IL169068A - Polypeptide or polypeptide construct comprising at least one single domain antibody directed against von willebrand factor, a composition comprising it, a nucleic acid encoding it and its uses in the preparation of a medicament for prevention and/or alleviation of conditions of platelet mediated aggregation - Google Patents

Description

169068/2 n ow >- naiMittn iimwsii o wpii A polypeptide or polypeptide construct comprising at least one single domain antibody directed against Von WiUebrand factor, a composition comprising it, a nucleic acid encoding it and uses of it in the preparation of a medicament for prevention and/or alleviation of conditions of platelate-mediated aggregation ABLYNX N.V.

C. 160608 1 169068/2 BACKGROUND TO THE INVENTION Upon damage to a blood vessel, subendothelial structures are exposed that mediate platelet adhesion through interaction with von Willebrand factor (vWF). vWF forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein lb (gplb), an interaction especially important under high shear conditions, leading to the formation of a haemostatic plug and thus preventing excessive bleeding (Bennett S, Thromb Haemost (2001) Mar;85(3):395-400). During normal haemostasis, these processes lead to wound healing of the damaged blood vessel wall. In pathological conditions however, excessive platelet function may lead to thrombus formation. The vWF subunit is composed of several homologues domains each covering different functions. vWF interacts through its A3 domain with fibrillar collagen fibers and through its A1 domain with the platelet receptor gplb. Under normal conditions platelets and vWF do not interact. However, when vWF is bound to collagen at high shear rate, it is believed to undergo a conformational change allowing its binding with the platelet receptor gplb. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion through the collagen receptors on the platelets (gpla/lla, gpVI, gpIV, p65, TIIICBP) resulting in platelet activation. This leads to activation of the gpllb/llla receptor, fibrinogen binding, and finally to platelet aggregation.

Platelet aggregation inhibitors have been isolated from blood sucking organisms such as leech. Saratin, derived from leech Hirudo medicinalis is described in WO 02/15919 A2 and in Cruz CP et al ref. Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model. Journal of Vascular Surgery, 2001 , 34: 724-729 and in Smith TP et al, Saratin, an inhibitor of collagen-platelet interaction, decreases venous anastomotic intimal hyperplasia in a canine dialysis access model, Vase Endovascular Surg. 2003 Jul-Aug;37(4):259-69.

Antibody-based therapeutics have been developed, some of which are currently used in therapy.

Abciximab (Chimeric 7E3 Fab; ReoPro; US 6,071 ,514, EP 0 882 453), the Fab fragment of the mouse human chimeric antibody 7E3 which inhibits ligand binding to the platelet gpllb/llla receptor, was approved for human use as adjunctive therapy to prevent ischemic complications of percutaneous coronary interventions in December 1994. The principle safety issue with gp llb/llla inhibitors is the risk of bleeding, as the potent anti-platelet effect of these drugs may adversely affect haemostasis.

A murine monoclonal antibody was developed against vWF A1 domain (US 2002/0028204 A1 ; US 6,280,731 and in WO 00/10601) and against its active conformation (US 6,251 ,393). The in vivo efficacy is described in Kageyama S, et al :"Effect of a humanized monoclonal antibody to von Willebrand factor in a canine model of coronary arterial thrombosis", Eur J Pharmacol. 2002 May 17;443(1-3):143-9, and in "Anti-human vWF monoclonal antibody, AJvW-2 Fab, inhibits repetitive coronary artery thrombosis without bleeding time prolongation in dogs". Thromb Res., 2001 Mar 1 ;101(5):395-404. and in "Anti-human von willebrand factor monoclonal antibody AJvW-2 prevents thrombus deposition and neointima formation after balloon injury in guinea pigs". Arterioscler Thromb Vase Biol. 2000 Oct;20(10):2303-8). AJvW-2 inhibited high shear stress induced aggregation of human platelets and had no effect on low shear stress induced platelet aggregation.

The effects in baboons of a murine antibody 82D6A3 raised against the A3 domain of human vWF, are disclosed in WO 02/051351 , and Dongmei Wu et al, "Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons". Hemostasis, thrombosis and vascular biology, 2002, 99: 3623-3628.

Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human gplb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. When injected into baboons, intact IgG and its F(ab')(2) fragments caused almost immediate thrombocytopenia, due to the bivalency of F(ab')(2) which mediates platelet crosslinking, or Fc:Fc receptor interactions which mediate activation of platelet aggregation (WO 0 091 ; Cauwenberghs N. et al, Arteriosclerosis, Thrombosis and Vascular biology, 2000, 20: 1347 and see, for example, Cadroy Y et al, Blood, 994, 83: 3218-3224, Becker BH 3 169068/2 et al, Blood, 1989, 74: 690-694, Ravanat C. et al, Thromb. Haemost. 1999 , 82 : 528a abstract). Platelet deposition onto collagen-rich bovine pericardium was inhibited when Fab fragments were injected into the baboons before a thrombus was generated. However, when the Fab fragments were injected after a thrombus was allowed to form, no inhibition of further thrombosis was observed. The yields of expression of said Fab molecules are very low and the method of production is very labour intensive.

US Patent Application US 2002/028204 by Nagano et al. describes an antithrombotic agent and anti-Von Willebrand Factor monoclonal antibody.

US Patent US 6,251 ,393 by Handin er a/., describes conformation specific anti-Von Willebrand Factor antibodies.

Arbabi Ghahroudi er al. (1997 FEBS Letters 414:521-526) describes the selection and identification of single domain antibody fragments from camel-heavy chain antibodies.

Muyldermans (2001 Reviews in Molecular Biotechnology 74-277-302) is a review article describing the contemporaneous status of single domain camel antibodies in 2001.

THE AIMS OF THE PRESENT INVENTION An aim of the present invention is to provide polypeptides comprising one or more single domain antibodies directed towards vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb and/or collagen, homologues of said polypeptides, and/or functional portions of said polypeptides, for the treatment for conditions which require a modulation of platelet-mediated aggregation and which overcomes the problems of the prior art. It is a further aim to provide methods of production of said polypeptides, methods to coat devices with such polypeptides used in medical procedures (e.g. PCTA, stenting), methods and kits for screening for agents that modulate platelet-mediated aggregation and kits for the diagnosis of diseases related to platelet-mediated aggregation SUMMARY OF THE INVENTION In one of its aspects the present invention provides a polypeptide or polypeptide construct comprising at least one single domain antibody directed against von Willebrand Factor (vWF), wherein the at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 3, 5 or 7, or to an homologous sequence of any of SEQ ID NOs: 3,5 or 7 with a sequence identity of more than 70% with the parent sequence and wherein said single domain antibody is able to inhibit at least 50% of platelet aggregation at high shear (1600 s"1) at a concentration of 1 pg/ml or lower. - 3a - 169068/1 In another of its aspects, the present invention provides a composition comprising a polypeptide or polypeptide construct according to the invention and a pharmaceutically acceptable vehicle.

Yet in another of its aspects, the present invention provides a composition comprising a polypeptide or polypeptide construct according to the invention adapted to the chosen route of administration, wherein the route of administration is orally or parenterally, by intra-nasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes; and a pharmaceutically acceptable vehicle.

In a further of its aspects the present invention provides a nucleic acid encoding a polypeptide or polypeptide construct according to the invention.

In another of its aspects the present invention provides a use of a polypeptide or polypeptide construct according to the invention, for preparation of a medicament for the prevention, treatment and/or alleviation of conditions of platelet-mediated aggregation wherein the conditions are any of the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, restenosis, restenosis after PCTA or stenting, thrombus formation in stenosed arteries, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries.

DETAILED DESCRIPTION OF THE INVENTION Single domain antibodies have been made which specifically recognize target molecules involved in the first and subsequent steps of platelet aggregation. This results in antithrombotic agents which are more efficacious and safer.

One embodiment of the present invention is a polypeptide construct comprising: - at least one single domain antibody directed against any of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb, or collagen.

Another embodiment of the present invention is a polypeptide construct as described above, wherein the single domain antibody directed against the A1 domain of activated vWF specifically recognizes the activated vWF conformation at the site of thrombus formation but does not bind to circulating unactivated forms of vWF. 4 Another embodiment of the present invention is a polypeptide construct as described above, further comprising at least one single domain antibody directed against one or more serum proteins.

Another embodiment of the present invention is a polypeptide construct as described above wherein said at least one serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.

Another embodiment of the present invention is a polypeptide construct as described above, wherein at least one single domain antibody directed against one or more serum proteins corresponds to a sequence represented by any of SEQ ID NO: 16 to 19 and 49 to 61.

Another embodiment of the present invention is a polypeptide construct as described above corresponding to a sequence represented by any of SEQ ID NOs: 13 to 15 and 42 to 45.

Another embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a humanised sequence.

Another embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 38 to 41 and 42 to 45 Another embodiment of the present invention is a polypeptide construct as described above corresponding to a sequence represented by any of SEQ ID NOs: 8 to 12, 20 to 22, 32 to 34, and 42 to 47.

Another embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a Camelidae VHH antibody.

Another embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 1 to 7, 23 to 31 , 35 to 37 and 62 to 65.

Another embodiment of the present invention is a polypeptide construct as described above, wherein said single domain antibody is an homologous sequence, a functional portion, or a functional portion of an homologous sequence of the full length single domain antibody.

Another embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion thereof, of an homologous sequence of a functional portion thereof.

Another embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

Another embodiment of the present invention is a composition comprising a polypeptide construct as described above and at least one thrombolytic agent, for simultaneous, separate or sequential administration to a subject.

Another embodiment of the present invention is a composition as described above wherein said thrombolytic agent is any of staphylokinase, tissue plasminogen activator, streptokinase, single chain streptokinase, urokinase and acyl plasminogen streptokinase complex.

Another embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above, or a composition as described above for use in the treatment, prevention and/or alleviation of disorders relating to platelet-mediate aggregation or dysfunction thereof.

Another embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above, or a composition as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to platelet-mediate aggregation or dysfunction thereof.

Another embodiment of the present invention is a polypeptide construct, nucleic acid or composition as described above or a use of a polypeptide construct, nucleic acid or composition as described above wherein said disorders are any arising from transient cerebral ischemic attack, unstable or stable angina, angina pectoris, cerebral infarction, 6 myocardial infarction, peripheral arterial occlusive disease, restenosis, coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, carotid endarterectomy or atherectomy.

Another embodiment of the present invention is a polypeptide construct, nucleic acid or composition as described above or a use of a polypeptide construct, nucleic acid or composition as described above wherein said disorders are any of the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, restenosis, restenosis after PCTA or stenting, thrombus formation in stenosed arteries, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries.

Another embodiment of the present invention is a polypeptide construct, nucleic acid or composition as described above or a use of a polypeptide construct, nucleic acid or composition as described above wherein said disorder is 'plaque or thrombus formation in high sheer environments.

Another embodiment of the present invention is a polypeptide construct, nucleic acid or composition as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, subcutaneously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

Another embodiment of the present invention is a composition comprising a polypeptide construct as described above or a nucleic acid encoding said polypeptide construct, or a composition as described above and a pharmaceutically acceptable vehicle.

Another embodiment of the present invention is a method of producing a polypeptide as described above, comprising (a) culturing host cells comprising nucleic acid capable of encoding a polypeptide as described above under conditions allowing the expression of the polypeptide, and, (b) recovering the produced polypeptide from the culture. 7 Another embodiment of the present invention is a method as described above, wherein said host cells are bacterial or yeast.

Another embodiment of the present invention is a method for treating invasive medical devices to prevent platelet-mediate aggregation around the site of invasion comprising the step of coating said device with a polypeptide construct as described above.

Another embodiment of the present invention is an invasive medical device for circumventing platelet-mediate aggregation around the site of invasion, wherein said device is coated with a polypeptide construct as described above.

Another embodiment of the present invention is a method of identifying an agent that modulates platelet-mediated aggregation comprising (a) contacting a polypeptide construct as described above with a polypeptide corresponding to Its target, or a fragment thereof, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptides, and (b) measuring the binding between the polypeptides of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identified said candidate modulator as an agent that modulate platelet-mediated aggregation.

Another embodiment of the present invention is a kit for screening for agents that modulate platelet-mediated aggregation according to the method as described above.

Another embodiment of the present invention is an unknown agent that modulates platelet-mediated aggregation identified according to the method as described above.

Another embodiment of the present invention is a method of diagnosing a disease or disorder characterised by dysfunction of platelet-mediated aggregation comprising the steps of: (a) contacting a sample with a polypeptide construct as described above, and (b) detecting binding of said polypeptide construct to said sample, and 8 169068/2 (c) comparing the binding detected in step (b) with a standard, wherein a difference in binding relative to said sample is diagnostic of a disease or disorder characterised by dysfunction of platelet-mediated aggregation.

Another embodiment of the present invention is a kit for screening for diagnosing a disease or disorder characterised by dysfunction of platelet-mediated aggregation according to the method as described above.

Another embodiment of the present invention is a kit as described above comprising a polypeptide construct as described above.

The present invention relates to a polypeptide construct comprising one or more single domain antibodies each directed against a target and the finding that the construct has a modulating effect on platelet-mediated aggregation.

Targets According to the invention, a target is any of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb or collagen. Said targets are mammalian, and are derived from species such as rabbits, goats, mice, rats, cows, calves, camels, llamas, monkeys, donkeys, guinea pigs, chickens, sheep, dogs, cats, horses, and preferably humans. The sequence of human vWF is provided in Table 30, SEQ ID NO: 48.

A target is also a fragment of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb or collagen, capable of eliciting an immune response. A target is also a fragment of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb or collagen, capable of binding to a single domain antibody raised against the 'parent' full length target.

A fragment as used herein refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 3, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more amino acids. A fragment is of sufficient length such that the interaction of interest is maintained with affinity of 1 x 10"6 M or better. 9 A fragment as used herein also refers to optional insertions, deletions and substitutions of one or more amino acids which do not substantially alter the ability of the target to bind to a single domain antibody raised against the wild-type target. The number of amino acid insertions deletions or substitutions is preferably up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

A single domain antibody directed against a target means single domain antibody that it is capable of binding to its target with an affinity of better than 10"6 M.

Single domain antibodies Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678 for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

VHHs, according to the present invention, and as known to the skilled addressee are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelidae as described in WO9404678 (and referred to hereinafter as VHH domains or nanobodies). VHH molecules are about 10x smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Cameiids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Cameiids (WO 9749805). As such, anti-albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein. As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into 'unusual' or non-conventional epitopes such as cavities (WO9749805), the affinity of such VHH's to circulating albumin may be increased.

Classes of VHH The present invention further relates to a polypeptide construct, wherein a single domain antibody is a VHH directed to a target mentioned herein, wherein the VHH belongs to a class having human-like sequences. The class is characterised in that the VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 according to the Kabat numbering. A VHH sequence represented by SEQ ID NO: 1 and SEQ ID NO: 3 which bind to vWF, belong to this human-like class of VHH polypeptides. As such, peptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.

Therefore, one aspect of the present invention allows for the direct administration of a polypeptide construct comprising one or more single domain antibodies corresponding to a sequence represented by any of SEQ ID NOs: 1 and 3 to a patient in need of the same.

Another human-like class of Camelidae single domain antibodies represented by SEQ ID No. 16 and 18 have been described in WO 03/035694 and contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but 11 compensating this loss in hydrophilicity by a number of residues such as the charged arginine residue, serine or uncharged residues such as glycine at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.

Any of the VHHs as used by the invention may be of the traditional class or of the classes of human-like Camelidae antibodies. Said antibodies may be directed against whole targets or a fragment thereof. These polypeptides include the full length Camelidae antibodies, namely Fc and VHH domains, chimeric versions of heavy chain Camelidae antibodies with a human Fc domain.

The one or more single domain antibodies of the polypeptide construct which are directed against a target may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a polypeptide construct comprises anti-target single domain antibodies which do not all share the same sequence, but which are directed against the same target, or fragment thereof, one or more antigens thereof.

It is another aspect of the invention that the polypeptide construct comprises two or more single domain antibodies, wherein any two single domain antibodies are directed against different targets i.e. against any of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gplb and collagen.

Another aspect of the invention is a bispecific polypeptide construct comprising a single domain antibody directed against vWF A1 domain, A1 domain of activated vWF, and another single domain antibody directed against vWF A3 domain. Said bispecific polypeptide construct inhibits the interaction between vWF and collagen, and the interaction between vWF and platelets.

According to an aspect of the present invention a polypeptide construct may comprise two or more single domain antibodies which have been joined. The single domain antibodies may be 12 identical in sequence and directed against the same target or antigen. Depending on the number of VHHs linked, a multivalent VHH may be bivalent (2 VHHs), trivalent (3 VHHs), tetravalent (4 VHHs) or have a higher valency molecules.

The present invention also relates to the finding that a polypeptide construct as disclosed herein further comprising one or more single domain antibodies each directed against a serum protein of a subject, surprisingly has significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target single domain antibody(ies) when not pari: of said construct. Furthermore, the said constructs were found to exhibit the same favourable properties of VHHs such as high stability remaining intact in mice, extreme pH resistance, high temperature stability and high target affinity.

Examples of such constructs are represented by SEQ ID No. 13 to 15, which comprise anti-vWF VHH and anti-mouse serum albumin VHH.

Therefore, another embodiment of the present invention is a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 13 to 15.

Other examples of such constructs are represented by SEQ ID No. 42 to 45, which comprise humanized anti-vWF VHH and anti-mouse serum albumin VHH.

Therefore, another embodiment of the present invention is a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 42 to 45.

The serum protein may be any suitable protein found in the serum of subject, or fragment thereof. In one aspect of the invention, the serum protein is serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen. Depending on the intended use such as the required half-life for effective treatment and/or compartimentalisation of the target antigen, the VHH-partner can be directed to one of the above serum proteins.

Examples of single domain antibodies directed against serum albumin are the sequences represented by the sequences corresponding to any of SEQ ID NOs: 16 to 19 and 49 to 61. 13 Therefore another aspect of the invention is a polypeptide construct further comprising one or more anti-serum single domain antibodes, wherein the sequence of a anti-serum single domain antibody corresponds to any represented by SEQ ID NOs: 16 to 19 and 49 to 61.

Such constructs are able to circulate in the subject's serum for several days, reducing the frequency of treatment, the inconvenience to the subject and resulting in a decreased cost of treatment. Furthermore, it is an aspect of the invention that the half-life of the polypeptide constructs disclosed herein may be controlled by the number of anti-serum protein single domain antibodies present in the construct. A controllable half-life is desirable in several circumstances, for example, in the application of a timed dose of a therapeutic polypeptide construct.

Another embodiment of the present invention is a polypeptide construct as mentioned herein, further comprising a thrombolytic agent.

Said thrombolytic agent may be non-covalently or covalently attached to a single domain antibody via covalent or non-covalent means. Such covalent means are described below. Non-covalent means include via a protein interaction such as biotin/strepavidin, or via an immunoconjugate.

Alternatively, the thrombolytic agent may be administered simultaneous, separate or sequential in respect of a polypeptide construct of the invention.

Another aspect of the invention is a composition comprising at least one polypeptide construct as disclosed herein and at least one thrombolytic agent, for simultaneous, separate or sequential administration to a subject.

One aspect of the invention is a method for treating autoimmune disease comprising administering to an individual an effective amount of at least one polypeptide construct of the invention and at least one thrombolytic agent, simultaneously, separately or sequentially.

Another aspect of the invention is a kit containing at least one polypeptide construct of the invention and at least one thrombolytic agent for simultaneous, separate or sequential 14 administration to a subject. It is an aspect of the invention that the kit may be used according to the invention. It is an aspect of the invention that the kit may be used to treat the diseases as cited herein.

By simultaneous administration means the polypeptide and thrombolytic agent are administered to a subject at the same time. For example, as a mixture or a composition comprising said components. Examples include, but are not limited to a solution administered intraveneously, a tablet, liquid, topical cream, etc., wherein each preparation comprises the components of interest.

By separate administration means polypeptide and thrombolytic agent are administered to a subject at the same time or substantially the same time. The components are present in the kit as separate, unmixed preparations. For example, the polypeptide and thrombolytic agent may be present in the kit as individual tablets. The tablets may be administered to the subject by swallowing both tablets at the same time, or one tablet directly following the other.

By sequential administration means the polypeptide and thrombolytic agent are administered to a subject sequentially. The polypeptide and thrombolytic agent are present in the kit as separate, unmixed preparations. There is a time interval between doses. For example, one component might be administered up to 336, 312, 288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1 , or 0.5 hours after the other component.

In sequential administration, one component may be administered once, or any number of times and in various doses before and/or after administration of another component. Sequential administration may be combined with simultaneous or sequential administration.

The medical uses of the polypeptide construct described below, also apply to the composition comprising a polypeptide construct as disclosed herein and at least one polypeptide thrombolytic agent, for simultaneous, separate or sequential administration to a subject as disclosed here above.

Thrombolytic agents according to the invention may include, for example, staphylokinase, tissue plasminogen activator, streptokinase, single chain streptokinase, urokinase and acyl plasminogen streptokinase complex.

The single domain antibodies may be joined to form any of the polypeptide constructs disclosed herein comprising more than one single domain antibody using methods known in the art or any future method. For example, they may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatisation agent such as described by Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the single domain antibody may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more anti-target single domain antibodies and one or more anti-serum protein single domain antibodies. A method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO 96/34103. One way of joining multiple single domain antibodies is via the genetic route by linking single domain antibody coding sequences either directly or via a peptide linker. For example, the C-terminai end of the first single domain antibody may be linked to the N-terminal end of the next single domain antibody. This linking mode can be extended in order to link additional single domain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.

The polypeptide constructs disclosed herein may be made by the skilled artisan according to methods known in the art or any future method. For example, VHHs may be obtained using methods known in the art such as by immunising a camel and obtaining hybridoma's therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by using phage display.

One aspect of the present invention relates to the finding that polypeptides represented by SEQ ID NOs: 1 to 7 as in Table 30 derived from Camelidae VHHs, bind to vWF and inhibit its interaction with collagen.

Therefore, one embodiment of the present invention is a polypeptide construct wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 1 to 7. 16 Another embodiment of the present invention is a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 8 to 12. Said sequences correspond to monospecific polypeptide constructs (such as in SEQ ID No. 8 and 11 ) or heterospecific polypeptide constructs comprising VHHs of different sequences (such as in SEQ ID No. 9, 10 and 12), both directed against vWF.

Another embodiment of the present invention a polypeptide construct comprising one or more single domain antibodies directed against vWF.

Platelet aggregation is a very complex phenomenon and in an in vivo situation, the interaction of vWF with collagen only takes place at high shear as observed in small arteries. To assess platelet aggregation under high shear, the inventors performed perfusion experiments. Example 16 represents shear data obtained with the specific vWF-A3 binders SEQ ID No. 1 to 12. This experiment is representative for the interactions that take place upon damage of the vessel wall in a small artery (for example during angioplasty).

Surprisingly, monovalent VHH's perform very well in a platelet aggregation experiment under high shear: 50% inhibition of platelet aggregation was obtained at a concentration between 0.08 and 0.3 μg/ml. In comparison, the IgG vWF-specific antibody inhibiting the interaction with collagen, 82D6A3, inhibits 50% of platelet aggregation at approximately a twenty-fold higher concentration (Vanhoorelbeke K. et al, Journal of Biological Chemistry, 2003, 278: 37815-37821). These results were unexpected given that the IC50 values for the monovalent VHH's are up to 7 times fold worse in ELISA then the IC50 value of the IgG of 82D6A3.

This clearly proves that the large size of said antibodies is not suited to interaction with macromolecules which are starting, or are in the process of aggregating, such as those involved in platelet-mediated aggregation. vWF forms multimers of up to 60 monomers (final multimers of up to 20 million dalton in size). Indeed, it has been shown that not all A3 domains are accessible to 82D6A3 (Dongmei WU, Blood, 2002, 99, 3623 to 3628). Furthermore the large size of conventional antibodies, would restrict tissue penetration, for example, during platelet-mediated aggregation at the site of a damaged vessel wall. 17 Nanobodies have a unique structure that consists of a single variable domain. VHH molecules derived from Camelidae antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense tissues and for accessing the limited space between macromolecules participating in or starting the process of platelet mediated aggregation.

To our knowledge, this is the first time that experiments show, that the small size of a nanobody is advantagous over a large intact antibody for inhibition of interactions between such large macromolecules.

Despite the small size of nanobodies, and thus advantages for penetration, it is still surprising that such a small molecule can inhibit interactions between large polymers such as vWF (up to 60 monomers) and collagen and with such a high efficiency. It has been described that only the large multimeric forms of vWF are hemostatically active (Furlan, M,. 1996, Ann. Hematol. 72:341-348). Binding of multimeric vWF to collagen occurs with ~100-fold higher affinity than binding of monomeric vWF fragments.

The results from the high shear experiments indicate that a lower dose may be administered to patients. Therefore, fewer side effects are expected (such as immunogenicity or bleeding problems).

The present invention also relates to the finding that the polypeptides corresponding to a sequence represented by any of SEQ ID NOs 23 to 31 from single domain llama antibodies, bind to the A1 domain of vWF.

Therefore, another embodiment of the present invention is a polypeptide construct comprising one or more single domain antibodies, wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 23 to 31.

Another embodiment of the present invention a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 32 to 34. Said sequences correspond to 18 bivalent polypeptide constructs comprising VHHs of the same sequences, both directed against vWF A1 domain.

The inventors have performed perfusion experiment a flow chamber, to study the effect of polypeptide constructs comprising sequences represented by SEQ ID NOs: 23 to 31 upon platelet aggregation under high shear. Example 25 provides shear data obtained with the specific vWF-A1 binders SEQ ID No. 23 to 31 The present invention also relates to the finding that the polypeptides corresponding to a sequence represented by any of SEQ ID NOs 62 to 65 from single domain llama antibodies, bind selectively to the A1 domain of the active conformation of vWF (such as after being bound to collagen) rather than to freely circulating unactivated vWF. This results in antithrombotic agents that are both safer and more efficacious. As used herein, "selective binding" in reference to vWF A1 domains means that the llama antibodies have at least a tenfold and preferably a hundredfold greater affinity for the active conformation of vWF compared to the unactivated form.

Therefore, another embodiment of the present invention is a polypeptide construct comprising one or more single domain antibodies, wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 62 to 65.

In another embodiment of the present invention, a polypeptide construct comprises one or more single domain antibodies directed to the same target, and further comprises one or more single domain antibodies directed to the same target but to a different epitope in the same domain.

For example, the sequences represented by SEQ ID NOs: 9, 10 and 12 are heterospecific polypeptide constructs comprising VHHs directed to different epitopes in the A3 domain of vWF. Therefore, another embodiment of the present invention a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 9, 10 and 12.

Another embodiment of the present invention Is a polypeptide construct wherein the number of single domain antibodies directed to the same target is two or more. 19 The sequences represented by SEQ ID NOs: 8 and 11 are polypeptide constructs comprising VHHs directed to the same epitopes in the A3 domain of vWF, wherein the both VHHs have identical sequences. Therefore, another embodiment of the present invention is a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 8 and 11.

In another embodiment of the present invention, a polypeptide construct comprises one or more single domain antibodies directed to one domain of the same target, and one or more single domain antibodies directed to the same target but to another domain of the same target. Examples of different domains might be the A1 and A3 domains of vWF In another example, the sequences represented by SEQ ID NOs: 20, 21 and 22 are heterospecific polypeptide constructs comprising VHHs directed to epitopes on different domains of vWF i.e. A1 and A3 of vWF. Therefore, another embodiment of the present invention is a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 20, 21 and 22.

It is aspect of the invention that at least one VHH directed to the A1 domain in a heterospecific polypeptide construct recognizes the active conformation of vWF. Such a VHH corresponds to a sequence represented by any of SEQ ID NOs: 62 to 65.

Such polypeptide constructs may have superior anti-thrombotic effects compared to the monomeric VHH's. Perfusion experiment were performed in a flow chamber, to study platelet aggregation under high shear to study the effects of these polypeptide constructs. Example 30 represents shear data obtained with the heterospecific polypeptide construct comprising anti vWF-A1 VHH and anti-vWF-A3 VHH.

The present invention also relates to the finding that the polypeptides represented by SEQ ID NOs 35 to 37 from single domain llama antibodies, bind to collagen type I and/or type III.

Therefore, another embodiment of the present invention is a polypeptide construct, wherein at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 35 to 37.

In another embodiment of the present invention, a polypeptide construct comprises one or more single domain antibodies directed to the collagen I and/or type III, and one or more single domain antibodies directed to the same target but to a different epitope in the same domain. The sequences represented by 3P1-31_3P2-31 and 3L-41_3P2-31 are heterospecific polypeptide constructs comprising VHHs directed to different epitopes in collagen type I. Therefore, another embodiment of the present invention a polypeptide construct corresponding to a sequence represented by any of SEQ ID NOs: 46 and 47.

Another aspect of the invention is a polypeptide construct comprising one or more single domain antibodies directed to the platelet glycoprotein lb.

A murine anti-human vWF monoclonal antibody, AJvW-2 (IgG), was developed that inhibited the interaction between platelet glycoprotein lb (gplb) and von Willebrand factor (vWF) during the ristocetin- and botrocetin- induced aggregation of human platelets (PCT application number WO 00/10601 ). AJvW-2 Fab, inhibits repetitive coronary artery thrombosis without bleeding time prolongation in dogs (Kageyama S et al, Thromb Res., 2001 Mar 1 ;101 (5):395-404) and prevents thrombus deposition and neointima formation after balloon injury in guinea pigs (Kageyama S, et al, Arterioscler Thromb Vase Biol. 2000 Oct;20(10):2303-8).

Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human gplb (PCT application number WO 01/10911 A2). When injected into baboons, intact IgG and its F(ab')2 fragments caused almost immediate thrombocytopenia, due to the bivalency of F(ab')2 which mediates platelet crosslinking, or Fc:Fc receptor interactions which mediate activation of platelet aggregation (Cauwenberghs N. et al, Arteriosclerosis, Thrombosis and Vascular biology, 2000, 20: 1347 and see, for example, Cadroy Y et al, Blood, 1994, 83: 3218-3224, Becker BH et al, blood, 1989, 74: 690-694, Ravanat C. et al, Thromb. Haemost. 1999 , 82 : 528a abstract). Platelet deposition onto collagen-rich bovine pericardium was inhibited when Fab fragments were injected into the baboons before a thrombus was generated. However, when the Fab fragments were injected after a thrombus was allowed to form, no inhibition of further thrombosis was observed.

It was shown that the affinity of the Fab fragment for the gplb receptor on the platelet dropped by a factor of 10 as compared to the intact IgG or F(ab')2 (KD= 49.2 nM, 4.7 nM and 6.4 nM 21 respectively). Also the IC50 value for ristocetirt-induced platelet aggregation was up to 10-fold worse for Fab as compared to IgG or F(ab')2 (IC50 of 40 nM, 4.5 nM and 7.7 nM respectively).

It might be expected that the undesirable thrombocytopenia caused by Fc:Fc receptor mediated activation of platelet aggregation and/or F(ab')2-mediated crosslinking of platelets which has been observed when using intact IgG or F(ab')2 therapeutically in vivo, will be avoided by the use of VHH, since VHH contains no Fc and it is not bivalent. No loss of affinity and activity will be obtained as observed with the Fab fragment of 6B4 as nanobodies are already single domain molecules.

Humanised antibodies The discovery of naturally occurring single domain antibodies in llama, dromedary and camel revealed a new class of therapeutic molecules which combine the advantages of monoclonal antibodies for example specificity, low toxicity with the advantages of small molecules for example tissue penetration and stability. Unfortunately, the development of appropriate therapeutic products based on these proteins has the drawback of being Camelidae derived, and thus not human. Non-human proteins contain amino acid residues that can be immunogenic when injected into a human patient. Although studies have shown that Came//c ae-derived VHH are not immunogenic when injected in mice, replacing Camelidae residues by human residues is preferable. These humanized polypeptides should be substantially non-immunogenic in humans, but retain the affinity and activity of the wild type polypeptide.

By humanised is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent. Humanising a polypeptide, according to the present invention, comprises a step of replacing one or more of the Camelidae amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, 'i.e. the humanisation does not significantly affect the antigen binding capacity of the resulting polypeptide.

The inventors have determined the amino acid residues of the antibody variable domain (VHH) which may be modified without diminishing the native affinity of the domain for antigen 22 and while reducing its immunogenicity with respect to a heterologous species; the use of VHHs having modifications at the identified residues which are useful for administration to heterologous species; and to the VHH so modified. More specifically, the invention relates to the preparation of modified VHHs, which are modified for administration to humans, the resulting VHH themselves, and the use of such "humanized" VHHs in the treatment of diseases in humans.

The inventor have also found that humanization of VHH polypeptides requires the introduction and mutagenesis of only a limited number of amino acids in a single polypeptide chain without dramatic loss of binding and/or inhibition activity. This is in contrast to humanization of scFv, Fab, (Fab)2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

A humanisation technique may be performed by a method comprising the replacement of any of the following residues either alone or in combination: FR1 positions 1, 5, 28 and 30, the hallmark amino acid at position 37, 44, 45 and 47 in FR2, FR3 residues 74, 75, 76, 83, 84, 93 and 94 and positions 103, 104, 108 and 111 in FR4 ; numbering according to the Kabat numbering. Examples of such humanized sequences are given in Table 30, SEQ ID No. 2, 38 to 41.

Polypeptides represented in example 63 and 64 have a high degree of homology to human germline VH DP-47. Further humanization required the introduction and mutagenesis of a limited amount of amino acids in a single polypeptide chain. This is in contrast to humanization of scFv, Fab, (Fab)2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

The polypeptides contain human-like residues in FR2. Humanization required mutagenesis of residues in FR1 at position 1 and 5 which were introduced by the primer used for repertoire cloning and do not occur naturally in the llama sequence. Mutagenesis of those residues did not result in loss of binding and/or inhibition activity. Humanization of FR1 also required mutagenesis of position 28 and 30. Mutagenesis of those residues also did not result in loss of binding and/or inhibition activity. 23 Humanization also required mutagenesis of residues in FR3 at position 74, 75, 76, 83, 84, 93, 94. Mutagenesis of those residues did not result in loss of binding and/or inhibition activity.

Humanization also required mutagenesis of residues in FR4 at position 104, 108 and 111. Mutagenesis of Q108L resulted in lower production level in Escherichia coli. Position 108 is solvent exposed in camelid VHH, while in human antibodies this position is buried at the VH-VL interface (Spinelli, 1996; Nieba, 1997). In isolated VHs position 108 is solvent exposed. The introduction of a non-polar hydrophobic Leu instead of polar uncharged Gin can have a drastic effect on the intrinsic foldability/stability of the molecule.

One embodiment of the present invention is a method for humanizing a VHH comprising the steps of: (a) replacing of any of the following residues either alone or in combination: FR1 positions 1 , 5, 28 and 30, the hallmark amino acid at position 37, 44, 45 and 47 in FR2, FR3 residues 74, 75, 76, 83, 84, 93 and 94 , and positions 103, 104, 108 and 111 in FR4 ; numbering according to the Kabat numbering.

Examples of such humanized sequences are given in Table 30, SEQ ID No. 2, 38 to 41.

The use of antibodies derived from sources such as mouse, sheep, goat, rabbit etc., and humanised derivatives thereof as a treatment for conditions which require a modulation of platelet-associated aggregation, is problematic for several reasons. Traditional antibodies are not stable at room temperature, and have to be refrigerated for preparation and storage, requiring necessary refrigerated laboratory equipment, storage and transport, which contribute towards time and expense. Refrigeration is sometimes not feasible in developing countries. The yields of expression of said Fab molecules are very low and the method of production is very labor intensive. Furthermore, the manufacture or small-scale production of said antibodies is expensive because the mammalian cellular systems necessary for the expression of intact and active antibodies require high levels of support in terms of time and equipment, and yields are very low. Furthermore, traditional antibodies have a binding activity 24 which depends upon pH, and hence are unsuitable for use in environments outside the usual physiological pH range such as, for example, in treating gastric bleeding, gastric surgery. Furthermore, traditional antibodies are unstable at low or high pH and hence are not suitable for oral administration. However, it has been demonstrated that camelid antibodies resist harsh conditions, such as extreme pH, denaturing reagents and high temperatures (Ewert S et al, Biochemistry 2002 Mar 19,41(11):3628-36), so making them suitable for delivery by oral administration. Furthermore, traditional antibodies have a binding activity which depends upon temperature, and hence are unsuitable for use in assays or kits performed at temperatures outside biologically active-temperature ranges (e.g. 37 ± 20°C).

The polypeptide constructs represented by SEQ ID NOs: 1 to 47 and 49 to 65 and their derivatives not only possess the advantageous characteristics of conventional antibodies, such as low toxicity and high selectivity, but they also exhibit additional properties. They are more soluble, meaning they may be stored and/or administered in higher concentrations compared with conventional antibodies. They are stable at room temperature meaning they may be prepared, stored and/or transported without the use of refrigeration equipment, conveying a cost, time and environmental savings (described in example 61 ). Other advantageous characteristics as compared to conventional antibodies include short half-life in the circulation which may be modulated according to the invention by, for example, albumin-coupling, a bispecific nanobody with one specificity against albumin and the other against the target, Fc coupling, VHH coupling (bivalent VHHs) or by pegylation (described in example 41 until 54). A short and controllable half-life is desirable for surgical procedures, for example, which require an inhibition of platelet-mediated aggregation for a limited time period. Also, when bleeding problems occur or other complications, dosage can be lowered immediately. The polypeptides of the present invention also retain binding activity at a pH and temperature outside those of usual physiological ranges, which means they may be useful in situations of extreme pH and temperature which require a modulation of platelet-mediated aggregation, such as in gastric surgery, control of gastric bleeding, assays performed at room temperature etc. The polypeptides of the present invention also exhibit a prolonged stability at extremes of pH, meaning they would be suitable for delivery by oral administration. The polypeptides of the present invention may be cost-effectively produced through fermentation in convenient recombinant host organisms such as Escherichia coli and yeast; unlike conventional antibodies which also require expensive mammalian cell culture facilities, achievable levels of expression are high. Examples of yields of the polypeptides of the present invention are 1 to 10 mg/ml (£. coli) and up to 1g/l (yeast). The polypeptides of the present invention also exhibit high binding affinity for a broad range of different antigen types, and ability to bind to epitopes not recognised by conventional antibodies; for example they display long CDR- based loop structures with the potential to penetrate into cavities and exhibit enzyme function inhibition. Furthermore, since binding often occurs through the CDR3 loop only, it is envisaged that peptides derived from CDR3 could be used therapeutically (Desmyter et al., J Biol Chem, 2001 , 276: 26285-90). The preparation of such peptide is described in Example 65. The polypeptides of the invention are also able to retain full binding capacity as fusion protein with an enzyme or toxin. Furthermore, it might be expected that the undesirable thrombocytopenia caused by Fc:Fc receptor mediated activation of platelet aggregation and/or F(ab')(2)-mediated crosslinking of platelets which has been observed when using intact IgG or F(ab')(2) therapeutically in vivo (see Cauwenberghs N. et al, Arteriosclerosis, Thrombosis and Vascular biology, 2000, 20: 1347), will be avoided in the use of VHH, since VHH contains no Fc and it is not bivalent. Thus the polypeptides represented by SEQ ID NOs: 1 to 15, 20 to 47, 62 to 65, homologues or functional portions thereof provide a considerable cost and time saving in the treatment and diagnosis of conditions related to platelet-mediated aggregation, and the patient in need of said polypeptides would encounter fewer of the problems associated with conventional agents.

Platelet-mediated aggregation is the process wherein vWF-bound collagen adheres to platelets and/or platelet receptors (examples of both are gpla/lla, gplb, or collagen), ultimately resulting in platelet activation. Platelet activation leads to fibrinogen binding, and finally to platelet aggregation. It is within the scope of the present invention to provide polypeptides which modulate the processes which comprise platelet-mediated aggregation such as vWF-collagen binding, vWF-platelet receptor adhesion, collagen-platelet receptor adhesion, platelet activation, fibrinogen binding and/or platelet aggregation. Said polypeptides are derived from Camelidae antibodies directed towards vWF, vWF A1 , A1 domain of activated vWF or A3 domains, gplb or collagen, and share the same advantages as the polypeptides represented by SEQ ID NOs: 1 to 15, 20 to 47 and 62 to 65, as described above.

According to an aspect of the invention a polypeptide construct may be a homologous sequence of a full-length polypeptide construct. According to another aspect of the invention, 26 a polypeptide construct may be a functional portion of a full-length polypeptide construct. According to another aspect of the invention, a polypeptide construct may be a homologous sequence of a full length polypeptide construct. According to another aspect of the invention, a polypeptide construct may be a functional portion of a homologous sequence of a full length polypeptide construct. According to an aspect of the invention a polypeptide construct may comprise a sequence of a polypeptide construct.

According to an aspect of the invention a single domain antibody used to form a polypeptide construct may be a complete single domain antibody (e.g. a VHH) or a homologous sequence thereof. According to another aspect of the invention, a single domain antibody used to form the polypeptide construct may be a functional portion of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the polypeptide construct may be a homologous sequence of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the polypeptide construct may be a functional portion of a homologous sequence of a complete single domain antibody.

Another aspect of the present invention are the single domain antibodies corresponding to any of SEQ ID NOs: 1 to 7, 16 to 19, 23 to 31 , 35 to 41 , and 49 to 65, a homologous sequence thereof, and/or a functional portion thereof.

According to another aspect of the invention a polypeptide construct may be an homologous sequence of the parent sequence. According to another aspect of the invention, a polypeptide construct may be a functional portion parent sequence. According to another aspect of the invention, a polypeptide construct may be a functional portion of a homologous sequence of the parent sequence.

As used herein, an homologous sequence may comprise additions, deletions or substitutions of one or more amino acids, which do not substantially alter the functional characteristics of the polypeptide. The number of amino acid deletions or substitutions is preferably up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids. 27 A homologous sequence according to the present invention includes polypeptides extended by the addition of amino acids to form human heavy chain antibody or human single domain heavy chain antibody, which do not substantially alter the functional characteristics of the unmodified polypeptide.

A homologous sequence of the present invention may include a polypeptide represented by any of SEQ ID NOs: 1 to 47 and 49 to 65, which has been humanised (as described in examples 63 and 64 .

A homologous sequence of the present invention may include a sequence corresponding to the sequence of any of SEQ ID NOs: 1 to 47 and 49 to 65 which exists in other Camelidae species such as, for example, camel, llama, dromedary, alpaca, guanaco etc.

Where homologous sequence indicates sequence identity, it means a sequence which presents a high sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with the parent sequence, and is preferably characterised by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.

Alternatively, an homologous sequence may also be any amino acid sequence resulting from allowed substitutions at any number of positions of the parent sequence according to the formula below: Ser substituted by Ser, Thr, Gly, and Asn; Arg substituted by one of Arg, His, Gin, Lys, and Glu; Leu substituted by one of Leu, lie, Phe, Tyr, Met, and Val; Pro substituted by one of Pro, Gly, Ala, and Thr; Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His, and Gin; Ala substituted by one of Ala, Gly, Thr, and Pro; Val substituted by one of Val, Met, Tyr, Phe, lie, and Leu; Gly substituted by one of Gly, Ala, Thr, Pro, and Ser; lie substituted by one of lie, Met, Tyr, Phe, Val, and Leu; Phe substituted by one of Phe, Trp, Met, Tyr, He, Val, and Leu; Tyr substituted by one of Tyr, Trp, Met, Phe, lie, Val, and Leu; 28 His substituted by one of His, Glu, Lys, Gin, Thr, and Arg; Gin substituted by one of Gin, Glu, Lys, Asn, His, Thr, and Arg; Asn substituted by one of Asn, Glu, Asp, Gin, and Ser; Lys substituted by one of Lys, Glu, Gin, His, and Arg; Asp substituted by one of Asp, Glu, and Asn; Glu substituted by one of Glu, Asp, Lys, Asn, Gin, His, and Arg; Met substituted by one of Met, Phe, lie, Val, Leu, and Tyr.

A homologous according to the present invention may refer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000 nucleotides able to hybridize to the reverse-complement of the nucleotide sequence capable of encoding a polypeptide under stringent hybridisation conditions (such as the ones described by SAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York).

As used herein, a functional portion refers to a single domain antibody of sufficient length such that the interaction of interest is maintained with affinity of 1 x 10"8 M or better.

Alternatively a functional portion of a single domain antibody of the invention comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target.

Alternatively a functional portion of any of SEQ ID NO: 1 to 7 is a polypeptide which comprises a partial deletion of the complete amino acid sequence and which still maintains the binding site(s) and protein domain(s) necessary for the inhibition of binding of vWF to collagen.

Alternatively a functional portion of any of SEQ ID NOs: 23 to 31 and 62 to 65 is a polypeptide which comprises a partial deletion of the complete amino acid sequence and which still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the A1 domain of vWF.

Alternatively a functional portion of any of SEQ ID NOs: 35 to 37 is a polypeptide which comprises a partial deletion of the complete amino acid sequence and which still maintains 29 the binding site(s) and protein domain(s) necessary for the binding of and interaction with collagen.

Alternatively a functional portion comprises a partial deletion of the complete amino acid sequence of a polypeptide and which still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the antigen against which it was raised. It includes, but is not limited to VHH domains.

As used herein, a functional portion as it refers to a polypeptide sequence refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 5 or more amino acids.

A portion as it refers to a nucleotide sequence encoding a polypeptide sequence refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 15 or more nucleotides.

An aspect of the present invention is the administration of a polypeptide construct according to the invention can avoid the need for injection. Conventional antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity, however, they have one important drawback: they are relatively unstable, and are sensitive to breakdown by proteases. This means that conventional antibody drugs cannot be administered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation because they are not resistant to the low pH at these sites, the action of proteases at these sites and in the blood and/or because of their large size. They have to be administered by injection (intravenously, subcutaneously, etc.) to overcome some of these problems. Administration by injection requires specialist training in order to use a hypodermic syringe or needle correctly and safely. It further requires sterile equipment, a liquid formulation of the therapeutic polypeptide, vial packing of said polypeptide in a sterile and stable form and, of the subject, a suitable site for entry of the needle. Furthermore, subjects commonly experience physical and psychological stress prior to and upon receiving an injection.

An aspect of the present invention overcomes these problems of the prior art, by providing the polypeptides constructs of the present invention. Said constructs are sufficiently small, resistant and stable to be delivered orally, sublingualis topically, nasally, vaginally, rectally or by inhalation substantial without loss of activity. The polypeptides constructs of the present invention avoid the need for injections, are not only cost/time savings, but are also more convenient and more comfortable for the subject.

One embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the gastric environment without the substance being inactivated.

As known by persons skilled in the art, once in possession of said polypeptide construct, formulation technology may be applied to release a maximum amount of polypeptide in the right location (in the stomach, in the colon, etc.). This method of delivery is important for treating, prevent and/or alleviate the symptoms of disorders whose targets are located in the gut system.

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the gastric environment without being inactivated, by orally administering to a subject a polypeptide construct as disclosed herein.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the gastric environment without being inactivated.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the gut system without said substance being inactivated, by orally administering to a subject a polypeptide construct as disclosed herein. 31 An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject without the substance being inactivated, by orally administering to a subject a polypeptide construct as disclosed herein.

Another embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms or disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the vaginal and/or rectal tract.

In a non-limiting example, a formulation according to the invention comprises a polypeptide construct as disclosed herein, in the form of a gel, cream, suppository, film, or in the form of a sponge or as a vaginal ring that slowly releases the active ingredient over time (such formulations are described in EP 707473, EP 684814, US 5629001).

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the vaginal and/or rectal tract, by vaginally and/or rectally administering to a subject a polypeptide construct as disclosed herein.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the vaginal and/or rectal tract.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the vaginal and/or rectal tract without being said substance being inactivated, by administering to the vaginal and/or rectal tract of a subject a polypeptide construct as disclosed herein.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject without said substance being inactivated, by administering to the vaginal and/or rectal tract of a subject a polypeptide construct as disclosed herein. 32 Another embodiment of the present invention is a polypeptide construct as disclosed herein, for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the nose, upper respiratory tract and/or lung.

In a non-limiting example, a formulation according to the invention, comprises a polypeptide construct as disclosed herein in the form of a nasal spray (e.g. an aerosol) or inhaler. Since the polypeptide construct is small, it can reach its target much more effectively than therapeutic IgG molecules.

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the upper respiratory tract and lung, by administering to a subject a polypeptide construct as disclosed herein, by inhalation through the mouth or nose.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the nose, upper respiratory tract and/or lung, without said polypeptide being inactivated.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the nose, upper respiratory tract and lung without inactivation, by administering to the nose, upper respiratory tract and/or lung of a subject a polypeptide construct as disclosed herein.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject without inactivation by administering to the nose, upper respiratory tract and/or lung of a subject a polypeptide construct as disclosed herein. 33 One embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa. Because of their small size, a polypeptide construct as disclosed herein can pass through the intestinal mucosa and reach the bloodstream more efficiently in subjects suffering from disorders which cause an increase in the permeability of the intestinal mucosa.

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa, by orally administering to a subject a polypeptide construct as disclosed herein.

This process can be even further enhanced by an additional aspect of the present invention -the use of active transport carriers. In this aspect of the invention, VHH is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this "carrier" is a second VHH which is fused to the therapeutic VHH. Such fusion constructs are made using methods known in the art. The "carrier" VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the intestinal mucosa without being inactivated, by administering orally to a subject a polypeptide construct of the invention. 34 An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject without being inactivated, by administering orally to a subject a polypeptide construct of the invention.

This process can be even further enhanced by an additional aspect of the present invention - the use of active transport carriers. In this aspect of the invention, a polypeptide construct as described herein is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this "carrier" is a VHH which is fused to said polypeptide. Such fusion constructs made using methods known in the art. The "carrier" VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.

One embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the tissues beneath the tongue effectively. A formulation of said polypeptide construct as disclosed herein, for example, a tablet, spray, drop is placed under the tongue and adsorbed through the mucus membranes into the capillary network under the tongue.

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the tissues beneath the tongue effectively, by sublingually administering to a subject a polypeptide construct as disclosed herein.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able to pass through the tissues beneath the tongue.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the tissues beneath the tongue without being inactivated, by administering sublingually to a subject a polypeptide construct as disclosed herein.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject without being inactivated, by administering orally to a subject a polypeptide construct as disclosed herein.

One embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the skin effectively.

A formulation of said polypeptide construct, for example, a cream, film, spray, drop, patch, is placed on the skin and passes through.

An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the skin effectively, by topically administering to a subject a polypeptide construct as disclosed herein.

Another embodiment of the present invention is a use of a polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a substance that controls platelet mediated aggregation which is able pass through the skin effectively.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the skin without being inactivated, by administering topically to a subject a polypeptide construct as disclosed herein.

An aspect of the invention is a method for delivering a substance that controls platelet mediated aggregation to the bloodstream of a subject, by administering topically to a subject a polypeptide construct as disclosed herein.

In another embodiment of the present invention, a polypeptide construct as disclosed herein further comprises a carrier single domain antibody (e.g. VHH) which acts as an active transport carrier for transport of said polypeptide construct via the lung lumen to the blood. 36 A polypeptide construct further comprising a carrier that binds specifically to a receptor present on the mucosal surface (bronchial epithelial cells) resulting in the active transport of the polypeptide from the lung lumen to the blood. The carrier single domain antibody may be fused to the polypeptide construct. Such fusion constructs made using methods known in the art and are describe herein. The "carrier" single domain antibody binds specifically to a receptor on the mucosal surface which induces an active transfer through the surface.

Another aspect of the present invention is a method to determine which single domain antibodies (e.g. VHHs) are actively transported into the bloodstream upon nasal administration. Similarly, a naive or immune VHH phage library can be administered nasally, and after different time points after administration, blood or organs can be isolated to rescue phages that have been actively transported to the bloodstream. A non-limiting example of a receptor for active transport from the lung lumen to the bloodstream is the Fc receptor N (FcRn). One aspect of the invention includes the VHH molecules identified by the method. Such VHH can then be used as a carrier VHH for the delivery of a therapeutic VHH to the corresponding target in the bloodstream upon nasal administration.

One embodiment of the present invention is a polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders relating to platelet-mediated aggregation or dysfunction thereof. Said disorders include .thrombotic thrombocytopenic purpura (TTP), transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis. Said disorders further include those arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.

Other disorders are any of the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, restenosis, restenosis after PCTA or stenting, thrombus formation in stenosed arteries, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries. 37 One aspect of the invention is a polypeptide construct as disclosed herein for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said polypeptide construct is administered intravenously, subcutaneously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

Another aspect of the invention is the use of a polypeptide construct as disclosed herein for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said polypeptide construct is administered intravenously, subcutaneously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

Another aspect of the invention is a method of treating, preventing and/or alleviating disorders or conditions relating to relating to platelet-mediated aggregation or dysfunction thereof, comprising administering to a subject a polypeptide construct as disclosed herein, wherein said heterospecific polypeptide construct is administered intravenously, subcutaneously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

Another aspect of the invention is a polypeptide construct as disclosed herein for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

Another aspect of the invention is a use of a polypeptide as disclosed herein for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

One can use a polypeptide construct of the present invention in order to screen for agents that modulate the binding of the polypeptide to a vWF (or gplb or collagen). When identified in an assay that measures binding or said polypeptide displacement alone, agents will have to be subjected to functional testing to determine whether they act as modulators of platelet-mediated aggregation. 38 In an example of a displacement experiment, phage or cells expressing vWF or a fragment thereof are incubated in binding buffer with, for example, a polypeptide represented by SEQ ID NO: 1 which has been labeled, in the presence or absence of increasing concentrations of a candidate modulator. To validate and calibrate the assay, control competition reactions using increasing concentrations of said polypeptide and which is unlabeled, can be performed. After incubation, cells are washed extensively, and bound, labelled polypeptide is measured as appropriate for the given label (e.g., scintillation counting, fluorescence, etc.). A decrease of at least 10% in the amount of labelled polypeptide bound in the presence of candidate modulator indicates displacement of binding by the candidate modulator. Candidate modulators are considered to bind specifically in this or other assays described herein if they displace 50% of labelled polypeptide (sub-saturating polypeptide dose) at a concentration of 1 μΜ or less. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptides represented by SEQ ID NOs: 2 to 15, 20 to 47 and 62 to 65 or the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

Alternatively, binding or displacement of binding can be monitored by surface plasmon resonance (SPR). Surface plasmon resonance assays can be used as a quantitative method to measure binding between two molecules by the change in mass near an immobilized sensor caused by the binding or loss of binding of , for example, the polypeptide represented by SEQ ID NO: 1 from the aqueous phase to a vWF, or fragment thereof immobilized in a membrane on the sensor. This change in mass is measured as resonance units versus time after injection or removal of the said polypeptide or candidate modulator and is measured using a Biacore Biosensor (Biacore AB). vWF, or fragment thereof can be for example immobilized on a sensor chip (for example, research grade CM5 chip; Biacore AB) in a thin film lipid membrane according to methods described by Salamon et al. (Salamon et a/., 996, Biophys J. 71: 283-294; Salamon et al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213-219, each of which is incorporated herein by reference.). Sarrio et al. demonstrated that SPR can be used to detect ligand binding to the GPCR A(1) adenosine receptor immobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20: 5164-5174, incorporated herein by reference). Conditions for the binding of a polypeptide construct of the invention in an SPR assay can be fine-tuned by one of skill in the art using 39 the conditions reported by Sarrio et al. as a starting point. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptide constructs disclosed herein and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

SPR can assay for modulators of binding in at least two ways. First, a polypeptide represented by SEQ ID NO: 1 , for example, can be pre-bound to immobilized vWF, or fragment thereof, followed by injection of candidate modulator at a concentration ranging from 0.1 nM to 1 μΜ. Displacement of the bound polypeptide can be quantitated, permitting detection of modulator binding. Alternatively, the membrane-bound vWF, or fragment thereof can be pre-incubated with a candidate modulator and challenged with, for example, a polypeptide represented by SEQ ID NO: 1. A difference in binding affinity between said polypeptide and vWF, or fragment thereof pre-incubated with the modulator, compared with that between said polypeptide and vWF, or fragment thereof in absence of the modulator will demonstrate binding or displacement of said polypeptide in the presence of modulator. In either assay, a decrease of 10% or more in the amount of said polypeptide bound in the presence of candidate modulator, relative to the amount of said polypeptide bound in the absence of candidate modulator indicates that the candidate modulator inhibits the interaction of vWF, or fragment thereof and said polypeptide. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptides represented by SEQ ID NOs: 2 to 15, 20 to 47 and 62 to 65 or the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb, or collagen, or a fragment thereof.

Another method of detecting inhibition of binding of, for example, a polypeptide represented by SEQ ID NOs: 1 to 15, 20 to 34, 38 to 45 or 62 to 65 to vWF, or fragments thereof uses fluorescence resonance energy transfer (FRET). FRET is a quantum mechanical phenomenon that occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close proximity to each other (usually < 100 A of separation) if the emission spectrum of D overlaps with the excitation spectrum of A. The molecules to be tested, e.g. a polypeptide represented by SEQ ID NO: 1 and a vWF, or fragment thereof , are labelled with a complementary pair of donor and acceptor fluorophores. While bound closely together by the 40 vWF : polypeptide interaction, the fluorescence emitted upon excitation of the donor fluorophore will have a different wavelength from that emitted in response to that excitation wavelength when the said polypeptide and vWF, or fragment thereof are not bound, providing for quantitation of bound versus unbound molecules by measurement of emission intensity at each wavelength. Donor fluorophores with which to label the vWF, or fragment thereof are well known in the art. Of particular interest are variants of the A. Victoria GFP known as Cyan FP (CFP, Donor (D)) and Yellow FP (YFP, Acceptor (A)). As an example, the YFP variant can be made as a fusion protein with vWF, or fragment thereof. Vectors for the expression of GFP variants as fusions (Clontech) as well as flurophore-labeled reagents (Molecular Probes) are known in the art. The addition of a candidate modulator to the mixture of fluorescently-labelled polypeptide and YFP-vWF will result in an inhibition of energy transfer evidenced by, for example, a decrease in YFP fluorescence relative to a sample without the candidate modulator. In an assay using FRET for the detection of vWF : polypeptide interaction, a 10% or greater decrease in the intensity of fluorescent emission at the acceptor wavelength in samples containing a candidate modulator, relative to samples without the candidate modulator, indicates that the candidate modulator inhibits the vWF:polypeptide interaction. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptides represented by any of SEQ ID NOs: 2 to 15, 20 to 47, 62 to 65 or the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

A variation on FRET uses fluorescence quenching to monitor molecular interactions. One molecule in the interacting pair can be labeled with a fluorophore, and the other with a molecule that quenches the fluorescence of the fluorophore when brought into close apposition with it. A change in fluorescence upon excitation is indicative of a change in the association of the molecules tagged with the fluorophore:quencher pair. Generally, an increase in fluorescence of the labeled vWF, or fragment thereof is indicative that the polypeptide molecule (e.g. a polypeptide construct of the invention) bearing the quencher has been displaced. For quenching assays, a 10% or greater increase in the intensity of fluorescent emission in samples containing a candidate modulator, relative to samples without the candidate modulator, indicates that the candidate modulator inhibits vWF : polypeptide interaction. Of course, the above method might easily be applied to screening for 41 candidate modulators which alter the binding between the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

In addition to the surface plasmon resonance and FRET methods, fluorescence polarization measurement is useful to quantitate binding. The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Complexes, such as those formed by vWF, or fragment thereof associating with a fluorescently labelled polypeptide (e.g. a fluorescently-labeled polypeptide represented by any of SEQ ID NOs: 1 to 15, 20 to 34, 38 to 45 and 62 to 65), have higher polarization values than uncomplexed, labeled polypeptide. The inclusion of a candidate inhibitor of the vWF:polypeptide interaction results in a decrease in fluorescence polarization, relative to a mixture without the candidate inhibitor, if the candidate inhibitor disrupts or inhibits the interaction of vWF, or fragment thereof with said polypeptide. Fluorescence polarization is well suited for the identification of small molecules that disrupt the formation of vWF: polypeptide complexes. A decrease of 10% or more in fluorescence polarization in samples containing a candidate modulator, relative to fluorescence polarization in a sample lacking the candidate modulator, indicates that the candidate modulator inhibits the vWF: polypeptide interaction. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

Another alternative for monitoring vWF : polypeptide interactions uses a biosensor assay. ICS biosensors have been described in the art (Australian Membrane Biotechnology Research Institute; Cornell B, Braach-Maksvytis V, King L, Osman P, Raguse B, Wieczorek L, and Pace R. "A biosensor that uses ion-channel switches" Nature 1997, 387, 580). In this technology, the association of vWF, or fragment thereof and a polypeptide (e.g. a polypeptide represented by any of SEQ ID NOs: 1 to 15, 20 to 34, 38 to 45 and 62 to 65) is coupled to the closing of gramacidin-facilitated ion channels in suspended membrane bilayers and thus to a measurable change in the admittance (similar to impedence) of the biosensor. This approach is linear over six orders of magnitude of admittance change and is ideally suited for large scale, high throughput screening of small molecule combinatorial libraries. A 42 % or greater change (increase or decrease) in admittance in a sample containing a candidate modulator, relative to the admittance of a sample lacking the candidate modulator, indicates that the candidate modulator inhibits the interaction of vWF, or fragment thereof and said polypeptide. It is important to note that in assays testing the interaction of vWF, or fragment thereof with a polypeptide (such as for example, a polypeptide represented by any of SEQ ID NOs: 1 to 15, 20 to 34, 38 to 45 and 62 to 65), it is possible that a modulator of the interaction need not necessarily interact directly with the domain(s) of the proteins that physically interact with said polypeptide. It is also possible that a modulator will interact at a location removed from the site of interaction and cause, for example, a conformational change in the vWF. Modulators (inhibitors or agonists) that act in this manner are nonetheless of interest as agents to modulate platelet-mediated aggregation. Of course, the above method might easily be applied to screening for candidate modulators which alter the binding between the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

Any of the binding assays described can be used to determine the presence of an agent in a sample, e.g., a tissue sample, that binds to vWF, or fragment thereof, or that affects the binding of, for example, a polypeptide represented by any of SEQ ID N0: 1 to 15, 20 to 34, 38 to 45 or 62 to 65 to the vWF. To do so a vWF, or fragment thereof is reacted with said polypeptide in the presence or absence of the sample, and polypeptide binding is measured as appropriate for the binding assay being used. A decrease of 10% or more in the binding of said polypeptide indicates that the sample contains an agent that modulates the binding of said polypeptide to the vWF, or fragment thereof. Of course, the above generalised method might easily be applied to screening for candidate modulators which alter the binding between the polypeptide constructs disclosed herein, and macromolecules involved in platelet-mediated aggregation such as, for example, vWF, gplb or collagen, or a fragment thereof.

Cells A cell that is useful according to the invention is preferably selected from the group consisting of bacterial cells such as, for example, E. coli, yeast cells such as, for example, S. cerevisiae, P. pastoris, insect cells or mammalian cells. 43 A cell that is useful according to the invention can be any cell into which a nucleic acid sequence encoding a polypeptide comprising any of SEQ ID NOs: 1 to 47 and 49 to 65 or a polypeptide construct of the invention according to the invention can be introduced such that the polypeptide is expressed at natural levels or above natural levels, as defined herein. Preferably a polypeptide of the invention that is expressed in a cell exhibits normal or near normal pharmacology, as defined herein. Most preferably a polypeptide of the invention that is expressed in a cell comprises the nucleotide sequence capable of encoding the amino acid sequences presented in Table 30 or capable of encoding a amino acid sequence that is at least 70% identical to the amino acid sequence presented in Table 30.

According to a preferred embodiment of the present invention, a cell is selected from the group consisting of COS7-cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321 1 astrocytoma cell but also other transferable cell lines.

In general, "therapeutically effective amount", "therapeutically effective dose" and "effective amount" means the amount needed to achieve the desired result or results (treating or preventing platelet aggregation). One of ordinary skill in the art will recognize that the potency and, therefore, an "effective amount" can vary for the various compounds that inhibit platelet-mediated aggregation used in the invention. One skilled in the art can readily assess the potency of the compound.

As used herein, the term "compound" refers the polypeptide constructs disclosed herein, or to a nucleic acid capable of encoding said polypeptide, or an agent identified according to the screening method described herein or said polypeptide comprising one or more derivatised amino acids.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

The invention disclosed herein is useful for treating or preventing a condition of platelet-mediated aggregation, in a subject and comprising administering a pharmaceutically effective 44 amount of a compound or composition that inhibits BTK and that inhibits platelet-mediated aggregation.

The invention disclosed herein is useful for treating or preventing the first steps of thrombus formation, in a subject and comprising administering a pharmaceutically effective amount of a compound or composition according to the invention.

The invention disclosed herein is useful for treating or preventing restenosis, in a subject and comprising administering a pharmaceutically effective amount of a compound or composition according to the invention.

One aspect of the present invention is the use of compounds of the invention for treating or preventing a condition of platelet-mediated aggregation, in a subject and comprising administering a pharmaceutically effective amount of a compound in combination with another, such as, for example, aspirin.

One aspect of the present invention is the use of compounds of the invention for treating or preventing a condition of platelet-mediated aggregation, in a subject and comprising administering a pharmaceutically effective amount of a compound in combination with another, such as, for example, a thrombolytic agent.

Another aspect of the present invention is a use of a compound of the invention for treating or preventing plaque or thrombus in an individual. Said plaque or thrombus formation may be under conditions of high sheer. In both thrombosis and reocclusion, the reversible adhesion or tethering of the platelets at high shear rate is followed by a firm adhesion through the collagen receptor on platelets resulting in platelet activation; the tethering of platelets by vWF to collagen exposed in the damaged vessel wall is especially important under high shear conditions. The inventors have found that polypeptide constructs of the present invention unexpected performed well under high sheer conditions (e.g. Example 16.) The present invention is not limited to the administration of formulations comprising a single compound of the invention. It is within the scope of the invention to provide combination 45 treatments wherein a formulation is administered to a patient in need thereof that comprises more than one compound of the invention.

Conditions of platelet-mediated aggregation include, but are not limited to, unstable angina, stable angina, angina pectoris, embolus formation, deep vain thrombosis, hemolytic uremic syndrome, hemolytic anemia, acute renal failure, thrombolytic complications, thrombotic thrombocytopenic purpura, disseminated intravascular comgelopathy, thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and atrial thrombosis formation in atrial fibrillation, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, pre-eclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic exposure to cardiovascular devices. Such conditions may also result from thromboembolism and reocculsion during and after thrombolytic therapy, after angioplasty, and after coronary artery bypass.

It is well known in the art how to determine the inhibition of platelet-mediated aggregation using the standard tests described herein, or using other similar tests. Preferably, the method would result in at least a 10% reduction in platelet-mediated aggregation, including, for example, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably by 90%.

Similarly, the method would result in at least a 10% reduction in intracellular calcium mobilisation including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. Similarly, the method would result in at least a 10% reduction in the level of phosphorylated PLCg 2 including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

The reduction can be measured, for example, by comparing the optical impedence in a chronology platelet aggregometer. Any other known measurement method may also be used. For example, (1 ) upon collagen stimulation, the level of collagen-induced intracellular calcium mobilization increases over time and so the measurement may include measuring the level of collagen-induced intracellular calcium or (2) upon collagen stimulation, the level of 46 phosphorylated PLCg 2 increases over time and so the measurement may include measuring the level of phosphorylated PLCg 2.

The cells can be contacted in vitro, for example, by adding a compound of the invention to the culture medium (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the compound) or by adding the compound to the extracellular fluid in vivo (by local delivery, systemic delivery, inhalation, intravenous injection, bolus delivery, or continuous infusion). The duration of "contact" with a cell or population of cells is determined by the time the compound is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell or cells. Preferably, the duration of contact is 1-96 hours, and more preferably, for 24 hours, but such time would vary based on the half life of the compound and could be optimized by one skilled in the art using routine experimentation.

The compound useful in the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or a domestic animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intra-nasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes.

The compound of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene for the compound of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells.

Thus, the present compound may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 47 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneal^ by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form 48 must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compound may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the present compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. 49 Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compound to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Workman (U.S. Pat. No. 4,820,508).

Useful dosages of the compound can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semisolid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the compound varies depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using 50 only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.

The invention provides for an agent that is a modulator of platelet-mediated aggregation.

The candidate agent may be a synthetic agent, or a mixture of agents, or may be a natural product (e.g. a plant extract or culture supernatant). A candidate agent according to the invention Includes a small molecule that can be synthesized, a natural extract, peptides, proteins, carbohydrates, lipids etc.

Candidate modulator agents from large libraries of synthetic or natural agents can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based agents. Synthetic agent libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, Wl). Combinatorial libraries are available and can be prepared. Alternatively, libraries of natural agents in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and agents are readily modified through conventional chemical, physical, and biochemical means.

Useful agents may be found within numerous chemical classes. Useful agents may be organic agents, or small organic agents. Small organic agents have a molecular weight of more than 50 yet less than about 2,500 daitons, preferably less than about 750, more preferably less than about 350 daitons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.

For primary screening, a useful concentration of a candidate agent according to the invention is from about 10 mM to about 100 μΜ or more (i.e. 1 mM, 10 mM, 100 m , 1 M etc.). The primary screening concentration will be used as an upper limit, along with nine additional concentrations, wherein the additional concentrations are determined by reducing the primary screening concentration at half-log intervals (e.g. for 9 more concentrations) for secondary screens or for generating concentration curves.

High throughput screening kit A high throughput screening kit according to the invention comprises all the necessary means and media for performing the detection of an agent that modulates platelet-mediated aggregation by interacting with a target of the invention, such as for example vWF, or fragment thereof in the presence of a polypeptide (for example, a polypeptide represented by SEQ ID NOs: 1 to 15, 20 to 34, 38 to 45, 62 to 65 or a polypeptide construct), preferably at a concentration in the range of 1 μΜ to 1 mM. The kit comprises the following. Recombinant cells of the invention, comprising and expressing the nucleotide sequence encoding vWF, or fragment thereof, which are grown according to the kit on a solid support, such as a microtiter plate, more preferably a 96 well microtiter plate, according to methods well known to the person skilled in the art especially as described in WO 00/02045. Alternatively vWF, or fragment thereof is supplied in a purified form to be immobilized on, for example, a 96 well microtiter plate by the person skilled in the art. Alternatively vWF, or fragment thereof is supplied in the kit pre-immobilized on, for example, a 96 well microtiter plate. Alternatively, in cases where the macromolecule to be screened against is gplb, gpla/lla, or collagen, the above embodiments would carry gplb, gpla/lla, or collagen polypeptide or polynucleic acid respectively in place of vWF. Kit may contain more than one macromolcule (e.g. vWF, gplb or collagen macromolecule and/or polynucleic acid). Modulator agents according to the invention, at concentrations from about 1 μΜ to 1 mM or more, are added to defined wells in the presence of an appropriate concentration of polypeptide construct said concentration of said polypeptide preferably in the range of 1 μΜ to 1 mM. Kits may contain more than one polypeptide 52 Binding assays are performed as according to the methods already disclosed herein and the results are compared to the baseline level of, for example vWF, or fragment thereof binding to a polypeptide, such as, for example, a polypeptide represented by any of SEQ ID NOs: 2 to 15, 20 to 34, 38 to 45 or 62 to 65, but in the absence of added modulator agent. Wells showing at least 2 fold, preferably 5 fold, more preferably 10 fold and most preferably a 100 fold or more increase or decrease in vWF-polypeptide binding (for example) as compared to the level of activity in the absence of modulator, are selected for further analysis.

Other Kits Useful According to the Invention The invention provides for kits useful for screening for modulators of platelet-mediated aggregation, as well as kits useful for diagnosis of diseases or disorders characterised by dysregulation platelet-mediated aggregation. Kits useful according to the invention can include an isolated vWF, or fragment thereof. Alternatively, or in addition, a kit can comprise cells transformed to express vWF, or fragment thereof. In a further embodiment, a kit according to the invention can comprise a polynucleotide encoding vWF, or fragment thereof . In a still further embodiment, a kit according to the invention may comprise the specific primers useful for amplification of vWF, or fragment thereof. Alternatively, in cases where the macromolecule to be screened against is gplb, or collagen, the above embodiments would carry gplb, gpla/lla, or collagen polypeptide or polynucleic acid, or fragment thereof respectively in place of vWF. Kit may contain more than one macromolcule (e.g. vWF, gplb, or collagen macromolecule or polynucleic acid, or fragment thereof). Kits useful according to the invention can comprise an isolated polypeptide represented by any of SEQ ID NOs: 1 to 15, 20 to 47 or 62 to 65, a homologue thereof, or a functional portion thereof, or a polypeptide construct according to the invention. A kit according to the invention can comprise cells transformed to express said polypeptide. Kits may contain more than one polypeptide. In a further embodiment, a kit according to the invention can comprise a polynucleotide encoding a macromolecule, for example, vWF, gplb, or collagen, or fragment thereof. In a still further embodiment, a kit according to the invention may comprise the specific primers useful for amplification of a macromolecule such as, for example, vWF gplb, or collagen, or fragment thereof. All kits according to the invention will comprise the stated items or combinations of items and packaging materials therefore. Kits will also include instructions for use. 53 Medical devices The invention also provides for invasive medical devices coated with a polypeptide construct of the invention or an agent resulting from a screening method of the invention for use in devices requiring the same. Non-limiting examples of devices include surgical tubing, occlusion devices, prosthetic devices. Application for said devices include surgical procedures which require a modulation of platelet-mediated aggregation around the site of invasion.

One embodiment of the present is a method for treating invasive medical devices to prevent platelet-mediate aggregation around the site of invasion comprising the step of coating said device with a polypeptide construct or agent according to the invention.

Another embodiment of the present is a invasive medical devices that circumvents platelet-mediate aggregation around the site of invasion, wherein said device is coated with a polypeptide construct or agent according to the invention. 54 EXAMPLES The invention is illustrated by the following non-limiting examples.

Legend to examples Example 1. Immunization of Ilama002 Example 2. Repertoire cloning Example 3. Rescue of the library, phage preparation Selection for binders for vWF inhibiting the interaction with collagen: Example 4. Selection for binders for vWF inhibiting the interaction with collagen first and second round of panning Example 5. Functional characterization of vWF binders Inhibition of binding of vWF to collagen by VHH Example 6. Expression and purification of VHH Example 7. ELISA binding to vWF Example 8. Specificity of the VHHs Example 9. Inhibition ELISA with purified VHH Example 10. Sequencing of the clones Example 11. Epitope mapping Example 12. Bivalent and bispecific VHHs expression and purification Example 13. Binding in ELISA to vWF Example 14. Inhibition ELISA with purified VHH Example 15. Stability of bivalent or bispecific constructs in human plasma Example 16. Evaluate inhibition by VHH at high shear.

Selection of binders for vWF inhibiting the interaction with platelets: Example 17. Selection of binders for vWF inhibiting the interaction with platelets panning Example 18. Screening for binding to the A1 domain of vWF Example 19. Selection of binders for vWF inhibiting the interaction with platelets MATCHM Example 20. ELISA binding to vWF of purified VHH Example 2 . Inhibition ELISA with purified VHH Example 22. Sequencing of the clones Example 23. Evaluate inhibition by VHH at high shear.

Example 24. Bivalent VHHs expression and purification Example 25. Evaluate inhibition by VHH at high shear. 55 Make bispecific constructs for vWF-specific VHH: Example 26. Construction and sequence of bispecific constructs Example 27. Expression and purification of bispecific constructs Example 28. Binding to vWF Example 29. Inhibition of binding of vWF to collagen by the bispecific constructs as compared to the monovalent VHHs Example 30. Evaluate inhibition by VHH at high shear.

Screening for binders for collagen type I and type III: Example 31. Selection of binders for collagen type I Example 32. Test VHH in ELISA for binding to collagen type I and type III.

Example 33. Sequencing of the clones Example 34. Binding of purified VHH to collagen type I and type III Example 35. Selection of binders for collagen type I inhibiting the interaction with vWF Example 36. Test VHH in ELISA for binding to collagen type I and type Ml.

Example 37. Sequencing of the clones Example 38. Binding of purified VHH to collagen type I and type III Example 39. Test inhibition of binding of vWF to collagen by collagen-specific VHH in ELISA Example 40. Test inhibition of platelet aggregation by collagen-specific VHH at low and at high shear Improved half-life of VHH: Example 41. Immunization of llamas Example 42. Repertoire cloning Example 43. Rescue of the library, phage preparation Example 44. Phage ELISA Example 45. Selection first and second round of biopanning Example 46. Screening of individual clones after biopanning Example 47. Hinfl patern and sequencing Example 48. Test cross-reactivity with albumin of different species Example 49. Expression and purification Example 50. ELISA on MSA of the purified nanobodies Example 51. Construction and sequence of bispecific constructs Example 52. Expression and purification of bispecific constructs Example 53. Functionality of both VHHs in the bispecific construct 56 Example 54. Inhibition of binding of vWF to collagen by the bispecific constructs as compared to the monovalent VHHs Selection of binders for gplb inhibiting the interaction with vWF: Example 55. Selection of binders for rgplb Example 56. Screening for binders in ELISA.

Example 57. Binding of purified VHH to rgplb Example 58. Sequencing of the clones Example 59. Test inhibitory properties of VHHs specific for gplb Example 60. Evaluate inhibition by VHH at high shear.

Coating of stents, tubings, balloons, catheters, transplantation material with VHH: Example 61. Stability of VHH Example 62. VHH immobilized in a polymer Humanisation of C37: Example 63. Alignment of C37 with DP-47 Example 64. Mutagenesis of C37 Fragments of anti-VWF VHHs Example 65. Expression of a VHH-CDR3 fragment of vWF-C37 Example 66. Selection via first and second round biopanning on recombinant A1 (rA1) Example 67. Screening of individual clones after biopanning Example 68. Hinfl pattern and sequencing Example 69. Inhibition ELISA Examples Example 1 : Immunization of Ilama002 One llama was immunized with a cocktail of vWF and collagen type I and type III. Those antigens are all involved in the first interactions leading to platelet aggregation (Figure 1 ). The immunization scheme is summarized in Table 1 Example 2: Repertoire cloning Peripheral blood lymphocytes (PBLs) were isolated by centrifugation on a density gradient (Ficoll-Paque Plus Amersham Biosciences). PBLs were used to extract total RNA (Chomczynski and Sacchi 1987). cDNA was prepared on 100 μg total RNA with MMLV 57 Reverse Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides. The cDNA was purified with a phenol/chloroform extraction, followed by an ethanol precipitation and subsequently used as template to amplify the VHH repertoire.

In a first PCR, the repertoire of both conventional (1.6 kb) and heavy-chain (1.3 kb) antibody gene segments were amplified using a leader specific primer (5' GGCTGAGCTCGGTGGTCCTGGCT- 3') (SEQ ID N° 66) and the oligo d(T) primer (5'- AACTG G A AG AATTC G C G G C C G C AG G AATTTTTTTTTTTTTTTTTT-3' ) (SEQ ID N° 67). The resulting DNA fragments were separated by agarose gel electrophoresis and the 1.3 kb fragment, encoding heavy-chain antibody segments was purified from the agarose gel. A second PCR was performed using a mixture of FR1 reverse primers and the same oligo d(T) forward primer. The PCR products were digested with Sfi\ (introduced in the FR1 primer) and BstEtt (naturally occurring in FR4). Following gel electrophoresis, the DNA fragment of approximately 400 basepairs were purified from gel and ligated into the corresponding restriction sites of phagemid pAX004 to obtain a library of cloned VHHs after electroporation of Escherichia coli TG1. The size of the library was 1.4 x 107 cfu, and all clones contained insert of the correct size.

Example 3: Rescue of the library, phage preparation The library was grown at 37°C in 10 ml 2xTY medium containing 2% glucose, and 100 pg/ml ampicillin, until the OD600nm reached 0.5. M13K07 phages (1012) were added and the mixture was incubated at 37°C for 2 x 30 minutes, first without shaking, then with shaking at 100 rpm. Cells were centrifuged for 10 minutes at 4500 rpm at room temperature. The bacterial pellet was resuspended in 50 ml of 2xTY medium containing 100 pg/ml ampicillin and 25 pg/ml kanamycin, and incubated overnight at 37°C with vigorously shaking at 250 rpm. The overnight cultures were centrifuged for 15 minutes at 10000 rpm at 4°C. Phages were PEG precipitated (20% poly-ethylene-glycol and 1.5 M NaCI) and centrifuged for 30 minutes at 10000 rpm. The pellet was resuspended in 20 ml PBS. Phages were again PEG precipitated and centrifuged for 30 minutes at 20000 rpm and 4°C. The pellet was dissolved in 5 ml PBS-1 % casein. Phages were titrated by infection of TG1 cells at OD600nm= 0.5 and plating on LB agar plates containing 100 pg/ml ampicillin and 2% glucose. The number of transformants indicates the number of phages (= pfu). The phages were stored at -80°C with 15% glycerol. 58 Selection for binders for vWF inhibiting the interaction with collagen (Figure 2) Example 4: Selection for binders for vWF inhibiting the interaction with collagen: first and second round of panning A well in a microtiterplate was coated with 2 μg/ml vWF or with PBS containing 1% casein. After overnight incubation at 4°C, the wells were blocked with PBS containing 1% casein, for 3 hours at RT. 200 μΙ phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10x with PBS-Tween and 10x with PBS. Phages were specifically eluted with 100 μΙ of 100 μg/ml collagen type III. Elutions were performed for overnight at room temperature. Eluted phages were allowed to infect exponentially growing TG1 cells, and were then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. This experiment was repeated for a second round of panning, under the same conditions as described above. The results from the panning are presented in Table 2.

Example 5: Functional characterization of vWF binders: Inhibition of binding of vWF to collagen by VHH A microtiter plate was coated overnight at 4°C with collagen type III at 25 pg/ml in PBS. The plate was washed five times with PBS-Tween and blocked for 2 hours at room temperature with PBS containing 1 % casein. The plate was washed five times with PBS-tween. 100 μΙ of 2 μg/ml vWF (vWF is pre-incubated at 37'C for 15 minutes) was mixed with 20 μΙ peripiasmic extract containing a VHH antibody (described in Example 6) and incubated for 90 minutes at room temperature in the wells of the microtiterplate. The plate was washed five times with PBS-tween. An anti-vWF-HRP monoclonal antibody (DAKO) was diluted 3,000-fold in PBS and incubated for 1 hour. The plate was washed five times with PBS-Tween and vWF-binding was detected with ABTS/H202. Signals were measured after 30 minutes at 405 nm. The results are presented in Table 3, showing that inhibitors are obtained after the first and second round of panning.

Example 6: Expression and purification of VHH Plasmid was prepared for binders for vWF inhibiting the interaction with collagen typelll and was transformed into WK6 electrocompetent cells. A single colony was used to start an overnight culture in LB containing 2% glucose and 100 μ9/ιηΙ ampicillin. This overnight culture was diluted 100-fold in 300 ml TB medium containing 100 μ9/ιηΙ ampicillin, and incubated at 59 37°C until OD600nm= 0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37°C or overnight at 28°C.

Cultures were centrifuged for 20 minutes at 10000 rpm at 4°C. The pellet was frozen overnight or for 1 hour at -20°C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4°C at 20000 rpm. The supernatant containing the VHH was loaded on Ni-NTA and purified to homogeneity. The yield of VHH was calculated according to the extinction coefficient. Results are summarized in Table 4.

Example 7: ELISA: binding to vWF A microtiter plate was coated with 2 μg/ml vWF, overnight at 4°C. Plates were blocked for two hours at room temperature with 300 μΙ 1% casein in PBS. The plates were washed three times with PBS-Tweeh. Dilution series of all purified samples were incubated for 2 hours at RT. Plates were washed six times with PBS-Tween, after which binding of VHH was detected by Incubation with mouse anti-myc mAB 1/2000 in PBS for 1 hour at RT followed by anti-mouse-HRP conjugate 1/1000 in PBS, also for 1 hour at RT. Staining was performed with the substrate ABTS/H202 and the signals were measured after 30 minutes at 405 nm. The binding as a function of concentration of purified VHH is indicated in Figure 3.

Example 8: Specificity of the VHHs Microtiterplates were coated with 2 μg/ml vWF and 3 other antigens not involved in platelet aggregation, but that were also immunized in llama 002. ELISA was performed as described in Example 7 with 670, 67 and 6.7 nM VHH. Results are summarized in Table 5. The results show that the inhibitory VHH are specific for vWF.

Example 9: Inhibition ELISA with purified VHH Inhibition ELISA was performed as described in Example 5 but with decreasing concentrations of VHH and with human plasma at a dilution of 1/60 instead of with purified vWF or with human undiluted plasma. Results are represented in figure 4. The concentration of VHH resulting in 50% inhibition (IC50) is given in Table 6. 60 Example 10: Sequencing of the clones Clones were sequenced with M13 universal reverse primer. Amino acid sequences are shown in Table 30 (SEQ ID numbers 1 , 3, 4, 5, 6 and 7).

Example 11 : Epitope mapping Cloning the A3 domain of vWF in pBAD-Oprl-ss The pBAD-Oprl-strep-spec vector was used to display the VWF A3 domain as a fusion with OprI on the surface of UT5600 E.coli cells (F- ara-14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbDI rpsL109 xyl-5 mtl-1 thil DompT fepC266) (Cote-Sierra et al, 1998, Gene, 221 : 25-34). The gene coding for the A3 domain of vWF (201 aa) was amplified by PCR using the A3for and A3back PCR primers.

A3for: CTG GTG CTG CAG AGG TGA AGC TTC GGA GAG GGG CTG CAG ATC (SEQ ID N° 68) A3back: ATC CAT GCA AAT CCT CTA GAA TCC AGA GCA CAG TTT GTG GAG (SEQ ID N° 69) Fragment and vector were digested with Hindlll and Xbal, ligated and transformed in UT5600 (= pBAD-vWFA1/pBAD-vWFA3). Transformed cells were plated on LB agar plates containing 20 pg/ml streptomycin, 50 pg/rnl spectinomycin.

The pBAD-vWFA3 plasmid was transformed in UT5600 F- cells and plated on LB agar plates with 20 pg/ml streptomycin, 50 pg/ml spectinomycin. A single colony was used to inoculate LB medium with 20 pg/ml streptomycin, 50 pg/ml spectinomycin. Cells were grown overnight at 37°C at 200 rpm. The next day, cells were induced with 0.2% arabinose and incubated for 1 more hour at 37°C at 150 rpm. Total cell lysates were boiled in reducing sample buffer, loaded on a 12% SDS-PAGE and transferred to nitrocellulose for Western blotting. Transferred proteins were detected using a monoclonal anti-Oprl antibody (SH2.2) (Cote-Sierra et al, 1998, Gene, 221 : 25-34). An anti-mouse IgG conjugated with alkaline phosphatase was applied (Sigma), and the blots were developed with BCIP/NBT (Figure 5). The pBAD-vWF-A3 plasmids were transformed in UT5600 F- cells and plated on LB agar plates with 20 μg/ml streptomycin, 50 pg/ml spectinomycin. A single colony was used to inoculate LB medium with 20 μg ml streptomycin, 50 g/ml spectinomycin. Cells were grown overnight at 37°C at 200 rpm. The next day, cells were induced with 0.2% arabinose and incubated for 1 more hour at 37°C at 150 rpm. A microtiter plate was coated overnight at 4°C with the monoclonal anti-Oprl antibody (SH2.2) diluted 1/1000 in PBS and blocked for 2 hours 61 at RT with PBS containing 1% casein. After induction, total cells were allowed to bind to the plate for 1 hour at room temperature. The plates were washed five times with PBS-Tween. Phage preparations of single colonies were allowed to bind for two hours at room temperature. The plates were washed five times with PBS-Tween. An anti-M13 HRP conjugate was used for detection of phage binding to E. coli cells expressing the A3 domain or to an irrelevant antigen on their surface. The plates were washed five times with PBS-Tween. Staining was performed with ABTS/H202 and signals were measured after 30 minutes at 405 nm. Results are summarized in Table 7.

Example 12: Bivalent and bispecific VHHs: expression and purification The E. coli production vector pAX11 was designed (Figure 6), which allows the two-step cloning of bivalent or bispecific VHH.

The carboxy terminal VHH is cloned first with Pstl and BstEII, while in the second step the other VHH is inserted by Sfil and Notl, which do not cut within the first gene fragment. The procedure avoids the enforcement of new sites by amplification and thus the risk of introducing PCR errors. The sequence is shown in Table 30 (SEQ ID numbers 8, 9, 10, 11 and 12).

Protein was expressed and purified as described In Example 6. An extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%). Yields obtained for 1 liter expression and purification of bivalent protein in E. coli are summarized in Table 8.

Example 13: Binding in ELISA to vWF Binding to vWF was tested in ELISA as described in Example 7 and compared to binding of monovalent VHH. The results are shown in Figure 7. It is clear from the results that bivalent and bispecific VHH show stronger binding to VWF when compared to monovalent VHH.

Example 14: Inhibition ELISA with purified VHH Inhibition for binding of vWF to collagen was tested for monovalent as compared to bivalent VHHs as described in Example 5. Instead of using purified vWF, human, baboon and pig plasma was used in parallel at a dilution of 1/60. IC50 values are summarized in Table 9. 62 Example 15: Stability of bivalent or bispecific constructs in human plasma Stability of bivalent constructs was tested by incubation at 37°C in human plasma. AM-4-15-3/AM2-75 was incubated in human plasma at a concentration of 38 μ9 ΓηΙ at 37°C. A sample was removed after 1 , 2, 3, 6 and 24 hours incubation. Samples were diluted 10-fold and analyzed by Western blot. Results are summarized in Figure 8 and show that the bivalent construct is stable for at least 24 hours at 37°C in human plasma.

Example 6: Evaluation of inhibition by VHH at high shear.

Glass coverslips (18x18 mm, Menzel Glaser) were cleaned overnight by a chromosulfuric acid (2% chromium trioxide) solution and rinsed with distilled water before spraying. Monomeric collagen type III was solubilized in 50 mmol/L acetic acid and sprayed with a density of 30 g/cm2 on glass coverslips with a retouching airbrush (Badger model 100, Badger Brush Co). After the spraying procedure, the collagen surface was blocked for 1 hour with 1% human albumin in PBS (10 mmol/L phosphate buffer, pH 7.4, and 0.15 mol/L NaCI) to prevent nonspecific protein binding during the subsequent perfusion. Perfusion studies over collagen type III were carried out in a specially devised small parallel-plate perfusion chamber with well-defined rheological characteristics accommodating a glass coverslip. Whole blood was obtained by venipuncture from volunteers. Blood was drawn through the perfusion chamber by a Harvard infusion pump (pump 22, model 2400-004; Harvard, Natick, MA). The perfusion time was 5 minutes. Triplicate coverslips were inserted in the chamber. Five milliliters of whole blood was pre-warmed at 37°C for 5 minutes with or without addition of VHH, and then recirculated through the chamber for 5 minutes at a wall shear rate of 300 s~1 or 1600 s~ The coverslips were removed, rinsed , fixed with 0.05% glutaraldehyde, dehydrated with methanol, and stained with May-Grunwald/Giemsa. Platelet adhesion was quantitated with a light microscope (1 ,000* magnification) connected to a computerized image analyzer (AMS 40-10, Saffron Walden, UK). Platelet adhesion was expressed as the percentage of the surface covered with platelets. Results are summarized in Table 10 and 11. t 63 Selection of binders for vWF inhibiting the interaction with platelets (figure 9).

Example 17: Selection of binders for vWF inhibiting the interaction with platelets: panning Immunotubes were coated with 2 μς/ιηΙ vWF or with PBS containing 1 % casein. After overnight incubation at 4°C, the tubes were blocked with PBS containing 1 % casein, for 3 hours at RT. 200 μΙ phages were added to the immunotubes with a final volume of 2 ml in PBS. After 2 hours incubation at RT, the immunotubes were washed 10x with PBS-Tween and 10x with PBS. Bound phages were eluted with 2 ml 0.2 M glycin buffer pH= 2.4. Elutions were performed for 20 minutes at room temperature. Eluted phages were allowed to infect exponentially growing TG1 cells, and were then plated on LB agar plates containing 100 μ π\\ ampicillin and 2% glucose. The results from the panning are presented in Table 12.

Example 18: Screening for binding to the A1 domain of vWF The pBAD-Oprl -strep-spec vector was used to display the VWF A1 domain as a fusion with Oprl on the surface of UT5600 E.coli cells (F- ara-14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thil DompT fepC266) (Cote-Sierra et al, 1998, Gene, 221 : 25-34). The gene coding for the A1 domain of vWF (2 9aa) was amplified by PCR using the A1for and A1 back PCR primers.

A1for: CCG GTG AGC CCC ACC ACT CTA AGC TG GAG GAC ATC TCG GAA CCG (SEQ ID N° 70) Al back: ccc CAG GGT CGA AAC CCT CTA GAG CCC CGG GCC CAC AGT GAC (SEQ ID N° 71) Fragment and vector were digested with Hindlll and Xbal, ligated and transformed in UT5600 (= pBAD-vWFA1/pBAD-vWFA3). Transformed cells were plated on LB agar plates containing 20 pg/ml streptomycin, 50 g/ml spectinomycin.

The pBAD-vWFA1 plasmid was transformed in UT5600 F- cells and plated on LB agar plates with 20 pg/ml streptomycin, 50 pg/ml spectinomycin. A single colony was used to inoculate LB medium with 20 pg/ml streptomycin, 50 g ml spectinomycin. Cells were grown overnight at 37°C at 200 rpm. The next day, cells were induced with 0.2% arabinose and incubated for 1 more hour at 37°C at 150 rpm. Total cell lysates were boiled in reducing sample buffer, loaded on a 12% SDS-PAGE and transferred to nitrocellulose for Western blotting. Transferred proteins were detected using a monoclonal anti-Oprl antibody (SH2.2) (Cote-Sierra et al, 1998, Gene, 221 : 25-34). An anti-mouse IgG conjugated with alkaline 64 phosphatase was applied (Sigma), and the blots were developed with BCIP/NBTas shown in Figure 10.

The ELISA was performed as described in Example 11. Results are summarized in Table 13. The results indicate that vWF-A1 domain-specific VHH are obtained.

Example 19: Selection of binders for vWF inhibiting the interaction with platelets: MATCH E.coli cells expressing the A1 domain of vWF (Example 18) were used for a MATCHM experiment: UT5600 cells transformed with pBAD-Oprl-A1 were grown and induced with 0.2% arabinose. Cells were washed and incubated with the phages for 1 hour at RT. This mixture was washed 7 times with PBS-Tween and phages were eluted with exponentially growing TG1 cells. We performed a first and a second round of selection. Results are summarized in Table 14.

Example 20: ELISA: binding to vWF of purified VHH VHH specific for the A1 domain of vWF were expressed and purified as described in Example 6. Binding in ELISA to vWF was measured as described in Example 7. Results are shown in Figure 11.

Example 21: Inhibition ELISA with purified VHH A microtiter plate was coated overnight at 4°C with an antibody specific for platelet receptor gplb at 5μg/ml in PBS. The plate was washed five times with PBS-Tween, and blocked with 300 μΙ PBS-1% casein for 2 hours at room temperature. The plate was washed 3 times with PBS-Tween. Platelet receptor gplb (gplb) was applied to the wells of the microtiter plate at a concentration of 1 pg/ml and allowed to bind for 2 hours at room temperature. The plate was washed five times with PBS-Tween. VHH (A38 (negative control) and A50 (vWF A1 binder)) was added at decreasing concentration. Plasma containing vWF was pre-incubated at a dilution of 1/ 28 at 37°C for 5 minutes. Risto was added at a final concentration of 760 pg/ml and added to the VHH. This mixture was incubated for 30 minutes at room temperature. 100 μΙ of this mixture was then applied to a microtiter plate well and incubated for 90 minutes at room temperature. The plate was washed five times with PBS-Tween. A anti-vWF-HRP monoclonal antibody was diluted 3.000-fold in PBS and incubated for 1 hour. The plate was 65 washed five times with PBS-tween and vWF-binding was detected with ABTS/H202. Signals were measured after 30 minutes at 405 nm. Results are summarized in Figure 12.

Example 22: Sequencing of the clones Clones were sequenced with M13 universal reverse primer. Amino acid sequences are shown in Table 30 (SEQ ID numbers 23, 24, 25, 26, 27, 281 (29, 30 and 31).

Example 23: Evaluate inhibition by VHH at high shear.

Shear experiments were performed as described in Example 16. Platelet adhesion was expressed as the percentage of the surface covered with platelets. Results are summarized in Table 15 and 16.

Example 24: Bivalent VHHs: expression and purification Bivalent molecules were constructed as described in Example 12. The sequence is shown in Table 30 (SEQ ID numbers 32, 33 and 34).

Protein was expressed and purified as described in Example 6. An extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%).

Example 25: Evaluate inhibition by VHH at high shear.

Shear experiments were performed as described in Example 16. Platelet adhesion was expressed as the percentage of the surface covered with platelets. Results are summarized in Table 17 and 18.

Make bispecific constructs for vWF-specific VHH (Figure 13) Example 26: Construction and sequence of bispecific constructs Constructs were made as described in Example 12, with one VHH specific for vWF and inhibiting the interaction with collagen, and the second VHH also specific for vWF but inhibiting the interaction with platelet receptor gplb: Sequences are shown in Table 30 (SEQ ID NOs: 20, 21 and 22) 66 Example 27: Expression and purification of bispecific constructs Protein was expressed and purified as described in Example 6. A extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%). Yields obtained for 1 liter expression and purification of bispecific protein in E. coli are summarized in Table 19.

Example 28: Binding to vWF Binding to vWF was tested in ELISA as described in example 7. Results are shown in Figure 14.

Example 29: Inhibition of binding of vWF to collagen by the bispecific constructs as compared to the monovalent VHHs Inhibition for binding of vWF to collagen was tested for monovalent as compared to bispecific constructs as described in example 5. IC50 values are summarized in Table 20.

Example 30: Evaluate inhibition by VHH at high shear.

Shear experiments were performed as described in Example 16. Platelet adhesion was expressed as the percentage of the surface covered with platelets. Results are summarized in Table 21 and 22.

Screening for binders for collagen type I and type III (Figure 15) Example 31: Selection of binders for collagen type I A microtiterplate was coated with 25 μg/ml collagen type I. Phages were prepared as described in Example 3 and allowed to bind to the well of a microtiterplate that was blocked for 2 hours. After washing, phages were eluted with 0.1 glycin buffer pH=4.5. Results are summarized in Table 23.

Example 32: Test VHH in ELISA for binding to collagen type I and type III.

Clones were tested for binding in ELISA as described in example 7 but then on collagen type I or type III coated wells at 25 μg ml in PBS. The results are summarized in Table 24. 67 Example 33: Sequencing of the clones Clones were sequenced with M13 universal reverse primer. Amino acid sequences are shown in Table 30 (SEQ ID numbers 35, 36 and 37).

Example 34: Binding of purified VHH to collagen type I and type III VHH were expressed and purified as described in Example 6. A microtiterpiate was coated with 25 μ9 ιτιΙ collagen typel or typelll and blocked. Binders were applied in duplo dilutions and binding was detected as described in Example 7. Results are summarized in Figure 16.

Example 35: Selection of binders for collagen type I inhibiting the interaction with vWF A microtiterpiate was coated with 25 μg/ml collagen type I. Phages were prepared as described in Example 3 and allowed to bind to the well of a microtiterpiate that was blocked for 2 hours. After washing, phages were eluted with 300 μg ml vWF. A second and third round of selection were performed in the same way.

Example 36: Test VHH in ELISA for binding to collagen type I and type III.

Clones were tested for binding to collagen type I and type III in ELISA as described in Example 34.

Example 37: Sequencing of the clones Clones were sequenced with M13 universal reverse primer.

Example 38: Binding of purified VHH to collagen type I and type III VHH were expressed and purified as described in example 6. A microtiterpiate was coated with 25 μg/ml collagen typel or typelll and blocked. Binders were applied in duplo dilutions and binding was detected as described in Example 34.

Example 39: Test inhibition of binding of vWF to collagen by collagen-specific VHH in ELISA Inhibition was tested as described in Example 5. 68 Example 40: Test inhibition of platelet aggregation by collagen-specific VHH at low and at high shear Shear experiments were performed as described in Example 16. Platelet adhesion was expressed as the percentage of the surface covered with platelets.

Improved half-life of VHH Example 41 : Immunization of llamas One llama was immunized with human serum albumin (HSA). The immunization scheme is summarized in Table 25.

Example 42: Repertoire cloning The library was prepared as described in Example 2. The size of the library was 2 x 107 cfu, and all clones contained insert of the correct size.

Example 43: Rescue of the library, phage preparation Phages were prepared as described in Example 3.

Example 44: Phage ELISA A microtiter plate (Maxisorp) was coated overnight at 4°C with PBS-1 % casein or with 5 μg/ml HSA (human serum albumin). The plate was washed 3 times with PBS-Tween (0.05% Tween20) and blocked for 2 hours at room temperature with 200 μΙ PBS-1 % casein. The plate was washed five times with PBS-Tween. Phages were prepared as described above and applied to the wells in consecutive twofold dilutions. Plates were washed five times with PBS-Tween. Bound phage were detected with a mouse monoclonal antibody anti-M13 conjugated with horse radish peroxidase (HRP) diluted 1/2000 in PBS. The plates were washed five times with PBS-Tween. Staining was performed with ABTS/H202 and signals were measured after 30 minutes at 405 nm. Results are shown in Figure 17 and indicate the presence of HSA-specific nanobodies in the library.

Example 45: Selection: first and second round of biopanning A well in a microtiterplate was coated with 10 μg/ml mouse serum albumin (MSA), or with PBS containing 1% casein. After overnight incubation at 4°C, the wells were blocked with PBS containing 1 % casein, for 3 hours at RT. 200 μΙ phages was added to the wells. After 2 69 hours incubation at RT, the wells were washed 10x with PBS-Tween and 10x with PBS. Bound phages were eluted with 100 μΙ 0.2 M glycin buffer pH= 2.4. Elutions were performed for 20 minutes at room temperature. Eluted phages were allowed to infect exponentially growing E. coli TG1 cells, and were then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. A second round was performed with the same conditions as described above. Results are summarized in Table 26.

Example 46: Screening of individual clones after biopanning ELISA: binding to human serum albumin (HSA) and mouse serum albumin (MSA) Periplasmic extract was prepared as described in Example 6.

A microtiter plate was coated with 5 μg/ml HSA, with 5 μg/ml mouse serum albumin (MSA) or with PBS-1 % casein, overnight at 4°C. Plates were blocked for two hours at room temperature with 300 μΙ 1 % casein in PBS. The plates were washed three times with PBS-Tween. Periplasmic fraction was prepared for 23 individual clones after the first and second round of selection, and allowed to bind to the wells of the microtiterplate. Plates were washed six times with PBS-Tween, after which binding of nanobody was detected by incubation with mouse anti-Histidine monoclonal antibody Serotec MCA 1396 (1/1000 dilution) in PBS for 1 hour at RT followed by anti-mouse-alkaline phosphatase conjugate 1/2000 in PBS, also for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg2S0 , pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are summarized in Table 27.

Example 47: Hinfl patern and sequencing A PCR was performed on positive clones after the second round of panning, with a set of primers binding to a sequence in the vector. The PCR product was digested with the restriction enzyme Hinfl and loaded on a agarose gel. 4 clones were selected with a different Hinfl-pattern for further evaluation. Those clones were sequenced, and results are summarized in Table 30 (SEQ ID numbers 16, 17, 18 and 19 ).

Example 48: Test cross-reactivity with albumin of different species A SDS-PAGE was run for plasma (1/10 dilution) from different species (baboon, pig, hamster, human, rat. mouse and rabbit) and blotted on a nitrocellulose membrane. Phages were prepared for clones MSA 21. MSA 24, MSA 210, MSA212 and a irrelevant nanobody as 70 described in Example 3. Phages were allowed to bind to the nitrocellulose blotted serum albumins and unbound phages were washed away. Binding was detected with a anti-M13 polyclonal antibody coupled to HRP. DAP was used as a substrate for detection. Results are shown in Figure 18.

From these results we can conclude that all 4 binders are cross-reactive between pig, human, mouse (less for MSA212) and hamster serum albumin. MSA 21 is also cross-reactive with rabbit serum albumin. With the irrelevant nanobody no binding was observed (not shown). As a control experiment, a SDS-PAGE was run with the different plasma samples diluted 1/100 in PBS. The gel was stained with coomassie. We can conclude from Figure 19 that albumin levels in all plasma samples are high except for rabbit plasma, with low levels of albumin.

Example 49: Expression and purification Protein was expressed and purified as described in Example 6.

Example 50: ELISA on MSA of the purified nanobodies A microtiterplate was coated with 5 μg/ml MSA overnight at 4C. After washing, the plate was blocked for 2 hours at RT with PBS-1 % casein. Samples were applied in duplicate starting at a concentration of 2500 nM at 1/3 dilutions and allowed to bind for 2 hours at RT. A polyclonal rabbit anti-nanobody serum was added at 1/1000 (K208) for one hour at RT. Detection was with anti-rabbit alkaline phosphatase conjugate at 1/1000 and staining with PNPP. Results are shown in Figure 20.

Example 51 : Construction and sequence of bispecific constructs Bispecific constructs were prepared with the first VHH specific for albumin (MSA21 ) and the second VHH specific for vWF (Figure 21). Constructs were made as described in Example 12. Sequences are shown in Table 30 (SEQ ID numbers 13, 14 and 15) Example 52: Expression and purification of bispecific constructs Protein was expressed and purified as described in Example 6. A extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%). 71 Example 53: Functionality of both VHHs in the bispecific construct A microtiterplate was coated with 5 μο/ιηΙ mouse serum albumin overnight at 4°C. After washing the plate, wells were blocked for 2 hours with PBS-1% casein. The bispecific proteins were allowed to bind to the wells for 2 hours at RT. After washing, human, dog and pig plasma was added at different dilutions and allowed to bind for 2 hours at RT. Binding of vWF was detected with anti-vWF-HRP from DAKO at 1/3000 dilution. Staining was performed with ABTS/H202. Results are shown in Figure 22 and indicate that functionality of both VHHs is retained in the bispecific construct.

Example 54: Inhibition of binding of vWF to collagen by the bispecific constructs as compared to the monovalent VHHs Inhibition for binding of vWF to collagen was tested for monovalent as compared to bispecific constructs as described in Example 5. IC50 values are summarized in Table 28. Results indicate that the inhibitory properties of the VHH are retained in the bispecific construct.

Selection of binders for gplb inhibiting the interaction with vWF (Figure 23) Immunization, repertoire cloning and phage preparation were performed as described in Examples 1 , 2, 3.

Example 55: Selection of binders for rgplb A microtiterplate was coated with a mouse mAb against rgplb. The plate was blocked and rgplb was allowed to bind for 2 hours at RT at 5 μg/ml. The plate was washed. Phages were prepared as described above and allowed to bind to the wells of the microtiterplate. After washing, phages were eluted with 0.1 M glycin buffer pH=4.5. A second round of panning was performed in the same way.

Example 56: Screening for binders in ELISA.

Periplasmic extract was prepared as described in Example 6.

The supernatant was applied to wells coated with mAb arid subsequently gplb, as described in Example 55. Dilution series of all purified samples were incubated for 2 hours at RT. Plates were washed six times with PBS-Tween, after which binding of VHH was detected by incubation with mouse anti-His-HRP mAB 1/2000 in PBS for 1 hour at RT followed by staining with the substrate ABTS/H202. The signals were measured after 30 minutes at 405 nm. 72 Example 57: Binding of purified VHH to rgplb Periplasmic fraction was prepared as described in Example 6. The supernatant containing the VHH was loaded on Ni-NTA and purified to homogeneity. The yield of VHH was calculated according to the extinction coefficient. ELISA was performed as described in Example 55.

Example 58: Sequencing of the clones Clones were sequenced with M13 universal reverse primer.

Example 59: Test inhibitory properties of VHHs specific for gplb VHHs were tested for inhibition in ELISA as described in Example 21.

Example 60: Evaluate inhibition by VHH at high shear.

Shear experiments were performed as described in Example 16. Platelet adhesion was expressed as the percentage of the surface covered with platelets.

Coating of stents, tubings, balloons, catheters, transplantation material with VHH Example 61 : Stability of VHH VHH C37 was incubated at 37°C and inhibition of binding of vWF to collagen was measured at different time points by ELISA as described in Example 7. Results were compared to VHH stored at -20°C and are presented in Figure 24. Shown for comparison are the activities of a scFv against B3 antigen (Reiter et al, Protein Engineering, 1994, 7: 697-704), and said scFv modified by the introduction of a disulphide bond between framework residues 44 and 105 to enhance its stability (dsFv). The dsFv lost 40% of its activity after 60 hours incubation at 37°C. After one year of incubation at 37°C, C37 was analyzed for its inhibitory properties as compared to C37 stored in the freezer. The ELISA was performed as described in Example 5 with human plasma at a final dilution of 1/200. The results are shown in Figure 25 and indicate that functionality is fully retained (IC50 value of 0.085 versus 0.1 μg/ml for C37 stored at 37°C versus -20°C). Therefore, it is expected that VHH will have a long shelf-life.

Example 62: VHH immobilized in a polymer A mixture was prepared of 0.5 ml of 30% acrylamide; 1 ml of 1 M Tris pH= 7.5; 3.5 ml H20; 35 μΙ of 10% APS; 3.5 μΙ TEMED. In some wells, VHH C37 was added at a final 73 concentration of 10 μg/ml. The mixture was allowed to polymerize in the wells of a 96-well plate for 3 hours at RT. Human plasma was added at different dilutions starting with undiluted plasma. After 1 hour incubation at RT, the plate was washed and anti-vWF-HRP (DAKO) was added at 1/2000, for 1 hour at RT. After washing the plate, substrate (ABTS/H202) was added and OD405nm was measured. The result is shown in Figure 26. The results indicate that VHH remain functional upon immobilization in a polymer.

Humanlsatlon of C37 Example 63: Alignment of C37 with DP-47 Alignment of the C37 nanobody (SEQ ID number 1) and a human VH3 germline (DP-47) revealed a high degree of homology: o 4 AA changes in FR1 on position 1 , 5, 28 and 30 o 4 AA changes in FR3 on position 74, 75, 84 and 94 o 3 AA changes in FR4 on position 104, 108 and 111 as is shown in Figure 27 Example 64: Mutagenesis of C37 C37 was mutated by using a non-PCR based site-directed mutagenesis method as described by Chen and Ruffner (Chen and Ruffner, Amplification of closed circular DNA in vitro, Nucleic Acids Research, 1998, 126-1127) and commercialized by Stratagene (Quickchange site-directed mutagenesis).

Plasmid DNA was used as template in combination with 2 mutagenic primers (table 29) introducing the desired mutation(s). The 2 primers are each complementary to opposite strands of the template plasmid DNA. In a polymerase reaction using the Pfu DNA polymerase each strand is extended from the primer sequence during a cycling program using a limited amount of cycles. This results in a mixture of wild type and mutated strands. Digestion with Dpnl results in selection of mutated In vitro synthesized DNA. The DNA was precipitated and transformed to E. coli and analyzed for the required mutation by sequence analysis. The clone with the correct sequence was named C37-hum, the amino acid sequence is in Table 30 SEQ ID number 2.

Expression and purification of C37-hum was performed as described in Example 6. Inhibition of binding of vWF to collagen for C37 was compared to C37-hum as described in Example 5. 74 Results are shown in Figure 28. It clearly shows that the humanized version of C37 remains fully functional.

The positions that still need to be humanized are: Q1 , Q5, D104, Q108 and 1111. We can humanize position 1 and 5 without loss of inhibition since these amino acids were introduced by the FR1 primer and do not occur naturally in the llama sequence. We can also humanize position 111 since we isolated a VHH identical to C37 except for 1111V (AM-2-75 SEQ ID number 3) with the same functional characteristics (Example 9 and Table 6).

Position 108 is solvent exposed in camelid VHH, while in human antibodies this position is buried at the VH-VL interface (Spinelli, 1996; Nieba, 1997). In isolated VHs position 108 is solvent exposed. The introduction of a non-polar hydrophobic Leu instead of polar uncharged Gin can have a drastic effect on the intrinsic foldability/stability of the molecule.

Fragments of anti-VWF VHHs Example 65: Expression of a VHH-CDR3 fragment of vWF-C37 The CDR3 region of C37 was amplified by using a sense primer located in the framework 4 region (Forward: CCCCTGGTCCCAGTTCCCTC) (SEQ ID N° 72) and an anti-sense primer located in the framework 3 region (Reverse: TGTGCTCGCGGGGCCGGTAC) (SEQ ID N° 73).

In order to clone the CDR-3 fragment in pAX10, a second round PCR amplification was performed with following primers introducing the required restriction sites: Reverse primer Sfi1 : GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCTCGCGGGGCCGGTAC (SEQ ID N° 74) Forward primer Not1: GTCCTCGCAACTGCGCGGCCGCCCCCTGGTCCCAGTTCCCTC (SEQ ID N" 75) The PCR reactions were performed in 50 ml reaction volume using 50pmol of each primer. The reaction conditions for the primary PCR were 11 min at 94 °C, followed by 30/60/120 sec at 94/55/72 °C for 30 cycles, and 5 min at 72°C. All reaction were performed wit 2.5 m MgCI2 , 200 mM dNTP and 1.25U AmpliTaq God DNA Polymerase (Roche Diagnostics, Brussels, Belgium).

After cleavage with Sfi1 and Not1 the PCR product was cloned in pAX10. 75 Isolation of conformation-specific anti-vWF VHH's Example 66: Selection via first and second round biopanning on recombinant A1 (rA1) A well in a microtiter plate was coated with 5 μg/ml recombinant A1 domain of vWF (rA1 ), or with PBS containing 1 % casein. After overnight incubation at 4°C, the wells were blocked with PBS containing 1% casein, for 3 hours at RT. 200 μΙ phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10x with PBS-Tween and 10x with PBS. Bound phages were eluted with 100 μΙ 0.2 M glycin buffer, pH 2.4. Elutions were performed for 20 minutes at room temperature. Eluted phages were allowed to infect exponentially growing E. coli TG1 cells, and were then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. A second round was performed with the same conditions as described above but phages were re-suspended in 10 μg/ml vWF. The wells of the microtiterplate were washed 7 times for 30 minutes with 10 μg/ml vWF. Results are summarized in Table 31.

Example 67: Screening of individual clones after biopanning ELISA: binding to rA1 and vWF A single colony was used to start an overnight culture in LB containing 2% glucose and 100 g/ml ampicillin. This overnight culture was diluted 100-fold in TB medium containing 100 μg/ml ampicillin, and incubated at 37°C until OD600nm= 0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37°C or overnight at 28°C. Cultures were centrifuged for 20 minutes at 10,000 rpm at 4°C. The pellet was frozen overnight or for 1 hour at -20°C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4"C at 20.000 rpm. The supernatant containing the VHH was used for further analysis.

A microtiter plate was coated with 2 μg/ml rA1 or with 1 μg/ml vWF, overnight at 4°C. Plates were blocked for two hours at room temperature with 300 μΙ 1 % casein in PBS. The plates were washed three times with PBS-Tween. Periplasmic fraction was prepared for 192 individual clones after the second round of selection, and allowed to bind to the wells of the microtiter plate. Plates were washed six times with PBS-Tween, after which binding of nanobody was detected by incubation with rabbit polyclonal anti-nanobody (1/2000 dilution) in PBS for 1 hour at RT followed by goat anti-rabbit-HRP conjugate 1/2000 in PBS, also for 1 76 hour at RT. Staining was performed with the substrate ABTS/H202 and the signals were measured after 30 minutes at 405 nm. Results are summarized in Table 32. We can conclude that 50 clones bind to rA1 and not to vWF.

Example 68: Hinfl pattern and sequencing A PCR was performed on positive clones for rA1 and negative for vWF, after the second round of panning, with a set of primers binding to a sequence in the vector. The PCR product was digested with the restriction enzyme Hinfl and loaded on a agarose gel. 30 clones were selected with a different Hinfl-pattern for further evaluation. Those clones were tested in more detail by ELISA as described in example 67. Out of the 30 clones, 4 were shown to clearly have a much higher affinity for rA1 than for vWF. The data are shown in Figures 29 (binding to rA1) and 30 (binding to vWF). These clones were sequenced, and results are summarized in Table 30 (SEQ ID numbers 62 to 65).

Example 69: Inhibition ELISA Inhibition by nanobodies for binding of vWF to gplb was determined by ELISA. A microtiter plate was coated overnight at 4°C with an antibody specific for platelet receptor gplb at 5μg/ml in PBS. The plate was washed five times with PBS-Tween, and blocked with 300 μΙ PBS-1 % casein for 2 hours at room temperature. The plate was washed 3 times with PBS-Tween. Plasma was applied to the wells of the microtiter plate at a 1/2 dilution and allowed to bind for 1.5 hours at 37C. The plate was washed five times with PBS-Tween. VHH was added at decreasing concentration. Plasma containing vWF was pre-incubated at a dilution of 1/50 at 37°C for 5 minutes. Ristocetin was added at a final concentration of 1 mg/ml and added to the VHH. This mixture was incubated for 1 hour 37C. 50 μΙ of this mixture was then applied to a microtiter plate well and incubated for 90 minutes at 37C. The plate was washed five times with PBS-Tween. An anti-vWF-HRP monoclonal antibody was diluted 3,000-fold in PBS and incubated for 1 hour. The plate was washed five times with PBS-tween and vWF-binding was detected with ABTS/H202. Signals were measured after 30 minutes at 405 nm. 77 FIGURES Figure 1. Interactions involved in the first steps of platelet aggregation.

Figure 2. Interactions involved in the first steps of platelet aggregation. A VHH is indicated inhibiting the interaction between vWF and collagen.

Figure 3. Binding to vWF as determined by ELISA, by purified VHH as described in Example 7.

Figure 4. ELISA to test inhibition by VHH of binding of vWF to collagen as described in Example 9.

Figure 5. Western blot showing expression of A3 domain of vWF as a fusion with Oprl on the surface of E.coli as described in Example 11.

Figure 6. Restriction map of multiple cloning site of PAX0 for construction of bivalent or bispecific nanobodies.

Figure 7. Binding in ELISA to purified vWF, for monovalent versus bivalent and bispecific VHH as described in Example 13.

Figure 8. Stability of bispecifc VHH in human plasma upon incubation at 37°C for up to 24 hours as described in Example 15.

Figure 9. Interactions involved in the first steps of platelet aggregation. A VHH is indicated inhibiting the interaction between vWF and platelets.

Figure 10. Western blot showing expression of A1 domain of vWF as a fusion with Oprl on the surface of E.coli as described in Example 18.

Figure 11. Binding to vWF as determined by ELISA, by purified VHH as described in Example 20.

Figure 12. Inhibition of binding of gplb to VWF by A50 and A38 (negative control) as described in Example 21.

Figure 13. Interactions involved in the first steps of platelet aggregation. A bispecific constructs is indicated with one VHH specific for vWF and inhibiting the interaction between vWF and collagen and the second VHH specific for vWF but inhibiting the interaction between vWF and platelets.

Figure 14. Binding in ELISA to vWF as described in Example 28.

Figure 15. Interactions involved in the first steps of platelet aggregation. A VHH is indicated specific for collagen and inhibiting the interaction between vWF and collagen. 78 Figure 16. Binding of purified VHH to collagen type I and type III in ELISA as described in Example 34.

Figure 17. Phage ELISA to show that HSA-specific nanobodies are present in the library as described in Example 44.

Figure 18. Binding of phages expressing the albumin binders, to plasma blotted on nitrocellulose as described in Example 48.

Figure 19. Coomassie staining of plasma samples on SDS-PAGE as described in Example 48.

Figure 20. Binding of purified nanobodies to mouse albumin as determined by ELISA as described in Example 50.

Figure 21. Bispecific constructs with one VHH binding to albumin and a second VHH binding to vWF for improvement of half-life as described in Example 51.

Figure 22. Sandwich ELISA showing the functionality of both VHHs in a bispecific construct as described in Example 53.

Figure 23. Interactions involved in the first steps of platelet aggregation. A VHH is indicated specific for gplb and inhibiting the interaction between vWF and platelets.

Figure 24. Residual activity for C37 stored at -20°C as compared to C37 incubated at 37°C for up to 194 hours. C37 stability is compared to stability of a scFv specific for B3 antigen and a stabilized form, dsFv (stabilized by 2 disulphide bonds) as described in Example 6 .

Figure 25. Inhibitory activity for C37 stored at -20°C as compared to C37 incubated at 37°C for 1 year as described in Example 6 .

Figure 26. Binding of vWF from human plasma to C37 immobilized in acrylamide as described in Example 62.

Figure 27. Amino acid alignment of C37 with human germline sequence DP-47 as described in Example 63.

Figure 28. Inhibition of binding of vWF to collagen as determined by ELISA for C37 and C37 hum as described in Example 64.

Figure 29. Binding of A11 , A12, A13, A14, A15 and A16 clones to rA1 as measured in ELISA Figure 30. Binding of A11 , A12, A13, A14, A15 and A16 clones to vWF as measured in ELISA 79 TABLES Table 1. Immunization scheme used for llama 002 according to Example 1.

Table 2. Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in Example 4. Pfu vWF (antigen) divided by pfu casein (a specific binding) = enrichment.

Table 3. Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 5.

Table 4. Yield (mg/liter culture) after expression and purification of VHH grown in WK6 E.coli cells as described in Example 6.

Table 5. OD 405 nm for binding of VHH in ELISA to vWF and 3 antigens that were also immunized in Ilama002 according to Example 8.

Table 6. Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 9.

Table 7. Epitope mapping of VHH binding to vWF and inhibiting the interaction with collagen as described in example 11.

Table 8. Yields of purified protein (mg) per liter of culture for bivalent and bispecific VHHs as described in Example 12.

Table 9. IC50 values for monovalent as compared to bivalent and bispecific VHHs. Inhibition was tested with human, pig and baboon plasma as described in Example 14.

Table 10. Inhibition of platelet aggregation at high shear (1600 s'1) as described in Example 16.

Table 11. Inhibition of platelet aggregation at low shear (300 s"1) as described in Example 16. Table 12. Plaque forming units (pfu) after one round of panning on vWF as described in Example 17. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment.

Table 13. Results of screening in ELISA of individual colonies for binding to vWF and to the A1 domain of vWF as described in Example 18.

Table 14. Results after one round of MATCHM on pBAD-Oprl-A1 cells as described in Example 19.

Table 15. Inhibition of platelet aggregation at high shear (1600 s"1) as described in Example 23.

Table 6. Inhibition of platelet aggregation at low shear (300 s'1) as described in Example 23. 80 Table 17. Inhibition of platelet aggregation at high shear (1600 s"1) as described in Example 25.

Table 18. Inhibition of platelet aggregation at low shear (300 s' ) as described in Example 25. Table 19. Yields after expression and purification of bispecific constructs as described in Example 27.

Table 20. IC50 values for bispecifici nanobodies for the A1 and A3 domain of vWF as described in Example 29.

Table 21. Inhibition of platelet aggregation at high shear (1600 s'1) as described in Example 30.

Table 22. Inhibition of platelet aggregation at low shear (300 s'1) as described in Example 30. Table 23. Plaque forming units (pfu) after one round of panning on collagen type I as described in Example 31. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment.

Table 24. Number of clones binding to collagen type I and type III after one round of selection as described in Example 32.

Table 25. Immunization scheme for human serum albumin according to example 41.

Table 26. Results after one and two rounds of panning on mouse serum albumin as described in Example 45.

Table 27. Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 46.

Table 28. IC50 values for bispecific nanobides against albumin and against vWF as described in Example 54.

Table 29. Sequences of the primers used for humanization of C37 as described in Example 64.

Table 30. Amino acid sequence listing of the peptides of the present invention and of human von Willebrand factor (vWF). The sequence of human vWF indicates A1 and A3 domains respectively in bold lettering.

Table 31. Results after two panning rounds on rA1 domain of vWF as described in Example 66.

Table 32. ELISA analyses of selected clones for binding to rA1 and vWF as described in Example 67. 81 Table 1: Immunization scheme used for llama 002 according to Example 1.

Llama002 vWF Collagen Collagen Day of immunization Type 1 Type III 0 100 μ9 100 μg 100 μ9 7 ^00 μg 100 μ9 100 μ9 14 50 μg 50 μg 50 μg 21 50 50 μ9 50 μ 28 50 μ9 50 μg 50 μ9 50 μ9 50 μ9 50 μθ Table 2: Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in example 4. Pfu vWF (antigen) divided by pfu casein (a specific binding) = enrichment. round Pfu vWF Pfu casein Enrichment First 1 x 10' 2.5 χ 0δ 40 Second 5 x 10" 2.5 x 106 200 Table 3: Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 5. round Number of inhibitors versus number of clones tested First 4/800 Second 4/96 Table 4: Yield (mg/liter culture) after expression and purification of VHH grown in WK6 E.coli cells as described in Example 6.

Name VHH Yield (mg/liter culture) 22-2L-34 1.4 82 T76 2.9 AM-4-15-3 2.2 22-4L-16 2.8 C37 3.8 AM-2-75 3.6 Table 5: OD 405 nm for binding of VHH in ELISA to vWF and 3 antigens that were also immunized in Ilama002 according to Example 8.

OD405 nm vWF Antigen 1 Antigen 2 Antigen 3 nM 670 67 6.7 670 67 6.7 670 67 6.7 670 67 6.7 T76 0.13 0.05 0.05 0.05 0.06 0.06 0.04 0.04 0.04 0.03 22-2L-34 m ipf 0.06 0.0 0.10 0.10 0.07 0.05 0.06 0.05 0.03 22-4L-16 0.08 0.10 0.11 0.15 0.11 0.05 0.08 0.07 0.03 C37 Hfmm* 0.10 0.10 0.12 0.12 0.11 0.08 0.10 0.08 0.06 AM-2-75 ¾« 0.09 0.11 0.12 0.14 0.11 0.09 0.10 0.13 0.05 AM-4-15-3 in m 0.09 0.12 0.12 0.12 0.11 0.10 0.10 0.10 0.08 Table 6: Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 9.

Name VHH IC50 (nM) human IC50 (nM) undiluted plasma 1/60 human plasma 22-2L-34 10 - T76 30 - AM-4-15-3 7 200 22-4L-16 4 000 C37 3 - AM-2-75 2 100 83 Table 7: Epitope mapping of VHH binding to vWF and inhibiting the interaction with collagen as described in Example 11.

Name VHH Binding to A3 domain of vWF 22-2L-34 Yes T76 No 22-4L- 6 No C37 Yes AM-2-75 Yes Table 8: Yields of purified protein (mg) per liter of culture for bivalent and bispecific VHHs as described in Example 12.

NH2-terminal VHH COOH-terminal VHH Yield mg/liter culture AM-2-75 AM-4-15-3 3.2 AM-4-15-3 AM-4-15-3 2.3 AM-4-15-3 AM-2-75 4.0 AM-2-75 AM-2-75 1.0 AM-2-75 22-4L-16 3.0 Table 9: IC50 values for monovalent as compared to bivalent and bispecific VHHs. Inhibition was tested with human, pig and baboon plasma as described in Example 14.

First VHH Second IC50 (ng/ml) IC50 (ng/ml) baboon IC50 (ng/ml) pig VHH human plasma plasma plasma AM-2-75 150 400 50 AM-4-15-3 50 200 40 22-4L-16 15 70 7 AM-2-75 AM-4-15-3 3 5 6 AM-4-15-3 AM-2-75 2 8 3 AM-4-15-3 AM-4-15-3 5 10 7 AM-2-75 22-4L-16 8 20 10 AM-2-75 AM-2-75 5 84 Table 10: Inhibition of platelet aggregation at high shear (1600 s' ) as described in Example 16.

Concentration % inhibition [μθ/ηΙ] AM-2-75 0.2 0 A -2-75 0.3 12 AM-2-75 0.4 56 AM-2-75 0.6 97 AM-2-75 0.8 96 A -4-15-3 0.05 0 A -4-15-3 0.1 75 AM -4-15-3 0.25 74 AM-4- 5-3 0.5 86 AM-4-15-3 1 91 22-4L-16 0.1 32 22-4L-16 0.5 54 22-4L-16 0.75 86 22-4L- 6 2 97 22-4L- 6 10 99 AM-4-15-3/AM-4-15-3 0.05 0 AM-4-15-3/AM-4-15-3 0.075 23 AM-4-15-3/AM-4- 5-3 0.1 37 AM-4-15-3/AM-4-15-3 0.15 56 AM-4-15-3/AM-4-15-3 0.2 98 AM-4-15-3/AM-4-15-3 1.9 100 AM-4-15-3/AM-2-75 1.9 100 AM-2-75/AM-4-15-3 0.05 2 AM-2-75/AM-4- 5-3 0.1 36 AM-2-75/AM-4-15-3 0.2 96 AM-2-75/AM-4-15-3 0.35 91 85 AM-2-75/AM-4-15-3 0.4 98 AM-2-75/AM-2-75 0.04 5 AM-2-75/AM-2-75 0.1 26 AM-2-75/A -2-75 0.2 52 AM-2-75/AM-2-75 0.3 80 AM-2-75/AM-2-75 0.4 99 AM-2-75/AM-2-75 0.83 100 A -2-75/22-4L-16 1.17 99 Table 11: Inhibition of platelet aggregation at low shear (300 s" ) as described in Example 16.

Concentration % inhibition [ 9 πιΙ] AM-2-75 10 20 AM-4-15-3 10 17 22-4L-16 10 22 AM-4-15-3/AM-4-15-3 10 23 AM-4-15-3/A -2-75 10 21 A -2-75/AM-4-15-3 10 18 AM-2-75/AM-2-75 2 32 AM-2-75/22-4L-16 10 13 Table 12: Plaque forming units (pfu) after one round of panning on vWF as described in Example 17. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment.

Pfu vWF Pfu casein Enrichment 1.5 x 107 1 x 104 1.500 Table 13: Results of screening in ELISA of individual colonies for binding to vWF and to the A1 domain of vWF as described in Example 18.

No. clones +ve for vWF / No. tested No. clones +ve for A1 / No. tested 344/380 5/570 Table 14: Results after one round of MATCHM on pBAD-Oprl-A1 cells as described in Example 19.

Round No. clones +ve for vWF / No. tested No. clones +ve for A1 / No. tested First 1/96 second 45/348 12/348 Table 15: Inhibition of platelet aggregation at high shear (1600 s"1) as described in Example 23.

Concentration % inhibition ^g/ml] 2A1-4L-129 13.5 26 2A1-4L-129 20 50 2L-A1-15 9.7 30 2L-A1-15 25 45 A50 10.2 20 2A1-4L-79 11.1 20 2A1-4L-34 11.1 3 Z29 10.6 0 I53 9.7 0 M53 10.6 0 Table 16: Inhibition of platelet aggregation at low shear (300 s'1) as described in Example 23.

Concentration % inhibition g/mil 2A1-4L-129 10 0 2L-A1-15 10 3 A50 25 0 2A1-4L-79 25 15 87 Table 17: Inhibition of platelet aggregation at high shear (1600 s"1) as described in Example 25.

Concentration % inhibition [μ9/ηΊΐ] 2A1-4L-79/2A1-4L-79 25 54 2LA1 -15/2LA1-15 25 45 Table 18: Inhibition of platelet aggregation at low shear (300 s'1) as described in Example 25.

Concentration % inhibition [ g/mi] 2A1-4L-79/2A1-4L-79 25 0 2LA1-15/2LA1-15 25 23 Table 19: Yields after expression and purification of bispecific constructs as described in Example 27.

NH2 terminal VHH COOH-terminal VHH Yield mg/liter culture 2A1-4L-79 AM-4-15-3 7.5 2A1-4L-79 AM-2-75 2 2A1-4L-79 22-4L-16 2.5 Table 20: IC50 values for bispecifici nanobodies for the A1 and A described in example 29.

NH2-terminal VHH COOH-terminal VHH IC50 (ng/ml) 2A1-4L-79 AM-4-15-3 10 AM-4-15-3 - 45 2A1-4L-79 AM-2-75 12 AM-2-75 - 40 2A1-4L-79 22-4L-16 10 22-4L-16 - 10 2A1-4L-79 - >10000 88 Table 21: Inhibition of platelet aggregation at high shear (1600 s"1) as described in Example 30.

Concentration % inhibition [ 9 ΓηΙ] 2A1-4L-79/A -4-15-3 12 00 2A1-4L-79/AM-2-75 0.02 0 2A1-4L-79/AM-2-75 0.1 28 2A1-4L-79/AM-2-75 0.5 79 2A1-4L-79/AM-2-75 1 95 2A1-4L-79/22-4L-16 12 96 Table 22: Inhibition of platelet aggregation at low shear (300 s'1) as described in Example 30.

Concentration % inhibition r^g/mi] 2A1-4L-79/AM-4-15-3 10 15 2A1-4L-79/A -2-75 10 25 2A1-4L-79/22-4L- 6 10 27 Table 23: Plaque forming units (pfu) after one round of panning on collagen type I as described in Example 31. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment.

Phages eluted from collagen type I 5 x 106 Phages eluted from casein 4 x 104 Enrichment 100 89 Table 24: Number of clones binding to collagen type I and type III after one round of selection as described in Example 32.

Collagen Type 1 15/32 Collagen Type III 7/32 Casein 0/32 Table 25: Immunization scheme for human serum albumin according to Example 41.

Day of immunization HSA LlamaOOe 0 100 7 100 IQ 14 50 iig 21 50 ixg 28 50 μ9 50 HQ Table 26: Results after one and two rounds of panning on mouse serum albumin as described in Example 45.

First round Second round Pfu mouse serum albumin 2.5 x 10' 2.5 x 10' Pfu casein 5 x 103 2.5 x 103 Enrichment 5.000 10.000 Table 27: Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 46.

First round Second round ELISA mouse serum albumin 1/16 15/16 ELISA human serum albumin 1/16 15/16 ELISA casein 0/16 0/16 90 Table 28: IC50 values for bispecific nanobides against albumin and against vWF as described in Example 54.

IC50 (ng/ml) AM-2-75 100 SA21/AM-2-75 60 AM -4-15-3 155 MSA21/AM-4-15-3 245 22-4L-16 100 MSA21/22-4L-16 140 Table 29: Sequences of the primers used for humanization of C37 as described in Example 64.

Mutation Template Primer sequence A74S+N75 Wild 5 ' -AGA GAC AAC TCC AAG AAC ACG CTG TAT CTG CAA K+P84A type ATG AAC AGC CTG AGA GCT GAG GAC ACG-3' (SEQ ID N° 76) Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr (SEQ ID N° 77) A74S+N75 A74S+N75 5 '-AT TAC TGT GCT AAA GGG GCC GGT ACT AGT T-3' K+P84A+R K+P84A (SEQ ID N° 78) 94 Tyr Cys Ala Lys Gly Ala Gly Thr Ser (SEQ ID N° 79) N28T+N30 A74S+N75 5' -TCC TGT GCA GCC TCC GGA TTC ACT TTC AGT TGG S K+P84A+R TA-3' (SEQ ID N° 80) A74S+N75 94K Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp K+P84A+R (SEQ ID N° 81) 94K Table 30: Amino acid sequence listing of the peptides of the present invention and of human von Willebrand factor (vWF). The sequence of human vWF indicates A1 and A3 domains respectively in bold lettering.

NAME SEQ SEQUENCE ID NO 91 C37 1 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYP S VRQAPG GLEWVSTIS TYGEPRYADSVKGRFTISRDNAN TLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTISS C37- 2 QVQLQESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTIS hum TYGEPRYADSVKGRFTISRDNSK TLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS AM-2- 3 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPG GLEWVSTIS 75 TYGEPRYADSV GRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTVSS 22-2L- 4 QVQLQDSGGGLVQAGGSLRLSCAASVRIFTSYA GWFRQAPGKEREFVAAIN 34 RSG STYYSDSVEGRFTISRDNAKNTVSLQMDSLKLEDTAVYYCAADYSGSY TSLWSRPERLDWGQGTQVTVFS 22-4L- 5 QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAIS 16 WSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVADTGGIS WI TQGYNYWGQGTQVTVSS T76 6 QVQLQESGGGLVQPGESLRLSCAASGSIFSINT GWYGQAPGKQRELVASIT FGGVTINT-TADSVKGRFTISRDNTODTVYLQ NSLKPEDTAVYICNAVTWGGLT NYWGQGTQVTVSS.

AM-4- 7 QVQLQDSGGGLVQPGGSLRLACAASGSIFSINSMGWYRQAPG QRELVAHAL 15-3 ADGSASYRDSVKGRFTISRDNA NTVYLQM SLKPEDTAVYYCNTVPSSVTK GYWGQGTQVTVSS --A¾ AM-4- 8 QVQLQDSGGGLVQPGGSLRIIACAASGSIFSINSMGWYRQAPGKQRELVAHAL 15- ADGSASYRDSVKGRFTISRDNAK TVYLQ NSLKPEDTAVYYCNTVPSSVTK 3/AM- GYWGQGTQVTVSSEPKTP PQPAAAQVQLQDSGGGLVQPGGSLRLACAASGS 4-15-3 IFSINSMGWRQAPG QRELVAHALADGSASYRDSVKGRFTISRDNA NTVY LQMNSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS AM-4- 9 QVQLQDSGGGLVQPGGSLRLACAASGSIFSINS GWYRQAPG QRELVAHAL 15- ADGSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTK 3/AM- GY GQGTQVTVSSEP TPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGF 2-75 NFNWYP SWWQAPGKGLEWVSTISTYGEPRYADSVKADSPSSETTPTTRCI CNEQPETEDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS AM-2- 10 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYP SWVRQAPGKGLEWVSTIS 75/AM- YGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY 4-15-3 LPQRGNWDQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSLRLACA ASGSIFSINSMGWYRQAPGKQRELVAHALADGSASYRDS KGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCNTVPSSVT GYWGQGTQVTVSS A -2- 11 QVQLQESGGGLVQPGGSLRLSCLAASGFNFNWYPMSWVRQAPGKGLEWVSTIS 75/A - YGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY 2-75 LIPQRGNWDQGTQVTVSSQVQLQESGGGLVQPGGSLRLSCAASGFNF WYP S WWQAPG GLEWVSTISTYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRP EDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS A -2- 12 QVQLQESGGGLVQPGGSLRLSCAASGF FNWYP SWVRQAPG GLEWVSTIS 75/22- TYGEPRYADSVKGRFTISRDNA¾NTLYLQ NSLRPEDTAVYYCARGAGTSSY 4L-16 LPQRGNWDQGTQVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSLRLSCA ASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS Anti-vWF VHH + ainti-mouse serum .al iOTxn,VHH . ;,/■·., ·',': · 92 SA21/ 13 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS AM-2- SLGDSTLYADSVKGRFTSRDNAK TLYLQMNSL PEDTAVYYCTIGGSLNPG 75 GQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFNFN WYPMSWVRQAPGKGLEWVSTISTYGEPRYADSV ADSPSSETTPTTRCICNE QPETEDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS MSA21/ 14 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVE VSGIS AM-4- SLGDSTLYADSV GRFTSRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPG -3 GQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSLRIiACAASGSIFS INSMG YRQAPG QRELVAHALADGSASYRDSVKGRFTISRDNA NTVYLQM NSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS MSA21/ 15 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTVfVRQAPGKGVEWVSGIS 22-4L- SLGDSTLYADSV GRFTSRDNAKNTLYLQM SLKPEDTAVYYCTIGGSI-NPG 16 GQGTQVTVSSEP TPKPQPAAAQVQLVESGGGLVQAGGSLRLSCAASGRTFS SYAMG FRQAPGKEREFVAAISWSGGSTY ADSVKGRF ISRDNAKNTVYLQ MNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS MSA21 16 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS SLGDSTLYADSVKGRFTISRDNAK TLYLQMNSLKPEDTAVYYCTIGGSLNP GGQGTQVTVSS SA24 17 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWVSSIS GSGSNTIYADSVKDRFTISRDNAKSTLYLQM SLKPEDTAVYYCTIGGSLSR SSQGTQVTVSS SA210 18 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWVSAIS SDSGTKNYADSVKGRFTISRDNAKKMLFLQM SLRPEDTAVYYCVIGRGSPS SQGTQVTVSS MSA212 19 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWVSAIS ADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPA SQGTQVTVSS MSAclS 49 AVQLVESGGGLVQAGDSLRLSCWSGTTFSSAAMGWFRQAPGKEREFVGAIK WSGTSTYYTDSVKGRFTISRDNVKNTVYLQMNNLKPEDTGVYTCAADRDRYR DRMGPMTTTDFRFWGQGTQVTVSS MSAc11 50 QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGWFRQAPGREREFVASIG SSGITT YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGLCYCAVNRYGIP 2 YRSGTQYQNWGQGTQVTVSS MSAcll 51 EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGWYRQAPGKERDMVATIS IGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCVAHRQTWR 0 GPYLLWGQGTQVTVSS MSAcll 52 QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAGSG RSNSYNYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASTNLWP 4 RDRNLYAYWGQGTQVTVSS MSAcll 53 EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMG FRQVPGKEREFVAAIS WSGGTTRYLDSVKGRFTISRDSTKNAVYLQMNSLKPEDTAVYYCAVDSSGRL 6 YWTLSTSYDYWGQGTQVTVSS 93 MSAc11 54 QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIY MA FRQVPG EREFVAAIS WSGGTTRYIDSV GRFTLSRDMTK MVYLQMNSLKPDDTAVYYCAVDSSGRL 9 YWTLSTSYD WGQGTQVTVSS MSAcl5 55 EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGWFRQAPGKEREFLAGVT WSGSSTFYGDSVKGRFTASRDSAK TVTLEMNSLNPEDTAVYYCAAAYGGGL YRDPRSYDYWGRGTQVTVSS MSclll 56 AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAWFRQAPGKEREGVSCIR DGTTYYADSV GRF ISSDNANNTVYLQTNSLKPEDTAVYYCAAPSGPATGS SHTFGI LRDDYDNWGQGTQVT SS MSAcll 57 EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGWFRQVPGKEREGVSCIS SSDGSTYYADSVKGRFTISSDNAK TVYLQMNTLEPDDTAVYYCAAGGLLLR VEELQASDYDYWGQGIQVTVSS MSAC18 58 AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGWFRQAPGKEREGVACIS NSDGSTYYGDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCATADRHYS ASHHPFADFAFNSWGQGTQVTVSS MSAcl7 59 EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMA FRRAPGKERELVVARN WGDGSTRYADSVKGRFTISRDNAKNTVYLQMNSL PEDTAVYYCAAVRTYGS ATYDI GQGTQV VSS MSAC12 60 EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYA GWFRQAPGKEREFVAAIN RNGGTTNYADALKGRFTISRDNT NTAFLQM SLKPDDTAVYYCAAREWPFS 0 TIPSGWRYWGQGTQVTVSS SAcl4 61 DVQLVESGGG VQPGGSLRLSCAASGPTASSHAIGWFRQAPGKEREFWGIN RGGVTRDYADSVKGRFAVSRDWK TVYLQMNRLKPEDSAIYICAARPEYSF TAMSKGDMDYWGKGTLVTVSS 2A1- 20 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNGKNTVYLQM NLKPEDTAVYYCAARSSAFSS 79/AM- GIYYREGSYAYWGQGTQVTVSSEP TPKPQPAAAQVQLQDSGGGLVQPGGSL 4-15-3 RLACAASGSIFSINSMGWYRQAPGKQRELVAHALADGSASYRDSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS 2A1- 21 QVQLQDSGGRLVKAGASLRLSCAASGRTFSS IiPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNG N1VYLQMMNLKPEDTAVYYCAARSSAFSS 79/AM- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL 2-75 RLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKADSPSS ETTPTTRCICNEQPETEDTAVYYCARGAGTSSYLPQRGN DQGTQVTVSS 2A1- 22 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTL TSSVRGRFTISRDNGNTVYLQMMiTLKPEDTAVYYClAARSSAFSS 79/22- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSL 4 RLSCAASGRTFSSYAMGWFRQAPG EREFVAAISWSGGSTYYADSV GRFTI L-16 SRDNA TVYLQ NSLKPEDTAVYYC ADTGGIS IRTQGYNYWGQGTQVTV SS A50 23 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAAIS RRGDNVYYADSVKGRFAISRDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSA ITLSTSTYDYWGQGTQVTVSS 153 24 QVQLQDSGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSD GSTLVAASVKGRFTISRDNAKNTVYLQMTSL PADTAVYYCATSRGYSGTYY STSRYDYWTGGTQVTVSS Z29 25 QVQLQESGGGSVQAGDSLTLSCAASGRTFSMHAMG FRQAPGKEREFVAAIS 94 PSAFTEYADSLKGRFTVSRDNAKKLVWLQ NGL PEDTAAYYCAARRGAPTA TTAPLYDYWGQGTQVTVSS M53 26 QVQLQDSGGGLVQAGESLRLSCGTSGRTFGRRA AWFRQAPGKERQFVAWIA RYDGSTLYADSV GRFTISRDDNKNT YLHM LTPEDTAVYYCAAGPRGLY YESRYEYWGQGTLVTVSS 2A1- 27 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLP AWFRQAPGKEREFVAFIG 4L-79 SDSSTLYTSSVRGRFTISRDNGKNTVYLQ MNLKPEDTAVYYCAARSSAFSS GIYYREGSYAYWGQGTQVTVSS 2A1- 28 QVQIJQESGGGLVQAGASLRLSCAASGRSFSSYPI^AWFRQAPGKEREFWFIG 4L-129 SDHSTLYSTSVRGRFTISRDNA NTVYLQ M LKPEDTAVYYCAARNSAWSS GIYYRETSYDYWGQGTQVTVSS 2A1- 29 QVQLQDSGGGSVQAGASLRLSCAASGGTFSSYAMAWFRQAPGKEREFVGFIG 4L-34 SDGSTLYSSSVRGRFTISRDNAKMTVALQMMNLKPEDTAVYYCAARARYSGI YYRETDYPYWGQGTQVTVSS 2A1- 30 QVQLQESGGGLVQAGASLRLSCTASGRSFGGFPMGWFRQAPGKEREFVSGLT 4L-78 RSLFTVYADSV GRFTVSTDNTKNTVYLQMNSLKPEDTAVYYCAARPDLYAY SRDPNEYDYWGQGTQVTVSS 2LA1- 31 QVQLQDSGGGLVQSGGSLRLACAASGRIVSTYAMGWFRQSPGKEREFVATVK 15 GRFTISRDNAK TLYLQMNSLKPSDTAVYYCAKTKRTGIFTTARMVDYWGQG TQVTVSS 2A1- 32 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSTOGRFTISRDNGI^TVYLQ NLKPEDTAVYYCAARSSAFSS 79/2A1 GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGRLVKAGASL -4L-79 RLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRFTIS RDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQV TVSS 2LA1- 33 QVQLQDSGGGLVQSGGSLRLACAASGRIVSTYAMGWFRQSPGKEREFVATVK /2LA GRFTISRDNAK TLYLQ NSLKPEDTAVYYCAKTKRTGIFTTARMVDYWGQG 1-15 TQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQSGGSLRLACAASGRIVSTYA MGWFRQSPGKEREFVATVKGRFTISRDNAK TLYLQMNSLKPEDTAVYYCAK TKRTGIFTTARMVDYWGQGTQVTVSS A50/A5 34 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAAIS 0 RRGDNVYYADSVKGRFAISRDNAESTLYLQ SLKPEDTAVYYCAAHVTVSA ITLSTSTYDYWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSLR LSCAASGRTFSSYRMGWFRQAPGKEREFVAAISRRGDNVYYADSVKGRFAIS RDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSAITLSTSTYDYWGQGTQVTV SS 3P1-31 35 QVQLQESGGGLVQAGGSLRLSCAASGRTFRRYA GWYRQAPGKQRELVAAIT SGGRTSVADTV GRFTI sSDNA NTVYLQMNSLKPEDAAVYYCTLYNSTTNY YNQSPSSWGQGTQVTVSS 3L-41 36 · QVQLQDSGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRVLVAAIT SNGRPSVADSVKGRFTISSDTAK TVYLQM SL PEDTALYYCTLY TSADY Y QSPSSWGQGTQVTVLS 3P2-31 37 QVQLQESGGGLVQAGDSLRLSCAASGRTFT GWFRQAPGKERQFVAALTWTG GSPVYADSVKGRFTTWRVLDIMTVYLH NSLKPEDTAVYHCAAARTYYGNIS EYYDYWGQGTQVTVSS Anti-yWE · VHH! humanized ' ; .····'· 'Vr'''-' · \ ";'·'',.'* ".'.;,' ·■"' " '"' "viu-i" ' :·ί 95 C37-3 38 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS TyGEPRYADSVKGRFTISRDNS NTLYLQMNSLRAEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTISS C37-4 39 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMS VRQAPGKGLE VSTIS TYGEPRYADSVKGRFTISRDNS TLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS C37-8 40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS C37-10 41 EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYP S VRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA GAGTSSY LPQRGNWDQGTLVTVSS MSA21/ 42 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS C37- SLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNP hum GGQGTQVTVSSEP TPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTF SWYP SWVRQAPGKGLEWVSTISTYGEPRYADSV GRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MSA24/ 43 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFG SWVRQAPGKEPEWVSSIS C37- GSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSR hum SSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTF SWYP SWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MS 210 44 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLE VSAIS / C37- SDSGTKNYADSVKGRFTISRDNAKK LFLQMNSLRPEDTAVYYCVIGRGSPS hum SQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTFS WYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNS TLYLQM NSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MSA212 45 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFG SWVRQAPGKGLEWVSAIS / C37- ADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSL PEDTAVYYCVIGRGSPA hum SQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTFS YP SWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSK TLYLQM NSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS ^!Anfci-collageri; Vipit':; bisvebifap^Xy ·¾ ,,■,' ' u¾ ¾;¾ ;; : -: .\i · :; 3P1- 46 QVQLQESGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRELVAAIT 31/3P2 SGGRTSVADTVI03RFTISSDNAKNTVYLQMNSLKPEDAAVYYCTLYNSTTNY -31 YNQSPSSWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGDSLRLSC AASGRTFTMGWFRQAPGKERQFVAALTWTGGSPVYADSVKGRFTT RVLDNN TVYLH NSLKPEDTAVYHCAAARTYYGNISEYYDYWGQGTQVTVSS 3L- 47 QVQLQDSGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPG QRVLVAAIT 41/3P2 SNGRPSVADSV GRFTISSDTAKNTVYLQMNSLKPEDTALYYCTLY TSADY -31 Y QSPSSWGQGTQVTVLSEPKTPKPQPAAAQVQLQESGGGLVQAGDSLRLSC AASGRTFT GWFRQAPG ERQFVAALT TGGSPVYADSVKGRFTTWRVLDN 96 TVYLHMNSLKPEDTAVYHCAAARTYYGNISEYYDYWGQGTQVTVSS ;Conformation- s ec^ VHH ·' ..·. ... , \ :^<^: '> All 62 BVQLVESGGRLVKAGASLRLSCAASGRTFSSLP AWFRQAPGKEREFVAFIG SDSSTLYTSSVRGRFTISRDNGKNTVYLQMM L PEDTAVYYCAARSSAFSS GIYYREGSYAYWGQGTQVTVSS A12 63 QVQLVESGGGLVQAGGSLRLSCTASGRTFSTYALGWFRQVPGKGREFIAVIY WRDGSSLYSDSVKGRFTISKDNAK TVYLQMNSLKPEDTAVYYCANRHDSRG TYYSSRGYDYWGQGTQVTVSS A13 64 QVQLVESGGGLVQAGGSLRLSCAASGRTKD AWFRQPPGKEREFVAVIYSSD GSTLVAASVKGRFTISRDNAKNTVYLQMTSLKPADTAVYYCATSRGYSGTYY STSRYDYWGQGTQVTVSS A15 65 QVQLVESGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSD GSTLVAASVTGRFTISRDNAKNMVYLQMTSLKPADTAVYYCASSRGYSGTYY STSRYDYWGQGTQVTVSS Human 48 MIPARFAGVII-ALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYS vWP FAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFV GTVTQ GDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFN KTCGLCGNFNIFAEDDF TQEGTLTSDPYDFANSWALSSGEQWCERASPPSS SCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCE TLCECAGG LECACPALLEYARTCAQEG VLYGWTDHSACSPVCPAGMEYRQCVSPCARTC QSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLS RDCNTCICRNSQ ICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLAR DCQDHSFSIVIEWQCADDRDAVCTRSVTVRLPGLH SLVKLKHGAGVAMDG QDIQLPLL GDLRIQHTVTASVRLSYGEDLQMD DGRGRLLV LSPVYAGKT CGLCGNY GNQGDDFLTPSGI-AEPRVEDFGNAW LHGDCQDLQKQHSDPCAL NPR TRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCG AliASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPD EEOJEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTM CYCEDGF HCT SGVPGSLLPDAVLSSPLSHRSKRSLSCRP MVKLVCPADN LRAEGLECTKTCQNYDLECMS GCVSGCLCPPGMVRHENRCVALERCPCFHQ GKEYAPGETVKIGC TCVCRDR WNCTDHVCDATCSTIGMAHYLTFDGLKYL FPGECQYVLVQDYCGSNPGTFRILVGN GCSHPSVKCKKRVTILVEGGEIEL FDGEVWVKRPMKDETHFEWESGRYIILLLG ALSWWDRHLSISWLKQTY QEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSW VSSQCADTR VPLD SSPATCH NIM QTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCE SIGDCACFCDTIAAYAHVCAQHGKWTVJRTATLCPQSCEER LRENGYECEW RYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDC PVCEVAGRRFASGKKVTLNPSDPEHCQICHCDWiJLTCEACQEPGGLWPPT DAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFV VD^ERLRISQ WVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGS QVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQR SRNFVRYVQGL KKK VIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSY LCDLAPEAPPPTLPPHMAQVTVGPGLRNSMVLDVAFVLEGSDKIGEADF RS KEFMEEVIQR DVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIR YQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVY VTGNPASDEIKRLPG DIQWPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGE GLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFA AFISKANIGP RLTQVSVLQYGSITTIDVPWIIVVPEKAHLLSLVDVMQREGGPSQIGDALGFA VRYLTSEMHGARPGAS AWILVTDVSVDSVDAAADAARSNRVTVFPIGIGD 97 169068/2 RYDAAQLRILAGPAGDSNVVKlQRIEDLPTMVTLGNSFLHKIjCSGiVRICMD EDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSP VKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLE VILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGN MEVNVYGAIMHEVRFNHLGHIF FTPQNNEFQLQLSPK FASK YGLCGICD ENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQV LLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDW RTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPD VMLE GSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCP TAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLT NPGECRPNFTCACR EECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCV S TVSCPLGYLASTATNDCGCTTTTCLPD VCVHRSTIYPVGQF EEGCDVCTC TDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEWTGS PRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVC PSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRC VQV GVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTL KRDETLQDGCDTHFCKV ERGEYF EKRVTGCPPFDEHKCLAEGGKIMKIPG TCCDTCEEPECNDITARLQYV VGSCKSEVEVDIHYCQGKCASKAMYSIDIN DVQDQCSCCSPTRTEPMQVALHCTNGSWYHEVLNAMECKCSPR CSK Table 31 : Results after two panning rounds on rA1 domain of vWF as described in Example 66 First library Second library Third library Pfu rA1 1 x 10B 2 x 107 4 x 108 Pfu casein 2 x 104 2 x 10* 2 x 104 Enrichment 5.000 1.000 200.000 Table 32: ELISA analyses of selected clones for binding to rA1 and vWF as described in example 67 First library Second library Third library ELISA rA1 54/64 51/64 49/64 ELISA vWF 36/64 35/64 33/64 ****** All passages of the description which are outside the scope of the claims do not constitute part of the claimed invention. 98 169068/5 SEQUENCE LISTING <110> ABLYNX N . V . <120> Therapeutic polypeptides, homologues thereof, fragments thereof and for use in modulating platelet-mediated aggregation <130> ABL-005-PCT3 <140> PCT/BE2004/000002 <141> 2004-01-09 <150> EP 03447005.4 <151> 2003-01-10 <150> PCT/EP03/06581 <151> 2003-06-23 <150> PCT/EP03/07313 <151> 2003-07-08 <150> PCT/BE03/00193 <151> 2003-11-07 <150> PCT/BE03/00189 <151> 2003-11-07 <150> PCT/BE03/00190 <151> 2003-11-07 <150> PCT/BE03/00192 <151> 2003-11-07 <150> PCT/BE03/00194 99 169068/5 <151> 2003-11-07 <150> PCT/BE03/00206 <151> 2003-12-01 <150> PCT/BE03/00191 <151> 2003-12-02 <160> 85 <170> Patentin version 3.1 <210> 1 <211> 121 <212> PRT <213> Lama glama <400> 1 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin Val Thr lie Ser ser 115 120 100 169068/5 <210> 2 <211> 121 <212> PRT <213> Lama glama <400> 2 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 25 30 Pro Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gly Ala Gly Thr ser ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr lie Ser ser 115 120 <210> 3 <211> 121 <212> PRT <213> Lama glama <400> 3 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 101 1 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Gys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin Val Thr val Ser Ser 115 120 <210> 4 <211> 126 <212> PRT <213> Lama glama <400> 4 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser al Arg lie Phe Thr Ser Tyr 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Ala lie Asn Arg Ser Gly Lys Ser Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Ser 65 70 75 80 Leu Gin Met Asp Ser Leu Lys Leu Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Ala Ala Asp Tyr Ser Gly Ser Tyr Thr Ser Leu Trp ser Arg Pro Glu 100 105 110 Arg Leu Asp Trp Gly Gin Gly Thr Gin Val Thr Val Phe Ser 115 120 125 <210> 5 <211> 124 102 169068/1 <212> PRT <213> Lama glama <400> 5 Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Ala lie Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ala Asp Thr Gly Gly lie Ser Trp lie Arg Thr Gin Gly Tyr Asn 100 105 110 Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 <210> 6 <211> 117 <212> PRT <213> Lama glama <400> 6 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu ser cys Ala Ala Ser Gly Ser lie Phe Ser lie Asn 25 30 Thr Met Gly Trp Tyr Gly Gin Ala Pro Gly Lys Gin Arg Glu Leu val 40 45 Ala Ser lie Thr Phe Gly Gly val Thr Asn Tyr Ala Asp Ser val Lys 50 55 60 103 169068/1 Gly Arg Phe Thr lie Ser Arg Asp Asn Thr Asn Asp Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr lie Cys Asn 85 90 95 Ala val Thr Trp Gly Gly Leu Thr Asn Tyr Trp Gly Gin Gly Thr Gin 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 117 <212> PRT <213> Lama glama <400> 7 Gin Val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser lie Phe Ser lie Asn 25 30 Ser Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val 40 45 Ala H s Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr cys Asn 85 90 95 Thr Val Pro Ser ser val Thr Lys Gly Tyr Trp Gly Gin Gly Thr Gin 100 105 110 val Thr val ser Ser 115 <210> 8 <211> 246 <212> PRT <213> Lama glama 104 169068/1 <400> 8 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser lie Phe Ser lie Asn 25 30 Ser Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val 40 45 Ala H s Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Asn 85 90 95 Thr Val Pro Ser ser Val Thr Lys Gly Tyr Trp Gly Gin Gly Thr Gin 100 105 110 val Thr val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala 115 120 125 Ala Gin Val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Pro Gly 130 135 140 Gly Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser lie Phe Ser lie 145 150 155 160 Asn Ser Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu 165 170 175 val Ala His Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser val 180 185 190 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr 195 200 205 Leu Gin Met Asn ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 210 215 220 Asn Thr Val Pro Ser Ser val Thr Lys Gly Tyr Trp Gly Gin Gly Thr 225 230 235 240 Gin val Thr val Ser ser 245 105 169068/1 <210> 9 <211> 249 <212> PRT <213> Lama glama <400> 9 Gin Val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser lie Phe ser lie Asn 25 30 Ser Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val 40 45 Ala His Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Asn 85 90 95 Thr Val Pro Ser Ser val Thr Lys Gly Tyr Trp Gly Gin Gly Thr Gin 100 105 110 Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala 115 120 125 Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 130 135 140 Gly Ser Leu Arg Leu Ser Cys Ala Ala ser Gly Phe Asn Phe Asn Trp 145 150 155 160 Tyr Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 Val Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val 180 185 190 Lys Ala Asp Ser Pro Ser Ser Glu Thr Thr Pro Thr Thr Arg Cys lie 195 200 205 Cys Asn Glu Gin Pro Glu Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 106 169068/1 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 225 230 235 240 Gin Gly Thr Gin val Thr val Ser ser 245 <210> 10 <211> 250 <212> P T <213> Lama glama <400> 10 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr val ser Ser Glu Pro Lys Thr Pro Lys Pro 115 120 125 Gin Pro Ala Ala Ala Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu 130 135 140 Val Gin Pro Gly Gly Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser 145 150 155 160 lie Phe Ser lie Asn Ser Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys 165 170 175 Gin Arg Glu Leu al Ala His Ala Leu Ala Asp Gly Ser Ala Ser Tyr 180 185 190 107 169068/1 Arg Asp Ser val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys 195 200 205 Asn Thr val Tyr Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Asn Thr Val Pro Ser Ser Val Thr Lys Gly Tyr Trp 225 230 235 240 Gly Gin Gly Thr Gin val Thr val Ser Ser 245 250 <210> 11 <211> 242 <212> PRT <213> Lama glama <400> 11 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr Val Ser Ser Gin Val Gin Leu Gin Glu Ser 115 120 125 Gly Gly Gly Leu val Gin pro Gly Gly Ser Leu Arg Leu Ser cys Ala 130 135 140 Ala Ser Gly Phe Asn Phe Asn Trp Tyr Pro Met Ser Trp val Arg Gin 145 150 155 160 108 169068/1 Ala Pro Gly Lys Gly Leu Glu Trp val ser Thr lie Ser Thr Tyr Gly 165 170 175 Glu Pro Arg Tyr Ala Asp Ser val Lys Gly Arg Phe Thr lie Ser Arg 180 185 190 Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Pro 195 200 205 Glu Asp Thr Ala val Tyr Tyr cys Ala Arg Gly Ala Gly Thr Ser Ser 210 215 220 Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin Gly Thr Gin val Thr val 225 230 235 240 Ser Ser <210> 12 <211> 257 <212> PRT <213> Lama glama <400> 12 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro 115 120 125 109 169068/1 Gin Pro Ala Ala Ala Gin Val Gin Leu val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gin Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 145 150 155 160 Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys 165 170 175 Glu Arg Glu Phe val Ala Ala lie Ser Trp ser Gly Gly Ser Thr Tyr 180 185 190 Tyr Ala Asp ser val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala 195 200 205 Lys Asn Thr Val Tyr Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr 210 215 220 Ala val Tyr Tyr Cys val Ala Asp Thr Gly Gly lie Ser Trp lie Arg 225 230 235 240 Thr Gin Gly Tyr Asn Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser 245 250 255 Ser <210> 13 <211> 246 <212> PRT <213> Lama glama <400> 13 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser cys Glu Ala ser Gly Phe Thr Phe Ser Arg Phe 25 30 Met Thr Trp val Arg Gin Ala Pro Gly Lys Gly val Glu Trp val 40 45 Ser Gly lie Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 110 169068/1 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Thr 85 90 95 lie Gly Gly Ser Leu Asn Pro Gly Gly Gin Gly Thr Gin val Thr val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin Val 115 120 125 Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr Pro Met 145 150 155 160 Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val Ser Thr 165 170 175 lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys Ala Asp 180 185 190 Ser Pro Ser Ser Glu Thr Thr Pro Thr Thr Arg Cys lie Cys Asn Glu 195 200 205 Gin Pro Glu Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala 210 215 220 Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin Gly Thr 225 230 235 240 Gin val Thr val Ser Ser 245 <210> 14 <211> 243 <212> P T <213> Lama glama <400> 14 Gin val Gin Leu Gin Glu ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 25 30 Gly Met Thr Trp val Arg Gin Ala Pro Gly Lys Gly val Glu Trp val 40 45 111 169068/1 Ser Gly lie Ser ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Thr 85 90 95 lie Gly Gly Ser Leu Asn Pro Gly Gly Gin Gly Thr Gin val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin Val 115 120 125 Gin Leu Gin Asp ser Gly Gly Gly Leu Val Gin Pro Gly Gly ser Leu 130 135 140 Arg Leu Ala Cys Ala Ala Ser Gly Ser lie Phe Ser lie Asn Ser Met 145 150 155 160 Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu val Ala His 165 170 175 Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser val Lys Gly Arg 180 185 190 Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu Gin Met 195 200 205 Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Asn Thr val 210 215 220 Pro Ser Ser Val Thr Lys Gly Tyr Trp Gly Gin Gly Thr Gin Val Thr 225 230 235 240 val Ser ser <210> 15 <211> 250 <212> PRT <213> Lama glama <400> 15 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 112 169068/1 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 25 30 Gly Met Thr Trp val Arg Gin Ala Pro Gly Lys Gly val Glu Trp val 40 45 Ser Gly lie Ser Ser Leu Gly Asp ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 lie Gly Gly Ser Leu Asn Pro Gly Gly Gin Gly Thr Gin val Thr val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin Val 115 120 125 Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val Ala Ala 165 170 175 lie Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gin 195 200 205 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala 210 215 220 Asp Thr Gly Gly lie Ser Trp lie Arg Thr Gin Gly Tyr Asn Tyr Trp 225 230 235 240 Gly Gin Gly Thr Gin val Thr val Ser ser 245 250 <210> 16 <211> 115 <212> PRT <213> Lama glama 113 169068/1 <400> 16 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 25 30 Gly Met Thr Trp val Arg Gin Ala Pro Gly Lys Gly val Glu Trp val 40 45 Ser Gly lie Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr lie Gly Gly Ser Leu Asn Pro Gly Gly Gin Gly Thr Gin val Thr 100 ' 105 110 val Ser Ser 115 <210> 17 <211> 115 <212> PRT <213> Lama glama <400> 17 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala ser Gly Phe Thr Phe Arg Asn Phe 25 30 Gly Met Ser Trp val Arg Gin Ala Pro Gly Lys Glu Pro Glu Trp val 40 45 Ser Ser lie Ser Gly Ser Gly Ser Asn Thr lie Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 114 1 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr lie Gly Gly ser Leu Ser Arg Ser Ser Gin Gly Thr Gin val Thr 100 105 110 val Ser Ser 115 <210> 18 <211> 114 <212> PRT <213> Lama glama <400> 18 Gin al Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 25 30 Gly Met Ser Trp val Arg Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Ala lie Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 val He Gly Arg Gly Ser Pro Ser Ser Gin Gly Thr Gin val Thr val 100 105 110 Ser ser <210> 19 <211> 114 <212> PRT <213> Lama glama <400> 19 115 1 Gin al Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 25 30 Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala lie Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Lys Met Leu Thr 65 70 75 80 Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Val lie Gly Arg Gly Ser Pro Ala Ser Gin Gly Thr Gin Val Thr Val 100 105 110 Ser Ser <210> 20 <211> 255 <212> PRT <213> Lama glama <400> 20 Gin val Gin Leu Gin Asp Ser Gly Gly Arg Leu val Lys Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Phe lie Gly Ser Asp Ser Ser Thr Leu Tyr Thr Ser Ser val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr cys Ala 85 90 95 116 169068/1 Ala Arg Ser ser Ala Phe Ser Ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin Val Thr val Ser ser Glu Pro 115 120 125 Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val Gin Leu Gin Asp 130 135 140 Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ala Cys 145 150 155 160 Ala Ala Ser Gly Ser lie Phe Ser lie Asn Ser Met Gly Trp Tyr Arg 165 170 175 Gin Ala Pro Gly Lys Gin Arg Glu Leu al Ala His Ala Leu Ala Asp 180 185 190 Gly Ser Ala Ser Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr lie Ser 195 200 205 Arg Asp Asn Ala Lys Asn Thr val Tyr Leu Gin Met Asn Ser Leu Lys 210 215 220 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr Val Pro Ser Ser Val 225 230 235 240 Thr Lys Gly Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 245 250 255 <210> 21 <211> 258 <212> PRT <213> Lama glama <400> 21 Gin val Gin Leu Gin Asp ser Gly Gly Arg Leu val Lys Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Phe lie Gly Ser Asp Ser ser Thr Leu Tyr Thr Ser Ser Val Arg 50 55 60 117 169068/1 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Ala Arg Ser Ser Ala Phe Ser Ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Glu Pro 115 120 125 Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val Gin Leu Gin Glu 130 135 140 Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr Pro Met Ser Trp val Arg 165 170 175 Gin Ala Pro Gly Lys Gly Leu Glu Trp val Ser Thr lie Ser Thr Tyr 180 185 190 Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys Ala Asp Ser Pro Ser Ser 195 200 205 Glu Thr Thr Pro Thr Thr Arg Cys lie Cys Asn Glu Gin Pro Glu Thr 210 215 220 Glu Asp Thr Ala val Tyr Tyr Cys Ala Arg Gly Ala Gly Thr Ser Ser 225 230 235 240 Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin Gly Thr Gin val Thr val 245 250 255 Ser Ser <210> 22 <211> 262 <212> PRT <213> Lama glama <400> 22 Gin val Gin Leu Gin Asp Ser Gly Gly Arg Leu Val Lys Ala Gly Ala 1 5 10 15 118 169068/1 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Phe lie Gly Ser Asp ser ser Thr Leu Tyr Thr ser ser val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr cys Ala 85 90 95 Ala Arg Ser ser Ala Phe Ser ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin Val Thr val Ser Ser Glu Pro 115 120 125 Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin Val Gin Leu Val Glu 130 135 140 Ser Gly Gly Gly Leu val Gin Ala Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg 165 170 175 Gin Ala Pro Gly Lys Glu Arg Glu Phe val Ala Ala lie Ser Trp Ser 180 185 190 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser val Lys Gly Arg Phe Thr lie 195 200 205 Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gin Met Asn Ser Leu 210 215 220 Lys Pro Glu Asp Thr Ala Val Tyr Tyr cys val Ala Asp Thr Gly Gly 225 230 235 240 lie Ser Trp lie Arg Thr Gin Gly Tyr Asn Tyr Trp Gly Gin Gly Thr 245 250 255 Gin val Thr val Ser ser 260 <210> 23 <211> 125 119 169068/1 <212> PRT <213> Lama glama <400> 23 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 25 30 Arg Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Ala lie Ser Arg Arg Gly Asp Asn val Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala lie Ser Arg Asp Asn Ala Glu Ser Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Ala Ala His val Thr val Ser Ala lie Thr Leu Ser Thr Ser Thr Tyr 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125 <210> 24 <211> 122 <212> PRT <213> Lama glama <400> 24 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Lys Asp Met Ala 25 30 Trp Phe Arg Gin Pro Pro Gly Lys Glu Arg Glu Phe val Ala val lie 40 45 Tyr ser ser Asp Gly Ser Thr Leu val Ala Ala Ser val Lys Gly Arg 50 55 60 120 169068/1 Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu Gin Met 65 70 75 80 Thr Ser Leu Lys Pro Ala Asp Thr Ala val Tyr Tyr Cys Ala fhr Ser 85 90 95 Arg Gly Tyr Ser Gly Thr Tyr Tyr Ser Thr Ser Arg Tyr Asp Tyr Trp 100 105 110 Thr Gly Gly Thr Gin Val Thr Val Ser Ser 115 120 <210> 25 <211> 123 <212> PRT <213> Lama glama <400> 25 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Ser val Gin Ala Gly Asp 1 5 10 15 Ser Leu Thr Leu Ser cys Ala Ala Ser Gly Arg Thr Phe Ser Met His 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Ala lie Ser Pro Ser Ala Phe Thr Glu Tyr Ala Asp Ser Leu Lys 50 55 60 Gly Arg Phe Thr val Ser Arg Asp Asn Ala Lys Lys Leu al Trp Leu 65 70 75 80 Gin Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Ala Tyr Tyr cys Ala 85 90 95 Ala Arg Arg Gly Ala Phe Thr Ala Thr Thr Ala Pro Leu Tyr Asp Tyr 100 105 110 Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 115 120 <210> 26 <211> 122 <212> PRT <213> Lama glama 121 169068/1 <400> 26 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Gly Thr Ser Gly Arg Thr Phe Gly Arg Arg 25 30 Ala Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Gin Phe val 40 45 Ala Trp lie Ala Arg Tyr Asp Gly Ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Asn Lys Asn Thr Met Tyr 65 70 75 80 Leu H s Met Asn Asn Leu Thr Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala Ala Gly Pro Arg Gly Leu Tyr Tyr Glu Ser Arg Tyr Glu Tyr Trp 100 105 110 Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 126 <212> PRT <213> Lama glama <400> 27 Gin val Gin Leu Gin Asp Ser Gly Gly Arg Leu Val Lys Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Phe lie Gly Ser Asp Ser Ser Thr Leu Tyr Thr Ser Ser val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr val Tyr Leu 65 70 75 80 122 169068/1 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Ser Ser Ala Phe Ser Ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125 <210> 28 <211> 126 <212> PRT <213> Lama glama <400> 28 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu ser cys Ala Ala Ser Gly Arg ser Phe Ser ser Tyr 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 val Phe lie Gly Ser Asp His ser Thr Leu Tyr ser Thr ser val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Ala Arg Asn Ser Ala Trp Ser Ser Gly lie Tyr Tyr Arg Glu Thr Ser 100 105 110 Tyr Asp Tyr Trp Gly Gin Gly Thr Gin Val Thr val Ser ser 115 120 125 <210> 29 <211> 124 <212> PRT <213> Lama glama <400> 29 123 169068/1 Gin Val Gin Leu Gin Asp Ser Gly Gly Gly Ser al Gin Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr 25 30 Ala Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Gly Phe lie Gly Ser Asp Gly Ser Thr Leu Tyr Ser Ser Ser Val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Ala Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Ala Arg Tyr Ser Gly lie Tyr Tyr Arg Glu Thr Asp Tyr Pro 100 105 110 Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 <210> 30 <211> 124 <212> PRT <213> Lama glama <400> 30 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg ser Phe Gly Gly Phe 25 30 Pro Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ser Gly Leu Thr Arg Ser Leu Phe Thr Val Tyr Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr Val Ser Thr Asp Asn Thr Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 124 169068/1 Ala Arg Pro Asp Leu Tyr Ala Tyr Ser Arg Asp Pro Asn Glu Tyr Asp 100 105 110 Tyr Trp Gly Gin Gly Thr Gin val Thr Val Ser ser 115 120 <210> 31 <211> 111 <212> PRT <213> Lama glama <400> 31 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu Val Gin Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Arg lie Val Ser Thr Tyr 25 30 Ala Met Gly Trp Phe Arg Gin Ser Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Thr Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn 50 55 60 Thr Leu Tyr Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val 65 70 75 80 Tyr Tyr Cys Ala Lys Thr Lys Arg Thr Gly lie Phe Thr Thr Ala Arg 85 90 95 Met val Asp Tyr Trp Gly Gin Gly Thr Gin Val Thr val Ser Ser 100 105 110 <210> 32 <211> 264 <212> PRT <213> Lama glama <400> 32 Gin val Gin Leu Gin Asp Ser Gly Gly Arg Leu val Lys Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 125 169068/1 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Phe lie Gly Ser Asp Ser Ser Thr Leu Tyr Thr Ser Ser Val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Ala Arg Ser ser Ala Phe Ser Ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser Glu Pro 115 120 125 Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val Gin Leu Gin Asp 130 135 140 Ser Gly Gly Arg Leu Val Lys Ala Gly Ala Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu Pro Met Ala Trp Phe Arg 165 170 175 Gin Ala Pro Gly Lys Glu Arg Glu Phe val Ala Phe lie Gly Ser Asp 180 185 190 Ser Ser Thr Leu Tyr Thr Ser Ser val Arg Gly Arg Phe Thr lie Ser 195 200 205 Arg Asp Asn Gly Lys Asn Thr val Tyr Leu Gin Met Met Asn Leu Lys 210 215 220 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Ser Ser Ala Phe 225 230 235 240 Ser ser Gly lie Tyr Tyr Arg Glu Gly Ser Tyr Ala Tyr Trp Gly Gin 245 250 255 Gly Thr Gin Val Thr Val Ser Ser 260 <210> 33 <211> 234 <212> P T <213> Lama glama 126 169068/1 <400> 33 Gin Val Gin Leu Gin Asp ser Gly Gly Gly Leu Val Gin Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Arg lie Val Ser Thr Tyr 25 30 Ala Met Gly Trp Phe Arg Gin ser Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Thr Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn 50 55 60 Thr Leu Tyr Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val 65 70 75 80 Tyr Tyr Cys Ala Lys Thr Lys Arg Thr Gly lie Phe Thr Thr Ala Arg 85 90 95 Met val Asp Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser ser Glu 100 105 110 Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val Gin Leu Gin 115 120 125 Asp Ser Gly Gly Gly Leu val Gin ser Gly Gly Ser Leu Arg Leu Ala 130 135 140 Cys Ala Ala Ser Gly Arg lie Val Ser Thr Tyr Ala Met Gly Trp Phe 145 150 155 160 Arg Gin Ser Pro Gly Lys Glu Arg Glu Phe Val Ala Thr Val Lys Gly 165 170 175 Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gin 180 185 190 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala Lys 195 200 205 Thr Lys Arg Thr Gly lie Phe Thr Thr Ala Arg Met Val Asp Tyr Trp 210 215 220 Gly Gin Gly Thr Gin val Thr Val Ser Ser 225 230 <210> 34 <211> 262 127 169068/1 <212> PRT <213> Lama glama <400> 34 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser cys Ala Ala Ser Gly Arg Thr Phe Ser ser Tyr 25 30 Arg Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Ala lie Ser Arg Arg Gly Asp Asn val Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala lie Ser Arg Asp Asn Ala Glu Ser Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His val Thr val Ser Ala lie Thr Leu Ser Thr Ser Thr Tyr 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser Glu Pro Lys 115 120 125 Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin Val Gin Leu Gin Glu Ser 130 135 140 Gly Gly Gly Leu val Gin Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 145 150 155 160 Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met Gly Trp Phe Arg Gin 165 170 175 Ala Pro Gly Lys Glu Arg Glu Phe val Ala Ala lie Ser Arg Arg Gly 180 185 190 Asp Asn Val Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala lie Ser 195 200 205 Arg Asp Asn Ala Glu Ser Thr Leu Tyr Leu Gin Met Asn Ser Leu Lys 210 215 220 Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala Ala His val Thr val Ser 225 230 235 240 128 1 Ala lie Thr Leu Ser Thr ser Thr Tyr Asp Tyr Trp Gly Gin Gly Thr 245 250 255 Gin val Thr val Ser Ser 260 <210> 35 <211> 122 <212> PRT <213> Lama glama <400> 35 Gin Val Gin Leu Gin 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Arg Tyr 25 30 Ala Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu val 40 45 Ala Ala lie Thr Ser Gly Gly Arg Thr Ser val Ala Asp Thr val Lys 50 55 60 Gly Arg Phe Thr lie Ser ser Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Thr 85 90 95 Leu Tyr Asn Ser Thr Thr Asn Tyr Tyr Asn Gin Ser Pro Ser Ser Trp 100 105 110 Gly Gin Gly Thr Gin Val Thr val Ser Ser 115 120 <210> 36 <211> 122 <212> PRT <213> Lama <400> 36 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 129 1 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Arg Tyr 25 30 Ala Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Val Leu val 40 45 Ala Ala lie Thr Ser Asn Gly Arg Pro Ser val Ala Asp Ser val Lys 50 55 60 Gly Arg Phe Thr lie Ser Ser Asp Thr Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr 85 90 95 Leu Tyr Asn Thr Ser Ala Asp Tyr Tyr Asn Gin Ser Pro Ser ser Trp 100 105 110 Gly Gin Gly Thr Gin val Thr val Leu Ser 115 120 <210> 37 <211> 120 <212> PRT <213> Lama glama <400> 37 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Met 25 30 Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Gin Phe val Ala Ala Leu 40 45 Thr Trp Thr Gly Gly Ser Pro val Tyr Ala Asp Ser val Lys Gly Arg 50 55 60 Phe Thr Thr Trp Arg val Leu Asp Asn Asn Thr Val Tyr Leu His Met 65 70 75 80 Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr His Cys Ala Ala Ala 85 90 95 Arg Thr Tyr Tyr Gly Asn lie Ser Glu Tyr Tyr Asp Tyr Trp Gly Gin 100 105 110 130 169068/1 Gly Thr Gin val Thr val Ser Ser 115 120 <210> 38 <211> 121 <212> PRT <213> Lama glama <400> 38 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin Val Thr lie Ser Ser 115 120 <210> 39 <211> 121 <212> PRT <213> Lama glama <400> 39 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 131 169068/1 Pro Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Lys Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr lie Ser Ser 115 120 <210> 40 <211> 121 <212> PRT <213> Lama glama <400> 40 Glu val Gin Leu Leu Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Lys Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr lie Ser ser 115 120 132 169068/1 <210> 41 <211> 121 <212> PRT <213> Lama glama <400> 41 Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Lys Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Leu val Thr val Ser Ser 115 120 <210> 42 <211> 248 <212> PRT <213> Lama glama <400> 42 Gin Val Gin Leu Gin Glu ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu ser cys Glu Ala ser Gly Phe Thr Phe Ser Arg Phe 25 30 Gly Met Thr Trp Val Arg Gin Ala Pro Gly Lys Gly Val Glu Trp Val 40 45 133 169068/1 Ser Gly lie Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Thr lie Gly Gly Ser Leu Asn Pro Gly Gly Gin Gly Thr Gin val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin 115 120 125 Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr Pro 145 150 155 160 Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val Ser 165 170 175 Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys Gly 180 185 190 Arg Phe Thr lie Ser Arg Asp Asn ser Lys Asn Thr Leu Tyr Leu Gin 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala Lys 210 215 220 Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin 225 230 235 240 Gly Thr Gin val Thr lie Ser Ser 245 <210> 43 <211> 248 <212> PRT <213> Lama glama <400> 43 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Asn 1 5 10 15 134 169068/1 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 25 30 Gly Met Ser Trp Val Arg Gin Ala Pro Gly Lys Glu Pro Glu Trp Val 40 45 Ser Ser lie Ser Gly Ser Gly Ser Asn Thr lie Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Thr lie Gly Gly Ser Leu Ser Arg Ser ser Gin Gly Thr Gin val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin 115 120 125 val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr Pro 145 150 155 160 Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val Ser 165 170 175 Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys Gly 180 185 190 Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 210 215 220 Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin 225 230 235 240 Gly Thr Gin val Thr lie Ser Ser 245 <210> 44 <211> 247 <212> PRT <213> Lama glama 135 169068/1 <400> 44 Gin al Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 25 30 Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Ala lie Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 val lie Gly Arg Gly Ser pro Ser ser Gin Gly Thr Gin val Thr val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val 115 120 125 Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser cys Ala Ala ser Gly Phe Thr Phe Ser Trp Tyr Pro Met 145 150 155 160 Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175 lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser val Lys Gly Arg 180 185 190 Phe Thr lie Ser Arg Asp Asn ser Lys Asn Thr Leu Tyr Leu Gin Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala Lys Gly 210 215 220 Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin Gly 225 230 235 240 Thr Gin Val Thr lie Ser Ser 245 136 169068/1 <210> 45 <211> 247 <212> PRT <213> Lama glama <400> 45 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 25 30 Gly Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Ala lie Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Lys Met Leu Thr 65 70 75 80 Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 val lie Gly Arg Gly Ser Pro Ala Ser Gin Gly Thr Gin val Thr val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gin Pro Ala Ala Ala Gin val 115 120 125 Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr Pro Met 145 150 155 160 Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val Ser Thr 165 170 175 lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys Ala Lys Gly 210 215 220 137 169068/1 Ala Gly Thr ser ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp Gin Gly 225 230 235 240 Thr Gin val Thr lie Ser Ser 245 <210> 46 <211> 254 <212> PRT <213> Lama glama <400> 46 Gin Val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Arg Tyr 25 30 Ala Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu val 40 45 Ala Ala lie Thr Ser Gly Gly Arg Thr ser val Ala Asp Thr val Lys 50 55 60 Gly Arg Phe Thr lie Ser ser Asp Asn Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Thr 85 90 95 Leu Tyr Asn Ser Thr Thr Asn Tyr Tyr Asn Gin Ser Pro Ser Ser Trp 100 105 110 Gly Gin Gly Thr Gin Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys 115 120 125 Pro Gin Pro Ala Ala Ala Gin val Gin Leu Gin Glu Ser Gly Gly Gly 130 135 140 Leu val Gin Ala Gly Asp Ser Leu Arg Leu ser Cys Ala Ala Ser Gly 145 150 155 160 Arg Thr Phe Thr Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 165 170 175 Gin Phe val Ala Ala Leu Thr Trp Thr Gly Gly Ser Pro val Tyr Ala 180 185 190 138 169068/1 Asp Ser val Lys Gly Arg Phe Thr Thr Trp Arg Val Leu Asp Asn Asn 195 200 205 Thr Val Tyr Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 210 215 220 Tyr His Cys Ala Ala Ala Arg Thr Tyr Tyr Gly Asn lie Ser Glu Tyr 225 230 235 240 Tyr Asp Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 245 250 <210> 47 <211> 254 <212> P T <213> Lama glama <400> 47 Gin val Gin Leu Gin Asp Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Arg Tyr 25 30 Ala Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg val Leu val 40 45 Ala Ala lie Thr Ser Asn Gly Arg Pro Ser Val Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser ser Asp Thr Ala Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr 85 90 95 Leu Tyr Asn Thr Ser Ala Asp Tyr Tyr Asn Gin Ser Pro Ser Ser Trp 100 105 110 Gly Gin Gly Thr Gin Val Thr Val Leu Ser Glu Pro Lys Thr Pro Lys 115 120 125 Pro Gin Pro Ala Ala Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Gly 130 135 140 Leu Val Gin Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 139 169068/1 Arg Thr Phe Thr Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg 165 170 175 Gin Phe Val Ala Ala Leu Thr Trp Thr Gly Gly Ser Pro Val Tyr Ala 180 185 190 Asp Ser val Lys Gly Arg Phe Thr Thr Trp Arg val Leu Asp Asn Asn 195 200 205 Thr al Tyr Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val 210 215 220 Tyr His Cys Ala Ala Ala Arg Thr Tyr Tyr Gly Asn lie Ser Glu Tyr 225 230 235 240 Tyr Asp Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 245 250 <210> 48 <211> 2804 <212> PRT <213> Homo Sapiens <400> 48 Met lie Pro Ala Arg Phe Ala Gly val Leu Leu Ala Leu Ala Leu lie 1 5 10 15 Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 25 30 Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 40 45 Ser Met Tyr ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60 Cys Gin Lys Arg Ser Phe Ser lie lie Gly Asp Phe Gin Asn Gly Lys 65 70 75 80 Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp lie His Leu 85 90 95 Phe val Asn Gly Thr val Thr Gin Gly Asp Gin Arg val Ser Met Pro 100 105 110 Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125 140 169068/1 Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg lie Asp Gly Ser Gly 130 135 140 Asn Phe Gin val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150 155 160 Leu Cys Gly Asn Phe Asn lie Phe Ala Glu Asp Asp Phe Met Thr Gin 165 170 175 Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190 Leu Ser Ser Gly Glu Gin Trp Cys Glu Arg Ala Ser Pro Pro Ser ser 195 200 205 Ser Cys Asn lie Ser ser Gly Glu Met Gin Lys Gly Leu Trp Glu Gin 210 215 220 Cys Gin Leu Leu Lys ser Thr Ser Val Phe Ala Arg Cys His Pro Leu 225 230 235 240 Val Asp Pro Glu Pro Phe val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255 Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270 Arg Thr Cys Ala Gin Glu Gly Met val Leu Tyr Gly Trp Thr Asp His 275 280 285 Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gin Cys 290 295 300 Val Ser Pro Cys Ala Arg Thr Cys Gin ser Leu His lie Asn Glu Met 305 310 315 320 Cys Gin Glu Arg Cys val Asp Gly Cys Ser Cys Pro Glu Gly Gin Leu 325 330 335 Leu Asp Glu Gly Leu Cys val Glu Ser Thr Glu Cys Pro Cys val His 340 345 350 Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365 Thr Cys lie Cys Arg Asn Ser Gin Trp lie Cys Ser Asn Glu Glu Cys 370 375 380 Pro Gly Glu Cys Leu Val Thr Gly Gin ser His Phe Lys Ser Phe Asp 385 390 395 400 141 169068/1 Asn Arg Tyr Phe Thr Phe Ser Gly lie Cys Gin Tyr Leu Leu Ala Arg 405 410 415 Asp Cys Gin Asp His Ser Phe Ser lie val lie Glu Thr val Gin Cys 420 425 430 Ala Asp Asp Arg Asp Ala val Cys Thr Arg Ser val Thr val Arg Leu 435 440 445 Pro Gly Leu His Asn ser Leu val Lys Leu Lys His Gly Ala Gly val 450 455 460 Ala Met Asp Gly Gin Asp lie Gin Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480 Arg lie Gin His Thr val Thr Ala Ser val Arg Leu Ser Tyr Gly Glu 485 490 495 Asp Leu Gin Met Asp Trp Asp Gly Arg Gly Arg Leu Leu val Lys Leu 500 505 510 Ser Pro val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525 Gly Asn Gin Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540 Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gin 545 550 555 560 Asp Leu Gin Lys Gin His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575 Thr Arg Phe ser Glu Glu Ala Cys Ala val Leu Thr ser Pro Thr phe 580 585 590 Glu Ala Cys His Arg Ala val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605 Arg Tyr Asp val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620 Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly val Arg val 625 630 635 640 Ala Trp Arg Glu Pro Gly Arg cys Glu Leu Asn Cys Pro Lys Gly Gin 645 650 655 val Tyr Leu Gin Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670 142 169068/1 Ser Tyr Pro Asp Glu Glu cys Asn Glu Ala Cys Leu Glu Gly cys Phe 675 680 685 Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys val Pro Lys 690 695 700 Ala Gin Cys Pro Cys Tyr Tyr Asp Gly Glu lie Phe Gin Pro Glu Asp 705 710 715 720 lie Phe Ser Asp His His Thr Met Cys Tyr cys Glu Asp Gly Phe Met 725 730 735 His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala val 740 745 750 Leu Ser ser Pro Leu Ser His Arg ser Lys Arg ser Leu ser cys Arg 755 760 765 Pro Pro Met val Lys Leu val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780 Gly Leu Glu Cys Thr Lys Thr Cys Gin Asn Tyr Asp Leu Glu Cys Met 785 790 795 800 Ser Met Gly Cys val ser Gly Cys Leu Cys Pro Pro Gly Met val Arg 805 810 815 His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gin 820 825 830 Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys lie Gly Cys Asn Thr 835 840 845 Cys val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860 Ala Thr Cys Ser Thr lie Gly Met Ala His Tyr Leu Thr Phe Asp Gly 865 870 875 880 Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gin Tyr Val Leu Val Gin Asp 885 890 895 Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg lie Leu Val Gly Asn Lys 900 905 910 Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg val Thr lie Leu 915 920 925 val Glu Gly Gly Glu lie Glu Leu Phe Asp Gly Glu val Asn val Lys 930 935 940 143 1 Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg 945 950 955 960 Tyr lie lie Leu Leu Leu Gly Lys Ala Leu Ser Val val Trp Asp Arg 965 970 975 His Leu Ser lie Ser val val Leu Lys Gin Thr Tyr Gin Glu Lys val 980 985 990 Cys Gly Leu Cys Gly Asn Phe Asp Gly lie Gin Asn Asn Asp Leu Th 995 1000 1005 Ser Ser Asn Leu Gin Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020 Ser Trp Lys val Ser Ser Gin Cys Ala Asp Thr Arg Lys val Pro 1025 1030 1035 Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn lie Met Lys Gin 1040 1045 1050 Thr Met Val Asp Ser Ser cys Arg lie Leu Thr Ser Asp Val Phe 1055 1060 1065 Gin Asp Cys Asn Lys Leu val Asp Pro Glu Pro Tyr Leu Asp val 1070 1075 1080 Cys lie Tyr Asp Thr Cys Ser Cys Glu Ser He Gly Asp Cys Ala 1085 1090 1095 Cys Phe Cys Asp Thr lie Ala Ala Tyr Ala His Val cys Ala Gin 1100 1105 1110 His Gly Lys val val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gin 1115 1120 1125 Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140 Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gin Val Thr cys Gin 1145 1150 1155 His Pro Glu Pro Leu Ala Cys Pro Val Gin Cys Val Glu Gly Cys 1160 1165 1170 His Ala His Cys Pro Pro Gly Lys lie Leu Asp Glu Leu Leu Gin 1175 1180 1185 Thr Cys Val Asp Pro Glu Asp Cys Pro val Cys Glu val Ala Gly 1190 1195 1200 144 169068/1 Arg Arg Phe Ala Ser Gly Lys Lys al Thr Leu Asn Pro Ser Asp 1205 1210 1215 Pro Glu His Cys Gin lie Cys His Cys Asp val Val Asn Leu Thr 1220 1225 1230 Cys Glu Ala cys Gin Glu Pro Gly Gly Leu Val Val Pro Pro Thr 1235 1240 1245 Asp Ala Pro val Ser Pro Thr Thr Leu Tyr val Glu Asp lie Ser 1250 1255 1260 Glu Pro Pro Leu His Asp Phe Tyr cys Ser Arg Leu Leu Asp Leu 1265 1270 1275 val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe 1280 1285 1290 Glu Val Leu Lys Ala Phe val val Asp Met Met Glu Arg Leu Arg 1295 1300 1305 lie Ser Gin Lys Trp val Arg val Ala val val Glu Tyr His Asp 1310 1315 1320 Gly Ser His Ala Tyr lie Gly Leu Lys Asp Arg Lys Arg Pro Ser 1325 1330 1335 Glu Leu Arg Arg lie Ala Ser Gin Val Lys Tyr Ala Gly Ser Gin 1340 1345 1350 Val Ala Ser Thr ser Glu Val Leu Lys Tyr Thr Leu Phe Gin lie 1355 1360 1365 Phe Ser Lys lie Asp Arg Pro Glu Ala Ser Arg lie Ala Leu Leu 1370 1375 1380 Leu Met Ala Ser Gin Glu Pro Gin Arg Met ser Arg Asn Phe val 1385 1390 1395 Arg Tyr val Gin Gly Leu Lys Lys Lys Lys val lie val lie Pro 1400 1405 1410 val Gly lie Gly pro His Ala Asn Leu Lys Gin lie Arg Leu lie 1415 1420 1425 Glu Lys Gin Ala Pro Glu Asn Lys Ala Phe val Leu Ser ser val 1430 1435 1440 Asp Glu Leu Glu Gin Gin Arg Asp Glu lie val Ser Tyr Leu Cys 1445 1450 1455 145 169068/1 Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro His Met 1460 1465 1470 Ala Gin Val Thr al Gly Pro Gly Leu Arg Asn Ser Met Val Leu 1475 1480 1485 Asp val Ala Phe Val Leu Glu Gly Ser Asp Lys lie Gly Glu Ala 1490 1495 1500 Asp Phe Asn Arg ser Lys Glu Phe Met Glu Glu val lie Gin Arg 1505 1510 1515 Met Asp val Gly Gin Asp Ser lie His Val Thr Val Leu Gin Tyr 1520 1525 1530 Ser Tyr Met Val Thr val Glu Tyr Pro Phe Ser Glu Ala Gin Ser 1535 1540 1545 Lys Gly Asp lie Leu Gin Arg val Arg Glu lie Arg Tyr Gin Gly 1550 1555 1560 Gly Asn Arg Thr Asn Thr Gly Leu Ala Leu Arg Tyr Leu ser Asp 1565 1570 1575 His Ser Phe Leu val Ser Gin Gly Asp Arg Glu Gin Ala Pro Asn 1580 1585 1590 Leu val Tyr Met val Thr Gly Asn Pro Ala ser Asp Glu lie Lys 1595 1600 1605 Arg Leu Pro Gly Asp lie Gin val al Pro lie Gly val Gly Pro 1610 1615 1620 Asn Ala Asn val Gin Glu Leu Glu Arg lie Gly Trp Pro Asn Ala 1625 1630 1635 Pro lie Leu lie Gin Asp Phe Glu Thr Leu Pro Arg Glu Ala Pro 1640 1645 1650 Asp Leu Val Leu Gin Arg Cys Cys Ser Gly Glu Gly Leu Gin lie 1655 1660 1665 Pro Thr Leu ser Pro Ala Pro Asp Cys Ser Gin Pro Leu Asp val 1670 1675 1680 lie Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe 1685 1690 1695 Asp Glu Met Lys ser Phe Ala Lys Ala Phe lie Ser Lys Ala Asn 1700 1705 1710 146 169068/1 lie Gly Pro Arg Leu Thr Gin val Ser val Leu Gin Tyr Gly Ser 1715 1720 1725 lie Thr Thr lie Asp val Pro Trp Asn Val Val Pro Glu Lys Ala 1730 1735 1740 His Leu Leu Ser Leu val Asp val Met Gin Arg Glu Gly Gly Pro 1745 1750 1755 Ser Gin lie Gly Asp Ala Leu Gly Phe Ala val Arg Tyr Leu Thr 1760 1765 1770 Ser Glu Met His Gly Ala Arg Pro Gly Ala Ser Lys Ala val val 1775 1780 1785 He Leu val Thr Asp val Ser val Asp Ser val Asp Ala Ala Ala 1790 1795 1800 Asp Ala Ala Arg Ser Asn Arg val Thr Val Phe Pro lie Gly lie 1805 1810 1815 Gly Asp Arg Tyr Asp Ala Ala Gin Leu Arg lie Leu Ala Gly Pro 1820 1825 1830 Ala Gly Asp Ser Asn val val Lys Leu Gin Arg lie Glu Asp Leu 1835 1840 1845 Pro Thr Met Val Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys 1850 1855 1860 Ser Gly Phe val Arg lie Cys Met Asp Glu Asp Gly Asn Glu Lys 1865 1870 1875 Arg Pro Gly Asp val Trp Thr Leu Pro Asp Gin Cys His Thr Val 1880 1885 1890 Thr cys Gin Pro Asp Gly Gin Thr Leu Leu Lys Ser His Arg val 1895 1900 1905 Asn cys Asp Arg Gly Leu Arg Pro Ser Cys Pro Asn Ser Gin Ser 1910 1915 1920 Pro val Lys val Glu Glu Thr cys Gly Cys Arg Trp Thr Cys Pro 1925 1930 1935 Cys Val Cys Thr Gly Ser ser Thr Arg His lie val Thr Phe Asp 1940 1945 1950 Gly Gin Asn Phe Lys Leu Thr Gly Ser Cys Ser Tyr Val Leu Phe 1955 1960 1965 147 169068/1 Gin Asn Lys Glu Gin Asp Leu Glu al lie Leu His Asn Gly Ala 1970 1975 1980 Cys Ser Pro Gly Ala Arg Gin Gly Cys Met Lys Ser lie Glu val 1985 1990 1995 Lys His Ser Ala Leu Ser val Glu Leu His Ser Asp Met Glu val 2000 2005 2010 Thr Val Asn Gly Arg Leu val Ser val Pro Tyr Val Gly Gly Asn 2015 2020 2025 Met Glu val Asn val Tyr Gly Ala lie Met H s Glu val Arg Phe 2030 2035 2040 Asn His Leu Gly His lie Phe Thr Phe Thr Pro Gin Asn Asn Glu 2045 2050 2055 Phe Gin Leu Gin Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr Tyr 2060 2065 2070 Gly Leu Cys Gly lie Cys Asp Glu Asn Gly Ala Asn Asp Phe Met 2075 2080 2085 Leu Arg Asp Gly Thr val Thr Thr Asp Trp Lys Thr Leu val Gin 2090 2095 2100 Glu Trp Thr val Gin Arg Pro Gly Gin Thr Cys Gin Pro lie Leu 2105 2110 2115 Glu Glu Gin Cys Leu val Pro Asp Ser ser His Cys Gin Val Leu 2120 2125 2130 Leu Leu Pro Leu Phe Ala Glu Cys H s Lys val Leu Ala Pro Ala 2135 2140 2145 Thr Phe Tyr Ala lie Cys Gin Gin Asp Ser Cys Hi s Gl n Glu Gin 2150 2155 2160 val cys Glu val lie Ala Ser Tyr Ala His Leu cys Arg Thr Asn 2165 2170 2175 Gly val Cys val Asp Trp Arg Thr Pro Asp Phe cys Ala Met Ser 2180 2185 2190 Cys Pro Pro Ser Leu val Tyr Asn His Cys Glu His Gly Cys Pro 2195 2200 2205 Arg His Cys Asp Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser 2210 2215 2220 148 Glu Gly Cys Phe Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser 2225 2230 2235 Cys val Pro Glu Glu Ala Cys Thr Gin Cys lie Gly Glu Asp Gly 2240 2245 2250 al Gin H s Gin Phe Leu Glu Ala Trp val Pro Asp His Gin Pro 2255 2260 2265 Cys Gin lie Cys Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr 2270 2275 2280 Thr Gin Pro Cys Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys 2285 2290 2295 Glu val Ala Arg Leu Arg Gin Asn Ala Asp Gin Cys Cys Pro Glu 2300 2305 2310 Tyr Glu Cys val Cys Asp Pro val Ser Cys Asp Leu Pro Pro Val 2315 2320 2325 Pro His Cys Glu Arg Gly Leu Gin Pro Thr Leu Thr Asn Pro Gly 2330 2335 2340 Glu Cys Arg Pro Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys 2345 2350 2355 Lys Arg Val Ser Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr 2360 2365 2370 Leu Arg Lys Thr Gin Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn 2375 2380 2385 Cys val Asn Ser Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser 2390 2395 2400 Thr Ala Thr Asn Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro 2405 2410 2415 Asp Lys Val Cys Val His Arg Ser Thr lie Tyr Pro val Gly Gin 2420 2425 2430 Phe Trp Glu Glu Gly Cys Asp val cys Thr cys Thr Asp Met Glu 2435 2440 2445 Asp Ala Val Met Gly Leu Arg Val Ala Gin Cys Ser Gin Lys Pro 2450 2455 2460 Cys Glu Asp Ser Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu 2465 2470 2475 149 169068/1 Gly Glu cys Cys Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val 2480 2485 2490 Thr Gly ser Pro Arg Gly Asp Ser Gin ser ser Trp Lys Ser Val 2495 2500 2505 Gly ser Gin Trp Ala Ser Pro Glu Asn Pro Cys Leu lie Asn Glu 2510 2515 2520 cys val Arg val Lys Glu Glu val Phe lie Gin Gin Arg Asn val 2525 2530 2535 Ser Cys Pro Gin Leu Glu val Pro Val Cys Pro Ser Gly Phe Gin 2540 2545 2550 Leu Ser Cys Lys Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu 2555 2560 2565 Arg Met Glu Ala Cys Met Leu Asn Gly Thr Val lie Gly Pro Gly 2570 2575 2580 Lys Thr val Met lie Asp val Cys Thr Thr cys Arg cys Met val 2585 2590 2595 Gin Val Gly val lie Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr 2600 2605 2610 Thr Cys Asn Pro Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr 2615 2620 2625 Gly Glu cys Cys Gly Arg cys Leu Pro Thr Ala Cys Thr lie Gin 2630 2635 2640 Leu Arg Gly Gly Gin lie Met Thr Leu Lys Arg Asp Glu Thr Leu 2645 2650 2655 Gin Asp Gly Cys Asp Thr His Phe Cys Lys val Asn Glu Arg Gly 2660 2665 2670 Glu Tyr Phe Trp Glu Lys Arg Val Thr Gly Cys Pro pro Phe Asp 2675 2680 2685 Glu His Lys Cys Leu Ala Glu Gly Gly Lys lie Met Lys lie Pro 2690 2695 2700 Gly Thr Cys Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp lie 2705 2710 2715 Thr Ala Arg Leu Gin Tyr Val Lys Val Gly Ser Cys Lys Ser Glu 2720 2725 2730 150 169068/1 val Glu val Asp lie His Tyr Cys Gin Gly Lys Cys Ala Ser Lys 2735 2740 2745 Ala Met Tyr ser lie Asp lie Asn Asp val Gin Asp Gin Cys Ser 2750 2755 2760 Cys cys ser Pro Thr Arg Thr Glu Pro Met Gin val Ala Leu His 2765 2770 2775 Cys Thr Asn Gly Ser val val Tyr His Glu val Leu Asn Ala Met 2780 2785 2790 Glu Cys Lys Cys Ser Pro Arg Lys Cys Ser Lys 2795 2800 <210> 49 <211> 128 <212> PRT <213> Lama glama <400> 49 val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Asp 10 15 Ser Leu Arg Leu Ser Cys val val Ser Gly Thr Thr Phe Ser ser Ala 25 , 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Gly Ala lie Lys Trp ser Gly Thr Ser Thr Tyr Tyr Thr Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn val Lys Asn Thr val Tyr 65 70 75 80 Leu Gin Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Thr Cys 85 90 95 Ala Ala Asp Arg Asp Arg Tyr Arg Asp Arg Met Gly Pro Met Thr Thr 100 105 110 Thr Asp Phe Arg Phe Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 124 151 169068/1 <212> P T <213> Lama glama <400> 50 Gin val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Phe 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Arg Glu Arg Glu Phe val 40 45 Ala Ser lie Gly Ser Ser Gly lie Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Leu Cys Tyr Cys 85 90 95 Ala val Asn Arg Tyr Gly lie Pro Tyr Arg Ser Gly Thr Gin Tyr Gin 100 105 110 Asn Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 115 120 <210> 51 <211> 120 <212> PRT <213> Lama glama <400> 51 Glu val Gin Leu Glu Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Asn Asp Tyr 25 30 Ala Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Glu Arg Asp Met val 40 45 Ala Thr lie Ser lie Gly Gly Arg Thr Tyr Tyr Ala Asp Ser val Lys 50 55 60 152 169068/1 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr al Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala lie Tyr Tyr Cys val 85 90 95 Ala His Arg Gin Thr val val Arg Gly Pro Tyr Leu Leu Trp Gly Gin 100 105 110 Gly Thr Gin val Thr val Ser ser 115 120 <210> 52 <211> 123 <212> PRT <213> Lama glama <400> 52 Gin Val Gin Leu Val Glu Ser Gly Gly Lys Leu val Gin Ala Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Val 40 45 Ala Gly Ser Gly Arg Ser Asn Ser Tyr Asn Tyr Tyr ser Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Asn Leu Trp Pro Arg Asp Arg Asn Leu Tyr Ala Tyr 100 105 110 Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 <210> 53 <211> 125 <212> PRT <213> Lama glama 153 169068/1 <400> 53 Glu al Gin Leu Val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Gly lie Tyr 25 30 Arg Met Gly Trp Phe Arg Gin val Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Ala lie Ser Trp ser Gly Gly Thr Thr Arg Tyr Leu Asp ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Ser Thr Lys Asn Ala val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala val Asp ser Ser Gly Arg Leu Tyr Trp Thr Leu Ser Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Gin val Thr Val Ser Ser 115 120 125 <210> 54 <211> 125 <212> PRT <213> Lama glama <400> 54 Gin val Gin Leu Val Glu Phe Gly Gly Gly Leu al Gin Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg ser Leu Gly lie Tyr 25 30 Lys Met Ala Trp Phe Arg Gin Val Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Ala lie Ser Trp Ser Gly Gly Thr Thr Arg Tyr lie Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Thr Lys Asn Met val Tyr 65 70 75 80 154 169068/1 Leu Gin Met Asn Ser Leu Lys Pro Asp Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala val Asp Ser Ser Gly Arg Leu Tyr Trp Thr Leu Ser Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Gin al Thr val Ser Ser 115 120 125 <210> 55 <211> 124 <212> PRT <213> Lama glama <400> 55 Glu val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr 25 30 Thr Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe Leu 40 45 Ala Gly val Thr Trp Ser Gly Ser ser Thr Phe Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Arg Asp Ser Ala Lys Asn Thr val Thr 65 70 75 80 Leu Glu Met Asn Ser Leu Asn Pro Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Ala Ala Ala Tyr Gly Gly Gly Leu Tyr Arg Asp Pro Arg Ser Tyr Asp 100 105 110 Tyr Trp Gly Arg Gly Thr Gin val Thr val 115 120 <210> 56 <211> 131 <212> PRT <213> Lama glama <400> 56 155 1 Ala al Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ala Trp 25 30 Pro lie Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Gly Val 40 45 Ser Cys lie Arg Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr lie Ser Ser Asp Asn Ala Asn Asn Thr val Tyr Leu Gin 65 70 75 80 Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 85 90 95 Pro Ser Gly Pro Ala Thr Gly Ser Ser His Thr Phe Gly lie Tyr Trp 100 105 110 Asn Leu Arg Asp Asp Tyr Asp Asn Trp Gly Gin Gly Thr Gin val Thr 115 120 125 val Ser ser 130 <210> 57 <211> 126 <212> PRT <213> Lama glama <400> 57 Glu val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp His Tyr 25 30 Thr lie Gly Trp Phe Arg Gin val Pro Gly Lys Glu Arg Glu Gly val 40 45 Ser Cys lie Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Ser Asp Asn Ala Lys Asn Thr val Tyr 65 70 75 80 156 169068/1 Leu Gin Met Asn Thr Leu Glu Pro Asp Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Leu Leu Leu Arg Val Glu Glu Leu Gin Ala Ser Asp 100 105 110 Tyr Asp Tyr Trp Gly Gin Gly lie Gin Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 128 <212> PRT <213> Lama glama <400> 58 Ala Val Gin Leu val Asp Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 25 30 Ala lie Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Gly val 40 45 Ala Cys lie Ser Asn ser Asp Gly Ser Thr Tyr Tyr Gly Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ala Asp Arg His Tyr Ser Ala ser His His Pro Phe Ala Asp 100 105 110 Phe Ala Phe Asn Ser Trp Gly Gin Gly Thr Gin val Thr Val Ser Ser 115 120 125 <210> 59 <211> 120 <212> PRT <213> Lama glama <400> 59 157 169068/1 Glu Val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Tyr Gly Leu Thr Phe Trp Arg Ala 25 30 Ala Met Ala Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Leu val 40 45 val Ala Arg Asn Trp Gly Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Thr Tyr Gly Ser Ala Thr Tyr Asp lie Trp Gly Gin 100 105 110 Gly Thr Gin Val Thr Val Ser Ser 115 120 <210> 60 <211> 123 <212> PRT <213> Lama glama <400> 60 Glu val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Asp Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser cys lie Phe Ser Gly Arg Thr Phe Ala Asn Tyr 25 30 Ala Met Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Ala lie Asn Arg Asn Gly Gly Thr Thr Asn Tyr Ala Asp Ala Leu 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Thr Lys Asn Thr Ala Phe 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Asp Asp Thr Ala val Tyr Tyr cys 85 90 95 158 169068/1 Ala Ala Arg Glu Trp Pro Phe Ser Thr lie Pro Ser Gly Trp Arg Tyr 100 105 110 Trp Gly Gin Gly Thr Gin Val Thr val 115 120 <210> 61 <211> 125 <212> PRT <213> Lama glama <400> 61 Asp val Gin Leu val Glu Ser Gly Gly Gly Trp Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu ser Cys Ala Ala Ser Gly Pro Thr Ala Ser ser His 25 30 Ala lie Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 al Gly lie Asn Arg Gly Gly val Thr Arg Asp Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Ala val Ser Arg Asp Asn val Lys Asn Thr val Tyr 65 70 75 80 Leu Gin Met Asn Arg Leu Lys Pro Glu Asp Ser Ala lie Tyr lie Cys 85 90 95 Ala Ala Arg Pro Glu Tyr Ser Phe Thr Ala Met ser Lys Gly Asp Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu val Thr val Ser Ser 115 120 125 <210> 62 <211> 126 <212> PRT <213> Lama glama <400> 62 Glu val Gin Leu val Glu Ser Gly Gly Arg Leu val Lys Ala Gly Ala 1 5 10 15 159 169068/1 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu 25 30 Pro Met Ala Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Phe val 40 45 Ala Phe lie Gly Ser Asp Ser Ser Thr Leu Tyr Thr Ser Ser val Arg 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Gly Lys Asn Thr val Tyr Leu 65 70 75 80 Gin Met Met Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Arg Ser Ser Ala Phe Ser Ser Gly lie Tyr Tyr Arg Glu Gly Ser 100 105 110 Tyr Ala Tyr Trp Gly Gin Gly Thr Gin val Thr val Ser Ser 115 120 125 <210> 63 <211> 125 <212> PRT <213> Lama glama <400> 63 Gin val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Thr Tyr 25 30 Ala Leu Gly Trp Phe Arg Gin val Pro Gly Lys Gly Arg Glu Phe lie 40 45 Ala Val lie Tyr Trp Arg Asp Gly Ser ser Leu Tyr Ser Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Lys Asp Asn Ala Lys Asn Thr val Tyr 65 70 75 80 Leu Gin Met Asn ser Leu Lys Pro Glu Asp Thr Ala val Tyr Tyr Cys 85 90 95 Ala Asn Arg His Asp Ser Arg Gly Thr Tyr Tyr Ser Ser Arg Gly Tyr 100 105 110 160 169068/1 Asp Tyr Trp Gly Gin Gly Thr Gin Val Thr val Ser Ser 115 120 125 <210> 64 <211> 122 <212> P T <213> Lama glama <400> 64 Gin val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Lys Asp Met Ala 25 30 Trp Phe Arg Gin Pro Pro Gly Lys Glu Arg Glu Phe Val Ala val lie 40 45 Tyr Ser Ser Asp Gly Ser Thr Leu val Ala Ala Ser val Lys Gly Arg 50 55 60 Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr val Tyr Leu Gin Met 65 70 75 80 Thr Ser Leu Lys Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr Ser 85 90 95 Arg Gly Tyr Ser Gly Thr Tyr Tyr Ser Thr Ser Arg Tyr Asp Tyr Trp 100 105 110 Gly Gin Gly Thr Gin val Thr val Ser Ser 115 120 <210> 65 <211> 122 <212> PRT <213> Lama glama <400> 65 Gin val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Ala Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Lys Asp Met Ala 25 30 161 169068/1 Trp Phe Arg Gin Pro Pro Gly Lys Glu Arg Glu Phe val Ala val lie 40 45 Tyr Ser Ser Asp Gly ser Thr Leu val Ala Ala Ser val Thr Gly Arg 50 55 60 Phe Thr lie ser Arg Asp Asn Ala Lys Asn Met val Tyr Leu Gin Met 65 70 75 80 Thr Ser Leu Lys Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala Ser Ser 85 90 95 Arg Gly Tyr Ser Gly Thr Tyr Tyr Ser Thr Ser Arg Tyr Asp Tyr Trp 100 105 110 Gly Gin Gly Thr Gin val Thr val Ser Ser 115 120 <210> 66 <211> 23 <212> DNA <213> Lama glama <400> 66 ggctgagctc ggtggtcctg get 23 <210> 67 <211> 45 <212> DNA <213> Lama glama <400> 67 aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt 45 <210> 68 <211> 42 <212> DNA <213> Lama glama <400> 68 ctggtgctgc agaggtgaag etteggagag gggctgeaga <210> 69 162 169068/1 <211> 42 <212> DNA <213> Lama glama <400> 69 atccatgcaa atcctctaga atccagagca cagtttgtgg ag 42 <210> 70 <211> 45 <212> DNA <213> Lama glama <400> 70 ccggtgagcc ccaccactct aagcttggag gacatctcgg aaccg 45 <210> 71 <211> 42 <212> DNA <213> Lama glama <400> 71 ccccagggtc gaaaccctct agagccccgg gcccacagtg ac 42 <210> 72 <211> 20 <212> DNA <213> Lama glama <400> 72 cccctggtcc cagttccctc <210> 73 <211> 20 <212> DNA <213> Lama glama <400> 73 tgtgctcgcg gggccggtac 20 163 169068/1 <210> 74 <211> 47 <212> DNA <213> Lama glama <400> 74 gtcctcgcaa ctgcggccca gccggcctgt gctcgcgggg ccggtac <210> 75 <211> 42 <212> DNA <213> Lama glama <400> 75 gtcctcgcaa ctgcgcggcc gccccctggt cccagttccc tc <210> 76 <211> 60 <212> DNA <213> Lama glama <400> 76 agagacaact ccaagaacac gctgtatctg caaatgaaca gcctgagagc tgaggacacg <210> 77 <211> 20 <212> PRT <213> Lama Glama <400> 77 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn ser Leu Arg 1 5 10 15 Ala Glu Asp Thr <210> 78 <211> 30 <212> DNA 164 169068/1 <213> Lama glama <400> 78 attactgtgc taaaggggcc ggtactagtt 30 <210> 79 <211> 9 <212> PRT <213> Lama Glama <400> 79 Tyr Cys Ala Lys Gly Ala Gly Thr Ser 1 5 <210> 80 <211> 35 <212> DNA <213> Lama glama <400> 80 tcctgtgcag cctccggatt cactttcagt tggta 35 <210> 81 <211> 11 <212> PRT <213> Lama Glama <400> 81 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp 1 5 10 <210> 82 <211> 253 <212> DNA <213> Lama glama <400> 82 aagcttgcat gcaaattcta tttcaaggag acagtcataa tgaaatacct attgcctacg 60 gcagccgctg gattgttatt actcgcggcc cagccggcca tggggcctaa taggcggccg 120 165 169068/1 cacaggtgca gctgcaggag tcataatgag ggacccaggt caccgtctcc tcagaacaaa aactcatctc agaagaggat ctgaatgggg ccgcacatca tcatcatcat cattaatgag aattcactgg ccg <210> 83 <211> 61 <212> PRT <213> Lama glama <400> 83 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gin Pro Ala Met Gly Pro Ala Ala Ala Gin val Gin Leu Gin Glu 25 30 Ser Gly Thr Gin val Thr val Ser Ser Glu Gin Lys Leu lie Ser Glu 40 45 Glu Asp Leu Asn Gly Ala Ala His His His His His His 50 55 60 <210> 84 <211> 98 <212> PRT <213> Homo sapiens <400> 84 Glu val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe ser Ser Tyr 25 30 Ala Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Ala lie Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 166 169068/1 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Ala Lys <210> 85 <211> 121 <212> PRT <213> Lama glama <400> 85 Gin val Gin Leu Gin Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu ser cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr 25 30 Pro Met Ser Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 40 45 Ser Thr lie Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu 65 70 75 80 Gin Met Asn Ser Leu Arg Pro Glu Asp Thr Ala val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ala Gly Thr Ser Ser Tyr Leu Pro Gin Arg Gly Asn Trp Asp 100 105 110 Gin Gly Thr Gin val Thr lie Ser Ser 115 120

Claims (22)

- 167 - 169068/1 CLAIMS:

1. A polypeptide or polypeptide construct comprising at least one single domain . antibody directed against von Willebrand Factor (vWF), wherein the at least one single - domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 3, 5 5 or 7, or to an homologous sequence of any of SEQ ID NOs: 3,5 or 7 with a sequence identity of more than 70% with the parent sequence and wherein said single domain antibody is able to inhibit at least 50% of platelet aggregation at high shear (1600 s"1) at a concentration of 1 μg/ml or lower.

2. The polypeptide or polypeptide construct according to claim 1 , wherein said 10 single domain antibody is a humanized VHH domain.

3. The polypeptide or polypeptide construct according to claims 1 to 2, wherein said single domain antibody corresponds to a sequence represented by any of SEQ ID NO: 3, or to an homologous sequence of SEQ ID NO: 3 with a sequence identity of more than 70% with the parent sequence. 15

4. The polypeptide or polypeptide construct according to claims 1 to 3, wherein said single domain antibody corresponds to a sequence represented by SEQ ID NO: 5, or to an homologous sequence of SEQ ID NO: 5 with a sequence identity of more than 70% with the parent sequence. ■:', ", ·

5. The polypeptide or polypeptide construct according to claims 1 to 4, wherein 20 said single domain antibody corresponds to a sequence represented by SEQ ID NO: 7, or to an homologous sequence of SEQ ID NO: 5 with a sequence identity of more than , 70% with the parent sequence. : v

6. The polypeptide or polypeptide construct according to claims 1 to 5 comprising two single domain antibodies directed against von Willebrand Factor; and 25 '.;·;', i) wherein at least one of the two single domain antibodies correspond to a sequence represented by SEQ ID NO: 3 with a sequence identity of more ■: ' : '· ■' ' than 70%, or ii) wherein . at least one of the two single domain antibodies correspond to a sequence represented by SEQ ID NO: 5, or to an homologous sequence . 30 of SEQ ID NO: 5 with a sequence identity of more than 70%, or iii) . wherein at least one of the two single domain antibodies correspond to a sequence represented by SEQ ID NO: 7, or to an homologous sequence V of SEQ ID NO: 7 with a sequence identity of more than 70%. - 168 - 169068/1 ,

7. The polypeptide or polypeptide construct according to claims 1 to 6, in which at , least one single domain antibody is a VHH domain. , .

8. . The polypeptide or polypeptide construct according to claims 1 to 7, in which at least one single domain antibody is a VHH domain comprising an amino acid at 5 position 45 according to the Kabat numbering that is selected from the group consisting of glycine, alanine, valine, leucine, isoleuc.ine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, and glutamine. .

9. The polypeptide or polypeptide construct according to claims 1 to 8, in which at least one single domain antibody is a VHH domain comprising an amino acid at 0 position 103 according to the Kabat numbering selected from the group consisting of arginine, serine or an uncharged residue, optionally glycine.

10. The polypeptide or polypeptide construct according to claims 1, to 9, in which at least one single domain antibody is a VHH domain that is obtained by immunising a camel and obtaining hybridoma's therefrom, or by cloning a library of single domain 5 antibodies and subsequently selecting the VHH using phage display. 1 1 . ...

11. The polypeptide or polypeptide construct according to claims 1 to 10, in which at least one single domain antibody is humanized. 1 ; ' ...

12. ; The polypeptide or polypeptide construct according to claims 1 to 1 1 , in which ■ at least one single domain antibody is a humanized VHH domain. 0

13. The polypeptide or polypeptide construct according to claims 1 to .12, in which at least one single domain antibody is humanized by replacing one or more of the Camelidae amino acids by their human counterparts as found in a human consensus sequence.

14. The polypeptide or polypeptide construct according to claims 1 to 13, in which 5 at least one single domain antibody is humanized by replacing any of the following residues either alone or in combination: FR1 positions 1 , 5, 28 and 30, the hallmark amino acids at FR2 positions 37, 44, 45 and 47, FR3 positions 74, 75, 76; 83, 84, 93 and 94 and FR4 positions 103, 104, 108 and 1 1 1 , wherein the numbering of the positions is according to the Kabat numbering. 0

15. ; The polypeptide or polypeptide construct according to claims 1 to 14 that comprises one or more single domain antibodies directed against the A1 domain of vWF or the A1 domain of activated vWF, and one or more single domain antibodies directed against the A3 domain of vWF.

16. The polypeptide or polypeptide construct according to claims 1 to 15, in which 5 the two or more single domain antibodies are of the same sequence. - 169 - 169068/1

17. The polypeptide or polypeptide construct according to claims 1 to 16, in which the C-terminal end of the first single domain antibody is linked to the N-terminal e rid of the next single domain antibody.

18. The polypeptide or polypeptide construct according to claims 1 to 17, wherein said homologous sequence is not able to inhibit 50% of platelet aggregation under low shear (300 s"1) condition at 10 pg/ml or at lower concentrations.

19. The composition comprising a polypeptide or polypeptide construct according to claims 1 to 17 and a pharmaceutically acceptable vehicle.

20. The. composition comprising a polypeptide or polypeptide construct according to claims 1 to 17 adapted to the chosen route of administration, wherein the route of administration is orally or parenterally, ; by intra-nasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes; and. a pharmaceutically acceptable vehicle. ". ; '-- :- ' ;

21. The nucleic acid encoding a polypeptide or polypeptide construct according to claims 1 to 17. ' ;

22. The use of a polypeptide or polypeptide construct according to claims 1 to 17 for the preparation of a medicament for the prevention, treatment and/or alleviation of conditions of platelet-mediated aggregation wherein said conditions are any of the formation of a non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, restenosis, restenosis after PCTA or stenting, thrombus formation in stenosed arteries, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries. For the Applicants, REINHOLD COHN AND PARTNERS