BINDING AGENT FOR INHIBITING OR PREVENTING COAGULATION

This invention relates to screening methods for thrombin binding agents, thrombin binding agents obtained or obtainable using the methods, and the use of such thrombin binding agents.

Blood coagulation is a key process in the prevention of bleeding from damaged blood vessels (haemostasis ) . However, a blood clot that obstructs the flow of blood through a vessel (thrombosis) or breaks away to lodge in a vessel elsewhere in the body

(thromboembolism) can be a serious health threat.

A number of anticoagulant therapies are available to treat pathological blood coagulation. A common drawback of these therapies is an increased risk of bleeding (see Mackman (2008) Nature 451: 914-918) . Many anticoagulant agents have a narrow therapeutic window between the dose that prevents thrombosis and the dose that induces bleeding. This window is often further restricted by variations in the response in individual patients.

International patent application no. PCT/GB2012/053140 published as WO2013/088164 (also referred to herein as the "reference disclosure") in the name of Cambridge Enterprise Limited, filed on 14 December 2012 and claiming priority from UK patent application no. GB1121513.4 filed on 14 December 2011, discloses a novel antibody molecule (also referred to herein as the

"reference antibody molecule (s) ") which recognises the exosite 1 epitope of thrombin and selectively inhibits thrombin without promoting bleeding. The reference antibody molecules are disclosed as being useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin.

According to a first aspect of the present invention there is provided a method of obtaining a thrombin binding agent which inhibits thrombin activity but causes minimal or no inhibition of haemostasis and/or causes minimal or no bleeding, the method comprising the steps of: (1) contacting a test antigen binding agent with a molecule comprising an epitope defined by: (i) at least three (for example, four or five) of the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin; and/or (ii) at least three (for example, four or five) of the amino acids R67, R73, Q38, R77A and S36A of human thrombin; and/or (iii) the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin; and

(2) determining whether the test antigen binding agent binds specifically to the epitope,

wherein, if the test antigen binding agent binds specifically to the epitope, the test antigen binding agent is a thrombin binding agent . In a second aspect of the present invention there is provided a method of obtaining a thrombin binding agent, comprising the steps of:

(1) contacting a test antigen binding agent with a molecule comprising an epitope defined by: (i) the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin; and/or (ii) at least four (for example, five) of the amino acids R67, R73, Q38, R77A and S36A of human thrombin; and/or (iii) the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin; and

(2) determining whether or not the test antigen binding agent binds specifically to the epitope,

wherein, if the test antigen binding agent binds specifically to the epitope, the test agent is a thrombin binding agent. According to the second aspect of the invention, the thrombin binding agent may inhibit thrombin activity but cause minimal or no inhibition of haemostasis and/or cause minimal or no bleeding.

The epitope as defined in the present invention is located within exosite 1 (also known as "anion binding exosite 1" and the

"fibrinogen recognition exosite") of thrombin. Exosite 1 is a well-characterised secondary binding site on the thrombin molecule (see for example James Ά. Huntington, 2008, Structural Insights into the Life History of Thrombin, in Recent Advances in Thrombosis and Hemostasis 2008, editors; K. Tanaka and E.W.

Davie, Springer Japan KK, Tokyo, pp. 80-106) . Exosite 1 is formed in mature thrombin but is not formed in prothrombin (see for example Anderson et al . (2000) JBC 2775 16428-16434). The sequence of human preprothrombin is set out in SEQ ID NO: 1.

Human prothrombin has the sequence of residues 44 to 622 of SEQ ID NO: 1. Mature human thrombin has the sequence of residues 314- 363 (light chain) and residues 364 to 622 (heavy chain) .

Exosite 1 is involved in recognising thrombin substrates, such as fibrinogen, but is remote from the catalytic active site. Various thrombin binding factors are known to bind to exosite 1, including the anticoagulant dodecapeptide hirugen (Naski et al . 1990 JBC 265 13484-13489), factor V, factor VIII, thrombomodulin (cofactor for protein C and TAFI activation) , fibrinogen, PARI and fibrin (the co-factor for factor XIII activation) .

However, as elaborated below, the epitope as defined in the present invention is distinct from the epitopes recognised by other thrombin binding factors known to bind to exosite 1. The epitope as defined in the present invention has been determined by detailed analysis of the binding interaction between thrombin and the antibody disclosed in the reference disclosure (see below) .

The numbering scheme for thrombin residues set out herein is conventional in the art and is based on the chymotrypsin template (Bode W et al. EMBO J. 1989 8: 3467-3475). Thrombin has insertion loops relative to chymotrypsin that are lettered sequentially using lower case letters.

As described in the reference disclosure, exosite 1 of mature human thrombin is underlined in SEQ ID NO: 1 and may include the following residues: M32, F34, R35, K36, S36a, P37, Q38, E39, L40, L65, R67, S72, R73, T74, R75, Y76, R77a, N78, E80, K81, 182, S83, M84, K109, K110, K149e, G150, Q151, S153 and V154. Other thrombin residues which are located close to (i.e. within 0.5nm or within lnm) of any one of these residue may also be considered to be part of exosite 1. The epitope a defined in the present invention may further include on or more of these exosite residues.

The method of the present invention may be used to screen for candidate agents for preventing or treating thrombosis, embolism or other conditions mediated by thrombin.

The molecule comprised an epitope (as defined herein) used in the method of the present invention may be a thrombin mimic.

The epitope in the present invention may be defined as the amino acid residues of the molecule which exhibit a negative solvation free energy gain (in kcal/mol) as determined using PISA analysis upon formation of a binding interface between the epitope and the test agent. The epitope for example may be defined by at least four, or five, of the amino acids R67, R73, Q38, R77A and S36A of human thrombin.

PISA (an acronym for "Protein interfaces, surfaces and

assemblies") is a method based on chemical thermodynamics for the detection and analysis of macromolecular assemblies using crystal structures. PISA is available at the European Bioinformatics

Institute (see http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) . The method is described in Krissinel & Henrick (2007) J. Mol. Biol. 372: 774-797. Preferably, PISA analysis is undertaken using default parameters of Version 1.49 updated 27 March 2013.

Additionally or alternatively, the epitope in the present invention may be defined as the five amino acid residues of the molecule which have the highest interface area (in A ² ) as determined using PISA analysis upon formation of a binding interface between the epitope and the test agent. Here, the epitope may be defined by the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin. Additionally or alternatively, the epitope may be defined as the amino acid residues of the molecule which exhibit a negative total free energy gain (in kcal/mol) calculated as the solvation free energy gain as determined using PISA analysis plus free energy gain attributable to any hydrogen bonds (additional -0.44 kcal/mol per bond), salt bridges (additional -0.15 kcal/mol per salt bridge) and/or disulphide bonds (additional -4 kcal/mol per bond) present upon formation of a binding interface between the epitope and the test agent. Here, the epitope may be defined by the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin . The methods of the present invention may further comprise a step of determining the effect of the test antigen binding agent or thrombin binding agent on thrombin activity, haemostasis and/or bleeding. The effect of the test antigen binding agent or thrombin binding agent on thrombin activity, haemostasis and/or bleeding may be compared with the effect of an anticoagulant standard (for example, a therapeutic anticoagulant) on thrombin activity, haemostasis and/or bleeding.

According to the methods of the present invention, the test antigen binding agent may be other than an antibody (as described in the reference disclosure; see below) having a VH domain comprising a HCDR1, HCDR2 and HCDR3 with the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising a LCDR1 , LCDR2 and LCDR3 with the sequences of SEQ ID NOs 7, 8 and 9, respectively.

In a further aspect of the present invention, there is provided a method of selecting a thrombin binding agent capable of binding to the same thrombin exosite 1 epitope as an antibody having a VH domain comprising a HCDR1, HCDR2 and HCDR3 with the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising a LCDR1, LCDR2 and LCDR3 with the sequences of SEQ ID NOs 7, 8 and 9, respectively, comprising the steps of:

(1) providing a test antigen binding agent; and

(2) selecting a test antigen binding agent capable of binding to the same epitope as the antibody based on reactivity of the test antigen binding agent with the epitope or based on competition between the test antigen binding agent and the antibody, wherein the test antigen binding agent selected in step (2) is a thrombin binding agent other than the antibody. The method of the present invention may further comprise a step of isolating and/or purifying the thrombin binding agent.

According to another aspect of the present invention, there is provided a novel thrombin binding agent obtained or obtainable according to the method of the invention.

Further provided is a thrombin binding agent that specifically binds to an epitope defined by: (i) the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin; and/or (ii) at least four (for example, five) of the amino acids R67, R73, Q38, R77A and S36A of human thrombin; and/or (iii) the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin, provided that the thrombin binding agent is not an antibody (as disclosed in

PCT/GB2012/053140 published as WO2013/08816 ; see below) having a VH domain comprising a HCDR1, HCDR2 and HCDR3 with the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain

comprising a LCDR1, LCDR2 and LCDR3 with the sequences of SEQ ID NOs 7, 8 and 9, respectively.

The thrombin binding agent as described above may inhibit thrombin activity but cause minimal or no inhibition of

haemostasis and/or cause minimal or no bleeding. Thrombin binding agents of the present invention and/or isolated anti-exosite 1 antibody molecules of the reference disclosure may inhibit thrombin in vivo without promoting or substantially promoting bleeding or haemorrhage, i.e. the thrombin binding agents of the present invention and/or the reference antibody molecules do not inhibit or substantially inhibit normal physiological responses to vascular injury (i.e. haemostasis ) . For example, haemostasis may not be inhibited or may be minimally inhibited by the thrombin binding agents of the present invention and/or the reference antibody molecules (i.e. inhibited to an insignificant extent which does not affect the well-being of patient or require further intervention) . Bleeding may not be increased or may be minimally increased by thrombin binding agents of the present invention and/or the reference antibody molecules. The thrombin binding agents of the present invention and/or the anti-exosite 1 antibody of the reference disclosure may bind to an epitope which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues of exosite 1. Preferably, thrombin binding agents of the present invention and/or the anti-exosite 1 antibody of the reference disclosure bind to an epitope which consists entirely of exosite 1 residues.

For example, thrombin binding agents of the present invention and/or the anti-exosite 1 antibody of the reference disclosure may bind to an epitope which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 residues selected from the group consisting of M32, F34, S36a, P37, Q38, E39, L40, L65, R67, R73, T74, R75, Y76, R77a, 182 and Q151 of human thrombin or the equivalent residues in thrombin from another species. In some embodiments, the epitope may comprise the thrombin residues Q38, R73, T74, Y76 and R77a and optionally one or more additional residues .

Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may 1 specific for thrombin exosite 1 and bind to this epitope with high affinity relative to other epitopes, for example epitopes from mammalian proteins other than mature thrombin. For example, thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may display a binding affinity for thrombin exosite 1 which is at least 500 fold, at least 1000 fold or at least 2000 fold greater than other epitopes.

Preferably, thrombin binding agents of the present invention and/or the reference antibody molecules which are specific for exosite 1 may bind to mature thrombin but display no binding or substantially no binding to prothrombin.

Without being bound by any theory, thrombin binding agents of the present invention and/or anti-exosite 1 antibodies of the reference disclosure may be unable to access thrombin within the core of a haemostatic clot, and are therefore unable to affect haemostasis by interrupting normal thrombin function at sites of vascular injury. However, because the thrombin binding agents of the present invention and/or anti-exosite 1 antibodies of the reference disclosure still bind to thrombin on the surface of the clot and in the outer shell of the clot, thrombosis is prevented, i.e. non-haemostatic clot extension is prevented. A thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure may have a dissociation constant for exosite 1 of less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than ΙΟηΜ, or less than InM. For example, an antibody molecule may have an affinity for exosite 1 of 0.1 to 50 nM, e.g. 0.5 to 10 nM. A suitable thrombin binding agent of the present invention and/or anti-exosite 1 antibody molecule of the reference disclosure may, for example, have an affinity for thrombin exosite 1 of about 1 nM.

Binding kinetics and affinity (expressed as the equilibrium dissociation constant, K _d ) of the thrombin binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be determined using standard techniques, such as surface plasmon resonanee e.g. using BIAcore analysis. The thrombin binding agents of the present invention and/or anti- exosite 1 antibodies of the reference disclosure may also bind to exosite 1 of mature thrombin from other species. Thrombin sequences from other species are known in the art and available on public databases such as Genbank. The corresponding residues in thrombin sequences from other species may be easily identified using sequence alignment tools.

The thrombin binding agent according to the present invention may be an antibody, an antibody fragment, or another agent

specifically binding to an antigen.

More generally, the terms "test antigen binding agent", "antigen binding agent" or "thrombin binding agent" (collectively "binding agent (s)") as used herein may refer to an antibody, an antibody fragment, an antigen binding fragment of an antibody, a mini- antibody or another agent specifically binding to an antigen. Each of the binding agents of the invention may be a whole antibody, a Fab, a F(ab') ₂ fragment, a Fd fragment, a disulfide- linked Fv (scFv) , an anti-idiotypic (anti-Id) antibody, a single chain antibody, an antibody fragment such as for example a Fab' fragment, an affibody, a trinectin, a monobody, an FN3 monobody, an anticalin, a Small Modular Immunopharmaceutical (SMIP), or a suitable antibody mimetic. The antibody or antibody fragment may include one or more of the components or domains found in whole antibodies comprising for example the heavy chain (HCDR) , the variable domain (V) of the complementarity determining region (CDR) of a heavy chain (HCDR, VH) and a light chain (LCDR, VL) . The antibody, antibody fragment and/or antigen binding fragment of the present invention may be polyclonal or monoclonal. The antibody, antibody fragment and/or antigen binding fragment of the invention may be derived from any species comprising but not limited to mouse, rat, dog, cat, sheep, goat, rabbit, hamster, opossum, humans, horse, apes, primates, cow, shark or whale. The antibody may comprise or consist of an antigenetically engineered antibody and/or an antibody generated in a transgenic animal, microorganism or plant, or an antibody generated synthetically. The antibody may be human, humanised or chimeric. Generally, any binding agent with specific binding affinity to a specified antigen may be used as an antigen binding agent according to the invention . Binding agents of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may be an

immunoglobulin or fragment thereof, and may be natural or partly or wholly synthetically produced, for example a recombinant molecule .

Binding agents of the present invention and/or the anti-exosite 1 antibody molecules of the reference disclosure may include any polypeptide or protein comprising an antibody antigen-binding site, including Fab, Fab ₂ , Fab ₃ , diabodies, triabodies,

tetrabodies, minibodies and single-domain antibodies, including nanobodies, as well as whole antibodies of any isotype or subclass. Antibody molecules and methods for their construction and use are described, in for example Holliger & Hudson, Nature Biotechnology 23 ( 9) : 1126-1136 (2005).

In some embodiments, the binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may be a whole antibody. For example, the binding agent of the present invention and/or the anti-exosite 1 antibody molecule may be an IgG, IgA, IgE or IgM or any of the isotype sub-classes, particularly IgGl and IgG4. The binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be monoclonal antibodies. In other embodiments, the binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may be an antibody fragment . Binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be chimeric, humanised or human antibodies. Binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides and/or serum components.

Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies may also be employed.

Binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or

immunoprecipitation may be used (Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal .

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or f agments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest. Other binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be

identified by screening patient serum for antibodies which bind to exosite 1 as defined herein.

In some embodiments, binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be produced by any convenient means, for example a method described above, and then screened for differential binding to mature thrombin relative to thrombin with an exosite 1 mutation, gamma thrombin (exosite 1 defective due to autolysis at R75 and R77a) or prothrombin. Suitable screening methods are well-known in the art.

An antibody which displays increased binding to mature thrombin, relative to non-thrombin proteins, thrombin with an exosite 1 mutation, gamma-thrombin or prothrombin, for example an antibody which binds to mature thrombin but does not bind to thrombin with an exosite 1 mutation, gamma thrombin or prothrombin, may be identified as an binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure.

After production and/or isolation, the biological activity of a binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be tested. For example, the ability of the binding agent and/or antibody molecule to inhibit thrombin substrate, cofactor or inhibitor binding and/or cleavage by thrombin may be determined and/or the ability of the binding agent and/or antibody molecule to inhibit thrombosis without promoting bleeding may be determined.

Suitable binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may be tested for activity using a fibrinogen clotting or thrombin time assay. Suitable assays are well-known in the art. The effect of a binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure on coagulation and bleeding may be determined using standard techniques. For example, the effect of a binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure on thrombosis may be determined in an animal model, such as a mouse model with ferric chloride induced clots in blood vessels . Effects on haemostasis may also be determined in an animal model, for example, by measuring tail bleed of a mouse.

Antibody molecules normally comprise an antigen binding domain comprising an immunoglobulin heavy chain variable domain (VH) an an immunoglobulin light chain variable domain (VL) , although antigen binding domains comprising only a heavy chain variable domain (VH) are also possible (e.g. camelid or shark antibodies)

Each of the VH and VL domains typically comprise three

complementarity determining regions (CDRs) responsible for antigen binding, interspersed by framework regions.

In some embodiments of the reference disclosure, binding to exosite 1 may occur wholly or substantially through the VHCDR3 of the anti-exosite 1 antibody molecule of the reference disclosure.

For example, an anti-exosite 1 antibody molecule of the reference disclosure may comprise a VH domain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 5 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions. In some embodiments of the reference disclosure, the HCDR3 may comprise the amino acid residues at positions 4 to 9 of SEQ ID NO: 5 (SEFEPF) , or more preferably the amino acid residues at positions 2, and 4 to 10 of SEQ ID NO: 5 (D and SEFEPFS)with substitutions, deletions or insertions at one or more other positions in SEQ ID NO : 5. The HCDR3 may be the only region of the antibody molecule that interacts with a thrombin exosite 1 epitope or substantially the only region.

HCDR3 may therefore determine the specificity and/or affinity the antibody molecule for the exosite 1 region of thrombin. The VH domain of an anti-exosite 1 antibody molecule of the reference disclosure may additionally comprise an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the sequence of SEQ ID NO: 4 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. In some embodiments of the reference disclosure, the HCDR2 may comprise the amino acid residues at positions 3 to 7 of SEQ ID NO: 4 (DPQDG) or the amino acid residues at positions 2 and 4 to 7 of SEQ ID NO: 4 (L and PQDG ) of SEQ ID NO: 4, with substitutions, deletions or

insertions at one or more other positions in SEQ ID NO: 4.

The VH domain of an anti-exosite 1 antibody molecule of the reference disclosure may further comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 3 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. In some embodiments of the reference disclosure, the HCDR1 may comprise amino acid residue T at position 5 of SEQ ID NO: 3 with substitutions, deletions or insertions at one or more other positions in SEQ ID NO: 3.

In some embodiments of the reference disclosure, an anti-exosite 1 antibody molecule may comprise a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5 respectively. For example, an antibody molecule of the reference disclosure may comprise a VH domain having the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 2.

The anti-exosite 1 antibody molecule of the reference disclosure may further comprise a VL domain, for example a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7, 8 and 9 respectively with, independently, 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or

insertions. The substitutions may be conservative substitutions. For example, an antibody molecule of the reference disclosure may comprise a VL domain having the sequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 6 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 6.

In some embodiments of the reference disclosure, the VL domain may comprise Tyr49.

A thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may for example comprise one or more amino acid substitutions, deletions or insertions which improve one or more properties of the antibody, for example affinity, functional half-life, on and off rates .

The techniques that are required in order to introduce

substitutions, deletions or insertions within amino acid

sequences of CDRs, antibody VH or VL domains and antibodies are generally available in the art. Variant sequences may be made, with substitutions, deletions or insertions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind exosite 1 of thrombin and/or for any other desired property.

In some embodiments of the reference disclosure, an anti-exosite 1 antibody molecule of the reference disclosure may comprise a VH domain comprising a HCDR1 , a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4, and 5, respectively, and a VL domain comprising a LCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9, respectively . For example, the VH and VL domains may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 respectively; or may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 comprising, independently 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions.

In some embodiments, an antibody of the present invention and/or of the reference disclosure may comprise one or more

substitutions, deletions or insertions which remove a

glycosylation site. For example in the reference disclosure, a glycosylation site in VL domain of SEQ ID NO 6 may be mutated out by introducing a substitution at either N28 or S30. The anti-exosite 1 antibody molecule of the reference disclosure may be in any format, as described above. In some embodiments, the thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may be a whole antibody, for example an IgG, such as IgGl or IgG4, IgA, IgE or IgM.

An anti-exosite 1 antibody molecule of the reference disclosure may be one which competes for binding to exosite 1 with an antibody molecule described above, for example an antibody molecule which

(i) binds thrombin exosite 1 and

(ii) comprises a VH domain of SEQ ID NO: 2 and/or VL domain of SEQ ID NO: 6; an HCDR3 of SEQ ID NO: 5; an HCDR1, HCDR2 , LCDR1, LCDR2, or LCDR3 of SEQ ID NOS : 3, 4, 7, 8 or 9

respectively; a VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 respectively; and/or a VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 and a VL domain comprising LCDR1, LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8 and 9 respectively.

Competition between binding agents such as antibody molecules may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules, to enable identification of antibody molecules which bind the same epitope or an overlapping epitope. Such methods are readily known to one of ordinary skill in the art .

Thus, a further aspect of the present invention and/or of the reference disclosure provides a thrombin binding agent such as an antibody molecule comprising an antibody antigen-binding site that competes with an antibody molecule, for example an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the parent antibody described above for binding to exosite 1 of thrombin. A suitable thrombin binding agent such as an antibody molecule may comprise an antibody antigen-binding site which competes with an antibody antigen-binding site for binding to exosite 1 wherein the antibody antigen-binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDR1, LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8, and 9 respectively, for example the VH and VL domains of SEQ ID NOS: 2 and 6.

The thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may inhibit the binding of thrombin-binding factors, including factors which bind to exosite 1. For example, a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may competitively or non- competitively inhibit the binding of one or more of fV, fVIII, thrombomodulin, fibrinogen or fibrin, PARI and/or hirugen and hirudin analogues to thrombin.

The thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may inhibit one or more activities of thrombin. For example, the thrombin binding agent of the present invention and/or the anti- exosite 1 antibody molecule of the reference disclosure may inhibit the hydrolytic cleavage of one or more thrombin

substrates, such as fibrinogen, platelet receptor PAR-1 and coagulation factor FVIII. For example, binding of the thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure to thrombin may result in an at least 5-fold, at least 10-fold, or at least 15- fold decrease in the hydrolysis of fibrinogen, PAR-1, coagulation factor FVIII and/or another thrombin substrates, such as factor V, factor XIII in the presence of fibrin, and protein C and/or TAFI in the presence of thrombomodulin. In some embodiments, binding of thrombin by the thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may result in no detectable cleavage of the thrombin substrate by thrombin.

Techniques for measuring thrombin activity, for example by measuring the hydrolysis of thrombin substrates in vitro are standard in the art and are described herein.

The thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure may be further modified by chemical modification, for example by PEGylation, or by incorporation in a liposome, to improve their pharmaceutical properties, for example by increasing in vivo half-life.

The effect of a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure on coagulation and bleeding may be determined using standard techniques. For example, the effect of thrombin binding agent and/or an antibody on a thrombosis model may be determined Suitable models include ferric chloride clot induction in blood vessels in a murine model, followed by a tail bleed to test normal haemostasis. Other suitable thrombosis models are well known in the art (see for example Westrick et al . ATVB (2007) 27 2079-2093) Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical

composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure, an increase in its or their lifespan and/or in its or their efficacy in the body and/or an increase in its or their solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure.

In some embodiments, thrombin binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be provided in a lyophilised form reconstitution prior to administration. For example, lyophi thrombin binding agents and/or antibody molecules may be re constituted in sterile water and mixed with saline prior to administration to an individual.

Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure will usually be administered in the form of a pharmaceutical

composition, which may comprise at least one additional component in addition. Thus pharmaceutical compositions may comprise, in addition to the thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

For parenteral, for example sub-cutaneous or intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives,

stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol ; 3 ' -pentanol ; and m-cresol) ; low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as

polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions, such as sodium; metal complexes (e.g. Zn-protein

complexes); and/or non-ionic surfactants, such as TWEEN™,

PLURONICS™ or polyethylene glycol (PEG) . A pharmaceutical composition comprising a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset) . The method of treatment may comprise administering a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure to an individual in need thereof.

Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient.

Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc, is within the

responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of binding agents and antibody molecules are well known in the art (Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al . (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922) . Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a thrombin binding agent and/or antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the binding agent and/or antibody (e.g. whole antibody, fragment) and the nature of any detectable label or other molecule attached to the binding agent and/or antibody.

A typical binding agent and/or antibody dose will be in the range 100 pg to 1 g for systemic applications, and 1 ]ig to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the binding agent and/or antibody will be a whole antibody, e.g. the IgGl or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmocokinetic and

pharmacodynamic properties of the antibody composition, the route of administration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g. about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks.

Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above. In some embodiments, thrombin binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be administered as sub-cutaneous injections. Sub-cutaneous injections may be administered using an auto-injector, for example for long term prophylaxis/treatment.

In some embodiments, the therapeutic effect of the thrombin binding agent of the present invention and/or anti-exosite 1 antibody molecule of the reference disclosure may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of the thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

Thrombin binding agents of the present invention and/or an anti- exosite 1 antibody molecules of the reference disclosure inhibit thrombin and may be useful in the treatment of thrombin-mediated conditions.

Haemostasis is the normal coagulation response i.e. the

prevention of bleeding or haemorrhage, for example from a damaged blood vessel. Haemostasis arrests bleeding and haemorrhage from blood vessels in the body.

Thrombin binding agents of the present invention and/or an anti- exosite 1 antibody molecules of the reference disclosure may have no effect or substantially no effect on haemostasis i.e. they do not promote bleeding or haemorrhage.

Aspects of the invention and/or reference disclosure provide: a thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure for use in a method of treatment of the human or animal body; a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure for use in a method of treatment of a thrombin-mediated disorder; the use of a thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure in the manufacture of a medicament for the treatment of a thrombin- mediated condition; and a method of treatment of a thrombin- mediated condition comprising administering a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure to an individual in need thereof .

Inhibition of thrombin by a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be of clinical benefit in the treatment of any thrombin-mediated condition. A thrombin-mediated condition may include disorders associated with the formation or activity of thrombin.

Thrombin plays a key role in haemostasis, coagulation and thrombosis. Thrombin-mediated conditions include thrombotic conditions, such as thrombosis and embolism.

Thrombosis is coagulation which is in excess of what is required for haemostasis (i.e. excessive coagulation), or which is not required for haemostasis (i.e. extra-haemostatic or non- haemostatic coagulation) .

Thrombosis is blood clotting within the blood vessel lumen. It is characterised by the formation of a clot (thrombus) that is in excess of requirement or not required for haemostasis. The clot may impede blood flow through the blood vessel leading to medical complications. A clot may break away from its site of formation, leading to embolism elsewhere in the circulatory system. In the arterial system, thrombosis is typically the result of

atherosclerotic plaque rupture.

In some embodiments, thrombosis may occur after an init

physiological haemostatic response, for example damage endothelial cells in a blood vessel. In other embodiments, thrombosis may occur in the absence of any physiological haemostatic response.

Thrombosis may occur in individuals with an intrinsic tendency t thrombosis (i.e. thrombophilia) or in "normal" individuals with no intrinsic tendency to thrombosis, for example in response to an extrinsic stimulus. Thrombosis and embolism may occur in any vein, artery or other blood vessel within the circulatory system and may include microvascular thrombosis.

Thrombosis and embolism may be associated with surgery (either during surgery or afterwards) or the insertion of foreign objects, such as coronary stents, into a patient.

For example, thrombin binding agents of the present invention and/or anti-exosite 1 antibody molecules of the reference disclosure may be useful in the surgical and other procedures in which blood is exposed to artificial surfaces, such as open heart surgery and dialysis.

Thrombotic conditions may include thrombophilia, thrombotic stroke and coronary artery occlusion.

Patients suitable for treatment as described herein include patients with conditions in which thrombosis is a symptom or a side-effect of treatment or which confer an increased risk of thrombosis or patients who are predisposed to or at increased risk of thrombosis, relative to the general population. For example, a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may also be useful in the treatment or prevention of venous thrombosis in cancer patients, and in the treatment or prevention of hospital-acquired thrombosis, which is responsible for 50% of cases of venous thromboembolism. Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may exert a therapeutic or other beneficial effect on thrombin- mediated conditions, such as thrombotic conditions, without substantially inhibiting or impeding haemostasis. For example the risk of haemorrhage in patients treated with a thrombin binding agent of the present invention and/or an anti-exosite antibody molecule of the reference disclosure may not be increased or substantially increased relative to untreated individuals .

Individuals treated with conventional anticoagulants, such as natural and synthetic heparins, warfarin, direct serine protease inhibitors (e.g. argatroban, dabigatran, apixaban, and

rivaroxaban) , hirudin and its derivatives (e.g. lepirudin and bivalirudin) , and anti-platelet drugs (e.g. clopidogrel, ticlopidine and abciximab) cause bleeding. The risk of bleeding in patients treated with a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure may be reduced relative to individuals treated with conventional anticoagulants .

Thrombin-mediated conditions include non-thrombotic conditions associated with thrombin activity, including inflammation, infection, tumour growth and metastasis, organ rejection and dementia (vascular and non-vascular, e.g. Alzheimer's disease) (Licari et al. J Vet Emerg Crit Care (San Antonio) . 2009

Feb; 19 (1) : 11-22; Tsopanoglou et al . Eur Cytokine Netw. 2009 Dec 1; 20 (4) : 171-9) .

Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may also be useful in in vitro testing, for example in the analysis and characterisation of coagulation, for example in a sample obtained from a patient. Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may useful in the measurement of thrombin generation. Assays of thrombin generation are technically problematic because the conversion of fibrinogen to fibrin causes turbidity, which precludes the use of a simple chromogenic end-point.

The addition of a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure to a sample of blood prevents or inhibits fibrin formation and hence turbidity and permits thrombin generation to be measured using a chromogenic substrate, without the need for a defibrination step.

For example, a method of measuring thrombin generation may comprise contacting a blood sample (for example, in vitro or ex vivo) with a chromogenic thrombin substrate in the presence of , thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure and measuring the chromogenic signal from the substrate;

wherein the chromogenic signal is indicative of thrombin generation in the sample.

The chromogenic signal may be measured directly witho

defibrination of the sample.

Suitable substrates are well known in the art and include S2238 (H-D-Phe-Pip-Arg-pNa ) , β-Ala-Gly-Arg-p-nitroanilide diacetate (Prasa, D. et al . (1997) Thromb. Haemost. 78, 1215; Sigma Aldrich Inc) and Tos-Gly-Pro-Arg-pNa .AcOH (Biophen CS-01(81); Aniara Inc OH USA) .

Thrombin binding agents of the present invention and/or anti- exosite 1 antibody molecules of the reference disclosure may also be useful in inhibiting or preventing the coagulation of blood as described above in extracorporeal circulations, such as

haemodialysis and extracorporeal membrane oxygenation. For example, a method of inhibiting or preventing blood

coagulation in vitro or ex vivo may comprise introducing a thrombin binding agent of the present invention and/or an anti- exosite 1 antibody molecule of the reference disclosure to a blood sample . The blood sample may be introduced into an extracorporeal circulation system before, simultaneous with or after the introduction of the thrombin binding agent of the present invention and/or the anti-exosite 1 antibody molecule of the reference disclosure and optionally subjected to treatment such as haemodialysis or oxygenation. In some embodiments, the treated blood may be subsequently administered to an individual. Other embodiments provide a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure for use in a method of inhibiting or preventing blood coagulation in a blood sample ex vivo and the use of a thrombin binding agent of the present invention and/or an anti-exosite 1 antibody molecule of the reference disclosure in the manufacture of a medicament for use in a method of inhibiting or preventing blood coagulation in a blood sample ex vi vo .

Other aspects of the invention and/or reference disclosure relate to the production of antibody molecules which bind to the exosite 1 epitope of thrombin and may be useful, for example in the treatment of pathological blood coagulation or thrombosis.

In another aspect of the invention there is provided a method for producing a thrombin binding agent (for example, a thrombin binding antibody or fragment thereof or other binding agent as defined herein), the method comprising the steps of:

(1) providing, by way of addition, deletion, substitution or insertion of one or more amino acids (for example, one, two, three or more amino acids) in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3 of a thrombin binding agent as defined herein, in which the parent VH domain HCDR1, HCDR2 and HCDR3 have amino acid sequences other than SEQ ID NOS: 3, 4 and 5, respectively;

(2) optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and (3) testing the VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify a thrombin binding agent which binds to a molecule comprising an epitope defined by: (i) at least three of the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin; and/or (ii) at least three of the amino acids R67, R73, Q38, R77A and S36A of human thrombin; and/or (iii) the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin. The reference disclosure describes a method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin, which may comprise:

providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1 , HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid sequence variant of the parent VH domain, and;

optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and testing the VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for the exosite 1 epitope of thrombin.

In the reference disclosure, a VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDR3 sequence of SEQ ID NO: 5 or a variant with the addition, deletion, substitution or insertion of one, two, three or more amino acids. In the reference disclosure, the VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDRl and HCDR2 sequences of SEQ ID NOS: 3 and 4 respectively, or variants of these sequences with the addition, deletion, substitution or insertion of one, two, three or more amino acids.

Also provided according to the present invention is a method for producing thrombin binding agent (for example, a thrombin binding antibody or fragment thereof or other binding agent as defined herein), the method comprising the steps of:

(1) providing a starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, in which the VH domain or VH domains either comprise a HCDRl, HCDR2 and/or HCDR3 to be replaced or lack a HCDRl, HCDR2 and/or HCDR3 encoding region;

(2) combining the starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDRl, HCDR2, and/or HCDR3 of a thrombin binding agent as defined herein, in which the HCDRl, HCDR2 and HCDR3 have amino acid sequences other than SEQ ID NOS: 3, 4 and 5, respectively, such that the donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains;

(3) expressing the nucleic acids of the product repertoire to produce product VH domains;

(4) optionally combining the product VH domains with one or more VL domains;

(5) selecting a thrombin binding agent which binds to a molecule comprising an epitope defined by: (i) at least three of the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin; and/or (ii) at least three of the amino acids R67, R73, Q38, R77A and S36A of human thrombin; and/or (iii) the amino acid Y76 and at least two, three, four, five or six of the amino acids R67, R73, Q38, R77A, T74 and S36A of human thrombin, which thrombin binding agent comprises a product VH domain and optionally a VL domain; and (6) recovering the thrombin binding agent or nucleic acid encoding it.

The reference disclosure also describes a method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin comprising:

providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region;

combining the starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID

NOS: 3, 4 and 5 respectively, such that the donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains;

expressing the nucleic acids of the product repertoire to produce product VH domains;

optionally combining the product VH domains with one or more VL domains;

selecting an antibody molecule that binds exosite 1 of thrombin, which antibody molecule comprises a product VH domain and optionally a VL domain; and

recovering the antibody molecule or nucleic acid encoding it.

Suitable techniques for the maturation and optimisation of antibody molecules are well-known in the art.

Antibody antigen-binding domains and antibody molecules for the exosite 1 epitope of thrombin may be tested as described above. For example, the ability to bind to thrombin and/or inhibit the cleavage of thrombin substrates may be determined. The thrombin binding agents produced according to the methods described above may be defined by one or more or all of the features of thrombin binding agents as described above. For example, the thrombin agent agent produced according to a method described above may inhibit thrombin activity but cause minimal or no inhibition of haemostasis and/or cause minimal or no bleeding . The effect of an antibody molecule on coagulation and bleeding may be determined using standard techniques. For example, a mouse thrombosis model of ferric chloride clot induction in a blood vessel, such as the femoral vein or carotid artery, followed by a tail bleed to test normal haemostasis, may be employed.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

Unless stated otherwise, antibody residues are numbered herein in accordance with the Kabat numbering scheme.

As used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and

definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Thus, the features set out above are disclosed in all combinations and permutations . As used herein, the term "amino acid" encompasses an amino acid analogue. The term "amino acid analogue" may be defined as any of the amino acid-like compounds that are similar in structure and/or overall shape to one or more of the twenty L-amino acids commonly found in naturally occurring proteins . These twenty L- amino acids are defined and listed in WIPO Standard ST.25 (1998), Appendix 2, Table 3 as alanine (Ala or A), cysteine (Cys or C) , aspartic acid (Asp or D) , phenylalanine (Phe or F) , glutamate (Glu or E), glycine (Gly or G) , histidine (His or H) , isoleucine (lie or I), lysine (Lys or K) , leucine (Leu or L), methionine (Met or M) , asparagine (Asn or N) , proline (Pro or P) , glutamine (Gin or Q) , arginine (Arg or R) , serine (Ser or S) , threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) , and tyrosine (Tyr or Y) .

Amino acid analogues may thus include natural amino acids with modified side chains or backbones. The analogue may share backbone structures, and/or even the most side chain structures of one or more natural amino acids, with the only difference ( s ) being containing one or more modified groups in the molecule. Such modification may include substitution of an atom (such as N) for a related atom (such as S) , addition of a group (such as methyl, or hydroxyl group, etc.) or an atom (such as CI or Br, etc.), deletion of a group (supra), substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. Amino acid analogues may include a-hydroxy acids, and β- amino acids, and can also be referred to as "modified amino acids". Amino acid analogues may either be naturally occurring or unnaturally occurring (e.g. synthesised) . As will be appreciated by those skilled in the art, any structure for which a set of rotamers is known or can be generated can be used as an amino acid analogue. The side chains may be in either the (R) or the (S) configuration (or D- or L-configuration) . Certain aspects and embodiments of the invention and reference disclosure will now be illustrated by way of example and with reference to the tables and reference figures described below. Figure 1 shows the binding and elution of the IgA on human thrombin-Sepharose column. Figure 1A shows an elution profile for IgA (narrow peak) from a thrombin-Sepharose column using a pH gradient (neutral to low, indicated by upward sloping line).

Figure IB shows a native blue gel showing total IgA load, flow- through from the human thrombin column and eluate following elution at low pH .

Figure 2 shows a non-reducing SDS-PAGE gel which indicates that the IgA binds thrombin but not prothrombin. In this pull-down assay, lectin agarose is used to bind to IgA in the presence of thrombin or prothrombin. The supernatant is then run on an SDS gel. Lane 1 is size standards; lane 2 shows a depletion of thrombin from the supernatant; Lane 3 shows that depletion is dependent on the presence of the IgA; Lanes 3 and 4 show that prothrombin is not depleted, and therefore does not bind to the IgA.

Figure 3 shows the relative rate of S2238 cleavage by thrombin in the presence or absence of IgA (i.e. a single slope of Abs405 with time for S2238 hydrolysis) . This indicates that the IgA does not bind at the thrombin active site.

Figure 4 shows the results of binding studies which indicate that the IgA competes with the fluorescently labelled dodecapeptide hirugen for binding to thrombin.

Figure 5 shows the effect of the IgA on the cleavage of S2238 by thrombin. This analysis allows the estimate of Kd for the IgA- thrombin interaction of 12nM.

Figure 6 shows an SDS-PAGE gel of whole IgA and Fab fragments under reducing and non-reducing (ox) conditions. The non-reduced IgA is shown to have a molecular weight of between 100-200 kDa and the non-reduced Fab has a molecular weight of about 50kDa.

Figure 7 shows the crystal structure of Thrombin-Fab complex showing interaction between the exosite 1 of thrombin and HCDR3 of the Fab fragment.

Figure 8 shows detail of crystal structure showing interaction between specific residues of thrombin exosite 1 and HCDR3 of the Fab fragment.

Figure 9 shows fluorescence microscopy images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen taken at between 2 and 30 minutes. lOOul of PBS was administered (vehicle control) .

Figure 10 shows fluorescence microscopy images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 40nM (final concentration in mouse blood, equivalent to a dose of approximately 0.6 mg/Kg) anti-exosite 1 IgA (ΙΟΟμΙ in PBS).

Figure 11 shows fluorescence microscopy images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 80nM (final concentration in mouse blood, equivalent to a dose of approximately 1.2 mg/Kg) anti-exosite 1 IgA(100 l in PBS), and a region outside of injury site for comparison. Figure 12 shows fluorescence microscopy) images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 200nM (final concentration in mouse blood, equivalent to a dose of approximately 3 mg/Kg) anti- exosite 1 IgA (ΙΟΟμΙ in PBS), and a region outside of injury site for comparison. Figure 13 shows fluorescence microscopy images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice injected with FITC labelled fibrinogen and 400nM (final concentration in mouse blood, equivalent to a dose of approximately 6 mg/Kg) ant exosite 1 IgA (ΙΟΟμΙ in PBS) .

Figure 14 shows fluorescence microscopy) images of FeCl ₃ induced blood clots in femoral vein injuries in C57BL/6 mice treated with FITC labelled fibrinogen and 4μΜ (final concentration in mouse blood, equivalent to a dose of approximately 60 mg/Kg) anti- exosite 1 IgA (ΙΟΟμΙ in PBS) .

Figure 15 shows a quantitation of the dose response to anti- exosite 1 IgA from the fluorescent images shown in figures 9 to 13.

Figure 16 shows tail bleed times in control C57BL/6 mice and in mice treated with increasing amounts of anti-exosite 1 IgA. The second average excludes the outlier.

Figure 17 shows the results of tail clip assays on wild-type male C57BL/6 mice (n=5) after injection into tail vein with either IgA or PBS. 15 mins after injection, tails were cut at diameter of 3mm and blood loss monitored over lOmin.

Figure 18A to 18D show the results of an FeCl ₃ carotid artery occlusion model on 9 week old WT C57BL/6 male mice injected as previously with 400nM anti-thrombin IgA (final concentration in blood, equivalent to a dose of approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCl ₃ for 2 min. Figure 18A shows results for a typical PBS-injected mice (occlusion in 20min) and figures 18B, 18C and 18D show examples of results for mice treated with 400nM anti-thrombin IgA (no occlusion). Figure 19 shows thrombin times (i.e. clotting of pooled plasma) with increasing concentrations of IgG and IgA of the invention, upon addition of 20nM human thrombin. Figure 20 shows the binding of synthetic IgG to immobilized thrombin (on ForteBio Octet Red instrument) . Figure 21 shows a typical Octet trace for the binding of 24nM S195A thrombin to immobilized IgG showing the on phase, followed by an off phase. The black line is the fit.

Figure 22 shows an Octet trace of 500nM prothrombin with a tip loaded with immobilized IgG. The same conditions were used as the experiment with thrombin in fig 21. There is no evidence of binding, even at this high concentration.

Experiments

Example 1. (Reference) Antibody Isolation and Characterisation Coagulation screening was carried out on a blood plasma sample from a patient. The coagulation tests were performed on a patient who suffered subdural haematoma following head injury. The haematoma spontaneously resolved without intervention. There was no previous history of bleeding and in the 4 years since the patient presented, there have been no further bleeding episodes. The results are shown in Table 1.

Table 1. Coagulation Screening Results.

The prothrombin time (PT), activated partial thromboplastin time (APTT) , and thrombin time (TT) were all prolonged in the patient compared to controls, but reptilase time was normal. Thrombin time was not corrected by heparinase, indicating that heparin treatment or contamination was not responsible.

Fibrinogen levels were normal in the patient, according to ELISA and Reptilase assays. The Clauss assay gave an artifactually low fibrinogen level due to the presence of the thrombin inhibitor. The PT and APTT clotting times were found to remain prolonged following a mixing test using a 50:50 mix with pooled plasma from normal individuals. This showed the presence of an inhibitor in the sample from the patient.

The patient' s blood plasma was found to have a high titre of an IgA. This IgA molecule was found to bind to a human thrombin column (Figure 1) . IgA binding lectin-agarose pulled down thrombin in the presence but not the absence of the IgA.

Prothrombin was not pulled down by the lectin-agarose in the presence of the IgA, indicating that the IgA specifically binds to thrombin but not prothrombin (Figure 2) . The binding site of the IgA on the thrombin molecule was then investigated .

A slightly higher rate of cleavage of S2238 by thrombin was measured in the presence of the IgA, indicating that the IgA does not block the active site of thrombin (Figure 3) .

The binding of fluorescently labelled hirugen to thrombin is inhibited by the presence of 700 nM of the IgA, indicating that the epitope for the antibody overlaps with the binding site of hirugen on thrombin, namely the exosite 1 of thrombin (Figure 4) .

The effect of the IgA on the hydrolysis of some of thrombin' s procoagulant substrates was tested. The results are shown in Table 2. These results demonstrate that the IgA molecule isolated from the patient sample inhibits multiple procoagulant activities of thrombin. Thrombin substrate Activity Antibody Effect

Fibrinogen Formation of fibrin No detectable

clot cleavage

Platelet receptor Activation of 15-fold decrease in PAR-1 peptide platelets hydrolysis

FVIII Feedback activation 7-fold decrease in of thrombin via hydrolysis

Xase complex

Table 2. Effect of anti-exosite 1 IgA on thrombin hydrolysis of procoagulant substrates.

Inhibition of thrombin by antithrombin (AT) in the presence the IgA was only marginally affected in both the absence and presence of heparin (Table 3).

Table 3. Effect of saturating concentration of anti-exosite 1 IgA (Fab) on thrombin inhibition by antithrombin (AT) in the absence and presence of InM heparin (Hep).

The dissociation constant (K _d ) of the IgA for thrombin was initially estimated based on rate of S2238 hydrolysis to be approximately 12nM (Figure 5) . The ]¾ for the binding of the IgA to S195A thrombin (inactivated by mutation of the catalytic serine) was determined to be 2nM using the ForteBio Octet Red instrument (Table 4) .

Table 4. Binding constants of anti-exosite 1 IgA (n=l under this precise condition), IgG (n=3) antibodies, and IgG-derived FAB to S195A thrombin (active site free, recombinant thrombin) . * Kd determined from steady-state analysis of response vs.

concentration. # Kd calculated from rates. ⁺ Determined using immobilised FAB.

The purified IgA was cleaved with papain (Figure 6), and the Fab fragment was isolated and combined with human PPACK-Thrombin (PPACK is a covalent active site inhibitor) . The human PPACK- Thrombin-FAB complex was crystallized and used for structural analysis. The statistics of the structure obtained were as follows: resolution is 1.9A; Rfactor = 19.43%; Rfree = 23.42%; one complex in the asymmetric unit; Ramachandran : favoured = 97.0%, outliers = 0%. The crystal structure revealed a close association between the HCDR3 of the IgA Fab and the exosite 1 of thrombin (Figure 7) . In particular, residues M32, F34, Q38, E39, L40, L65, R67, R73, T74, R75, Y76, R77a and 182 of the exosite 1 all directly interact with the HCDR3 loop of the IgA Fab (Figure 8) .

PISA analysis of the antibody-thrombin interface showed that the total buried surface area in the complex is 1075 A ² . The contact residues in the IgA heavy chain were (Kabat numbering) : 30, 51, 52a, 53-55, 96, 98, 99, 100, 100a, 100b, 100c, lOOd) . These are all in CDRs : CDRH1- GYTLTEAAIH; CDRH2- GLDPQDGETVYAQQFKG; CDRH3- GDFSEFEPFS DYFHF (underlined residues contacting) . CDRH3 was found to be the most important, providing 85% of the buried surface area on the antibody. The light chain made one marginal contact with Tyr49, right before CDRL2 (with Ser36a of thrombin) . Some individual contributions to buried surface were: Glu99 54A ² , PhelOO 134.8 A ² , GlulOOa 80.6 A ² , PhelOOc 141.7 A ² . The contact residues in thrombin were found to be ( chymotrypsin numbering) : 32, 34, 36a-40, 65, 67, 73-76, 77a, 82, and 151. The most important individual contributors to the buried surface were: Gln38 86.4 A ² , Arg73 44.5 A ² , Thr74 60.1 A ² , Tyr76 78.4 A ² , Arg77a 86.9 A ² .

The patient did not display increased or abnormal bleeding or haemorrhage, in spite of 3g/l circulating levels of this IgA, demonstrating that the antibody inhibits thrombin without affecting normal haemostasis.

Example 2. (Reference) The effect of IgA on Animal Thrombosis Models

C57BL/6 mice were anaesthetized. A catheter was inserted in the carotid artery (for compound injection). FITC labelled fibrinogen (2mg/ml) was injected via the carotid artery. PBS (control) or IgA was also injected via the carotid artery. The femoral vein was exposed and 10% FeCl ₃ applied (saturated blotting paper 3mm in length) for 3 min to induce clotting.

Fluorescence microscopy images were taken along the length injury site at 0, 5, 10, and 20 min post FeCl ₃ injury using fluorescence microscopy techniques.

Clots (fibrin deposits) in the femoral vein were clearly visible as bright areas (figure 9) . The lowest dose of the antibody was observed to cause significant inhibition of clotting but as the dose increased, clotting was abolished (figures 10 to 15 ) . The bleeding times of the mice were also measured. Bleeding times were assessed as time to cessation of blood flow after a tail cut. Despite the presence of a single outlier sample, the bleeding time was found to be unaffected by treatment with anti- exosite 1 IgA (figure 16) .

These results show that the anti-exosite 1 IgA antibody is a potent inhibitor of thrombosis but has no effect on bleeding time.

Example 3. (Reference) Tail clip assays

A tail clip assay was performed on wild-type male C57BL/6 mice injected with either 400nM IgA (final concentration in blood, equivalent to a dose of approximately 6 mg/Kg) or PBS. Blood loss was monitored over lOmins after the tail was cut at 3mm diameter 15 minutes after the injection. Total blood loss was found to be unaffected by treatment with anti-exosite 1 IgA (figure 17).

Example 4. (Reference) FeCl ₃ injury carotid artery occlusion FeCl ₃ injury carotid artery occlusion studies were performed on 9 week old WT C57BL/6 male mice. Mice were injected with 400nM anti-IIa IgA (final concentration in blood, equivalent to a dose of approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCl ₃ for 2 min. Blood flow was then monitored by Doppler and the time to occlusion measured. A "clot" was defined as stable occlusive thrombus where blood flow was reduced to values typically less than O.lml/min and stayed reduced. In the control mice, a stable clot was observed to form about 20mins after injury (Figure 18A) . However, the majority of mice treated with 400nM anti-IIa IgA were unable to form stable clots and gave traces in which the clots were quickly resolved, repeatedly resolved or never formed. Three representative traces are shown in Figures 18B to 18D.

Example 5. (Reference) Anti-exosite 1 IgG The IgA molecule identified in the patient described above was re-formatted as an IgG using standard techniques .

The clotting time of pooled human plasma spiked with increasing amounts of the original IgA and the new IgG was tested upon addition of human thrombin to 20nM (Figure 19) . Both parent IgA and the synthetic IgG increased time to clot formation in an identical concentration-dependent manner, implying identical affinities for thrombin.

This was confirmed by measuring the binding of synthetic IgG to immobilized S195A thrombin using a ForteBio™ Octet Red

instrument. Thrombin was attached to the probe and the binding of the antibodies (at various concentrations) was monitored. On- rates and off-rates were determined. Both antibodies gave similar on-rates of approximately 3xl0 ⁵ M ^_1 s ^_1 and off-rates of

approximately 5xlCT ⁴ s ^-1 , and dissociation constants (Kd) of approximately 2nM. Kds of approximately 2nM were also obtained for the IgA and the IgG by steady-state analysis (Table 4) . A representative steady state curve is shown in Figure 20. The properties of the IgA were therefore reproduced on an IgG framework .

Binding of prothrombin to the IgG antibody was tested using the Octet system by immobilizing IgG. Thrombin bound to the

immobilized IgG with comparable rates and affinities as those obtained using immobilized thrombin (Table 4); prothrombin did not bind to the IgG. Figure 21 is a trace of 24nM thrombin binding to and dissociating from the immobilized IgG. Figure 22 is the same experiment using 500nM prothrombin, and shows no evidence of binding.

Example 6. Analysis of binding interface between anti-exosite 1 Fab and thrombin

An anti-exosite 1 Fab fragment was made using standard techniques from the anti-exosite 1 IgA molecule described in Example 1. Binding of the anti-exosite 1 Fab fragment to human thrombin was analysed using the predicted crystal structure for each molecule. Binding analysis to identify key human thrombin epitope residues was performed using PISA analysis using default parameters of PISA Version 1.49 updated 27 March 2013. The results are shown in Table 5 below. The data in Table 5 suggest that the most important individual residue contributors to the buried surface in human thrombin when binding to the anti-exosite 1 Fab fragment were: Arg77a, Gln38, Tyr76, Thr74, and Arg73. However, when ranked according to solvation free energy gain as shown in Table 5, middle column, the most important individual residue contributors in human thrombin when binding to the anti- exosite 1 Fab fragment were: Arg67, Arg73, Gln38, Arg77a, and Ser36a .

The solvation deltaG (A ¹ G) middle column in Table 5 indicates the solvation energy of the corresponding residue, in kcal/M. The solvation energy gain of the interface is calculated as

difference in solvation energies of all residues between

dissociated and associated (interfacing) structures. Therefore, positive solvation energy (Δ¾) of a residue makes a negative contribution to the solvation energy gain of the interface, which corresponds to hydrophobic effect. Solvation energy estimates in PISA do not include the effect of satisfied hydrogen bonds, salt bridges or disulphide bonds across the interface. The effect of hydrogen bonds (-0.44 kcal/mol per bond), salt bridges

(additional -0.15 kcal/mol per salt bridge) and disulphide bonds (-4 kcal/mol per bond) is calculated separately and used to rank the epitope residues by total free energy gain (see Table 5, right column) . When ranked according to total free energy gain, the most important individual residue contributors in human thrombin when binding to the anti-exosite 1 Fab fragment were: Arg67, Arg73, Gln38, Arg77a, Tyr76, Thr74 and Ser36a.

Accordingly, the epitope in human thrombin to which the anti- exosite 1 Fab fragment binds can be defined by at least three, four, or five, of the amino acids R77A, Q38, Y76, T74 and R73 of human thrombin. The epitope defined in this way is determined using PISA analysis by calculating the amino acid residues of thrombin which have the highest interface area (in A ² ), for example the third, fourth or fifth highest interface area, upon formation of a binding interface between human thrombin and the anti-exosite 1 Fab fragment.

Alternatively, the epitope in human thrombin to which the anti- exosite 1 Fab fragment binds can be defined by at least three, four, or five, of the amino acids R67, R73, Q38, R77A and S36A of human thrombin. The epitope defined in this way is determined using PISA analysis by calculating solvation free energy gain (in kcal/mol), particularly amino acids contributing a meaningful negative solvation free energy gain, upon formation of a binding interface between human thrombin and the anti-exosite 1 Fab fragment .

Alternatively, the epitope in human thrombin to which the anti- exosite 1 Fab fragment binds can be defined by at least three, four, five, six or seven of the amino acids R67, R73, Q38, R77A, Y76, T74 and S36A of human thrombin, provided that the epitope includes the amino acid Y76. The epitope defined in this way is determined by calculating total free energy gain (in kcal/mol), which is a combination of solvation free energy gain (in

kcal/mol) as calculated using PISA analysis plus the effect of hydrogen bonds (additional -0.44 kcal/mol per bond), salt bridges (additional -0.15 kcal/mol per salt bridge) and disulphide bonds (additional -4 kcal/mol per bond) which is calculated separately, particularly amino acids contributing a meaningful negative total free energy gain, upon formation of a binding interface between human thrombin and the anti-exosite 1 Fab fragment.

The uniqueness of the human thrombin epitope bound by the anti- exosite 1 Fab fragment was assessed using comparative PISA analysis of human thrombin when binding to the known thrombin binding agents hirugen (1HAH), thrombomodulin (IDX5, complex of chains M and I) and fibrinogen E domain. The results are shown in Tables 6-8 below.

The data shown in Tables 5-8 confirm that the anti-exosite 1 Fab fragment binds to a different epitope on human thrombin to the epitopes bound by hirugen, thrombomodulin and fibrinogen E domain .

Table 5. Key amino acids of human thrombin when binding to anti-exosite 1 Fab fragment, sorted by buried surface area (SA, in A ² ), by solvation free energy gain (deltaGs) in solvation energy in

kcal/mol, or by total free energy gain ( deltaGtotal ) in solvation energy in kcal/mol. Negative values indicate favourable solvation energetics. Solvation energy estimates in PISA do not includ the effect of satisfied hydrogen bonds and salt bridges across the interface. The effect of hydrogen bonds (-0.44 kcal/mol per bond) and salt bridges (additional -0.15 kcal/mol per salt bridge) are included in total delta G in the binding interface. H refers to a hydrogen bond, and refers to a salt-bridge.

Table 6. Key amino acids of human thrombin when binding to hirugen, sorted by buried surface area (SA, in A ² ), by solvation free energy gain (deltaGs) in solvation energy in kcal/mol, or by total free energy gain ( deltaGtotal ) in solvation energy in kcal/mol. See Table 5 legend for further 5 information.

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Table 7. Key amino acids of human thrombin when binding to thrombomodulin, sorted by buried surface area (SA, in A ² ), by solvation free energy gain (deltaGs) in solvation energy in kcal/mol, or by total free energy gain ( deltaGtotal ) in solvation energy in kcal/mol. See Table 5 legend for further information.

Table 8. Key amino acids of human thrombin when binding to fibrinogen E domain, sorted by buried surface area (SA, in A ² ), by solvation free energy gain (deltaGs) in solvation energy in kcal/mol, or by total free energy gain ( deltaGtotal ) in solvation energy in kcal/mol. See Table 5 legend for further information.

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Sequences

Amino acid sequence of human preprothrombin (SEQ ID NO: 1;

GenelD: 2147; NP_000497.1 GI : 4503635; exosite 1 residues underlined)

1 mahvrglqlp gclalaalcs lvhsqhvfla pqqarsllqr vrrantflee vrkgnlerec 61 veetcsyeea fealesstat dvfwakytac etartprdkl aaclegncae glgtnyrghv 121 nitrsgiecq lwrsryphkp einstthpga dlqenfcrnp dssttgpwcy ttdptvrrqe 181 csipvcgqdq vtvamtprse gssvnlsppl eqcvpdrgqq yqgrlavtth glpclawasa 241 qakalskhqd fnsavqlven fcrnpdgdee gvwcyvagkp gdfgycdlny ceeaveeetg

301 dgldedsdra iegrtatsey qtffnprtfg sgeadcglrp lfekksledk terellesyi 361 dgrivegsda eigmspwqvm 1 frkspqell cgaslisdr vltaahclly ppwdknften 421 dllvrigkhs rtryerniek ismlekiyih prynwrenld rdialmklkk pvafsdyihp 481 vclpdretaa sllqagykgr vtgwgnlket wtanvgkgqp svlq vnlpi verpvckdst 541 riritdnmfc agykpdegkr gdacegdsgg pfvmkspfnn rwyqmgivsw gegcdrdgky

601 gfythvfrlk kwiqkvidqf ge

Amino acid sequence of anti-exosite 1 IgA and IgG VH domain with Rabat Numbering (CDRs underlined) : (SEQ ID NO: 2).

QVQLIQSGSAVKKPGASVRVSCKVSGYTLTEAAIHWVRQAPGKGLEWMGG

10 20 30 40 50

LDPQDGETVYAQQFKGRVTMTEDRSTDTAYMEVNNLRSEDTATYYCTTGD

52a 60 70 8082abc 90

FSEFEPFSMDYFHFWGQGTVVTVAS

lOOabcdefgh 110 Amino acid sequence of anti-exosite 1 IgA and IgG HCDR1 (SEQ ID NO: 3) .

GYTLTEAAIH

Amino acid sequence of anti-exosite 1 IgA and IgG HCDR2 (SEQ NO: 4) .

GLDPQDGETVYAQQFKG Amino acid sequence of anti-exosite 1 IgA and IgG HCDR3 (SEQ ID NO: 5) .

GDFSEFEPFS DYFHF

Amino acid sequence of anti-exosite 1 IgA and IgG VL domain with Rabat Numbering: (SEQ ID NO: 6) .

EIVLTQSPATLSLSPGERATLSCRASQNVSSFLAWYQHKPGQAPRLLIYD

10 20 30 40 50

ASSRATDIPIRFSGSGSGTDFTLTISGLEPEDFAVYYCQQRRSWPPLTFG

60 70 80 90 95a GGTKVEIKR

100 108

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR1 (SEQ ID NO: 7) .

RASQNVSSFLA

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR2 (SEQ ID NO: 8) .

DASSRAT

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR3 (SEQ ID NO: 9) .

QQRRSWPPLT SEQUENCE LISTING

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Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

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Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

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Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

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Arg Arg Gin Glu Cys Ser lie Pro Val Cys Gly Gin Asp Gin Val Thr

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Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

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Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin Gin Tyr Gin Gly Arg 210 215 220

Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala 225 230 235 240

Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe Asn Ser Ala Val Gin

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

260 265 270

Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu

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Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp 290 295 300

Glu Asp Ser Asp Arg Ala lie Glu Gly Arg Thr Ala Thr Ser Glu Tyr 305 310 315 320

Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

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Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

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Arg Glu Leu Leu Glu Ser Tyr lie Asp Gly Arg lie Val Glu Gly Ser

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Asp Ala Glu lie Gly Met Ser Pro Trp Gin Val Met Leu Phe Arg Lys

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Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

405 410 415

Phe Thr Glu Asn Asp Leu Leu Val Arg He Gly Lys His Ser Arg Thr

420 425 430

Arg Tyr Glu Arg Asn He Glu Lys He Ser Met Leu Glu Lys He Tyr 435 440 445

He His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp He Ala 450 455 460

Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr He His Pro 465 470 475 480

Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gin Ala Gly

485 490 495

Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr

500 505 510

Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu Gin Val Val Asn Leu 515 520 525

Pro He Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg He Arg He 530 535 540

Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg 545 550 555 560

Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser

565 570 575

Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly He Val Ser Trp Gly Glu

580 585 590

Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg 595 600 605

Leu Lys Lys Trp He Gin Lys Val He Asp Gin Phe Gly Glu

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Gly Gly Leu Asp Pro Gin Asp Gly Glu Thr Val Tyr Ala Gin Gin Phe 50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Arg Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Val Asn Asn Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Thr Gly Asp Phe Ser Glu Phe Glu Pro Phe Ser Met Asp Tyr Phe

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His Phe Trp Gly Gin Gly Thr Val Val Thr Val Ala Ser

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Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Asn Val Ser Ser Phe

20 25 30

Leu Ala Trp Tyr Gin His Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45

Tyr Asp Ala Ser Ser Arg Ala Thr Asp lie Pro lie Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Gly Leu Glu Pro

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Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Arg Ser Trp Pro Pro

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