5 1605PRTartificial sequencesynthetic peptide
160Tyr Asn Pro Met Gly 1 5 1615PRTartificial sequencesynthetic
peptide 161Tyr Asn Pro Met Gly 1 5 1625PRTartificial
sequencesynthetic peptide 162Tyr Asn Pro Met Gly 1 5
1635PRTartificial sequencesynthetic peptide 163Tyr Asn Pro Met Gly
1 5 1645PRTartificial sequencesynthetic peptide 164Tyr Asn Pro Met
Gly 1 5 1655PRTartificial sequencesynthetic peptide 165Tyr Asn Pro
Met Gly 1 5 1665PRTartificial sequencesynthetic peptide 166Tyr Asn
Pro Met Gly 1 5 1675PRTartificial sequencesynthetic peptide 167Tyr
Asn Pro Met Gly 1 5 1685PRTartificial sequencesynthetic peptide
168Tyr Asn Pro Met Gly 1 5 1695PRTartificial sequencesynthetic
peptide 169Tyr Asn Pro Met Gly 1 5 1705PRTartificial
sequencesynthetic peptide 170Tyr Asn Pro Met Gly 1 5
1715PRTartificial sequencesynthetic peptide 171Ile Gly Ala Met Gly
1 5 1725PRTartificial sequencesynthetic peptide 172Ile Gly Ala Met
Gly 1 5 1735PRTartificial sequencesynthetic peptide 173Ile Gly Ala
Met Gly 1 5 17414PRTartificial sequencesynthetic peptide 174Met Tyr
Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10
17514PRTartificial sequencesynthetic peptide 175Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val Thr 1 5 10 17614PRTartificial
sequencesynthetic peptide 176Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Asp Val Val Ala 1 5 10 17714PRTartificial sequencesynthetic
peptide 177Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Gly
1 5 10 17814PRTartificial sequencesynthetic peptide 178Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Thr 1 5 10
17914PRTartificial sequencesynthetic peptide 179Trp Tyr Arg Gln Ala
Ser Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 18014PRTartificial
sequencesynthetic peptide 180Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val Ala 1 5 10 18114PRTartificial sequencesynthetic
peptide 181Leu Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala
1 5 10 18214PRTartificial sequencesynthetic peptide 182Leu Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10
18314PRTartificial sequencesynthetic peptide 183Leu Tyr Arg Gln Ala
Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 18414PRTartificial
sequencesynthetic peptide 184Leu Tyr Arg Gln Ala Pro Gly Lys Gln
Arg Glu Leu Val Ala 1 5 10 18514PRTartificial sequencesynthetic
peptide 185Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Leu Val Ala
1 5 10 18614PRTartificial sequencesynthetic peptide 186Trp Phe Arg
Gln Ala Pro Gly Arg Glu Arg Asp Val Val Ala 1 5 10
18714PRTartificial sequencesynthetic peptide 187Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Asp Val Val Ala 1 5 10 18814PRTartificial
sequencesynthetic peptide 188Trp Phe Arg Gln Ala Pro Gly Lys Gly
Arg Asp Val Val Ala 1 5 10 18914PRTartificial sequencesynthetic
peptide 189Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Val Val Ala
1 5 10 19014PRTartificial sequencesynthetic peptide 190Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Asp Val Val Ala 1 5 10
19114PRTartificial sequencesynthetic peptide 191Trp Phe Arg Gln Ala
Pro Gly Lys Gly Arg Asp Val Val Ala 1 5 10 19214PRTartificial
sequencesynthetic peptide 192Trp Phe Arg Gln Ala Pro Gly Lys Gly
Arg Glu Leu Val Ala 1 5 10 19314PRTartificial sequencesynthetic
peptide 193Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala
1 5 10 19414PRTartificial sequencesynthetic peptide 194Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala 1 5 10
19514PRTartificial sequencesynthetic peptide 195Trp Phe Arg Gln Ala
Pro Gly Lys Gly Arg Glu Leu Val Ala 1 5 10 19614PRTartificial
sequencesynthetic peptide 196Trp Phe Arg Gln Ala Pro Gly Lys Gly
Arg Glu Leu Val Ala 1 5 10 19714PRTartificial sequencesynthetic
peptide 197Met Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala
1 5 10 19814PRTartificial sequencesynthetic peptide 198Met Tyr Arg
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala 1 5 10
19914PRTartificial sequencesynthetic peptide 199Met Tyr Arg Gln Ala
Pro Gly Lys Gly Arg Glu Leu Val Ala 1 5 10 20016PRTartificial
sequencesynthetic peptide 200Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 20117PRTartificial
sequencesynthetic peptide 201Ser Ile Ser Trp Ser Gly Thr Tyr Thr
Ala Tyr Ser Asp Asn Val Lys 1 5 10 15 Gly 20217PRTartificial
sequencesynthetic peptide 202Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly 20317PRTartificial
sequencesynthetic peptide 203Gly Ile Ser Trp Ser Gly Val Ser Thr
Asp Tyr Ala Glu Phe Ala Lys 1 5 10 15 Gly 20417PRTartificial
sequencesynthetic peptide 204Ser Ile Ser Trp Ser Gly Ser Tyr Thr
Ala Tyr Ala Asp Asn Val Lys 1 5 10 15 Gly 20516PRTartificial
sequencesynthetic peptide 205Thr Ile Thr Ser Gly Gly Arg Thr Ser
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 20617PRTartificial
sequencesynthetic peptide 206Ala Ile Ser Trp Ser Gly Gly Leu Thr
Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 20716PRTartificial
sequencesynthetic peptide 207Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 20816PRTartificial
sequencesynthetic peptide 208Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 20916PRTartificial
sequencesynthetic peptide 209Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 21016PRTartificial
sequencesynthetic peptide 210Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 21117PRTartificial
sequencesynthetic peptide 211Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 21217PRTartificial
sequencesynthetic peptide 212Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 21317PRTartificial
sequencesynthetic peptide 213Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 21417PRTartificial
sequencesynthetic peptide 214Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly 21517PRTartificial
sequencesynthetic peptide 215Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly 21617PRTartificial
sequencesynthetic peptide 216Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly 21717PRTartificial
sequencesynthetic peptide 217Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Ala Arg Ser Val Glu 1 5 10 15 Gly 21817PRTartificial
sequencesynthetic peptide 218Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 21917PRTartificial
sequencesynthetic peptide 219Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 22017PRTartificial
sequencesynthetic peptide 220Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 22117PRTartificial
sequencesynthetic peptide 221Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 22217PRTartificial
sequencesynthetic peptide 222Ala Ile Ser Arg Thr Gly Gly Ser Thr
Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15 Gly 22316PRTartificial
sequencesynthetic peptide 223Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 22416PRTartificial
sequencesynthetic peptide 224Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 22516PRTartificial
sequencesynthetic peptide 225Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 22632PRTartificial
sequencesynthetic peptide 226Arg Phe Thr Ile Ser Arg Asp Gly Pro
Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25
30 22732PRTartificial sequencesynthetic peptide 227Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asp
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
22832PRTartificial sequencesynthetic peptide 228Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ala
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
22932PRTartificial sequencesynthetic peptide 229Arg Phe Thr Ile Ser
Arg Asp His Ala Ala Asn Thr Val Tyr Leu Glu 1 5 10 15 Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
23032PRTartificial sequencesynthetic peptide 230Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asp Ser
Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
23132PRTartificial sequencesynthetic peptide 231Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Asp Cys Asn Phe 20 25 30
23232PRTartificial sequencesynthetic peptide 232Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asp Met Val Tyr Leu Gln 1 5 10 15 Met Ala Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
23332PRTartificial sequencesynthetic peptide 233Arg Phe Thr Ile Ser
Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
23432PRTartificial sequencesynthetic peptide 234Arg Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
23532PRTartificial sequencesynthetic peptide 235Arg Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
23632PRTartificial sequencesynthetic peptide 236Arg Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
23732PRTartificial sequencesynthetic peptide 237Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Asn
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
23832PRTartificial sequencesynthetic peptide 238Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Asn
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
23932PRTartificial sequencesynthetic peptide 239Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Glu 1 5 10 15 Met Asn Asn
Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24032PRTartificial sequencesynthetic peptide 240Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24132PRTartificial sequencesynthetic peptide 241Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24232PRTartificial sequencesynthetic peptide 242Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24332PRTartificial sequencesynthetic peptide 243Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24432PRTartificial sequencesynthetic peptide 244Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24532PRTartificial sequencesynthetic peptide 245Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24632PRTartificial sequencesynthetic peptide 246Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24732PRTartificial sequencesynthetic peptide 247Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Arg Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24832PRTartificial sequencesynthetic peptide 248Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30
24932PRTartificial sequencesynthetic peptide 249Arg Phe Thr Ile Ser
Arg Asp Gly Pro Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
25032PRTartificial sequencesynthetic peptide 250Arg Phe Thr Ile Ser
Arg Asp Gly Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
25132PRTartificial sequencesynthetic peptide 251Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala 20 25 30
25214PRTartificial sequencesynthetic peptide 252Asn Leu Lys Gln Gly
Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr 1 5 10 25317PRTartificial
sequencesynthetic peptide 253Gln Ser Arg Tyr Arg Ser Asn Tyr Tyr
Asp His Asp Asp Lys Tyr Ala 1 5 10 15 Tyr 25419PRTartificial
sequencesynthetic peptide 254Ala Gly Val Arg Ala Glu Asp Gly Arg
Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Asn Phe
25516PRTartificial sequencesynthetic peptide 255Leu Gly Arg Tyr Arg
Ser Asn Trp Arg Asn Ile Gly Gln Tyr Asp Tyr 1 5 10 15
25617PRTartificial sequencesynthetic peptide 256Gln Ser Arg Tyr Ser
Ser Asn Tyr Tyr Asp His Asp Asp Lys Tyr Ala 1 5 10 15 Tyr
2578PRTartificial sequencesynthetic peptide 257Val Val Asp Gly Lys
Arg Ala Pro 1 5 25816PRTartificial sequencesynthetic peptide 258Asn
Arg Arg Gln Lys Thr Val Gln Met Gly Glu Arg Ala Tyr Asp Tyr 1 5 10
15 25914PRTartificial sequencesynthetic peptide 259Asn Leu Lys Gln
Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr 1 5 10 26014PRTartificial
sequencesynthetic peptide 260Asn Leu Lys Gln Gly Asp Tyr Gly Tyr
Arg Phe Asn Asp Tyr 1 5 10 26114PRTartificial sequencesynthetic
peptide 261Asn Leu Lys Gln Gly Asp Tyr Gly Tyr Arg Phe Asn Asp Tyr
1 5 10 26214PRTartificial sequencesynthetic peptide 262Asn Leu Lys
Gln Gly Asp Tyr Gly Tyr Arg Phe Asn Asp Tyr 1 5 10
26319PRTartificial sequencesynthetic peptide 263Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
26419PRTartificial sequencesynthetic peptide 264Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Ser Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
26519PRTartificial sequencesynthetic peptide 265Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
26619PRTartificial sequencesynthetic peptide 266Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Asn Phe
26719PRTartificial sequencesynthetic peptide 267Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Asn Phe
26819PRTartificial sequencesynthetic peptide 268Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Asn Phe
26919PRTartificial sequencesynthetic peptide 269Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Asn Phe
27019PRTartificial sequencesynthetic peptide 270Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
27119PRTartificial sequencesynthetic peptide 271Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
27219PRTartificial sequencesynthetic peptide 272Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
27319PRTartificial sequencesynthetic peptide 273Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
27419PRTartificial sequencesynthetic peptide 274Ala Gly Val Arg Ala
Glu Asp Gly Arg Val Arg Thr Leu Pro Ser Glu 1 5 10 15 Tyr Thr Phe
27514PRTartificial sequencesynthetic peptide 275Asn Leu Lys Gln Gly
Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr 1 5 10 27614PRTartificial
sequencesynthetic peptide 276Asn Leu Lys Gln Gly Ser Tyr Gly Tyr
Arg Phe Asn Asp Tyr 1 5 10 27714PRTartificial sequencesynthetic
peptide 277Asn Leu Lys Gln Gly Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr
1 5 10 27811PRTartificial sequencesynthetic peptide 278Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 1 5 10 27911PRTartificial
sequencesynthetic peptide 279Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 28011PRTartificial sequencesynthetic peptide 280Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 28111PRTartificial
sequencesynthetic peptide 281Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 28211PRTartificial sequencesynthetic peptide 282Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 28311PRTartificial
sequencesynthetic peptide 283Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 28411PRTartificial sequencesynthetic peptide 284Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 28511PRTartificial
sequencesynthetic peptide 285Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 28611PRTartificial sequencesynthetic peptide 286Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 28711PRTartificial
sequencesynthetic peptide 287Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 28811PRTartificial sequencesynthetic peptide 288Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 28911PRTartificial
sequencesynthetic peptide 289Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 29011PRTartificial sequencesynthetic peptide 290Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 29111PRTartificial
sequencesynthetic peptide 291Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 29211PRTartificial sequencesynthetic peptide 292Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 29311PRTartificial
sequencesynthetic peptide 293Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 29411PRTartificial sequencesynthetic peptide 294Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 29511PRTartificial
sequencesynthetic peptide 295Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 29611PRTartificial sequencesynthetic peptide 296Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 29711PRTartificial
sequencesynthetic peptide 297Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 29811PRTartificial sequencesynthetic peptide 298Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 29911PRTartificial
sequencesynthetic peptide 299Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 30011PRTartificial sequencesynthetic peptide 300Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 30111PRTartificial
sequencesynthetic peptide 301Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10 30211PRTartificial sequencesynthetic peptide 302Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 30311PRTartificial
sequencesynthetic peptide 303Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 1 5 10
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