This invention relates to inhibitors of anticoagulants, in particular inhibitors of direct thrombin inhibitors (DTIs) such as thrombin exosite 1 binding agents, and medical uses thereof.

The serine protease thrombin plays a critical role in the maintenance and regulation in haemostasis. Thrombin can act as a procoagulant, an anticoagulant, a mitogen and a chemotactic agent. Insufficient thrombin formation and function results in bleeding disorders, and excessive thrombin activity causes thrombosis (the formation of a blood clot that obstructs the flow of blood through a vessel) and thromboembolism (the formation of a blood clot that breaks away to lodge in a vessel elsewhere in the body) .

Thrombosis and thromboembolism are major causes of morbidity and mortality in the industrialised world, and their treatment is a major aim of the pharmaceutical industry. There are several anticoagulants currently available on the market, all of which have the unwanted side-effect of inducing bleeding in a fraction of patients. Other factors, such as kidney disease, may result in the accumulation of anti-thrombin activity resulting in a high risk of bleeding.

Anticoagulants can be divided into two classes: direct thrombin inhibitors (DTIs) and indirect thrombin inhibitors. Examples of indirect thrombin inhibitors include heparin and dermatan, which inactivate thrombin by catalysing the activation of endogenous thrombin inhibitors such as antithrombin or heparin cofactor II. In contrast, DTIs bind directly to thrombin to mediate their inhibitory effect. Examples of DTIs include the bivalent compound hirudin and its derivatives bivalirudin, lepirudin and desirudin, and the monovalent compounds argatroban, melagatran (with its prodrug ximelagatran) and dabigatran. The use of DTIs in a clinical setting has developed in recent years due to limitations and risks associated with indirect thrombin inhibitors. However, underdosing of DTIs can result in uncontrolled thrombin generation and/or disseminated

intravascular coagulation, while overdosing anticoagulant can lead to bleeding diatheses. For DTIs (and other anticoagulants) there is accordingly a narrow therapeutic window between a dose that prevents thrombosis and a dose that induces bleeding. This window is often further restricted by variations in the response in individual patients. To diminish or prevent side-effects of overdosing or underdosing of DTIs, the availability of suitable antidotes is critical. Although various specific DTI antidotes have been developed and investigated, their in vivo use has been limited and none are clinically available at present.

Thrombin mutants are known in the prior art. For example, Wu et al. (1991, Proc. Natl Acad. Sci. USA 88: 6775-6779) studied the two key thrombin functions of fibrinogen clotting (procoagulant ) and protein C activation in a thrombomodulin-dependent process

(anticoagulant) in various single amino acid thrombin mutants. W095/13385 describes a number of thrombin mutants with single, double or triple amino acid mutations (see Tables 1 and la of W095/13385) . Thrombin mutants which were capable of protein C activation without significant fibrinogen clotting activity, or vice versa, were deemed to be anticoagulants or procoagulants , respectively. In a paper related to W095/13385, Tsiang et al.

(1995, J. Biol. Chem. 270: 16854-16863) describes the use of thrombin mutants to deduce a functional map of surface residues of thrombin. A further study of thrombin residues responsible fo] its activity is described in Hall et al . (2001; Thromb. Haemost. 86: 1466-1474; see Table 1 listing of thrombin mutants) .

Specific thrombin mutants have been presented as antidotes for thrombin inhibitors. US6,060,300 describes a prothrombin mutant (a "thrombin mutein") with the mutation G226A and at least one further replacement or deletion at residues H57, D102 or S195 (using equivalent thrombin chymotrypsinogen numbering; see below) . The mutant is stated to be an antidote for hirudin or its derivatives. US2013/0064807 describes a prothrombin or thrombin mutant with the mutations W215A and/or W217A. The mutants were stated to reverse the effects of the thrombin inhibitors

argatroban, bivalidurin, lepirudin and heparin sodium.

According to a first aspect of the present invention, there is provided a method of neutralising the activity of a DTI,

comprising contacting the DTI with an antidote molecule selected from the following:

(1) a thrombin mutant (including a prothrombin mutant) comprising a mutation, deletion or substitution at one or more or all of the thrombin amino acid residues S36a, K109 and/or K110; and

(2) a polypeptide comprising an amino acid sequence having at least 50% sequence identity, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to the amino acid sequence of the thrombin mutant, in which the amino residues at positions equivalent to thrombin amino acid residues S36a, K109 and/or K110 are absent or other than serine (S) and/or lysine (K) , respectively.

As described in further detail below, the present invention derives from a novel antidote molecule as a thrombin mutant which neutralises the activity of a DTI . Target thrombin residues for mutation were selected based on crystal structures of binding between (i) thrombin and thrombomodulin and (ii) thrombin and a specific DTI antibody molecule (also referred to herein as the "reference antibody molecule (s)" or "reference antibody") disclosed in 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. The reference antibody recognises the exosite 1 epitope of thrombin and selectively inhibits thrombin without promoting bleeding. Thrombin mutants as putative antidote molecules of the present invention were tested inter alia for their ability to bind to the reference antibody molecule with similar affinity to wild-type thrombin but to bind with less affinity to thrombomodulin. Novel and useful antidote molecules were identified, as described herein.

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

As used herein, the term "neutralising" encompasses diminishing or inhibiting either partially or completely the thrombin inhibitory activity of a DTI.

In one embodiment of the present method, the thrombin mutant may comprise a mutation, deletion or substitution at thrombin amino acid residue S36a; and/or the polypeptide amino acid residue equivalent to thrombin amino acid residue S36a may be absent or other than serine (S) .

In another embodiment, the thrombin mutant may comprise a mutation, deletion or substitution at thrombin amino acid residues K109 and K110; and/or the polypeptide amino acid residues equivalent to thrombin amino acid residues K109 and K110 may be absent or other than lysine (K) .

In yet a further embodiment, the thrombin mutant may comprise a mutation, deletion or substitution at thrombin amino acid residues S36a, K109 and K110; and/or the polypeptide amino acid residues equivalent to thrombin amino acid residues S36a, K109 and K110 may be absent or other than serine (S) or lysine (K) , respectively.

According to the method, the thrombin mutant may further comprise a mutation, deletion or substitution at one or more or all of the thrombin amino acid residues selected from N78, K81 and/or M84; and/or the polypeptide amino acid residues equivalent to thrombin amino acid residue N78, K81 and/or M84 may be absent or other than asparagine (N) , lysine (K) and/or methionine (M) ,

respectively.

In particular, the antidote molecule may comprise a mutation, deletion or substitution at the following thrombin amino acid residues of the thrombin mutant: (i) S36, K109 and K110; (ii) S36, N78, K81, K109 and K110; or (iii) S36, N78, K81, M84, K109 and K110, and/or equivalent residues of the polypeptide.

The amino acid residues mutated , deleted or substituted in the antidote molecule may be based or neutral amino acids . A basic amino acid (such as histidine, lysine or arginine) or neutral amino acid (such as alanine or glycine) may be replaced with an acid amino acid (such as aspartate, glutamate, asparagine or glutamine ) .

In accordance with the method of the invention, the catalytic domain (responsible for protease activity) of the thrombin mutant and/or corresponding domain of the polypeptide may be

inactivated, mutated or deleted. The thrombin catalytic domain, also referred to as the "active site", is well defined in the art (see for example James A. 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).

For example, the antidote molecule may comprise a mutation, deletion or substitution at one or more or all of the thrombin amino acid residues H57, G193 and/or S195 in the thrombin mutant and/or equivalent residues in the polypeptide. An S195 mutation (or mutation of the equivalent residue in the polypeptide) is one embodiment . Inactivation of the catalytic domain may additionally or

alternatively be inactivated using D-Phe-Pro-Arg- chloromethylketone ("PPACK") or another covalent active site inhibitor .

According to the method, the antidote molecule may bind with reduced affinity to thrombomodulin compared with wild-type thrombin binding to thrombomodulin. By "reduced" is meant at least about a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or an even higher binding reduction, or no detectable binding. For example, the antidote molecule may have a about a 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or 1,000-fold reduced affinity to thrombomodulin compared with wild- type thrombin binding to thrombomodulin.

Binding kinetics and affinity (expressed as the equilibrium dissociation constant, K _d ) of the anti-exosite 1 antibody molecules may be determined using standard techniques, such as surface plasmon resonance, e.g. using BIAcore analysis, or the ForteBio Octet Red system.

The antidote molecule may bind to the DTI with not less than about 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% affinity than wild-type thrombin binding to the DTI .

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 DTI may bind to the exosite 1 domain of thrombin.

Exosite 1 (also known as "anion binding exosite 1" and the

"fibrinogen recognition exosite") is a well-characterised secondary binding site on the thrombin molecule (see for example James A. Huntington, 2008, above) . 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 bind to exosite 1, including the anticoagulant dodecapeptide hirugen (Naski et al., 1990, J. Biol. Chem. 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) .

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. In some embodiments, other thrombin residues which are located close to (i.e. within 0.5nm or within 1 nm) of any one of these residues may also be considered to be part of exosite 1.

The DTI may be an antibody, an antibody fragment, an antigen binding fragment of an antibody, a mini-antibody or another agent specifically binding to an antigen. The DTI 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 may be polyclonal or monoclonal. The antibody, antibody fragment and/or antigen binding fragment 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. The DTI may be an immunoglobulin or fragment thereof, and may be natural or partly or wholly synthetically produced, for example a recombinant molecule. For example, the DTI may be an IgG, IgA, IgE or IgM or any of the isotype sub-classes, particularly IgGl and IgG . The DTI may include any polypeptide or protein comprising an antibody antigen-binding site, including Fab, Fab2 , Fab3, diabodies, triabodies, tetrabodies, minibodies and single-domain antibodies, including nanobodies, as well as whole antibodies of any isotype or sub-class.

In one embodiment, the DTI may be an antibody or a fragment thereof 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 particular, the DTI may be the reference antibody molecule as disclosed in

PCT/GB2012/053140 published as WO2013/088164 (see below) .

The method of the present invention may be an in vitro method, or alternatively an in vivo method.

The effect of antidote molecule of the present invention may be tested for activity in the pres t or absence of the DTI using a fibrinogen clotting or thrombin ime assay. Suitable assays are well-known in the art.

The ability of an antidote molecule of the invention neutralising the DTI and thereby the DTI's effect on coagulation and bleeding effect may be determined using standard techniques. For example, the ability of the antidote molecule to neutralise the DTI' s effect on thrombosis may be determined in an animal model, such as a mouse model with ferric chloride induced clots in blood vessels. Neutralising the DTI' s effect on haemostasis may also be determined in an animal model, for example, by measuring tail bleed of a mouse. Other suitable thrombosis models are well known in the art (see for example Westrick et al., 2007, Arterioscler . Thromb. Vase. Biol. 27: 2079-2093).

In another aspect of the present invention, there is provided a method of treating a side-effect of anticoagulant therapy, comprising administering to a patient in need thereof an

effective amount of the antidote molecule as defined herein. The treatment may include 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 side-effect may be thrombin generation and/or disseminated intravascular coagulation (which may be caused by underdosing of a DTI) or bleeding such as bleeding diatheses (which may be caused by overdosing of a DTI) .

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 antibody molecules as DTIs 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) , appropriate for the type of medicament being administered may used .

A therapeutically effective amount or suitable dose of an antidote molecule according to the invention 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 antidote molecule and the nature of any detectable label or other molecule attached to the antidote molecule.

A typical DTI antibody dose will be in the range 100 pq to 1 g for systemic applications, and 1 pg to 1 mg for topical

applications. Antidote molecules of the invention may be used a similar dose. Doses for treatment of an adult patient may be proportionally adjusted for children and infants, and also adjusted for other antidote molecules in proportion to molecula weight. The treatment schedule for an individual may be

dependent on the pharmocokinetic and pharmacodynamic properties of the antidote, the route of administration and the nature of the condition being treated.

In some embodiments, antidote molecules as described herein 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 antidote molecule may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of antidote molecule 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.

Also provided is a method of inhibiting the effect of a DTI in a mammal, comprising administering to the mammal an effective amount of the antidote molecule as defined herein.

Further provided is an antidote molecule as defined herein for use in a method of treating or preventing a side-effect of anticoagulant therapy.

In another aspect of the invention there is provided the use of an antidote molecule as defined herein in the manufacture of a medicament for treating or preventing a side-effect of

anticoagulant therapy.

In a further aspect of the invention, there is provided a molecule which is capable of neutralising the activity of a DTI, wherein the molecule is an antidote molecule per se as defined herein. In this aspect of the invention, known thrombin mutants such as those disclosed in Wu et al . (1991, above), W095/13385, Tsiang et al. (1995, above), Hall et al . (2001, above),

US6,060,300 and US2013/ 006 807 , all of which documents are herein incorporated by reference in their entirety, are specifically excluded from the scope of the invention as an antidote molecule per se.

The antidote molecule per se of the invention or as used in the methods of the invention may be a polypeptide comprising an amino acid sequence having least 50% sequence identity, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to the amino acid sequence of the thrombin mutant of the invention, with mutations, deletions or substitutions at the corresponding thrombin amino acid residues .

Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties .

Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector.

Examples include MatGat (Campanella et al . , 2003, BMC

Bioinformatics 4: 29; program available from

http://bitincka.com/ledion/matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FAS A (Altschul et al . , 1990, J. Mol. Biol. 215: 403-410; program available from

http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise

Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules : the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from

http://www.ebi.ac.uk/tools/emboss/align). All programs may be run using default parameters.

For example, sequence comparisons may be undertaken using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons ("Protein Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.

In one aspect of the invention, the sequence comparison may be performed over the full length of the reference sequence.

Additionally provided according to the invention is a nucleic acid encoding an antidote molecule per se as defined herein.

Also provided is a pharmaceutical composition comprising an antidote molecule per se as defined herein and a pharmaceutically acceptable excipient.

Antidote molecules of the invention 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.

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 antidote molecule, an increase in its lifespan and/or in its efficacy in the body or an increase in its 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 antidote molecule.

In some embodiments, antidote molecules of the invention may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antidote molecules may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

Antidote molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antidote molecule. Thus

pharmaceutical compositions may comprise, in addition to the antidote molecule, 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 antidote molecule. 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 antidote molecule 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 antidote molecule o the invention may be administered alone in combination with other treatments, either simultaneously sequentially depende upon the condition to be treated.

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 initial physiological haemostatic response, for example damage to 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.

Thus, DTIs such as the reference antibody molecule 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.

Any patient being treated with a DTI may suffer a side-effect caused by underdosing (such as uncontrolled thrombin generation and/or disseminated intravascular coagulation) or overdosing (such as bleeding diatheses).

Although the reference antibody molecule has been shown to inhibit thrombin activity but cause minimal or no inhibition of haemostasis and/or causes minimal or no bleeding, an antidote molecule which neutralises the activity of the reference antibody molecule is still desirable, for example for therapeutic and/or regulatory purposes .

Patients suitable for treatment as described herein include patients being treated with a DTI for conditions in which thrombosis is a symptom or a side-effect of treatment or which confer an increased risk of thrombosis or patients being treated with a DTI because they are predisposed to or at increased risk of thrombosis, relative to the general population. For example, patients may be treated with a DTI, and therefore the antidote molecule as described herein, for venous thrombosis in cancer patients, or hospital-acquired thrombosis which is responsible for 50% of cases of venous thromboembolism.

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 . , 2009, 19(1) : 11-22; Tsopanoglou et al . , 2009, Eur. Cytokine Netw. 20(4) : 171-179) .

Antidote molecules as described herein may also be useful in in vitro testing, for example in the analysis and characterisation of the effect of a DTI on coagulation, for example in a sample obtained from a patient . The invention thus encompases a method of analysing the effect of a DTI on coagulation, comprising the steps of:

(1) contacting the DTI (for example, in a blood sample obtained from a patient) with an antidote molecule of the invention; and

(2) measuring coagulation activity of the DTI.

The antidote molecule of the invention may be useful in

regulating DTI-mediated inhibition or prevention of the

coagulation of blood in extracorporeal circulations, such as haemodialysis and extracorporeal membrane oxygenation.

The invention also encompasses a method of regulating DTI- mediated inhibition or prevention of coagulation in a blood sample, comprising contacting the DTI with an antidote molecule of the present invention. The blood sample may be introduced into an extracorporeal circulation system before, simultaneously with or after the introduction of the antidote molecule.

For example, a method of inhibiting or preventing blood

coagulation in vitro or ex vivo may comprise introducing a DTI such as the reference antibody molecule to a blood sample. The blood sample may be introduced into an extracorporeal circulation system before, simultaneous with or after the introduction of the DTI and optionally subjected to treatment such as haemodialysis or oxygenation. The levels of inhibiting or preventing blood coagulation by the DTI may be regulated by an antidote molecule of the invention. In some embodiments, the treated blood may be subsequently administered to an individual. 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

(He 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 tyrosin<e (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) . 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 .

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 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) anti- 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. Figure 23 is a histogram showing Prothrombin Time (in s) to demonstrate a neutralising effect of a thrombin mutant according to an embodiment of the invention ( "Ila (MM+195A) " ) on an anti- exosite 1 antibody-derived VH5VK6 Fab.

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 hydrolysi procoagulant substrates .

Inhibition of thrombin by antithrombin (AT) in the presence the IgA was only marginally affected in both the absence an 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 (¾) of the IgA for thrombin was initially estimated based on rate of S2238 hydrolysis to be approximately 12nM (Figure 5) . The K _d 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 (SEQ ID NO: 3); CDRH2- GLDPQDGETVYAQQFKG (SEQ ID NO: 4); CDRH3- GDFSEFEPFSMDYFHF (SEQ ID NO: 5) (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 of 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. The use of thrombin variants as an antidote to the Anti-exosite 1 antibody

Six sites in the thrombin molecule were chosen for mutation to Glu: S36a, N78, K81, M84, K109 and K110. They were selected by comparing the structure of thrombomodulin and an IgA-derived Fab (designated "VH5VK6") of the DTI reference antibody molecule described in the reference examples above, each bound to PPACKed thrombin. It is noted that the reference disclosure and reference antibody are not publically available at the priority filing date of the present application.

The following thrombin mutants with single or multiple mutations in the above-mentioned chosen residues were constructed using standard techniques:

- S36aE, K109E or K110E single mutants;

- K109E/K110E double mutant;

- S36aE/K109E/K110E triple mutant

- S36aE/N78E/K81E/K109E/K110E pentuple mutant;

- S36aE/N78E/K81E/M84E/K109E/K110E hextuple (or "MM") mutant; and

- S36aE/N78E/K81E/M84E/K109E/K110E/S195A (or "MM + S195A") mutant . Expression vectors encoding the thrombin mutants were transformed into E. coli strain BL21STAR ( DE3 ) pLysS (Invitrogen) for

expression. Expressed mutants were refolded using established protocols, activated, and purified by Heparin Sepharose affinity chromatography .

Thrombin mutants were tested for: 1) binding to VH5VK6 (measured using the ForteBio Octet Red system [see above reference

examples] and/or BIAcore analysis); 2) binding to thrombomodulin (TM456, EGF domains 5 and 6 bind to thrombin); 3) ability to reverse prolongation of clotting time by VH5VK6 (prothrombin time); and 4) ability to clot plasma (thrombin time) . The results are shown in Table 5 below.

The Table 5 data shows that thrombin mutants with at least K109E and K110E mutations have a suitable profile for a DTI antidote, with weak binding to TM456 and strong binding to the DTI VH5VK6. The suitable DTI antidote profile was also found with the S36aE/K109E/K110E triple thrombin mutant, the

S36aE/N78E/K81E/K109E/K110E pentuple thrombin mutant, and the S36aE/N78E/K81E/M84E/K109E/K110E hextuple thrombin mutant. The S36aE thrombin mutant also had a suitable profile, when comparing results derived from the Octet Red and BIAcore systems .

Neutralisation of the effect of VH5VK6 ("Ab") on PT by a thrombin mutant was tested. VH5VK6 (Ι.βμΜ) was incubated with Tris buffered saline (TBS) control, thrombin I Ia (S195A) (3.2μΜ) or thrombin IIa (MM+S195A) ( 3.2-25.6μΜ) in Tris buffered saline for 15 minutes and added to an equal volume of normal pooled plasma. Clotting was initiated by addition of thromboplastin. The results are shown in Figure 23.

The data in Figure 23 demonstrate that addition of VH5VK6 alone significantly increases time to clot of normal plasma that is corrected by preincubation with thrombin and neutralised by the thrombin mutant MM+S195A.

The ability of the thrombin mutants to clot plasma (thrombin time) was then tested. ΙΟΟμΙ of 30nM thrombin or various thrombin mutants was added to ΙΟΟμΙ of normal pooled plasma and clot time was monitored. The results are shown in Table 6 below, and demonstrate the thrombin mutants tested significantly increased thrombin times. This shows that thrombin mutants had reduced ability to bind to and cleave fibrinogen. Thrombin mutants used in therapy should be active site inhibited, for example either by PPACK or the S195A mutation.

Table 5. Binding data from ForteBio Octet Red and/or BIAcore system. ND = note determined.

Molecule t (sec )

Ila (WT) 16.6

Ila-PPAck >300

Ila (S195A) >300

Ila (MM) >300

Ila (K109E/K110E) >300

Ila (K109E) 75.2

Ila (K109E/K110E) 30nM >300

Ila (K109E/K110E) 60nM >300

Ila (K109E/K110E) 150nM 73.8

Ila (K109E/K110E) 300nM 31.4

Table 6. Clot time of wild-type thrombin and thrombin mutants .

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 Iwrsryphkp 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 Ifrkspqell cgaslisdrw vltaahclly ppwdknften

421 dllvrigkhs rtryerniek ismlekiyih prynwrenld rdialmklkk pvafsdyihp

481 vclpdretaa sllqagykgr vtgwgnlket wtanvgkgqp svlqvvnlpi 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 ID NO: ) :

GLDPQDGETVYAQQFKG

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

GDFSEFEPFSMDYFHF

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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