WO2013123591A1 - Multimerized single domain antibody ligands of prothrombin as inhibitors of blood and extravascular coagulation - Google Patents

Abstract

A multimer of an antibody V_HH domain, where the V_HH domain has binding affinity to prothrombin, is useful for inhibiting generation of thrombin from prothrombin. Such a multimer is useful in a method of inhibiting thrombin production in a blood stream or in a tissue whereby the multimer is either introduced into the blood stream or into the tissue directly. Inhibiting thrombin production from prothrombin leads to inhibiting blood or extra-vascular coagulation. Thus, such multimers are particularly useful for inhibiting coagulation in applications where blood clotting can lead to fibrin deposition and thrombotic complications, for example, at sites of vascular or tissue injury, such as atherosclerotic lesions and arthritic joints.

Description

MULTIMERIZED SINGLE DOMAIN ANTIBODY LIGANDS OF PROTHROMBIN AS INHIBITORS OF BLOOD AND EXTRAVASCULAR COAGULATION

Field of the Invention

This invention relates to proteins for inhibiting blood coagulation and extra-vascular fibrin deposition, in particular to proteins comprising multimerized single domain antibody for inhibiting the generation of active thrombin by the prothrombinase.

Background of the Invention

Thrombotic complications constitute major life-threatening conditions for both the aging population and young adults (Hansson 2006; Libby 2005). One underlining cause is the activation of the blood coagulation cascade and fibrin deposition, which can generate occlusive blood clots and impede blood flow, leading to thromboembolism, deep-vein thrombosis, ischemic heart diseases or stroke (Libby 2005). Elevated levels of thrombin resulting from an activated coagulation cascade are associated with almost all inflammatory conditions ranging from arthritis (Morris 1994; Busso 2002), pulmonary fibrosis (Ludwicka-Bradley 2004; Vergnolle 2009) to inflammatory bowel diseases (Vergnolle 2009; Saibeni 2010). Extravascular fibrin deposition is a major pathogenic factor for chronic synovial inflammation in arthritis, especially in osteoarthritis and rheumatoid arthritis (Busso 2002). Thrombosis also increases the lethality of many human cancers (Agorogiannis 2002; Khorana 2004; Rak 2006; Ornstein 2002) and infectious diseases (Marsden 2003; Opal 2003). Such widespread occurrence and unmet medical needs have fueled major advances in anti-thrombotic and anti-coagulant therapies (Gross 2008; Hoppensteadt 2008) and a better understanding of blood coagulation biochemistry (Mann 2006; Bock 2007). These latest research advances present a unique opportunity for the development of more efficacious anti-thrombotic agents that take advantage of the localized characteristics of pathogenic thrombin generation and fibrin deposition induced by vascular lesions, atherosclerotic plaques, inflammatory joint diseases and other inflammatory conditions.

The blood coagulation cascade is triggered by the expression of the tissue factor on injured vasculatures or tissue cells (Mann 2006; Libby 2005; Busso 2002; Mann 1988), for example at sites of atherosclerotic lesions (Libby 2005) or within inflamed joints (Busso 2002). All coagulation pathways converge on the prothrombinase assembly, which rapidly converts prothrombin into the ultimate protease, thrombin, responsible for the formation of the blood (fibrin) clot (Mann 1988). Efficient generation of thrombin requires finely orchestrated cleavages of two peptide bonds in prothrombin by the prothrombinase composed of the serine protease factor (F) Xa, and the protein cofactor Va, which are assembled on phospholipid membranes in the presence of Ca²⁺ ions (Mann 1988; Mann 1987). Most recent research points to another level of complexity of the prothrombin activation process, in that an anti-coagulant form of thrombin (called meizo-thrombin or mlla) may be accumulated under certain physiological environments, while the procoagulant thrombin appears to be generated by activated platelets (Wood 2011 ) and by activated platelets under blood flow (Haynes 2012). One interesting therapeutic strategy is the potential combined use of FXa and thrombin inhibitors to enable fast-acting anti-coagulation, for example for use during coronary interventions (Fareed 2008). Docking interactions between prothrombin and the prothrombinase can also be targeted to reduce the production of thrombin at vascular or tissue sites decorated with elevated prothrombinase activity (Kalafatis 2003; Kretz 2006; Bock 2007). Indeed, monoclonal antibodies raised against a domain of human prothrombin can attenuate prothrombin turnover by the prothrombinase (Church 1991 ). DNA aptamers for thrombin were also shown to exhibit a potent inhibitory activity toward the prothrombinase-catalyzed generation of thrombin from prothrombin (Kretz 2006). However, the current generation of such antibody- or DNA-based ligands of prothrombin may suffer from significantly reduced in vivo efficacy as a result of high-affinity binding to and depletion by high levels of circulating prothrombin at micromolar concentrations. For example, an aptamer-based anticoagulant targeting prothrombin had only a moderate in vivo activity (IC₅₀ > 2 μΜ) (Griffin 1993) despite an intrinsically high affinity (K_d < 100 nM) of binding toward prothrombin (Kretz 2006). In fact, this is an intrinsic limitation for all prothrombin-specific ligands unless they can be made to act only on prothrombin- prothrombinase interactions at sites of prothrombinase accumulation.

The current generation of coagulation inhibitors, of which many are direct thrombin or FXa inhibitors, are administered and active systemically (Vorchheimer 2002; Hoppensteadt 2008; Gross 2008), and as such can cause either bleeding side effects or rebound coagulation and re-occlusion after cessation of anticoagulant therapy (Fareed 2008; Weitz 2002; Vorchheimer 2002). Mechanistically, most direct thrombin inhibitors target both the procoagulant thrombin and the anti-coagulant variant meizo-thrombin through their inhibitory actions on the catalytic active site. These complications underline the need for more selective thrombin inhibitors, especially for locally-active agents to prevent pathogenic thrombin accumulation at sites of vascular and/or tissue injury (Khrenov 2002; Libby 2002; Busso 2002). There also remains a need for potent agents to reduce extra-vascular coagulation, particularly for localized inhibition of fibrin deposition in inflamed tissues and organs.

Summary of the Invention The present invention relates to proteins for inhibiting blood coagulation and extra- vascular fibrin deposition, in particular to proteins comprising multimerized single domain antibodies for inhibiting the generation of active thrombin by the prothrombinase.

Accordingly, there is provided a polypeptide comprising a multimer of a single domain antibody (sdAb), the sdAb having binding affinity to prothrombin, to inhibit generation of thrombin from prothrombin by prothrombinase. The sdAb may have an IC₅₀ for prothrombin binding affinity in the mM to sub-mM range. For example, the sdAb may have an IC₅₀ for prothrombin binding affinity in a range from 0.1-900 mM.

In the polypeptide described above, the sdAb may comprise: a complementarity determining region (CDR) 1 selected from GRTFDRYGWF (SEQ ID NO: 1 ) and GRTFSSLSIAWF (SEQ ID NO: 4); a CDR2 selected from SIGTRLHYADSVKG (SEQ ID NO: 2) and G I RWTAGSKTYANWVKG (SEQ ID NO: 5); and a CDR3 selected from CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3) and CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6). In one specific, non-limiting example, the sdAb may comprise a CDR1 sequence of GRTFDRYGWF (SEQ ID NO: 1 ), a CDR2 sequence of SIGTRLHYADSVKG (SEQ ID NO: 2), and a CDR3 sequence of CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3). In another non-limiting example, the sdAb may comprise a CDR1 sequence of GRTFSSLSIAWF (SEQ ID NO: 4), a CDR2 sequence of G I RWTAGSKTYANWVKG (SEQ ID NO: 5), and a CDR3 sequence of CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6).

The sdAb may be a VH domain. The VH domain of the polypeptide of the present invention may comprise the amino acid sequence

DVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPGKEREFVASIGTRL HYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAESTRNWYYKMSN DYDYWGQGTQVTVSS (SEQ ID NO:7), DVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (SEQ ID NO:8),

DVQLQASGGGLVQAGGSLRLTCAASGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (SEQ ID N0:9), or a sequence substantially identical thereto.

In one example, the polypeptide comprises two or more sdAb. The sdAb may be linked together by one or more than one disulfide bond. The disulfide bonds may be between cysteine residues in the sdAb. The disulfide bonds may be between cysteine residues in a leading sequence. In one example, the leading sequence may be CHNDGGGGS (SEQ ID NO:12).

Alternatively, the sdAb may comprise the sequence

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPG KEREFVASIGTRLHYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAE STRNW YYKMSN D YDYWGQGTQ VTVSSLEH H H H H H (modified VHpro#10, SEQ ID NO:13),

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAP G KE RE F VAG I RWTAG SKT YANWVKG RF Al S KDN AKN KVYLQ M N YL KPE DTAVY YCAADNISDWGISKQLRTYHYWGQGTQVTVSSLEHHHHHH (modified VHpro#5,

SEQ ID NO:14), or a sequence substantially identical thereto.

In one example, in the polypeptide of the present invention, the multimer may be a dimer.

There is further provided a method of inhibiting thrombin production in a blood stream comprising introducing into the blood stream a polypeptide comprising a multimer of a sdAb, the sdAb having binding affinity to prothrombin.

There is further provided a method of inhibiting thrombin production in an inflamed tissue of a subject, the method comprising introducing into the tissue or the blood stream of the subject a polypeptide comprising a multimer of a sdAb, the sdAb having binding affinity to prothrombin. There is further provided a method of inhibiting blood coagulation in a subject comprising identifying a subject in need of an anti-coagulant; and, administering to the subject a polypeptide comprising a multimer of a sdAb, the sdAb having binding affinity to prothrombin. The subject may have an atherosclerotic lesion. There is further provided a method of inhibiting extra-vascular coagulation in a subject comprising identifying a subject in need of an extra-vascular anti-coagulant; and, administering to the subject a polypeptide comprising a multimer of a sdAb, the sdAb having binding affinity to prothrombin. The subject may have arthritic joint.

As described herein, sdAb are presently described that are specific for prothrombin. VHpro#5 (SEQ ID NO:1 1 ) and VHpro#10 (SEQ ID ΝΟ. 0) demonstrated low-affinity specific binding to human prothrombin. In blood coagulation inhibition assays, the antibody V_HH domain dimers exhibited potent inhibition of blood coagulation. The dimeric form of VHpro#10, in particular, exhibited potent inhibitory activities in the first few minutes of prothrombin activation; the inhibitory action was prolonged by a change of the composition of phospholipid membrane. This appears indicative of a unique mechanism of action by the VHpro#10 dimer, as it remains intact under the emerging thrombin activity. In addition to inhibiting the interaction of prothrombin with the prothrombinase, the VHpro#10 dimer also binds to meizo-thrombin. In view of recent studies relating to the anti-coagulant intermediate meizo-thrombin and procoagulant thrombin in prothrombin activation, polypeptides as described herein, such as the VHpro#10 dimer, may be more effective inhibitors of thrombin generation under the in vivo conditions of blood flow.

Further features of the invention will be described or will become apparent in the course of the following detailed description. Brief Description of the Drawings

In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings.

FIGURE 1A depicts alignment of modified antibody VH domain sequences with the antibody VH domain sequences from which the modified sequences are constructed. The original sequences are indicated by the suffix -mo_ while the modified sequences which dimerize are indicated by the suffix -di_. FIGURE 1 B is the HPLC profile indicating the elution position (indicated by *) of the modified VHpro#10 protein. FIGURE 1C is the mass spectra of the modified VHpro#10 (VHpro#10-di) identifying the formation of a dimeric molecule following protein purification. FIGURE 1 D is the result of the SDS- PAGE characterization of VHpro#10-di from various sources (1=from IMAC; 2=from HPLC; 3=from NMR; 4=from precipitate of dialysis) showing the response of the dimeric species to disulfide reduction by DTT and the co-existence of the dimeric protein with the monomeric species. FIGURE 1 E is the proton NMR spectra of VHpro#10-di demonstrating the proper folding of VHpro#10 in the form of a dimeric molecule.

FIGURE 2A depicts graphs of prothrombin time (PT) assays for VHpro#10 dimer (ii), IC₅₀=5.6pM) compared to VHpro#10 monomer (i), IC₅₀=10pM). FIGURE 2B depicts graphs of prothrombin time (PT) assays for VHpro#5 dimer (ii), ΙΟ₅₀=4.2μΜ) compared to VHpro#5 monomer (i), concentrations of 0, 15, 30, and 50 μΜ).

FIGURE 3A depicts graphs of inhibition activities using a coagulation proteome assay (i) and a thromboplastin assay (prothrombin time assay, (ii) on VHpro#10 dimer. The samples for both the coagulation proteome and prothrombin time (PT) assays were the same. There is not a clear dose-dependency for the delay of thrombin burst, but clearly a delay in thrombin peak level. This lack of burst delay for thrombin generation is consistent with prior experiments where the protein sample is extensively dialyzed to remove phosphate ions (>1000x, data not shown). FIGURE 3B depicts graphs of inhibition activities using a coagulation proteome assay (i) and a thromboplastin assay (ii) VHpro#5 dimer. The dose-dependent delay was not very evident with VHpro#5-di. However, there remained some indication of the peak level of thrombin generation shifting to the right with increasing concentration of VHpro#5-di remained. Importantly, prothrombin time was not affected in a significant way by increasing concentrations of phosphate up to 0.5 m , indicating that minor amounts of phosphate ions (up to 0.5 mM) not removed after buffer exchange do not significantly contribute to the observed inhibitory effects of VHpro# 0-di and VHpro#5-di.

FIGURE 4 depicts graphs of inhibition activities using a coagulation proteome assay on a known hirudin-based thrombin inhibitor, bivalirudin and MH2-wZIP4, another thrombin inhibitor described in WO 2012/142696. The assay was performed at two experimental temperatures, 37 °C (i) and 42 °C (ii).

FIGURE 5 depicts graphs illustrating dependence of VHpro#10 dimer inhibitory activities on the time of prothrombin activation by prothrombinase reconstituted in 75% / 25% phophatidylcholine/phophatidylserine (PC/PS), where thrombin generation is measured by fibrinogen clotting assay. Data show 1 (i), 2 (ii), and 3 (iii) minutes of prothrombin activation.

FIGURE 6 illustrates dependence of VHpro#10 dimer inhibitory activities on composition of a phospholipid membrane, where 1/CT is the inverse of clotting time, i) 75% / 25% PC/PS; ii) 95% / 5% PC/PS.

FIGURES 7A to 7C illustrate three methods of following prothrombin activation by prothrombinase, with S2238 and DAPA measuring the emergence of catalytic activity of meizo-thrombin and thrombin, and fibrinogen clotting measuring the level of active thrombin. FIGURE 7B shows DAPA kinetics, while FIGURE 7C shows DAPA fluorescence emission.

Description of Preferred Embodiments

The present invention relates to proteins for inhibiting blood coagulation and extra- vascular fibrin deposition, in particular to proteins comprising multimerized single domain antibodies for inhibiting the generation of active thrombin by the prothrombinase. Prothrombin is the circulating inactive precursor of the clot-making enzyme thrombin (Gross 2008; Mann 2006). Prothrombin circulates in the blood stream and the extra- vascular space in primarily a monomeric state, especially at a low concentration of Ca²⁺ ions. However, prothrombin appears to have a strong tendency to dimerize in the presence of Ca²⁺ ions and phospholipid membranes simultaneously (Anderson 1998). Such studies suggest the involvement of a dimeric or multimeric state of prothrombin for the efficient generation of thrombin from prothrombin by the prothrombinase enzyme complex. Such molecular interactions involving a dimeric or multimeric prothrombin provide an avenue for the modulation of thrombin production and thus the modulation of blood and extra-vascular coagulation through the multimerization of low-affinity ligands of prothrombin with decreased binding to circulating (or fluid-phase) prothrombin. The new coagulation inhibitors of the present invention therefore comprise multimeric single domain antibodies having low-affinity binding to prothrombin to enable specific targeting and inhibition of functionally-active membrane-bound prothrombin.

The present invention provides a polypeptide comprising a multimer of a sdAb, the sdAb having binding affinity to prothrombin, to inhibit generation of thrombin from prothrombin by prothrombinase. The term "antibody", also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (V_L) and a constant (C_L) domain, while the heavy chain folds into a variable (V_H) and three constant (C_H, C_H2, C_H3) domains. Interaction of the heavy and light chain variable domains (V_H and V_L) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.

The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy and light chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape and chemistry of the surface they present to the antigen. One commonly used scheme for identification of the regions of hypervariability is that of Kabat et al (1991a; 1991 b), which defines the "complementarity-determining regions" (CDR) based on sequence variability at the antigen-binding regions of the VH and VL domains. For this reason, the regions forming the antigen-binding site are referred to as CDR L1 , CDR L2, CDR L3, CDR H1 , CDR H2, CDR H3 in the case of antibodies comprising a VH and a VL domain; or as CDR1 , CDR2, CDR3 in the case of the antigen-binding regions of either a heavy chain or a light chain. A "single domain antibody" as referred to herein may include any suitable antigen-binding single domain antibody known in the art. The sdAb may be a naturally-occurring antibody fragment, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. In a non-limiting example, the sdAb may be derived from heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993), which lack light chains and thus their antigen binding sites consist of one domain, termed V_HH. sdAb have also been observed in shark and are termed V_NAR (Nuttall et al, 2003). Other sdAb may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term "sdAb" includes those sdAb directly isolated from V_H, V_HH, V_L) or V_NAR reservoir of any origin through phage display or other technologies, sdAb derived from the aforementioned sdAb, recombinantly produced sdAb, as well as those sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, e.g., camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.

A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). A sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDR may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1 , CDR2, and CDR3, and numbered as defined by Kabat et al (1991b). The sdAb in the polypeptide of the present invention has binding affinity for prothrombin, and may show no affinity for thrombin alone. Binding of the sdAb as described herein may inhibit generation of active thrombin by prothrombinase. The sdAb may bind prothrombin with low-affinity, thus enabling specific targeting and inhibition of functionally- active membrane-bound prothrombin. The sdAb monomer as described above may have low-affinity binding to prothrombin. By the term "low-affinity binding", it is meant that the prothrombin binding affinity may have a K_d in the mM to sub-mM range. For example, and without wishing to be limiting in any manner, the may be in the range of 0.1-900 mM, or any value therebetween; more specifically, the K_d may be 0.1 , 0.5, 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, or 900 mM, or any value therebetween or any range defined by any two values just defined.

The low-affinity binding of the sdAb monomer to prothrombin allows the multimeric sdAb of the present invention to specifically target membrane-bound prothrombin. Without wishing to be bound by theory in any manner, due to the low affinity of the sdAb, the antibody may show decreased binding to circulating (circulating) prothrombin; however, the avidity of the multimeric sdAb increases its interaction to membrane-bound prothrombin, such as that found in atherosclerotic lesions or inflamed subcutaneous tissues (e.g. arthritic joints) or other sites of vascular injury. In the polypeptide of the invention as described above, the sdAb may comprise: a complementarity determining region (CDR) 1 selected from GRTFDRYGWF (SEQ ID NO: 1 ) and GRTFSSLSIAWF (SEQ ID NO: 4); a CDR2 selected from SIGTRLHYADSVKG (SEQ ID NO: 2) and

GIRWTAGSKTYANWVKG (SEQ ID NO: 5); and a CDR3 selected from CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3) and CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6).

In one specific, non-limiting example, the sdAb may comprise a CDR1 sequence of GRTFDRYGWF (SEQ ID NO: 1 ), a CDR2 sequence of SIGTRLHYADSVKG (SEQ ID NO: 2), and a CDR3 sequence of CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3). In another non-limiting example, the sdAb may comprise a CDR1 sequence of GRTFSSLSIAWF (SEQ ID NO: 4), a CDR2 sequence of GIRWTAGSKTYANWVKG (SEQ ID NO: 5), and a CDR3 sequence of CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6). The term "sdAb" is as defined above. The sdAb may be of camelid origin or derived from a camelid V_HH, and thus may be based on camelid framework regions; alternatively, the CDR described above may be grafted onto V_NAR, V_HH, V_h, or V_L framework regions. The present embodiment further encompasses an antibody fragment that is "humanized" using any suitable method known in the art, for example, but not limited to CDR grafting and veneering. Humanization of an antibody or antibody fragment comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or fragment thereof when introduced into human subjects. In the process of CDR grafting, one or more than one of the heavy chain CDR defined herein may be fused or grafted to a human variable region (V_H, or V_L). In such a case, the conformation of said one or more than one hypervariable loop is preserved, and the affinity and specificity of the sdAb for its target (i.e., prothrombin) is also preserved. CDR grafting is known in the art and is described in at least the following: US Patent No. 6180370, US Patent No. 5693761 , US Patent No. 6054297, US Patent No. 5859205, and European Patent No. 626390. Veneering, also referred to in the art as "variable region resurfacing", involves humanizing solvent-exposed positions of the antibody or fragment; thus, buried non-humanized residues, which may be important for CDR conformation, are preserved while the potential for immunological reaction against solvent-exposed regions is minimized. Veneering is known in the art and is described in at least the following: US Patent No. 5869619, US Patent No. 5766886, US Patent No. 5821123, and European Patent No. 519596. Persons of skill in the art would be amply familiar with methods of preparing such humanized antibody fragments. In one specific, non-limiting example, the sdAb may comprise a sequence selected from:

DVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPGKEREFVASIGTRL HYADSVKGRFTISRDNAKSTAFLE NSLKPEDTAVYYCAAAESTRNWYYKMSN DYDYWGQGTQVTVSS (SEQ ID NO:7),

DVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (SEQ ID NO:8),

DVQLQASGGGLVQAGGSLRLTCAASGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (SEQ ID NO:9), or a sequence substantially identical thereto.

A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially identical" polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). These conservative amino acid mutations may be made to the framework regions of the sdAb while maintaining the CDR sequences listed above.

In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBl BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at http://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art.

The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any percentage therebetween) identical at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s) occurring in the framework regions.

The sdAb of the polypeptide of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection tag (for example, but not limited to c-Myc), a purification tag (for example, but not limited to a His₅ or His₆), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, linker sequences, which may include amino acids encoded by nucleotides bordering restriction sites, may be used in conjunction with the additional sequences or tags. Multimers of the sdAb may be produced by linking two or more of the sdAb together by any suitable method. Multimers may be homo- or hetero-multimers, and may be dimers, trimers, tetramers, pentamers, hexamers or compounds having even a larger number of mers. Methods of producing multimers are well-known to those of skill in the art. For example, multimers may be obtained by direct linking connection (Nielson et al, 2000), c- jun/Fos interaction (de Kruif & Logtenberg, 1996), "Knob into holes" interaction (Ridgway et al, 1996). In a particular non-limiting example, the multimer may comprise two or more sdAb linked together by one or more disulfide bonds. The disulfide bonds may be formed through one or more than one cysteine residues in the sdAb. The cysteine residue(s) may be naturally-occurring, or the sdAb may be modified to include one or more than one cysteine residue; the engineered cysteine residue(s) as just described may be contained within a leading or appending sequence to the sdAb. The naturally-occurring or engineered cysteine(s) can then form disulfide bonds, preferably spontaneously, between mers to form the multimer. For example and without wishing to be limiting in any manner, dimers may be formed by the inclusion of one additional cysteine residue in the sdAb or a leading or appending sequence, whereby two of these so modified sdAb are then linked together by reaction between their respective cysteine residues to form the dimer. Inclusion of more than one cysteine residue in the sdAb may lead to the formation of trimers and higher order multimers. Other methods of multimerizing the sdAb may include, but are not limited to addition of residues enabling transglutamination followed by cross-linking catalyzed by transglutaminases (similar to cross-linking of polymerized fibrin by factor XIII, see Griffin 2002).

When one or more than one disulfide-forming cysteine residue is included by modification of the sdAb (i.e. an engineered cysteine(s)), the one or more than one disulfide-forming cysteine residue may be included in a leading or appending sequence to the sdAb, as described above. Any suitable leading or appending sequence may be used, provided said sequence is structurally flexible and has no predominent interactions with the sdAb. In one particular example, the sequence may be CHNDGGGGS (SEQ ID NO: 12). The sequence may be a leading sequence; the leading sequence may be inserted at the very beginning of the sequence, or within the first few amino acids. In one specific, non-limiting example, the sdAb may comprise the sequence MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPG KEREFVASIGTRLHYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAE STRNWYYKMSNDYDYWGQGTQVTVSSLEHHHHHH (modified VHpro#10, SEQ ID NO:13), MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAP GKEREFVAGIRWTAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVY YCAADNISDWGISKQLRTYHYWGQGTQVTVSSLEHHHHHH (modified VHpro#5, SEQ ID NO: 14), or a sequence substantially identical thereto. The present invention also encompasses nucleic acid sequences encoding the molecules as described herein. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described. Polypeptides of the present invention may be particularly useful for inhibiting blood or extra-vascular coagulation in applications where fibrin deposition can lead to either thrombotic complications or tissue inflammation. For example, the polypeptides of the present invention can be utilized to specifically target prothrombin accumulated at sites of vascular injury, for example atherosclerotic lesions or inflamed subcutaneous tissues (e.g. arthritic joints). Such a method may comprise identifying a subject in need and administering to the subject a polypeptide of the present invention. A subject in need of an anti-coagulant may have an atherosclerotic lesion. A subject in need of an extra- vascular anti-coagulant may have arthritic joint.

The present invention also provides methods of inhibiting thrombin production in the blood stream or in inflamed tissue of a subject; the method comprises introducing into the blood stream a polypeptide as described herein.

In the methods described herein, the polypeptides may be administered "as is" to a subject in need of such anti-coagulation therapy, or may be formulated into a pharmacologically acceptable composition for administration to a subject in need of such anti-coagulation therapy. The composition or formulation may comprise pharmaceutically acceptable diluents, carriers, or excipients. By the term "pharmaceutically acceptable", it is meant that the diluent, carrier, or excipient is compatible with the compound of the present invention, and is not deleterious to the recipient of the composition. The diluent, carrier, or excipient may be any suitable diluent, carrier, or excipient known in the art, and may vary based on the route of administration and dosage required.

Any suitable administration method known in the art may be used, for example, parenteral injection (e.g. intradermal, intramuscular, intraosseous, intraperitoneal, intravenous, intra-arterial, subcutaneous), topical (e.g. cream, gel, liniment, balm, lotion, ointment, ear drops, eye drops, skin patch) or suppository (e.g. rectal, vaginal).

The polypeptides of the present invention may form part of a kit or commercial package together with instructions for use in inhibiting coagulation. Polypeptides are typically used in an amount effective to inhibit coagulation. Dosing regimes for a particular subject may be readily determined by a treating physician.

Subjects that may be treated include animals, for example, mammals. Mammalian subjects include, but are not limited to humans, horses, cows, sheep, goats, pigs, dogs, cats, rabbits, mice, hamsters, guinea pigs, monkeys, chimpanzees and rats.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Screeninp for Antibody VH Domains with Prothrombin-Binding Activity

Selection of prothrombin-binding phages: A phage-display library containing about 10⁸ different antibody V_HH fragments derived from heavy chain antibody mRNA of non- immunized llama (Tanha 2002; Tanha 2006) was screened for potentially high-affinity ligands of human prothrombin. Thus, 10 pg of prothrombin in PBS was coated overnight to Nunc Maxisorp™ 8-well strips at 4°C. Prothrombin coated wells were rinsed three times with PBS and then blocked with 400 μΙ_ PBS-2% skim milk (2% MPBS). 1 x10¹² plaque-forming units of phages in 2% MPBS were added and incubated at room temperature for 1.5 hours. Wells were washed ten times with PBS-0.1 % Tween™ 20 (PBS-T) and then ten times with PBS. Bound phages were eluted with 200 μΙ_ of freshly prepared 100 mM triethylamine, followed immediately by neutralization with 100 pL of 1 M Tris Tris-HCL, pH 7.4. Serial dilutions of eluted phages were used to infect 300 μΙ_ of exponentially growing TG1 cells for titer determination. A larger aliquot of eluted phages was used to infect 10 mL of exponentially growing TG1 culture for phage amplification. Infected cells were spun down and resuspended in LB. The cells were mixed in 300 μΙ_ aliquots with 3 mL top agarose and spread on LB plates. Phages were propagated overnight at 37°C, purified the next day (as described in Tanha 2002) and the titer determined. 2.5x10¹¹ pfu of phages were used for the next round of selection. Three rounds of selection were done and 20 phage clones were selected from round 3 for phage ELISA. Phage ELISA: The 20 phage clones from above were tested for potential binding to prothrombin using a phage ELISA assay. Thus, 200 pL LB was inoculated with single plaques and incubated at 37°C overnight in 96-well plates. The plates were then centrifuged and the supernatants containing phage particles were assayed for binding to prothrombin and PPACK-alpha-thrombin. 1.5 pg prothrombin or PPACK-alpha-thrombin was immobilized to Nunc Immuno Maxisorp™ 8-well strips and blocked with 2% MPBS for 2 hours at 37°C. 150 pL of phage-containing supernatant in 2% MPBS were added and incubated at room temperature for 1.5 hours, followed by washing with PBS-0.05% Tween™ 20 (0.05% PBS-T). 100 pL of 1 :5000 dilution of HRP/Anti-M13 monoclonal antibody conjugate (Amersham Pharmacia Biotech) in 2% MPBS were added and incubated at room temperature for 1 hour. The wells were then washed 5 times with 0.05% PBS-T and 5 times PBS. Bound phages were detected by addition of 100 pL of a 50:50 mixture of TMB peroxidase substrate and H₂0₂. The reaction was stopped by 100 pL of 1 M H3PO4 and absorbance at 450 nm was measured. An absorbance value greater than 0.2 is considered a positive signal. All of the phage clones tested positive for prothrombin but not for PPACK-alpha-thrombin. The V_HH of four phage clones (VHpro#1 , VHpro#5, VHpro#10 and VHpro#15) were then expressed and produced for further study by activity assays and NMR spectroscopy.

Identifying Low-affinity Prothrombin-Binding VH proteins by NMR spectroscopy: ¹⁵N- labelled VHpro#1 , VHpro#5, VHpro#10 and VHpro#15 were studied by proton and two- dimensional H/¹⁵N-HSQC NMR titration. Briefly, 0 pL, 10 pL and 20 or 22 pL of a concentrated solution of human prothrombin was added to each VH protein in an approximate volume of 450 pL at a concentration of 165 μΜ to identify which of these VH proteins have specific and low-affinity binding to prothrombin. Only VHpro#5 (SEQ ID NO:1 1 ) and VHpro#10 (SEQ ID NO: 10) demonstrated specific binding to human prothrombin, as evidenced by differential perturbations on the NMR spectra of these ¹ N- labelled proteins (data not shown). The affinity of the underlining binding interaction was weak, in the mM range, as estimated by the variation of the proton NMR signal perturbations with increasing concentrations of added prothrombin. Sequences for each of VHpro#5 and VHpro#10 is shown below, with complementarity determining regions (CDR) underlined. In addition to the V_HH sequences, the clone sequences contained a two-residue extension of MG at the N-terminus and a C-terminal LEHHHHHH tag, which was used for metal-affinity (IMAC) purification. VHpro#10 (SEQ ID NO: 10)

MGDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAPGKEREFVAGI RWTAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVYYCAADNISDW GISKQLRTYHYWGQGTQVTVSSLEHH H H H H

VHpro#5 (SEQ ID NO:1 1 )

MGDVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPGKEREFVASIG

TRLHYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAESTRNWYYKM

SNDYDYWGQGTQVTVSSLEHHHHHH

Example 2: Dimerization of Prothrombin-Binding Antibody VH Domains

VHpro#5 and VHpro#10 described in Example 1 were modified by adding an N-terminal sequence extension containing a cysteine residue near the beginning. This additional sequence CHNDGGGGS (SEQ ID NO: 12) was inserted following the initial MG residues of the V_HH protein and before the start of the V_HH framework sequences. The resulting modified VHpro#5 and VHpro#10 sequences are:

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPG KEREFVASIGTRLHYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAE STRNWYYKMSNDYDYWGQGTQVTVSSLEH H H H H H (modified VHpro#10, SEQ ID NO:13), and

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAP GKEREFVAGIRWTAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVY YCAADNISDWGISKQLRTYHYWGQGTQVTVSSLEHHHHHH (modified VHpro#5,

SEQ ID NO:14)

Genes encoding the modified VHpro#10 and modified VHpro#5 were amplified by PCR using the plasmids expressing the original VHpro#10 and VHpro#5 as the template, respectively. The two synthetic primers: 5'-tac atg cca tgg gtt gtc ata acg acg gcg gtg gcg gtt ctg atg tec age tgc agg cg-3' (SEQ ID NO: 15)

5'-aat egg etc gag tga gga gac ggt gac ctg-3' (SEQ ID NO: 16) contained Nco I and Xho I restriction sites. The PCR products were gel-purified, cleaved with Ncol and Xhol restriction endonucleases, and ligated to the Ncol/Xhol-digested pET- 22b vector. Each plasmid construct was confirmed by DNA sequencing. The expression constructs were transformed into E. coli strain BL21 (DE3) to enable periplasmic expression of the modified V_HH proteins. The modified V_HH proteins spontaneously dimerize through the formation of a disulfide bond between cysteine residues on two protein monomers. Fig. 1A provides alignment of the modified sequences with the original VH polypeptides. Dimeric V_HH proteins were isolated from E. coli cells by one of the following procedures.

In Procedure 1 , cells were lysed with 4 M Gdn-HCI, 50 mM NaPi and 300 mM NaCI at pH 8. The lysed cells were eluted on a column washed with 4 M urea, 50 mM NaPi and 300 mM NaCI at pH 8, with the protein being eluted with an aqueous solution of 20 mM immidazole, 4 M urea, 50 mM NaPi and 300 mM NaCI at pH 8. Recovered protein was dialyzed by a solution of 2 M urea, 50 mM NaPi and 50 mM NaCI at pH 6.8.

In Procedure 2, cells were lysed with 4 M Gdn-HCI, 10 mM Tris-HCI and 300 mM NaCI at pH 8. The lysed cells were eluted on a column washed with 4 M Gdn-HCI, 10 mM Tris- HCI and 300 mM NaCI at pH 8, with protein being eluted with an aqueous solution of 250 mM immidazole, 4 M Gdn-HCI, 10 mM Tris-HCI and 300 mM NaCI at pH 8. Recovered protein was dialyzed by a solution of 2 M Gdn-HCI, 10 mM Tris-HCI and 100 mM NaCI at pH 6.8.

In another (more detailed) procedure (Procedure 3), the cell pellet from a 1 L bacterial culture was suspended in a 100 mL solution of 6 M urea (or 4 M Gdn-HCI in some preparations) with 50 mM sodium phosphate and 300 mM NaCI at pH 8, followed by sonication (a total duration of 2 min with 15 seconds of on-time and 15 seconds of off- time at a power setting of 5). After spinning down the lysate, the supernatant was mixed with 4 mL of the Ni-NTA resin followed by gentle shaking for 1 hour and by the final transfer to a chromatographic column. The column of the resin suspensions was washed by a 100 mL solution of 4 M urea in 50 mM sodium phosphate, 300 mM NaCI, and 20 mM immidazole at pH 8. Resin-bound protein was eluted with 250 mM immidazole in 4 M urea, 50 mM sodium phosphate and 300 mM NaCI at pH 8, until the UV absorption (A₂8o) reaches ~ 0.1. Recovered protein was dialyzed by gradually reducing concentration of urea starting at 2 M in the buffer of 40 mM Tris.HCI, 150 mM NaCI at pH 7.6.

Dimers of the V_HH domain proteins were further purified using HPLC (Fig. 1B) and characterized using mass spectrometry (Fig. 1C), SDS PAGE analysis (Fig. 1D) and proton NMR spectroscopy (Fig. 1 E).

Example 3: Determination of Blood Coagulation Inhibition

Thromboplastin (Prothrombin Time) and Activated Partial Thromboplastin Time Tests: Prothrombin time (PT) clotting or activated partial thromboplastin time (APTT) clotting assays were carried out (at certain concentrations of the VH proteins) using normal human plasma with addition of thromboplastin or activated partial thromboplastin to initiate clotting. Inhibition activities of the prothrombin-binding V_HH proteins were expressed as fold prolongation of the prothrombin time in the absence of the V_HH proteins similar to the method of Maraganore et al. (Maraganore 1990). The assays employed kits from Biopool International (Ventura, CA, USA). Briefly, 50 μΙ_ of a coagulation control plasma (Pacific Hemostasis) solution was added to 100 pL HBS (20 mM HEPES and 150 mM NaCI at pH 7.4) containing varying concentrations of the V_HH proteins. After incubation at 37 °C for 5 min, the mixture was added to 50 pL of the thromboplastin or activated partial thromboplastin reagent. Clot formation was monitored by a change in absorbance at 420 nm using a SpectraMax™ plate reader. The results for VHpro#10 and VHpro#5 dimers, compared to their respective monomers, are shown in Figs. 2A and 2B, respectively, and are summarized in Table 1. Table 1 includes comparative results for a known hirudin-based thrombin inhibitor bivalirudin (Angiomax™), which was assayed under the same conditions as described above. It is evident from the results that the antibody V_HH domain dimers exhibit potent inhibition of blood coagulation.

Table 1 - Prothrombin Time Assay

Synthetic Coagulation Proteome Assay: This assay is based on a known procedure as reported previously (Brummel-Ziedens 2008). Briefly, a pro-cofactor solution containing re-lipidated tissue factor (10 pM; molar ratio PCPS.TF = 5000) was incubated with 4 μΜ PCPS in HBS (20 mM HEPES and 150 mM NaCI at pH 7.4) and 2 mM CaCI₂ for 8 min at 37 °C. Prior to initiation of the reaction, Factor V (40 nM) and Factor VIII (1.4 nM) were added to the mixture. The reaction was initiated by addition of the profactor solution to a zymogen-inhibitor solution containing prothrombin at 2.8 μΜ. Factor VII (20 nM), Factor Vila (0.2 nM), FX (340 nM), Factor IX (180 nM), Factor XI (60 nM), TFPI (5 nM), antithrombin II (6.8 μΜ) and varied concentrations of the V_HH proteins in HBS and 2 mM CaCI₂ were equilibrated at 37°C.

After initiation of the reaction, at selected time points, 10 pL aliquots were withdrawn from the reaction mixture and quenched in 20 mM EDTA in HBS (pH 7.4) containing 0.2 mM Spectrozyme™ TH and assayed immediately for the proteolytic activity of thrombin. The hydrolysis of Spectrozyme™ TH was monitored by the change in absorbance at 405 nm using a SpectraMax™ plate reader (Molecular Devices Corp., Menlo Park, CA, USA). Concentration of thrombin generated by the reaction was calculated from a standard curve prepared by serial dilutions of known concentrations of a-thrombin.

The results for VHpro#10 and VHpro#5 dimers are shown in Figs. 3A and 3B, respectively. The VHpro#10 and VHpro#5 dimer samples were exactly the same for the coagulation proteome assays (Figs. 3A and 3B) as for the prothrombin time (PT) assays (Figs. 2A and 2B, bottom panels). Referring to Fig. 3, there is not a clear dose- dependency for the delay of the burst for thrombin generation, but clearly a delay in the peak levels of thrombin. However, for VHpro#5 dimer (Fig. 3B), there is still some indication for the peak level of thrombin generation shifting to the right as concentration of the VHpro#5 dimer increases. Similar data were obtained in experiments (data not shown) where the antibody V_HH domain samples were extensively dialyzed to remove phosphate ions (>1000x). Further, as evidenced by the prothrombin time assays (Figs. 2A and 2B), prothrombin time was not affected in a significant way by increasing concentrations of phosphate up to 0.5 mM. This shows that minor amounts of phosphate ions not completely removed by buffer exchange do not contribute in any major way to the observed inhibitory effects of the V_HH domain dimers.

Comparing Fig. 3 to Fig. 4, peak levels of thrombin are consistently lower with the prothrombin-specific antibody V_HH domain dimers (Fig. 3) than bivalirudin, a hirudin- based thrombin inhibitor (Fig. 4). Thus, dimerized V_HH domains appear to be selective as inhibitors of the initiation phase of blood coagulation, while bivalirudin (Angiomax™) may be more specific for delaying the burst phase of thrombin generation. As such, multivalent targeting of prothrombin represents a potentially more selective strategy for inhibiting the generation of thrombin from prothrombin by the prothrombinase. Example 4: Prothrombinase Assay

Preparation of phospholipid vesicles: Five pmoles of 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) and 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were mixed at molar ratios of 75/25 or 95/5 in chloroform, and dried in a stream of nitrogen gas. The remaining traces of chloroform were removed by placing the formed lipid film under vacuum for 1 hour. The lipids were re-suspended in 5 ml_ of 20 mM HEPES, 150 mM NaCI, pH 7.4, and sonicated for 15 minutes on ice until the suspension is clear and only slightly opalescent. The suspension was transferred into a centrifuge tube and centrifuged at 100,000 g for 30 minutes followed by 60,000 g for 4 hours. The top 2 ml_ of the vesicle suspension were carefully withdrawn and stored under nitrogen gas at 4°C before being used for the prothrombinase assay.

The lipid concentration was determined by a colorimetric assay based on complex formation with ammonium ferrothiocyanate as described by Stewart (Stewart 1980). Briefly, 100 pL of vesicle suspension was vortexed together with 1 ml. of chloroform until lipid extraction was complete (several minutes), 1 mL of 0.1 N ammonium ferrothiocyanate solution in water was added and vortexed vigorously for 1 minute. On separation, the chloroform was removed with a Pasteur pipette for optical density determination. The extinction coefficient at 488 nm was calibrated as 3.565 cm^"1mM^~1.

Assaying Thrombin Generation by the Prothrombinase: The prothrombinase was reconstituted at ambient (room) temperature in 20 mM HEPES, 50 mM NaCI, 0.1 % (w/v) polyethylene glycol (PEG-8000), pH 7.4. The reaction mixture contained 1.4 μΜ prothrombin, 10 nM FVa, 5 mM CaCI₂, 30 μΜ phospholipid vesicles. The activation reaction was initiated with 0.1 nM FXa with or without a peptide inhibitor to be tested. Aliquots of the reaction mixture (typically 40 pl_) were withdrawn every minute and quenched on ice with 1/10th volume of 10x quenching solution containing 20 mM HEPES, 150 mM NaCI, 0.1 % (w/v) polyethylene glycol (PEG-8000), 150 mM EDTA, 1 mg/mL soybean trypsin inhibitor (type IS, Sigma), pH 7.4.

The time course of thrombin accumulation was collected using a fibrinogen clotting assay (DiMaio 1990). Fibrinogen solution was freshly prepared by dissolving about 0.5% (w/v) fibrinogen in 50 mM Tris-HCI, 100 mM NaCI, 0.1 % PEG-8000, pH 7.6 followed by filtering through a hydrophilic 0.45 pm polyvinylidene fluoride (PVDF) membrane with minimized protein binding. The concentration of fibrinogen after filtration was determined by using the extinction coefficient of 15.0 for 1 % fibrinogen at 280 nm. Fibrinogen clotting was initiated by diluting the quenched solutions of the prothrombinase reaction (containing activated thrombin) 100-300 times into 0.1 % fibrinogen in 50 mM Tris-HCI, 100 mM NaCI, 0.1 % PEG-8000, pH 7.6. Thrombin-induced clotting of fibrinogen was followed at 25°C by measuring the optical absorbance at 420 nm. The clotting time was obtained from extrapolation of the slope at the point of inflection to the zero absorbance baseline. The inverse clotting time was used as a measure of thrombin concentration.

Using the prothrombinase assay described above, it is evident that the dimeric prothrombin-specific ligand, VHpro#10 dimer, exhibits potent inhibitory activities particularly within the first few minutes of prothrombin activation (Fig. 5). Furthermore, the inhibitory action is prolonged by a change of the composition of phospholipid membrane, i.e. with a decrease of the content of the negatively-charged phophatidylserine (Fig. 6), as measured by the fibrinogen clotting assay.

This dependence of inhibitory activity on the time of prothrombin activation is apparently indicative of a unique mechanism of action of VHpro#10 dimer, as the protein remains intact and free from proteolytic degradation for at least 6 minutes post-prothrombin activation; in other words, the dimeric V_HH protein remains intact under the emerging thrombin activity. This time window for maximal inhibitory activity also matches the lifetimes of prothrombin and the transient species, meizo-thrombin (see Figs. 7A-7C), an intermediate of prothrombin activation by the prothrombinase, which indicates that the VHpro#10 dimer also binds to meizo-thrombin in addition to inhibiting the interaction of prothrombin with the prothrombinase. The most recent studies point to a differential mechanism of prothrombin activation whereby the anti-coagulant intermediate meizo- thrombin is produced and accumulated from the prothrombinase assembled on phospholipid membranes (Haynes 201 1 ), while the generation of the procoagulant thrombin or Ha is substantially enhanced by the prothrombinase assembled by activated platelets under blood flow (Wood 201 1 ; Haynes 2012). Multimerized antibody V_HH domain ligands of prothrombin, such as the VHpro#10 dimer, are therefore expected to be more effective inhibitors of thrombin generation under the in vivo conditions of blood flow, whereby the conversion of prothrombin to active thrombin does not appear to involve the steady-state accumulation of meizo-thrombin (Haynes 201 1 ; Wood 201 1 ; Haynes 2012). Example 6: ln-vivo experiments using a rat model of venous thrombosis

Male Sprague-Dawley rats (250-310 g; Charles River Laboratories, St-Constant, QC, Canada) are acclimated for at least 3 days prior to the start of the study. Animals are housed in microisolator cages and are kept on a 12-hr light/dark cycle with constant temperature and humidity. Food and water are provided ad libitum. The dimerized prothrombin-binding V_HH domains of this invention are dissolved in saline (0.9% sodium chloride) or phosphate-buffered saline (PBS) prior to use.

The FeCI₃-induced venous model of thrombosis in rats is generated as described by Wang (Wang 2008) with some modifications (Couture 2011 ). Briefly, rats are anesthetized with a 2.5% isoflurane/oxygen mixture and placed on a heat source (35- 37°C). The vena cava is then exposed via a midline incision and the region between the renal and iliolumbar veins is isolated. Saline (N = 20, where N is the number of rats), heparin (130 U/kg; N=8), argatroban (4.5 mg/kg; N=6) or bivalirudin (1.3 mg/kg; N=4) and dimeric V_HH domain proteins of this invention are then administered intravenously (2.8 mL/kg) via a catheter placed in the tail vein. One minute after drug administration, a piece of filter paper (Gel Blot Paper, GB003, Whatman, Piscataway, NJ, USA; 7 mm diameter) saturated with 10% FeCI₃ (EMD Chemicals Inc., Gibbstown, NJ, USA) is placed on the exposed surface of the vena cava and incubated for 3.5 minutes. During the application of FeCI₃, the abdominal region is covered with aluminum foil. At the end of the incubation period, the filter paper is removed and the exposed viscera covered with a saline-soaked gauze. Sixty minutes after the initial application of FeCI₃, a blood sample is collected via cardiac puncture using sodium citrate tubes. The vena cava is dissected and the thrombus removed and weighed. Preparing and processing each animal took approximately 1 hr and therefore 7 animals are normally treated each day. Control animals are always included when the other treatments are being administered. For this reason, the number of animals in the control group is usually higher than that of the treated-groups.

Protein content of the thrombus is also measured according to protocol by Wang (Wang 2005). Cleaned thrombus is digested for 16 hrs at 50°C in 200 μΙ of 100 mM Tris, pH 7.5 containing 400 pg proteinase K (Invitrogen). Contents of amino acids and small peptides are measured at OD₂so with digestion buffer used as a blank. Protein contents of the thrombus are found to parallel the dry weights of the thrombus (Conture 201 ), hence thrombus weights are used for routine measurements. Statistical analysis is conducted using GraphPad™ Prism (GraphPad Software Inc., San Diego CA, USA). Data are analyzed using one-way ANOVA with post-hoc Bonferroni correction for multiple comparisons. All data are given as mean ± standard error of the mean (SEM). Statistical significance was set at p≤ 0.05, in other words, the efficacy of a drug is significantly higher if p≤ 0.05 when comparing the drug-treated group with those administered only with the saline vehicle. A reduction in thrombus weights of the group that receives the VH domain proteins of the present invention compared to the saline group indicates efficacy of the prothrombin-binding V_HH domains in treating thrombotic conditions. Example 7: Collagen-induced Arthritis (CIA) Assay

The collagen-induced arthritis (CIA) model is used to determine the effect of the dimerized prothrombin-binding V_HH domains on arthritis, similarly to the evaluation of the thrombin inhibitor hirudin in the treatment of arthritis (Marty 2001 ).

Briefly, male DBA 1 J mice between 8 and 10 weeks of age (Charles River Labs) are acclimated under standard light and temperature conditions with food and water ad libitum for 1 week. Twelve mice are randomly assigned to a test group, twelve randomly assigned to an antigen (collagen)-only group and twelve randomly assigned to a no- antigen group. The mice are weighed to determine the average body weight of each group, and ankles and paws (maximal lateral) are evaluated to establish baseline measurements.

On day 1 , lightly anesthetized mice in the test group and the collagen-only group receive intradermally (i.d.) a 0.1 ml injection of an emulsion (1 :1 mixture) of bovine type II collagen (1 mg/ml) plus complete Freund's adjuvant (CFA, 1 mg/ml_) in mineral oil. Lightly anesthetized no-collagen mice are injected with an equal volume of mineral oil alone. Mice in the test group then receive an injection of a V_HH domain of the present invention. The test group is further divided into sub-groups, which receive different doses of the polypeptide. Mice are weighed and their ankles and paws evaluated daily for 18 days using a scoring system ranging from 0-4 (0=no sign of arthritis; 1 =swelling or redness of the paw or one digit; 2=two joints involved; 3=more than two joints involved; 4=severe arthritis of the entire paw). The onset of arthritis is indicated by contralateral paw swelling, which appears about 10 days post-injection. Clinical signs of inflammation are evaluated by the intensity of the edema in the paws and ankles. Potential biochemical assays can include, when needed, fibrin immunohistochemistry, measurements of TAT (thrombin-anti-thrombin III complex) in synovial fluids, and levels of chemokines (MIP- 1alfa) and pro-inflammatory cytokines (IL-12 and TNF-alfa) expression in joints. On day 18, mice are euthanized in a carbon dioxide atmosphere.

A reduction in ankle and paw swelling of the group that receive the antibody V_HH domain proteins of the present invention compared to the collagen-only group indicates efficacy of the polypeptide in treating arthritis.

Table 2 - Listing of Sequences:

SEQ ID Description Sequence

NO:

1 CDR1 , VHpro#10 GRTFDRYGWF

2 CDR2, VHpro#10 SIGTRLHYADSVKG

3 CDR3, VHpro#10 CAAAESTRNWYYKMSNDYDYWG

4 CDR1 , VHpro#5 GRTFSSLSIAWF

5 CDR2, VHpro#5 G I RWTAGSKTYANWVKG

6 CDR3, VHpro#5 CAADNISDWGISKQLRTYHYWG

7 VHpro#10 DVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFR

V_HH sequence QAPGKEREFVASIGTRLHYADSVKGRFTISRDNAKSTAF

LEMNSLKPEDTAVYYCAAAESTRNWYYKMSNDYDYW GQGTQVTVSS

8 VHpro#5 DVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWF

V_HH sequence (1 ) RQAPGKEREFVAGIRWTAGSKTYANWVKGRFAISKDN

AKNKVYLQMNYLKPEDTAVYYCAADNISDWGISKQLRT YHYWGQGTQVTVSS

9 VHpro#5 DVQLQASGGGLVQAGGSLRLTCAASGRTFSSLSIAWF

V_HH sequence (2) RQAPGKEREFVAGIRWTAGSKTYANWVKGRFTISRDN

AKSTAFLEMNSLKPEDTAVYYCAADNISDWGISKQLRT YHYWGQGTQVTVSS

10 VHpro#10 MGDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIA

Phage clone WFRQAPGKEREFVAGIRWTAGSKTYANWVKGRFAISK sequence DNAKNKVYLQMNYLKPEDTAVYYCAADNISDWGISKQL

RTYHYWGQGTQVTVSSLEHHHHHH

11 VHpro#5 M G D VQ LQ AS G G G L VQ AG G S L RLTCAAS G RT FD RYGW

Phage clone FRQAPGKEREFVASIGTRLHYADSVKGRFTISRDNAKS sequence TAFLEMNSLKPEDTAVYYCAAAESTRNWYYKMSNDYD

YWGQGTQVTVSSLEHHHHHH

12 Leader sequence CHNDGGGGS

13 Modified MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLTCAAS

VHpro#10 GRTFDRYGWFRQAPGKEREFVASIGTRLHYADSVKGR

FTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAESTRNW YYKMSNDYDYWGQGTQVTVSSLEHHHHHH

14 Modified VHpro#5 MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLSCTVS

GRTFSSLSIAWFRQAPGKEREFVAGIRWTAGSKTYAN

WVKGRFAISKDNAKNKVYLQMNYLKPEDTAVYYCAAD

Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.

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US Patent No. 6180370

US Patent No. 5693761

US Patent No. 6054297

US Patent No. 5859205

European Patent No. 626390

US Patent No. 5869619

US Patent No. 5766886

US Patent No. 5821 123

European Patent No. 519596

Claims

Claims:

1. A polypeptide comprising a multimer of a single domain antibody (sdAb), the sdAb having binding affinity to prothrombin to inhibit generation of thrombin from prothrombin.

2. The polypeptide according to claim 1 , wherein the sdAb has an IC₅₀ for prothrombin binding affinity in a mM to sub-mM range.

3. The polypeptide according to claim 1 , wherein the sdAb has an IC₅o for prothrombin binding affinity in a range from 0.1-900 mM.

4. The polypeptide according to any one of claims 1 to 3, wherein the sdAb comprises: a complementarity determining region (CDR) 1 selected from GRTFDRYGWF (SEQ ID NO: 1) and GRTFSSLSIAWF (SEQ ID NO: 4); a CDR2 selected from SIGTRLHYADSVKG (SEQ ID NO: 2) and G I RWTAGSKTYANWVKG (SEQ ID NO: 5); and a CDR3 selected from CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3) and

CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6).

5. The polypeptide according to any one of claims 1 to 3, wherein the sdAb comprises a CDR1 sequence of GRTFDRYGWF (SEQ ID NO: 1), a CDR2 sequence of SIGTRLHYADSVKG (SEQ ID NO: 2), and a CDR3 sequence of CAAAESTRNWYYKMSNDYDYWG (SEQ ID NO: 3).

6. The polypeptide according to any one of claims 1 to 3, wherein the sdAb comprises a CDR1 sequence of GRTFSSLSIAWF (SEQ ID NO: 4), a CDR2 sequence of G I RWTAG S KT YAN W VKG (SEQ ID NO: 5), and a CDR3 sequence of CAADNISDWGISKQLRTYHYWG (SEQ ID NO: 6).

7. The polypeptide according to any one of claims 1 to 3, wherein the sdAb is a V_HH.

8. The polypeptide according to any one of claims 1 to 3, wherein the sdAb comprises the amino acid sequence DVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPGKEREFVASIGTRL HYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAESTRNWYYKMSN DYDYWGQGTQVTVSS (SEQ ID NO:7),

DVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (SEQ ID N0:8),

DVQLQASGGGLVQAGGSLRLTCAASGRTFSSLSIAWFRQAPGKEREFVAGIRW TAGSKTYANWVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAADNISDWGIS KQLRTYHYWGQGTQVTVSS (5 seq, SEQ ID N0:9), or a sequence substantially identical thereto.

9. The polypeptide according to any one of claims 1 to 8, wherein the multimer comprises two or more sdAb linked together by one or more disulfide bonds.

10. The polypeptide according to claim 9, wherein the disulfide bonds are between cysteine residues in the sdAb.

11. The polypeptide according to claim 10, wherein the disulfide bonds are between cysteine residues in a leading sequence.

12. The polypeptide according to claim 11 , wherein the leading sequence is CHNDGGGGS (SEQ ID NO:12).

13. The polypeptide according to any one of claims 1 to 12, wherein the sdAb comprises the amino acid sequence

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLTCAASGRTFDRYGWFRQAPG KEREFVASIGTRLHYADSVKGRFTISRDNAKSTAFLEMNSLKPEDTAVYYCAAAE STRNWYYKMSNDYDYWGQGTQVTVSSLEHHHHHH (SEQ ID NO: 13),

MGCHNDGGGGSDVQLQASGGGLVQAGGSLRLSCTVSGRTFSSLSIAWFRQAP GKEREFVAGIRWTAGSKTYANWVKGRFAISKDNAKNKVYLQMNYLKPEDTAVY YCAADNISDWGISKQLRTYHYWGQGTQVTVSSLEHHHHHH (SEQ ID NO: 14), or a sequence substantially identical thereto.

14. The polypeptide according to any one of claims 1 to 13, wherein the multimer is a dimer.

15. A method of inhibiting thrombin production in a blood stream comprising contacting prothrombin in the blood stream with a polypeptide as defined in any one of claims 1 to 14.

16. A method of inhibiting blood coagulation in a subject comprising identifying a subject in need of an anti-coagulant; and, administering to the subject a polypeptide as defined in any one of claims 1 to 14.

17. A method of inhibiting thrombin production in an inflamed tissue of a subject, the method comprising introducing into the tissue or a blood stream of the subject a polypeptide as defined in any one of claims 1 to 14.

18. A method of inhibiting extra-vascular coagulation in a subject comprising identifying a subject in need of an extra-vascular anti-coagulant; and, administering to the subject a polypeptide as defined in any one of claims 1 to 14.

19. The method according to claim 16, wherein the subject has an atherosclerotic lesion.

20. The method according to claim 18, wherein the subject has an arthritic joint.