Indian Patents
Title of Invention

SINGLE DOMAIN VHH ANTIBODIES AGAINST VAN WILLEBRAND FACTOR
Abstract The present invention relates to improved Nanobodies™ 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.
Full Text FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13)
1. SINGLE DOMAIN VHH ANTIBODIES AGAINST VAN WILLEBRANDFACTOR
2.
(A) Ablynx N.V.
(B) A Company incorporated under the laws of BELGIUM
(C) Technologiepark 4,
B-9052 Zwijnaarde,
Belgium.
The following specification particularly describes the invention and the manner in which it is to be performed.


The present invention relates to improved Nanobodies™ against von Willebrand Factor (vWF), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. [Note: Nanobody™, Nanobodies™ and Nanoclone™ are trademarks of Ablynx N. V]
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.
Other aspects, embodiments, advantages and applications of the invention will become clear from the further description hereinbelow.
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.
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.
The anti-vWF Nanobodies described in WO 04/062551 may be directed against any epitope or conformation of vWF (such as the Al domain or A3 domain), but are preferably directed against the Al domain, and in particular against the activated conformation of the A1 domain.
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.


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..
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
It is a general object of the present invention to provide Nanobodies against vWF, in particular against human vWF.
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.
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.
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:
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); better suitability for formatting in a multivalent format (for example in a bivalent format);
better suitability for formatting in a multispecific format (for example one of the multispecific formats described in WO 04/062551 or hereinbelow); improved suitability or susceptibility for "humanizing" substitutions (as defined herein); and/or
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;
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;
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;
decreased or where desired increased cross-reactivity with vWF from different species; and/or


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).
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 Al domain of vWF, either in its activated or non-activated confirmation.
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: i) CDR1 comprises or essentially consists of an amino acid sequence chosen from the group consisting of:





YNPMG [SEQ ID NO: 22]
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
ii) CDR2 comprises or essentially consist of an amino acid sequence chosen from the group consisting of:
SISWSGTYTAYSDNVKG [SEQ ID NO: 23]
GISWSGVSTDYAEFAKG [SEQ ID NO: 24]
TSISWSGSYTAYADNVKG [SEQ ID NO: 25]
SISWSGMSTYYTDSVKG [SEQ ID NO: 26]
TITSGGRTSYADSVKG [SEQ ID NO: 27]
AISWSGGLTYYADSVKG [SEQ ID NO: 28]
TITSGGSTNYADPVKG [SEQ ID NO: 29]
TITSGGSTNYADSVKG [SEQ ID NO: 30]
AISRTGGSTYYARSVEG ' [SEQ ID NO: 31]
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]


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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
ni) CDR3 comprises or essentially consists of an amino acid sequence chosen from the

group consisting of:
QSRYRSNYYDHDDKYAY [SEQ ID NO: 33]
LGRYRSNWRNIGQYDY [SEQ ID NO: 34]
QSRYSSNYYDHDDKYAY [SEQ ID NO: 35]
SNRYRTHTTQAMYNY [SEQ ID NO: 36]
WDGKRAP [SEQ ID NO: 37]
NRRQKTVQMGERAYDY [SEQ ID NO: 38]
NLKQGSYGYRFNDY [SEQ ID NO: 39]
NLKQGDYGYRFNDY [SEQ ID NO: 40]
AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]


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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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).
In another aspect, the invention relates to a Nanobody (as defined herein), against
vWF, which consist of 4 framework regions (FRl to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in which:
i) CDR1 is an amino acid sequence chosen from the group consisting of:

NYGMG [SEQ ID NO: 15]
SYTLG [SEQ ID NO: 16]
NYNMG [SEQ ID NO: 17]
SSAMA [SEQ ID NO: 18]
YYNTG [SEQ ID NO: 19]
IGAMG [SEQ ID NO: 20]
IGTMG [SEQ ID NO: 21J
YNPMG [SEQ ID NO: 22]
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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);
in which:
CDR2 is an amino acid sequence chosen from the group consisting of:
SISWSGTYTAYSDNVKG [SEQ ID NO: 23]
GISWSGVSTDYAEFAKG [SEQ ID NO: 24]
TSISWSGSYTAYADNVKG [SEQ ID NO: 25]
SISWSGMSTYYTDSVKG [SEQ ID NO: 26]
TITSGGRTSYADSVKG [SEQ ID NO: 27]
AISWSGGLTYYADSVKG [SEQ ID NO: 28]
TITSGGSTNYADPVKG [SEQ ID NO: 29]
TITSGGSTNVADSVKG [SEQ ID NO: 30]
AISRTGGSTYYARSVEG [SEQ ID NO: 31 ]
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]
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%o sequence identity (as defined herein) with one of the "above amino acid sequences; in which


(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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);
which:
CDR3 is an amino acid sequence chosen from the group consisting of:
QSRYRSNYYDHDDKYAY [SEQ ID NO: 33]
LGRYRSNWRNIGQYDY [SEQ ID NO: 34]
QSRYSSNYYDHDDKYAY [SEQ ID NO: 35]
SNRYRTHTTQAMYNY [SEQ ID NO: 36]
VVDGKRAP [SEQ ID NO: 3 7]
NRRQKTVQMGERAYDY [SEQ ID NO: 38]
NLKQGSYGYRFNDY [SEQ ID NO: 39]
NLKQGDYGYRFNDY [SEQ ID NO: 40]
AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41 ]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]
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


(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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).
The Nanobodies against vWF as described above and as further described hereinbelow are also referred to herein as Nanobodies of the invention.
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.
In another aspect, the invention relates to a Nanobody against vWF, which consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), which is chosen from the group consisting of Nanobodies with the one of the following combinations of CDRl, CDR2 and CDR3, respectively:
CDRl :NYGMG;CDR2: SIS WSGTYTAYSDNVKG;
CDR3:QSRYRSNYYDHDDKYAY
CDR1:SYTLG;CDR2:G1SWSGVSTDYAEFAKG;
CDR3:LGRYRSNWRNIGQYDY
CDRl :NYGMG;CDR2:TSIS WSGSYTAYADNVKG;
CDR3:QSRYSSNYYDHDDKYAY
CDRl :NYNMG;CDR2:SIS WSGMSTYYTDSVKG;


CDR3:SNRYRTHTTQAMYNY
CDR1 :SSAMA;CDR2:TITSGGRTSYADSVKG;CDR3: VVDGKRAP
CDR1:YYNTG;CDR2:AISWSGGLTYYADSVKG;
CDR3:NRRQKTVQMGERAYDY
CDR1:IGAMG;CDR2:TITSGGSTNYADPVKG;CDR3:NLKQGSYGYRFNDY
CDR1:IGAMG;CDR2:T1TSGGSTNYADSVKG;CDR3:NLKQGSYGYRFNDY
CDR1:IGAMG;CDR2:TITSGGSTNYADSVKG;
CDR3.-NLKQGDYGYRFNDY
CDR1:IGTMG;CDR2:TITSGGSTNYADSVKG;CDR3:NLKQGDYGYRFNDY
CDR1:YNPMG;CDR2:AISRTGGSTYYARSVEG;
CDR3.-AGVRAEDGRVRTLPSEYNF
CDR1:YNPMG;CDR2:AISRTGGSTYYPDSVEG;
CDR3:AGVRAEDGRVRTLPSEYTF
CDR1:YNPMG;CDR2:AISRTGGSTYYPDSVEG;
CDR3:AGVRAEDGRVRSLPSEYTF
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or


(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).
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 I: Preferred combinations of CDR's, of CDR's and framework sequences, and of CDR's and humanized FR's

1 iCLONE D FR1 C CDR1 I I FR2 D CDR1 D FR3 I CDR3 D FR4
1 12A5 2 AVQLVESGGG 1> LVQPGGSLRL ASCLASGRIFS E 1 27 MYRQAPGK C IGAMG 4 QRELVA C 2 TITSGGSTNY 2 ADPVKG E 2 RFTISRDGPKNTVYLQ J MNSLKPEDTAVYYCYA 2 2 ) 7 j NLKQGSYGYRFNDY 8 WGQGTQVTVSS
1 QVQLVESGGG 112 2 2 22 LVQAGGSLRL 4 7 WFRQAPGK 0 SISWSGTYT 2 RFTISRDNAKNTVYLQ 5 QSRYRSNYYDHDDKY 712B1 3 SCAASGRTFS 9 NYGMG 5 EREFVT 1 AYSDNVKG 7 MDSLKPEDTAVYYCAA 3 AY 9 WGQGTQVTVSS
I ■ _II 12B6 1 QVQLVESGGG2 LVQAGGALRL '4 SCAASGRTFS ) YNPMG 17 WFRQAPGK (5 ERDVVA j i :3 AISRTGGST , I YYARSVEG 22 RFTISRDNAKRMVYLQ3 MNALKPEDTAVYYCAA 25 AGVRAEDGRVRTLPS4 EYNF 2 33 WGQGTQVTVSS
i ji| 12D11 1 AVQLVDSGGG2 LVQAGGSLRL5 SCTASERTTF 151 SSYTLG 17 WFRQAPGK7 EREFVG 20 GISWSGVST3 DYAEFAKG 22 RFTISRDHAANTVYLEM9 NSLKPEDTAVYYCAA 25 LGRYRSNWRNIGQYD5 Y 28 I 1 WGQGTQVTVSS
! 12-E3 1 EVQLVESGGG2 LVQAGGSLRL6 SCAASGRTFN 1 5 2 NYGMG 17 WFRQAPGK8 EREFVT 20 SISWSGSYT4 AYADNVKG 23 RFTISRDNAKNTVYLQ0 MDSLKPGDTAVYYCAA 2,5 QSRYSSNYYDHDDKY6 AY 282 WGQGTQVTVSS
12C9 1 AVQLVESGGG2 LVQPGGSLKL7 SCATSGSiFS 1 5 3 SSAMA 17 WYRQASGK9 QRELVA 20 TITSGGRTSY5 ADSVKG 23 RFTISRDNAKNTVYLQ1 MNSLKPEDTAVYDCNF 2 5 7 VVDGKRAP 2 8 3 WGQGTQVTVSS
14F8 1 AVQLVESGGG2 LVQAGESLRLB SCTSSGRAFS 1 5 4 YYNTG 18 WFRQAPGK0 EREFVA 20 AISWSGGLT6 YYADSVKG 23 RFTISRDNAKDMVYLQ2 MASLKPEDTAVYYCAA 25 NRRQKTVQMGERAYD8 Y 2 8 4 WGQGTQVTVSS
12B4 1 QVQLVESGGG2 LVQPGGSLRL9 SCLASGRIFS 1 5 5 IGAMG 18 LYRQAPGK1 QRELVA 20 TITSGGSTNY7 ADSVKG 23 RFTISRDGPKNTVYLQ3 MNSLKPEDTAVYYCYA 2 5 9 NLKQGSYGYRFNDY 2 85 WGQGTQVTVSS
12-E8 1 AVQLEESGGG 3 LVQPGGSLRL 0 SCLASGRIFS 1 5 6 IGAMG 18 LYRQAPGK2 QRELVA 20 TITSGGSTNY8 ADSVKG 23 RFTISRDGAKNTVYLQ4 MNSLKPEDTAVYYCYA 2 6 0 NLKQGDYGYRFN DY 2 8 6 WGQGTQVTVSS

TABLE I (continued):

1CLONE C I FR1 D CDR1 I FR2 C I ) CDR1 D FR3 I CDR3 D FR4
1312A6 1 QVQLVESGGG 1 LVQPGGSLRL 5 SCLASGRIFS 7 1 IGTMG C 2 LYRQAPGK C QRELVA 9 2TITSGGSTNY 3 ADSVKG E 2 RFTISRDGAKNTVYLQ 6 MNSLRPEDTAVYYCYA 1 2fi NLKQGDYGYRFN DY 7 WGQGTQVTVSS
1! 2 j 12D8 2 AVQLVESGGG 1 LVQPGGSLRL J SCLASGRIFS i 12 2 2 2 > 8 LYRQAPGK 1 TITSGGSTNY 3 RFTISRDGAKNTVYLQ 6 8 I IGTMG 4 QRELVA 0 ADSVKG 6 MNSLRPEDTAVYYCYA 2 NLKQGDYGYRFNDY j 8 WGQGTQVTVSS
1 12A2 j C QVKLEESGGG 112 2 2 2 LVQAGGALRL 5 8 WFRQAPGK 1 AISRTGGST 3 RFTISRDNAKRMVYLQ 6 AGVRAEDGRVRTLPS 8 1 SCAASGRTFS 9 YNPMG 5 ERDLVA 1| YYPDSVEG 7 MNNLKPEDTAVYYCAA 3 EYTF 9 WGQGTQVTVSS
12F2 QVKLVESGGG3 LVQAGGALRL4 SCAASGRTFS \ 53 YNPMG 13 WFRQAPGR6 ERDVVA 21 AISRTGGST2 YYPDSVEG 23 RFTISRDNAKRMVYLQ3 MNNLKPEDTAVYYCAA 13 AGVRAEDGRVRSLPS4 EYTF 2 9 0 WGQGTQVTVSS
I i J 14H10 1 QVKLEESGGG 3 LVQAGGALRL 5 SCAASGRTFS 1 6 1 YNPMG 18 WFRQAPGK7 ERDVVA 21 AISRTGGST 3 YYPDSVEG j 23 RFTISRDNAKRMVYLE9 MNNLKPDDTAVYYCAA 26 AGVRAEDGRVRTLPS5 EYTF 2 9 1 WGQGTQVTVSS
12B6H1 1 EVQLVESGGG 3 LVQPGGSLRL 6 SCAASGRTFS 1 6 2 YNPMG 18 WFRQAPGK8 GRDVVA 21 AISRTGGST 41 YYARSVEG 24 RFTISRDNAKRMVYLQ0| MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS 6 EYNF 2 9 2 WGQGTQVTVSS
12B6H2 1 EVQLVESGGG 3 LVQPGGSLRL 7 SCAASGRTFS 1 6 3 YNPMG 18 WFRQAPGK9 GREVVA 21 AISRTGGST5 YYARSVEG 24 RFTISRDNAKRMVYLQ1 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS7 EYNF 2 9 3 WGQGTQVTVSS
-i2B6H3 1 EVQLVESGGG 3 LVQPGGSLRL 8 SCAASGRTFS 1 6 4 YNPMG 19 WFRQAPGK0 GRDVVA 21 AISRTGGST 6 YYARSVEG 24 RFTISRDNAKNMVYLQ 2 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS8 EYNF 2 94 WGQGTQVTVSS
12B6H4 1 EVQLVSsGGG 3 LVQPGGSLRL 9 SCAASGRTFS 1 6 5 YNPMG 19 WFRQAPGK1 GRDVVA 21 AISRTGGST7 YYARSVEG 24 RFTISRDNAKRSVYLQ3 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS9 EYNF 2 95 WGQGTQVTVSS
12A2H1 1 EVQLVESGGG ' 4 LVQPGGSLRL 0 SCAASGRTFS | 1 6 6 YNPMG 19 WFRQAPGK2 GRELVA 21 AISRTGGST 8 YYPDSVEG 24 RFTISRDNAKRMVYLQ4 MNSLRAEDTAVYYCAA 27 AGVRAEDGRVRTLPS0 EYTF 2 96| WGQGTQVTVSS

" ■ ■ [CLONE 1 ! D IFR1 D CDR1 FR2 D CDR1 D FR3 | CDR3 D I FR4 |
12A2H3 1 4 1 EVQLVESGGG 11 2 2 Il LVQPGGSLRL 6 9 WFRQAPGK 1 AISRTGGST 4 RFTISRDNAKNMVYLQ 7 SCAASGRTFS 7 YNPMG 3 GRELVA 9 YYPDSVEG 5 MNSLRAEDTAVYYCAA ' AGVRAEDGRVRTLPS EYTF 2 97 !WGQGTQVTVSS
12A2H4 1 4 2 EVQLVESGGG LVQPGGSLRL SCAASGRTFS 1 3 8 YNPMG 19 WFRQAPGK4 GRELVA >2 AISRTGGST3 YYPDSVEG I 24 RFTISRDNAKRSVYLQ 7 AGVRAEDGRVRTLPS6 MNSLRAEDTAVYYCAA 2 EYTF 2 9 8 WGQGTQVTVSS
12A2H11 1 4 3 EVQLVESGGG LVQPGGSLRL SCAASGFTFS 1 5 9 YNPMG 19 WFRQAPGK5 GRELVA 22 AISRTGGST1 YYPDSVEG 24 RFTISRDNAKRMVYLQ7 MNSLRAEDTAVYYCAA I7 AGVRAEDGRVRTLPS3 EYTF 29 9 WGQGTQVTVSS
!I 12A2H13 1 4 4 EVQLVESGGG LVQPGGSLRL SCAASGFTFS 1 7 0 YNPMG 19 WFRQAPGK6 GRELVA 22 AISRTGGST2 YYPDSVEG 24 RFTISRDNAKNSVYLQ8 MNSLRAEDTAVYYCAA 27 AGVRAEDGRVRTLPS4 EYTF 3 0 0 WGQGTLVTVSS
12A5H1 1 4 5 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 1 IGAMG 19 MYRQAPGK7 GRELVA 22 TITSGGSTNY3 ADPVKG 24 RFTISRDGPKNTVYLQ 9 MNSLRAEDTAVYYCYA 275 NLKQGSYGYRFNDY 301 WGQGTQVTVSS
12A5H2 1 4 6 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 2 IGAMG 19 MYRQAPGK8 GRELVA 22 TITSGGSTNY 4 ADPVKG 25 RFTISRDGAKNTVYLQ 0 MNSLRAEDTAVYYCYA 276 NLKQGSYGYRFNDY 3 0 2 WGQGTQVTVSS
12A5H3 1 4 7 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 3 IGAMG 19 MYRQAPGK9 GRELVA 22 TITSGGSTNY5 ADPVKG 25 RFTISRDNAKNTVYLQ 1| MNSLRAEDTAVYYCYA 2 7 7 NLKQGSYGYRFNDY 3 02 -WGQGTQVTVSS

Thus, in the Nanobodies of the invention, at least one of the CDRl, CDR2 and CDR3 sequences present is suitably chosen 'from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; or from the group of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I. In this context, by "suitably chosen" is meant that, as applicable, a CDRl sequence is chosen from suitable CDRl 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.
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.
Preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I or from the group consisting of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.


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 CDRl and CDR2 sequences present is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table I or from the group of CDRl 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 CDRl and CDR2 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table 1.
Most preferably, in the Nanobodies of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I or from the group of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only I amino acid difference(s) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.
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 CDRl, 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.
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 CDRl and CDR2 sequences present are suitably chosen from the groups of CDRl 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 CDRl and CDR2 sequences, respectively, listed in listed in Table I; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table I.
Even more preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table 1. 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.
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 CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl 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.


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.
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 I 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.
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.
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 CDRl sequences mentioned in Table I; a CDR2 sequence, and one of the CDR3 sequences listed in Table I; or (3) a CDRl 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.
Some particularly preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl 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 CDRl 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).
Some even more preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl 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 [ that belongs to a different combination; or (2) a CDRl 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.
Particularly preferred Nanobodies of the invention may for example comprise a CDRl 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.
In the most preferred in the Nanobodies of the invention, the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the one of the combinations of CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.
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:
i) any amino acid substitution is preferably a conservative amino acid
substitution (as defined herein); and/or
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.
According to a non-limiting but preferred embodiment of the invention, the CDR sequences in the 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 (KD) of 10"5to 10'12 moles/liter (M) or less, and preferably 10"7to 10"12 moles/liter (M) or less and more preferably 10-5 to 10-12 moles/liter (M), and/or with an association constant (KA) of at least 107 M ', preferably at least 10s M-1, more preferably at least 109 M-1, such as at least 1012 M-1;and in particular with a KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The KD and KA 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.
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.
According to a specific, but non-limiting embodiment, the latter amino acid sequences have been "humanized", as further described below.


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.
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).
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.
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:
a) CDR1 comprises or essentially consists of:
the amino acid sequence YNPMG; or
an amino acid sequences that has 2 or only I amino acid difference(s) with the amino acid sequence YNPMG; and
b) CDR2 comprises or essentially consists of:
the amino acid sequence AISRTGGSTYYPDSVEG; or
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; or
an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG;
and
c) CDR3 comprises or essentially consists of:
the amino acid sequence AGVRAEDGRVRTLPSEYTF; or
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; or
an amino acid sequences that has only 1 amino acid difference with the
amino acid sequence AGVRAEDGRVRTLPSEYTF.
In particular, the invention relates to such a Nanobody, in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; or in which:
CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; or in which
CDR3 comprises or essentially consists of the amino acid sequence
AGVRAEDGRVRTLPSEYTF.
For example, the invention relates to such Nanobodies, in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; and
CDR3 comprises or essentially consists of the amino acid sequence
AGVRAEDGRVRTLPSEYTF; or in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; and
CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; or in which:


CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; and CDR3 comprises or essentially consists of the
amino acid sequence AGVRAEDGRVRTLPSEYTF
In one aspect, the invention relates to such a Nanobody, in which CDR1 comprises or essentially consists of the amino acid sequence YNPMG; and CDR3 comprises or essentially consists of the amino acid sequence AGVRAEDGRVRTLPSEYTF.
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).
Anotherone 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:
d) CDR1 is:
the amino acid sequence YNPMG; or
an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence YNPMG; and
e) CDR2 is:
the amino acid sequence AISRTGGSTYYPDSVEG; or 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; or


an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence A1SRTGGSTYYPDSVEG; and 0 CDR3 is:
the amino acid sequence AGVRAEDGRVRTLPSEYTF; or 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; or
an amino acid sequences that has only 1 amino acid difference with the amino acid sequence AGVRAEDGRVRTLPSEYTF. In particular, the invention relates to such a Nanobody, in which: CDR1 is the amino acid sequence YNPMG; or in which:
CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; or in which
CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF. For example, the invention relates to such Nanobodies, in which: CDR1 is the amino acid sequence YNPMG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF; or in which:
CDR1 is the amino acid sequence YNPMG; and CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; or in which:
CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF
In one aspect, the invention relates to such a Nanobody, in which CDR1 is the amino acid sequence YNPMG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF.
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).
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.
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.
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).
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).
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).


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).
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.
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).
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).
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 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.
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 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).
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.
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.
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.
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.
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.
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.
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.
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.
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 of 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.
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.
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.
Other aspects, embodiments, advantages and applications of the invention will become clear from the further description hereinbelow.
Detailed description of the invention
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" (6l 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 VHH domains or VH/VL domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH 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 1: one-letter and three-letter amino acid code

Nonpolar,uncharged(at pH 6,0 -7.0)(3) Alanine 1 Ala A
Valine Val V
Leucine Leu L
Isoleucine lie I
Phenylalanine Phe F
Methionine*1' Met M
Tryptophan Trp W
Proline Pro P
Polar, uncharged (at pH 6,0-7,0) Glycine-1" Gly G
Serine Ser S
Threonine Thr T
Cysteine Cys C
Asparagine Asn N
Glutamine Gin Q
Tyrosine Tyr Y
Polar,charged(at pH 6,0-7.0) Lysine Lys K
Arginine Arg R
Histidine(4; His H
Aspartate Asp D
Glutamate Glu E
Notes:(1) Sometimes also considered to be a polar uncharged amino acid.(2) Sometimes also considered to be a nonpolar uncharged amino acid.(3) As will be clear to the skilled person, the fact that an amino acid residue is referred to inthis Table as being either charged or uncharged at pH 6,0 to 7,0 does not reflect in any wayon the charge said amino acid residue may have at a pH lower than 6,0 and/or at a pH higherthan 7,0; the amino acid residues mentioned in the Table can be either charged and/oruncharged at such a higher or lower pH, as will be clear to the skilled person.(4) As is known in the art, the charge of a His residue is greatly dependant upon evensmall shifts in pH, but a His residu can generally be considered essentially uncharged at a pHof 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.
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.
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.
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.
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.
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.
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 Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin .into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; 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 He or into Leu.


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 VHH 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

FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13)
1. SINGLE DOMAIN VHH ANTIBODIES AGAINST VAN WILLEBRANDFACTOR
2.
(A) Ablynx N.V.
(B) A Company incorporated under the laws of BELGIUM
(C) Technologiepark 4,
B-9052 Zwijnaarde,
Belgium.
The following specification particularly describes the invention and the manner in which it is to be performed.


The present invention relates to improved Nanobodies™ against von Willebrand Factor (vWF), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. [Note: Nanobody™, Nanobodies™ and Nanoclone™ are trademarks of Ablynx N. V]
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.
Other aspects, embodiments, advantages and applications of the invention will become clear from the further description hereinbelow.
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.
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.
The anti-vWF Nanobodies described in WO 04/062551 may be directed against any epitope or conformation of vWF (such as the Al domain or A3 domain), but are preferably directed against the Al domain, and in particular against the activated conformation of the A1 domain.
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.


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..
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
It is a general object of the present invention to provide Nanobodies against vWF, in particular against human vWF.
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.
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.
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:
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); better suitability for formatting in a multivalent format (for example in a bivalent format);
better suitability for formatting in a multispecific format (for example one of the multispecific formats described in WO 04/062551 or hereinbelow); improved suitability or susceptibility for "humanizing" substitutions (as defined herein); and/or
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;
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;
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;
decreased or where desired increased cross-reactivity with vWF from different species; and/or


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).
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 Al domain of vWF, either in its activated or non-activated confirmation.
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: i) CDR1 comprises or essentially consists of an amino acid sequence chosen from the group consisting of:





YNPMG [SEQ ID NO: 22]
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
ii) CDR2 comprises or essentially consist of an amino acid sequence chosen from the group consisting of:
SISWSGTYTAYSDNVKG [SEQ ID NO: 23]
GISWSGVSTDYAEFAKG [SEQ ID NO: 24]
TSISWSGSYTAYADNVKG [SEQ ID NO: 25]
SISWSGMSTYYTDSVKG [SEQ ID NO: 26]
TITSGGRTSYADSVKG [SEQ ID NO: 27]
AISWSGGLTYYADSVKG [SEQ ID NO: 28]
TITSGGSTNYADPVKG [SEQ ID NO: 29]
TITSGGSTNYADSVKG [SEQ ID NO: 30]
AISRTGGSTYYARSVEG ' [SEQ ID NO: 31]
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]


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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
ni) CDR3 comprises or essentially consists of an amino acid sequence chosen from the

group consisting of:
QSRYRSNYYDHDDKYAY [SEQ ID NO: 33]
LGRYRSNWRNIGQYDY [SEQ ID NO: 34]
QSRYSSNYYDHDDKYAY [SEQ ID NO: 35]
SNRYRTHTTQAMYNY [SEQ ID NO: 36]
WDGKRAP [SEQ ID NO: 37]
NRRQKTVQMGERAYDY [SEQ ID NO: 38]
NLKQGSYGYRFNDY [SEQ ID NO: 39]
NLKQGDYGYRFNDY [SEQ ID NO: 40]
AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]


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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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).
In another aspect, the invention relates to a Nanobody (as defined herein), against
vWF, which consist of 4 framework regions (FRl to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in which:
i) CDR1 is an amino acid sequence chosen from the group consisting of:

NYGMG [SEQ ID NO: 15]
SYTLG [SEQ ID NO: 16]
NYNMG [SEQ ID NO: 17]
SSAMA [SEQ ID NO: 18]
YYNTG [SEQ ID NO: 19]
IGAMG [SEQ ID NO: 20]
IGTMG [SEQ ID NO: 21J
YNPMG [SEQ ID NO: 22]
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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);
in which:
CDR2 is an amino acid sequence chosen from the group consisting of:
SISWSGTYTAYSDNVKG [SEQ ID NO: 23]
GISWSGVSTDYAEFAKG [SEQ ID NO: 24]
TSISWSGSYTAYADNVKG [SEQ ID NO: 25]
SISWSGMSTYYTDSVKG [SEQ ID NO: 26]
TITSGGRTSYADSVKG [SEQ ID NO: 27]
AISWSGGLTYYADSVKG [SEQ ID NO: 28]
TITSGGSTNYADPVKG [SEQ ID NO: 29]
TITSGGSTNVADSVKG [SEQ ID NO: 30]
AISRTGGSTYYARSVEG [SEQ ID NO: 31 ]
AISRTGGSTYYPDSVEG [SEQ ID NO: 32]
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%o sequence identity (as defined herein) with one of the "above amino acid sequences; in which


(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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);
which:
CDR3 is an amino acid sequence chosen from the group consisting of:
QSRYRSNYYDHDDKYAY [SEQ ID NO: 33]
LGRYRSNWRNIGQYDY [SEQ ID NO: 34]
QSRYSSNYYDHDDKYAY [SEQ ID NO: 35]
SNRYRTHTTQAMYNY [SEQ ID NO: 36]
VVDGKRAP [SEQ ID NO: 3 7]
NRRQKTVQMGERAYDY [SEQ ID NO: 38]
NLKQGSYGYRFNDY [SEQ ID NO: 39]
NLKQGDYGYRFNDY [SEQ ID NO: 40]
AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41 ]
AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]
AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]
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


(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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 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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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).
The Nanobodies against vWF as described above and as further described hereinbelow are also referred to herein as Nanobodies of the invention.
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.
In another aspect, the invention relates to a Nanobody against vWF, which consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), which is chosen from the group consisting of Nanobodies with the one of the following combinations of CDRl, CDR2 and CDR3, respectively:
CDRl :NYGMG;CDR2: SIS WSGTYTAYSDNVKG;
CDR3:QSRYRSNYYDHDDKYAY
CDR1:SYTLG;CDR2:G1SWSGVSTDYAEFAKG;
CDR3:LGRYRSNWRNIGQYDY
CDRl :NYGMG;CDR2:TSIS WSGSYTAYADNVKG;
CDR3:QSRYSSNYYDHDDKYAY
CDRl :NYNMG;CDR2:SIS WSGMSTYYTDSVKG;


CDR3:SNRYRTHTTQAMYNY
CDR1 :SSAMA;CDR2:TITSGGRTSYADSVKG;CDR3: VVDGKRAP
CDR1:YYNTG;CDR2:AISWSGGLTYYADSVKG;
CDR3:NRRQKTVQMGERAYDY
CDR1:IGAMG;CDR2:TITSGGSTNYADPVKG;CDR3:NLKQGSYGYRFNDY
CDR1:IGAMG;CDR2:T1TSGGSTNYADSVKG;CDR3:NLKQGSYGYRFNDY
CDR1:IGAMG;CDR2:TITSGGSTNYADSVKG;
CDR3.-NLKQGDYGYRFNDY
CDR1:IGTMG;CDR2:TITSGGSTNYADSVKG;CDR3:NLKQGDYGYRFNDY
CDR1:YNPMG;CDR2:AISRTGGSTYYARSVEG;
CDR3.-AGVRAEDGRVRTLPSEYNF
CDR1:YNPMG;CDR2:AISRTGGSTYYPDSVEG;
CDR3:AGVRAEDGRVRTLPSEYTF
CDR1:YNPMG;CDR2:AISRTGGSTYYPDSVEG;
CDR3:AGVRAEDGRVRSLPSEYTF
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
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or
(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:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or


(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).
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 I: Preferred combinations of CDR's, of CDR's and framework sequences, and of CDR's and humanized FR's

1 iCLONE D FR1 C CDR1 I I FR2 D CDR1 D FR3 I CDR3 D FR4
1 12A5 2 AVQLVESGGG 1> LVQPGGSLRL ASCLASGRIFS E 1 27 MYRQAPGK C IGAMG 4 QRELVA C 2 TITSGGSTNY 2 ADPVKG E 2 RFTISRDGPKNTVYLQ J MNSLKPEDTAVYYCYA 2 2 ) 7 j NLKQGSYGYRFNDY 8 WGQGTQVTVSS
1 QVQLVESGGG 112 2 2 22 LVQAGGSLRL 4 7 WFRQAPGK 0 SISWSGTYT 2 RFTISRDNAKNTVYLQ 5 QSRYRSNYYDHDDKY 712B1 3 SCAASGRTFS 9 NYGMG 5 EREFVT 1 AYSDNVKG 7 MDSLKPEDTAVYYCAA 3 AY 9 WGQGTQVTVSS
I ■ _II 12B6 1 QVQLVESGGG2 LVQAGGALRL '4 SCAASGRTFS ) YNPMG 17 WFRQAPGK (5 ERDVVA j i :3 AISRTGGST , I YYARSVEG 22 RFTISRDNAKRMVYLQ3 MNALKPEDTAVYYCAA 25 AGVRAEDGRVRTLPS4 EYNF 2 33 WGQGTQVTVSS
i ji| 12D11 1 AVQLVDSGGG2 LVQAGGSLRL5 SCTASERTTF 151 SSYTLG 17 WFRQAPGK7 EREFVG 20 GISWSGVST3 DYAEFAKG 22 RFTISRDHAANTVYLEM9 NSLKPEDTAVYYCAA 25 LGRYRSNWRNIGQYD5 Y 28 I 1 WGQGTQVTVSS
! 12-E3 1 EVQLVESGGG2 LVQAGGSLRL6 SCAASGRTFN 1 5 2 NYGMG 17 WFRQAPGK8 EREFVT 20 SISWSGSYT4 AYADNVKG 23 RFTISRDNAKNTVYLQ0 MDSLKPGDTAVYYCAA 2,5 QSRYSSNYYDHDDKY6 AY 282 WGQGTQVTVSS
12C9 1 AVQLVESGGG2 LVQPGGSLKL7 SCATSGSiFS 1 5 3 SSAMA 17 WYRQASGK9 QRELVA 20 TITSGGRTSY5 ADSVKG 23 RFTISRDNAKNTVYLQ1 MNSLKPEDTAVYDCNF 2 5 7 VVDGKRAP 2 8 3 WGQGTQVTVSS
14F8 1 AVQLVESGGG2 LVQAGESLRLB SCTSSGRAFS 1 5 4 YYNTG 18 WFRQAPGK0 EREFVA 20 AISWSGGLT6 YYADSVKG 23 RFTISRDNAKDMVYLQ2 MASLKPEDTAVYYCAA 25 NRRQKTVQMGERAYD8 Y 2 8 4 WGQGTQVTVSS
12B4 1 QVQLVESGGG2 LVQPGGSLRL9 SCLASGRIFS 1 5 5 IGAMG 18 LYRQAPGK1 QRELVA 20 TITSGGSTNY7 ADSVKG 23 RFTISRDGPKNTVYLQ3 MNSLKPEDTAVYYCYA 2 5 9 NLKQGSYGYRFNDY 2 85 WGQGTQVTVSS
12-E8 1 AVQLEESGGG 3 LVQPGGSLRL 0 SCLASGRIFS 1 5 6 IGAMG 18 LYRQAPGK2 QRELVA 20 TITSGGSTNY8 ADSVKG 23 RFTISRDGAKNTVYLQ4 MNSLKPEDTAVYYCYA 2 6 0 NLKQGDYGYRFN DY 2 8 6 WGQGTQVTVSS

TABLE I (continued):

1CLONE C I FR1 D CDR1 I FR2 C I ) CDR1 D FR3 I CDR3 D FR4
1312A6 1 QVQLVESGGG 1 LVQPGGSLRL 5 SCLASGRIFS 7 1 IGTMG C 2 LYRQAPGK C QRELVA 9 2TITSGGSTNY 3 ADSVKG E 2 RFTISRDGAKNTVYLQ 6 MNSLRPEDTAVYYCYA 1 2fi NLKQGDYGYRFN DY 7 WGQGTQVTVSS
1! 2 j 12D8 2 AVQLVESGGG 1 LVQPGGSLRL J SCLASGRIFS i 12 2 2 2 > 8 LYRQAPGK 1 TITSGGSTNY 3 RFTISRDGAKNTVYLQ 6 8 I IGTMG 4 QRELVA 0 ADSVKG 6 MNSLRPEDTAVYYCYA 2 NLKQGDYGYRFNDY j 8 WGQGTQVTVSS
1 12A2 j C QVKLEESGGG 112 2 2 2 LVQAGGALRL 5 8 WFRQAPGK 1 AISRTGGST 3 RFTISRDNAKRMVYLQ 6 AGVRAEDGRVRTLPS 8 1 SCAASGRTFS 9 YNPMG 5 ERDLVA 1| YYPDSVEG 7 MNNLKPEDTAVYYCAA 3 EYTF 9 WGQGTQVTVSS
12F2 QVKLVESGGG3 LVQAGGALRL4 SCAASGRTFS \ 53 YNPMG 13 WFRQAPGR6 ERDVVA 21 AISRTGGST2 YYPDSVEG 23 RFTISRDNAKRMVYLQ3 MNNLKPEDTAVYYCAA 13 AGVRAEDGRVRSLPS4 EYTF 2 9 0 WGQGTQVTVSS
I i J 14H10 1 QVKLEESGGG 3 LVQAGGALRL 5 SCAASGRTFS 1 6 1 YNPMG 18 WFRQAPGK7 ERDVVA 21 AISRTGGST 3 YYPDSVEG j 23 RFTISRDNAKRMVYLE9 MNNLKPDDTAVYYCAA 26 AGVRAEDGRVRTLPS5 EYTF 2 9 1 WGQGTQVTVSS
12B6H1 1 EVQLVESGGG 3 LVQPGGSLRL 6 SCAASGRTFS 1 6 2 YNPMG 18 WFRQAPGK8 GRDVVA 21 AISRTGGST 41 YYARSVEG 24 RFTISRDNAKRMVYLQ0| MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS 6 EYNF 2 9 2 WGQGTQVTVSS
12B6H2 1 EVQLVESGGG 3 LVQPGGSLRL 7 SCAASGRTFS 1 6 3 YNPMG 18 WFRQAPGK9 GREVVA 21 AISRTGGST5 YYARSVEG 24 RFTISRDNAKRMVYLQ1 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS7 EYNF 2 9 3 WGQGTQVTVSS
-i2B6H3 1 EVQLVESGGG 3 LVQPGGSLRL 8 SCAASGRTFS 1 6 4 YNPMG 19 WFRQAPGK0 GRDVVA 21 AISRTGGST 6 YYARSVEG 24 RFTISRDNAKNMVYLQ 2 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS8 EYNF 2 94 WGQGTQVTVSS
12B6H4 1 EVQLVSsGGG 3 LVQPGGSLRL 9 SCAASGRTFS 1 6 5 YNPMG 19 WFRQAPGK1 GRDVVA 21 AISRTGGST7 YYARSVEG 24 RFTISRDNAKRSVYLQ3 MNSLRAEDTAVYYCAA 26 AGVRAEDGRVRTLPS9 EYNF 2 95 WGQGTQVTVSS
12A2H1 1 EVQLVESGGG ' 4 LVQPGGSLRL 0 SCAASGRTFS | 1 6 6 YNPMG 19 WFRQAPGK2 GRELVA 21 AISRTGGST 8 YYPDSVEG 24 RFTISRDNAKRMVYLQ4 MNSLRAEDTAVYYCAA 27 AGVRAEDGRVRTLPS0 EYTF 2 96| WGQGTQVTVSS

" ■ ■ [CLONE 1 ! D IFR1 D CDR1 FR2 D CDR1 D FR3 | CDR3 D I FR4 |
12A2H3 1 4 1 EVQLVESGGG 11 2 2 Il LVQPGGSLRL 6 9 WFRQAPGK 1 AISRTGGST 4 RFTISRDNAKNMVYLQ 7 SCAASGRTFS 7 YNPMG 3 GRELVA 9 YYPDSVEG 5 MNSLRAEDTAVYYCAA ' AGVRAEDGRVRTLPS EYTF 2 97 !WGQGTQVTVSS
12A2H4 1 4 2 EVQLVESGGG LVQPGGSLRL SCAASGRTFS 1 3 8 YNPMG 19 WFRQAPGK4 GRELVA >2 AISRTGGST3 YYPDSVEG I 24 RFTISRDNAKRSVYLQ 7 AGVRAEDGRVRTLPS6 MNSLRAEDTAVYYCAA 2 EYTF 2 9 8 WGQGTQVTVSS
12A2H11 1 4 3 EVQLVESGGG LVQPGGSLRL SCAASGFTFS 1 5 9 YNPMG 19 WFRQAPGK5 GRELVA 22 AISRTGGST1 YYPDSVEG 24 RFTISRDNAKRMVYLQ7 MNSLRAEDTAVYYCAA I7 AGVRAEDGRVRTLPS3 EYTF 29 9 WGQGTQVTVSS
!I 12A2H13 1 4 4 EVQLVESGGG LVQPGGSLRL SCAASGFTFS 1 7 0 YNPMG 19 WFRQAPGK6 GRELVA 22 AISRTGGST2 YYPDSVEG 24 RFTISRDNAKNSVYLQ8 MNSLRAEDTAVYYCAA 27 AGVRAEDGRVRTLPS4 EYTF 3 0 0 WGQGTLVTVSS
12A5H1 1 4 5 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 1 IGAMG 19 MYRQAPGK7 GRELVA 22 TITSGGSTNY3 ADPVKG 24 RFTISRDGPKNTVYLQ 9 MNSLRAEDTAVYYCYA 275 NLKQGSYGYRFNDY 301 WGQGTQVTVSS
12A5H2 1 4 6 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 2 IGAMG 19 MYRQAPGK8 GRELVA 22 TITSGGSTNY 4 ADPVKG 25 RFTISRDGAKNTVYLQ 0 MNSLRAEDTAVYYCYA 276 NLKQGSYGYRFNDY 3 0 2 WGQGTQVTVSS
12A5H3 1 4 7 EVQLVESGGG LVQPGGSLRL SCAASGRIFS 1 7 3 IGAMG 19 MYRQAPGK9 GRELVA 22 TITSGGSTNY5 ADPVKG 25 RFTISRDNAKNTVYLQ 1| MNSLRAEDTAVYYCYA 2 7 7 NLKQGSYGYRFNDY 3 02 -WGQGTQVTVSS

Thus, in the Nanobodies of the invention, at least one of the CDRl, CDR2 and CDR3 sequences present is suitably chosen 'from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; or from the group of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I. In this context, by "suitably chosen" is meant that, as applicable, a CDRl sequence is chosen from suitable CDRl 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.
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.
Preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I or from the group consisting of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 "amino acid difference(s)" with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.


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 CDRl and CDR2 sequences present is suitably chosen from the group consisting of the CDRl and CDR2 sequences, respectively, listed in Table I or from the group of CDRl 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 CDRl and CDR2 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table 1.
Most preferably, in the Nanobodies of the invention, all three CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I or from the group of CDRl, 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 CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I; and/or from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only I amino acid difference(s) with at least one of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.
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 CDRl, 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.
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 CDRl and CDR2 sequences present are suitably chosen from the groups of CDRl 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 CDRl and CDR2 sequences, respectively, listed in listed in Table I; and/or from the group consisting of the CDRl and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDRl and CDR2 sequences, respectively, listed in Table I.
Even more preferably, in the Nanobodies of the invention, at least two of the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDRl, CDR2 and CDR3 sequences, respectively, listed in Table 1. 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.
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 CDRl sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDRl 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.


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.
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 I 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.
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.
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 CDRl sequences mentioned in Table I; a CDR2 sequence, and one of the CDR3 sequences listed in Table I; or (3) a CDRl 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.
Some particularly preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl 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 CDRl 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).
Some even more preferred Nanobodies of the invention may for example comprise: (1) a CDRl sequence that has more than 80 % sequence identity with one of the CDRl 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 [ that belongs to a different combination; or (2) a CDRl 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.
Particularly preferred Nanobodies of the invention may for example comprise a CDRl 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.
In the most preferred in the Nanobodies of the invention, the CDRl, CDR2 and CDR3 sequences present are suitably chosen from the one of the combinations of CDRl, CDR2 and CDR3 sequences, respectively, listed in Table I.
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:
i) any amino acid substitution is preferably a conservative amino acid
substitution (as defined herein); and/or
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.
According to a non-limiting but preferred embodiment of the invention, the CDR sequences in the 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 (KD) of 10"5to 10'12 moles/liter (M) or less, and preferably 10"7to 10"12 moles/liter (M) or less and more preferably 10-5 to 10-12 moles/liter (M), and/or with an association constant (KA) of at least 107 M ', preferably at least 10s M-1, more preferably at least 109 M-1, such as at least 1012 M-1;and in particular with a KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The KD and KA 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.
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.
According to a specific, but non-limiting embodiment, the latter amino acid sequences have been "humanized", as further described below.


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.
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).
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.
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:
a) CDR1 comprises or essentially consists of:
the amino acid sequence YNPMG; or
an amino acid sequences that has 2 or only I amino acid difference(s) with the amino acid sequence YNPMG; and
b) CDR2 comprises or essentially consists of:
the amino acid sequence AISRTGGSTYYPDSVEG; or
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; or
an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG;
and
c) CDR3 comprises or essentially consists of:
the amino acid sequence AGVRAEDGRVRTLPSEYTF; or
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; or
an amino acid sequences that has only 1 amino acid difference with the
amino acid sequence AGVRAEDGRVRTLPSEYTF.
In particular, the invention relates to such a Nanobody, in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; or in which:
CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; or in which
CDR3 comprises or essentially consists of the amino acid sequence
AGVRAEDGRVRTLPSEYTF.
For example, the invention relates to such Nanobodies, in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; and
CDR3 comprises or essentially consists of the amino acid sequence
AGVRAEDGRVRTLPSEYTF; or in which:
CDRl comprises or essentially consists of the amino acid sequence YNPMG; and
CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; or in which:


CDR2 comprises or essentially consists of the amino acid sequence
AISRTGGSTYYPDSVEG; and CDR3 comprises or essentially consists of the
amino acid sequence AGVRAEDGRVRTLPSEYTF
In one aspect, the invention relates to such a Nanobody, in which CDR1 comprises or essentially consists of the amino acid sequence YNPMG; and CDR3 comprises or essentially consists of the amino acid sequence AGVRAEDGRVRTLPSEYTF.
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).
Anotherone 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:
d) CDR1 is:
the amino acid sequence YNPMG; or
an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence YNPMG; and
e) CDR2 is:
the amino acid sequence AISRTGGSTYYPDSVEG; or 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; or


an amino acid sequences that has 2 or only 1 amino acid difference(s) with the amino acid sequence A1SRTGGSTYYPDSVEG; and 0 CDR3 is:
the amino acid sequence AGVRAEDGRVRTLPSEYTF; or 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; or
an amino acid sequences that has only 1 amino acid difference with the amino acid sequence AGVRAEDGRVRTLPSEYTF. In particular, the invention relates to such a Nanobody, in which: CDR1 is the amino acid sequence YNPMG; or in which:
CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; or in which
CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF. For example, the invention relates to such Nanobodies, in which: CDR1 is the amino acid sequence YNPMG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF; or in which:
CDR1 is the amino acid sequence YNPMG; and CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; or in which:
CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF
In one aspect, the invention relates to such a Nanobody, in which CDR1 is the amino acid sequence YNPMG; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF.
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).
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.
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.
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).
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).
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).


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).
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.
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).
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).
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 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.
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 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).
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.
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.
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.
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.
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.
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.
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.
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.
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 of 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.
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.
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.
Other aspects, embodiments, advantages and applications of the invention will become clear from the further description hereinbelow.
Detailed description of the invention
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" (6l 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 VHH domains or VH/VL domains, respectively). In addition, the term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH 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 1: one-letter and three-letter amino acid code

Nonpolar,uncharged(at pH 6,0 -7.0)(3) Alanine 1 Ala A
Valine Val V
Leucine Leu L
Isoleucine lie I
Phenylalanine Phe F
Methionine*1' Met M
Tryptophan Trp W
Proline Pro P
Polar, uncharged (at pH 6,0-7,0) Glycine-1" Gly G
Serine Ser S
Threonine Thr T
Cysteine Cys C
Asparagine Asn N
Glutamine Gin Q
Tyrosine Tyr Y
Polar,charged(at pH 6,0-7.0) Lysine Lys K
Arginine Arg R
Histidine(4; His H
Aspartate Asp D
Glutamate Glu E
Notes:(1) Sometimes also considered to be a polar uncharged amino acid.(2) Sometimes also considered to be a nonpolar uncharged amino acid.(3) As will be clear to the skilled person, the fact that an amino acid residue is referred to inthis Table as being either charged or uncharged at pH 6,0 to 7,0 does not reflect in any wayon the charge said amino acid residue may have at a pH lower than 6,0 and/or at a pH higherthan 7,0; the amino acid residues mentioned in the Table can be either charged and/oruncharged at such a higher or lower pH, as will be clear to the skilled person.(4) As is known in the art, the charge of a His residue is greatly dependant upon evensmall shifts in pH, but a His residu can generally be considered essentially uncharged at a pHof 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.
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.
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.
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.
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.
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.
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 Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin .into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; 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 He or into Leu.


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 VHH 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

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Patent Number 255610
Indian Patent Application Number 1914/MUMNP/2007
PG Journal Number 11/2013
Publication Date 15-Mar-2013
Grant Date 08-Mar-2013
Date of Filing 15-Nov-2007
Name of Patentee ABLYNX N. V.
Applicant Address TECHNOLOGIEPARK 4, B-9052 ZWIJNAARDE,
Inventors:
# Inventor's Name Inventor's Address
1 SILENCE KAREN JEAN-TOMBEURSTRAAT 21 B-3090 OVERIJSE
PCT International Classification Number C07K16/36 A61K38/36
PCT International Application Number PCT/EP2006/004773
PCT International Filing date 2006-05-19
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/683,474 2005-05-20 U.S.A.