Binding molecules

Abstract

The present invention relates to binding molecules which target PAR-1. The invention also relates to methods and uses of said binding molecules.

Description

[0001] Binding Molecules

[0002] Field of Invention

[0003] The present invention relates to binding molecules which target PAR-1. The invention also relates to methods and uses of said binding molecules.

[0004] Background

[0005] The PAR1 receptor (proteinase-activated 1 receptor) is a G-protein-coupled receptor (GPCR). GPCRs are a large family of plasma membrane receptors that transmit signals from the outside to the inside of the cell. GPCRs share a common structural domain of seven membrane-spanning a-helices. This domain undergoes a conformational change when the receptor is activated by its extracellular endogenous ligand, which results in intracellular activation of G-proteins. The PAR1 receptor activation mechanism is unusual (Coughlin., Proc Natl Acad Sci. U S A. 1999). Thrombin binds to the large extracellular N-terminal domain of PAR1 and proteolytically cleaves this domain. This creates a new N-terminal region of the receptor, and this new region binds to the membrane spanning a-helices to activate the receptor. The small molecule therapeutic vorapaxar binds the membrane spanning domain to block receptor activation (Zhang et al., Nature, 2012). An alternative potential mechanism to block PAR1 receptor activation is to use an antibody that binds the extracellular N-terminal domain and so block the action of thrombin on the receptor.

[0006] Once activated, the PAR1 receptor activates Gq G-proteins, which results in stimulation of a signal transduction cascade that results in platelet activation (Soh et al., Br J Pharmacol. 2010). This cascade includes the inositol phosphate signalling pathway; Gq activates phospholipase C which generates inositol phosphates. PAR1 activation by thrombin can be measured by assaying the level of inositol phosphates. The activated PAR1 receptor also recruits arrestin, a protein that interacts with the receptor which can block G-protein signalling and also result in arrestin-mediated signal transduction (Soh et al., Br J Pharmacol. 2010). Arrestin interaction provides another method for quantifying PAR1 activation by thrombin.

[0007] The PAR1 receptor is a therapeutic target for treating cardiovascular disease, inflammatory disorders and certain cancers (Flaumenhaft and Ceunyck., Trends Pharmacol Sci. 2017). The receptor is activated by the blood clotting agent thrombin, a principle enzyme in the coagulation process, which activates platelets by stimulating the PAR1 receptor. As a result, blocking the action of thrombin on PAR1 is an attractive strategy for treating disorders involving inflammatory responses and vascular endothelium dysfunction. The antiplatelet medication vorapaxar, a small molecule used to treat cardiovascular disease (Tricoci et al., N Engl J Med. 2012), acts by inhibiting thrombin-mediated activation of the PAR1 receptor (Zhang et al., Nature, 2012). However, there is a need to develop further therapies which target PAR1 in particular in particular biologic such as antibodies or antigen binding fragments. Summary of Invention

[0008] An aspect of the invention relates to an antibody or antigen binding fragment thereof, that binds to Proteinase-Activated Receptor-1 (PAR-1) comprising; ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 10 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 11 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 14 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 15 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 18 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 19 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and cii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 22 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 23 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 26 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 27 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 30 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 31 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or ei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 34 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 35 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and eii). a variable light chain (VL) CDR1 comprising SEQ ID NO. 37 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 38 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 39 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or

[0009] An aspect of the invention relates to an immunoconjugate comprising the antibody or antigen binding fragment according to the invention, conjugated to a therapeutic moiety, a half-life extending moiety, and / or a label.

[0010] An aspect of the invention relates to an isolated nucleic acid encoding the antibody or antigen binding fragment according to the invention.

[0011] An aspect of the invention relates to a vector comprising the nucleic acid according to the invention.

[0012] An aspect of the invention relates to pharmaceutical composition comprising the antibody or antigen binding fragment, or the immunoconjugate of the invention and a pharmaceutically acceptable excipient, diluent and or carrier.

[0013] An aspect of the invention relates to the antibody of the invention for use in therapy. An aspect of the invention also relates to a method of treatment comprising administering a therapeutically effective amount of the antibody of the invention. An aspect also relates to the use of the antibody of the invention in the manufacture of a medicament.

[0014] An aspect of the invention relates to binding molecule comprising the antibody or antigen binding fragment according to the invention.

[0015] An aspect of the invention relates to multispecific binding molecule comprising the antibody or antigen binding fragment according to the invention.

[0016] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of detecting PAR-1 in a biological sample comprising contacting said biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0017] An aspect of the invention relates to a method for reducing platelet activation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0018] An aspect of the invention relates to a method for reducing cell proliferation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention. An aspect of the invention relates to a method for blocking thrombin induced beta-arrestin 1 recruitment comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0019] An aspect of the invention relates to a method for reducing platelet aggregation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0020] An aspect of the invention relates to kit comprising the antibody or antigen binding fragment according to the invention and optionally instructions for use.

[0021] Figures

[0022] Figure 1. Antibody binding to the PAR1 receptor. Antibody binding to cells expressing the human PAR1 receptor (CHO-PAR1) or to control cells not transfected with the receptor (CHO) was measured by flow cytometry using a fluorescent FITC-labelled secondary antibody. The higher signal for CHO-PAR1 cells than for the control CHO cells demonstrates binding of the antibodies to the PAR1 receptor. The binding evident at 1 and 10 nM concentrations indicates the antibodies bind with high affinity to the PAR1 receptor. Data points and error bars are the mean ± SEM of two replicate measurements (except Panel F, four technical replicates). Binding was measured for the five antibodies AV-002 (A), AV-004 (B), AV-005 (C), AV-013 (D), AV-015 (E), and the negative control IgG HG1 K (F). Note in Panel F the data for CHO- PAR1 and CHO overlie one another. The experiment was repeated once with similar results.

[0023] Figure 2. Inhibition of thrombin-stimulated IP1 generation by PAR1 receptor antibodies. IP1 is generated by thrombin-mediated activation of the PAR1 receptor followed by activation of Gq and PLC. The data demonstrate the antibodies inhibit thrombin-mediated signalling by the PAR1 receptor. The thrombin concentration was 0.9 nM. The IC50 values for inhibition are given in Table 1. Data points and error bars are the mean ± SEM of two replicate measurements. Binding was measured for the five antibodies AV-002 (A), AV-004 (B), AV-005 (C), AV-013 (D) and AV-015 (E). The experiment was performed three times for AV-002, AV- 004, AV-005 and AV-013, and twice for AV-015.

[0024] Figure 3. Inhibition of thrombin-stimulated arrestin recruitment by PAR1 receptor antibodies. Arrestin is recruited to the PAR1 receptor following activation of the receptor by thrombin. The time course of arrestin recruitment is shown in the figure. The data demonstrate the antibodies strongly inhibit thrombin-mediated arrestin recruitment to the PAR1 receptor. The antibody concentration was 1 pM and thrombin concentration was 1 nM. Cells were incubated with antibody for 10 minutes followed by application of thrombin. Inhibition of arrestin recruitment was measured for the five antibodies AV-002 (A), AV-004 (B), AV-005 (C), AV-013 (D) and AV-015 (E). Data points are the mean calculated net BRET value. The inhibition values are given in Table 2. Figure 4. Inhibition of human platelet aggregation by PAR1 antibodies. Platelet aggregation stimulated by thrombin (0.5 nM) was measured as a decrease of absorbance of light, quantified in an aggregometer. The time course of aggregation was measured in the absence of antibody (“Vehicle”) condition or in the presence of antibody at 130 nM final concentration. The data show the antibodies inhibit thrombin-stimulated platelet aggregation, evident as a delay in the onset of aggregation. Platelet aggregation was measured for the five human antibodies AV-002 (A), AV-004 (B), AV-005 (C), AV-013 (D) and AV-015 (E), and the mouse antibody ATAP-2 (F). Data points are single determinations. The experiment was performed three times. The inhibition data are shown in Table 3, expressed as a percentage inhibition of the thrombin-stimulated AUC for platelet aggregation.

[0025] Figure 5. Inhibition of human platelet aggregation by PAR1 antibodies at three concentrations - 27 nM, 130 nM and 670 nM. Platelet aggregation stimulated by thrombin (0.5 nM) was measured as a decrease of absorbance of light, quantified in an aggregometer. The results show the antibodies almost completely inhibit aggregation and are similarly active at the low concentration of 27 nM, intermediate concentration of 130 nM, and high concentration of 670 nM. The extent of inhibition is similar for the human antibodies, for ATAP-2, and for a high concentration of the small molecule PAR1 inhibitor vorapaxar (170 nM). Platelet aggregation was measured for four human antibodies AV-002 (A), AV-004 (B), AV-013 (C) and AV-015 (D), for the mouse antibody ATAP-2 (E), and vorapaxar (F). Data points are single determinations. The experiment was performed three to five times (each experiment a different human donor). The inhibition data were expressed as a percentage inhibition of the thrombin-stimulated AUC for platelet aggregation, and these results are shown in in Figure 6 and Table 4.

[0026] Figure 6. Human platelet aggregation % inhibition AUC for PAR1 antibodies at three concentrations - 27 nM, 130 nM and 670 nM. Platelet aggregation stimulated by thrombin (0.5 nM) was measured as a decrease of absorbance of light, quantified in an aggregometer. The data show a similar degree of inhibition for the three concentrations tested; that the degree of inhibition is strong (from 60 - 98% inhibition); and that the inhibition is similar to that of a high concentration of the small molecule PAR1 inhibitor vorapaxar (83%, dashed horizontal line, 170 nM concentration). IgG is the negative isotype control, showing a nonspecific antibody has no effect in platelet aggregation at the concentrations tested. The AUC of the absorbance decrease, reflecting the degree of platelet aggregation, was quantified for the no-antibody vehicle control and for the antibody-treated sample (Figure 5). Then the AUC for the antibody- treated sample was expressed as a percentage inhibition of the AUC for the no-antibody vehicle control. Bar height is the mean aggregation % inhibition (See Table 4 for values), error bars are the SEM, and the symbols are the values from three to five independent experiments (each experiment a different human donor).

[0027] Detailed Description The embodiments of the invention will now be described. In the following passages, different embodiments are described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0028] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012) Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0029] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties of the molecules disclosed herein are also described in the examples.

[0030] The present inventors have developed binding molecules which can target PAR-1. PAR-1 is a G-protein coupled receptor (GPCR) and in general it has been difficult to develop antibodies to this type of target due to cells with low receptors count and antibody libraries with poor specificity. Herein the inventors use a machine learning approach to develop antibodies with excellent characteristics. In particular the machine learning approach allowed for the production of a small library of molecules to be screened and still identify a range of molecules with excellent functional binding characteristics.

[0031] The antibodies or binding molecules of the invention are capable of binding to PAR-1 , in some embodiment that antibodies are capable of reversibly binding to PAR-1 . Upon binding to PAR- 1 the antibodies or antigen binding fragments may be capable of modulate one or more effect of PAR-1 signalling. For example the antibodies or binding molecules described herein are capable of one or more of the following: Inhibiting, reducing or blocking PAR-1 -stimulated cellular signalling,

[0032] • Inhibiting, reducing or blocking p-arrestin recruitment,

[0033] • Inhibiting, reducing or blocking platelet aggregation,

[0034] • Inhibiting, reducing or blocking platelet activation, and / or

[0035] • Reducing cell proliferation.

[0036] PAR-1 -stimulated cellular signalling refers to the intracellular signalling that occurs once PAR- 1 is activated. Upon activation PAR-1 stimulates signalling networks vias classical G-protein mediated pathways and p-arrestin signalling, as such “PAR-1 -stimulated cellular signalling” may refer to either of the G-protein mediated pathways and / or p-arrestin signalling. Inhibition, reduction or blockade of PAR-1 stimulated cellular signalling may be measured by any suitable method. For example, upon activation by thrombin, the PAR1 receptor stimulates intracellular signalling, primarily via the Gq G-protein pathway, this can be measured by quantifying the levels of inositol-1 -phosphate (IPi), which is generated via Gq-mediated activation of phospholipase C. As such inhibition, reduction or blockade of PAR-1 stimulated cellular signalling may be measured by quantifying IPi in a cellular assay such as that set out in Example 6.

[0037] P-arrestins are molecules that are ubiquitously expressed in all cell types and play a role in the signalling of GPCRs, for example desensitizing GPCRs, control of GPCR intracellular trafficking, and activation of GPCRs to multiple signalling pathways. Inhibition, reduction or blockade of p-arrestin 1 recruitment may be measured by any suitable method, for example by measuring the interaction of p-arrestin with PAR-1 as set out in Example 7.

[0038] The term platelet aggregation refers to the process of platelets sticking together in order to form a clot. The term platelet activation refers to the process in which platelets become activated in order to aggregate, in general platelet activation comprises a platelet binding von Willebrand factor which stimulates the platelet to degranulate, inducing the expression of the GPIIb / llla receptor which along with thromboxane A2 (TXA2) produced by platelet enzyme cyclooxygenase, allows for aggregation to occur. The present antibodies or antigen binding fragments thereof of the invention have been shown to achieve a similar level of platelet aggregation inhibitor compared to the approved small molecule Vorapaxar. Advantageously the antibodies or antigen binding fragments thereof of the invention have demonstrated a significant level of platelet aggregation inhibition even when used at low concentration for example approx. 27 nM see Example 9.

[0039] The antibody or antigen binding fragment of the invention are inhibitors of PAR-1 i.e., the antibody or antigen binding fragment is capable of inhibiting or otherwise decreasing the one or more of the biological activities or PAR-1 . The biological activities of PAR-1 may refer to any downstream effects of PAR-1 signalling. The terms “inhibiting”, “reducing” and “blocking” have their usual interpretation in the art. For example where a biological activity is inhibited by an antibody or antigen binding fragment of the invention the biological activity occurs at a decreased level compared to if the antibody or antigen binding fragment was not present. The inhibition or reduction may be expressed as a % inhibition or % reduction as appropriate.

[0040] The antibody or antigen binding fragment is able to bind PAR-1 , as such in an embodiment the antibody or antigen binding fragment may comprise a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 80%, 90% or 95% sequence identity thereto.

[0041] In an embodiment the antibody or antigen binding fragment may comprise a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 10 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 11 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 13 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR2 comprising SEQ ID NO. 14 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR3 comprising SEQ ID NO. 15 or a sequence comprising 80%, 90% or 95% sequence identity thereto.

[0042] In an embodiment the antibody or antigen binding fragment may comprise a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 18 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 19 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR2 comprising SEQ ID NO. 22 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR3 comprising SEQ ID NO. 23 or a sequence comprising 80%, 90% or 95% sequence identity thereto.

[0043] In an embodiment the antibody or antigen binding fragment may comprise a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 26 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 27 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 29 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR2 comprising SEQ ID NO. 30 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR3 comprising SEQ ID NO. 31 or a sequence comprising 80%, 90% or 95% sequence identity thereto.

[0044] In an embodiment the antibody or antigen binding fragment may comprise a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 34 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 35 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 37 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR2 comprising SEQ ID NO. 38 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VL CDR3 comprising SEQ ID NO. 39 or a sequence comprising 80%, 90% or 95% sequence identity thereto

[0045] The VH CDR1 may be selected from SEQ ID NO: 1 , 9, 17, 25 or 33, or a sequence comprising 80%, 90% or 95% sequence identity thereto. The VH CDR2 may be selected from SEQ ID NO: 2, 10, 18, 26, or 34, or a sequence comprising 80%, 90% or 95% sequence identity thereto. The VH CDR3 may be selected from SEQ ID NO: 3, 11 , 19, 27, or 35 or a sequence comprising 80%, 90% or 95% sequence identity thereto. The VL CDR1 may be selected from SEQ ID NO: 5, 13, 21 , 29, 37, or a sequence comprising 80%, 90% or 95% sequence identity thereto. The VL CDR2 may be selected from SEQ ID NO: 6, 14, 22, 30, 38 or a sequence comprising 80%, 90% or 95% sequence identity thereto. The VL CDR3 may be selected from SEQ ID NO: 7, 15, 23, 31 , 39 or a sequence comprising 80%, 90% or 95% sequence identity thereto.

[0046] In an embodiment the antibody or antigen binding fragment thereof, according to the invention comprises; ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 , a VH CDR2 comprising SEQ ID NO. 2, a VH CDR3 comprising SEQ ID NO. 3, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5, VL CDR2 comprising SEQ ID NO. 6, VL CDR3 comprising SEQ ID NO. 7, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9, a VH CDR2 comprising SEQ ID NO. 10, a VH CDR3 comprising SEQ ID NO. 11 , and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13, VL CDR2 comprising SEQ ID NO. 14, VL CDR3 comprising SEQ ID NO. 15, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17, a VH CDR2 comprising SEQ ID NO. 18, a VH CDR3 comprising SEQ ID NO. 19, and cii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 , VL CDR2 comprising SEQ ID

[0047] NO. 22, VL CDR3 comprising SEQ ID NO. 23, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25, a VH CDR2 comprising SEQ ID NO. 26, a VH CDR3 comprising SEQ ID NO. 27, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29, VL CDR2 comprising SEQ ID NO. 30, VL CDR3 comprising SEQ ID NO. 31 , or ei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33, a VH CDR2 comprising SEQ ID NO. 34, a VH CDR3 comprising SEQ ID NO. 35, and eii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 37, VL CDR2 comprising SEQ ID NO. 38, VL CDR3 comprising SEQ ID NO. 39.

[0048] In an embodiment the antibody or antigen binding fragment comprises a VH sequence. The VH sequence may comprise SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto. The VH sequence may comprise SEQ ID NO. 12 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto. The VH sequence may comprise SEQ ID NO. 20 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto. The VH sequence may comprise SEQ ID NO. 28 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto. The VH sequence may comprise SEQ ID NO. 36 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0049] In an embodiment the antibody or antigen binding fragment comprises a VL sequence. The VL sequence may comprise SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto. The VL sequence may comprise SEQ ID NO. 16 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto. The VL sequence may comprise SEQ ID NO. 24 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto. The VL sequence may comprise SEQ ID NO. 32 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto. The VL sequence may comprise SEQ ID NO. 40 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

[0050] The antibody or antigen binding fragment thereof may comprise a VH comprising a sequence selected from SEQ ID NO. 4, 12, 20, 28, 36 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto. The antibody or antigen binding fragment thereof may comprise a VL comprising a sequence selected from SEQ ID NO. 8, 16, 24, 32, 40 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0051] In an embodiment the antibody or antigen binding fragment thereof comprises a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0052] In an embodiment the antibody or antigen binding fragment thereof comprises a VH CDR1 comprising SEQ ID NO. 9 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 10 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 11 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a VL CDR1 comprising SEQ ID NO. 13 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 14 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 15 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0053] In an embodiment the antibody or antigen binding fragment thereof comprises a VH CDR1 comprising SEQ ID NO. 17 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 18 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 19 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a VL CDR1 comprising SEQ ID NO. 21 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 22 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 23 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0054] In an embodiment the antibody or antigen binding fragment thereof comprises a VH CDR1 comprising SEQ ID NO. 25 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 26 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 27 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a VL CDR1 comprising SEQ ID NO. 19 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 30 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 31 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0055] In an embodiment the antibody or antigen binding fragment thereof comprises a VH CDR1 comprising SEQ ID NO. 33 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 34 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 35 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a VL CDR1 comprising SEQ ID NO. 37 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 38 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 39 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise VH CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise VH CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2, or 3 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 1 , 2, 3, or 4 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR1 SEQ ID NO. 5 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR1 SEQ ID NO. 5 or a sequence comprising 1 , 2 or 3 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR1 comprising SEQ ID NO. 5 or a sequence comprising 1 or 2 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR1 comprising SEQ ID NO. 5 or a sequence comprising 1 amino acid substitution. The antibody or antigen binding fragment thereof may comprise a VL CDR1 comprising SEQ ID NO. 5. The antibody or antigen binding fragment thereof may comprise a VL CDR2 SEQ ID NO. 6 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR2 SEQ ID NO. 6 or a sequence comprising 1 , 2 or 3 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 1 or 2 amino acid substitutions. The antibody or antigen binding fragment thereof may comprise a VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 1 amino acid substitution. The antibody or antigen binding fragment thereof may comprise a VL CDR2 comprising SEQ ID NO. 6. The antibody or antigen binding fragment thereof may comprise a VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0056] In an embodiment the antibody or antigen binding fragment thereof comprises a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2, or 3 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and a variable light chain (VL) CDR1 comprising SEQ ID NO. 5, VL CDR2 comprising SEQ ID NO. 6, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0057] In an embodiment the antibody or antigen binding fragment thereof comprises: ai) a VH comprising SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and aii) a VL comprising SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or bi) a VH comprising SEQ ID NO. 12 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and bii) a VL comprising SEQ ID NO. 16 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ci) a VH comprising SEQ ID NO. 20 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and cii) a VL comprising SEQ ID NO. 24 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or di) a VH comprising SEQ ID NO. 28 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and dii) a VL comprising SEQ ID NO. 32 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ei) a VH comprising SEQ ID NO. 36 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and eii) a VL comprising SEQ ID NO. 40 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

[0058] As used herein, the terms "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and in some embodiments not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percentage identity between two amino acid sequences can be determined using well known mathematical algorithms.

[0059] As described above, the antibody or fragment defined by reference to the sequence may comprise one or more amino acid substitutions. In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within one or more the CDR region and / or one or more framework region the antibody or fragment of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (using the functional assays described herein or known in the art.

[0060] Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity.

[0061] Abbreviations for the amino acid residues of the polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 1 below.

[0062] Table 1 . Amino Acid Residues and Examples of Conservative Amino Acid Substitutions

[0063] The antibodies or antigen binding fragments of the present invention are isolated antibodies or antigen binding fragments. The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to an antibody, e.g. lgG1 or binding molecule that is substantially free of other antibodies or binding molecule, antibodies or antibody fragments. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0064] The binding molecules of the invention are capable of targeting PAR-1. In an embodiment the binding molecules specifically bind to PAR-1. PAR-1 is a GPCR, it comprises an integral membrane protein coupled to a G-protein. PAR-1 is activated by proteolytic cleavage of the amino terminal extracellular sequence. Activation of PAR-1 is mediated by serine proteases in particular thrombin. The serine protease, for example thrombin, cleaves the N-terminus of the PAR-1 receptor, which in turn acts as a tethered ligand. In the cleaved state, part of the PAR-1 receptor itself acts as the agonist, causing a physiological response. Protease-activated receptors play important roles in vascular physiology, neural tube closure, hemostasis, and inflammation. In particular PAR-1 and PAR-2 are thought to promote cancer invasion and metastasis at least in part by facilitating tumour cell migration, angiogenesis, and interactions with host vascular cells, including platelets, fibroblasts, and endothelial cells lining blood vessels.

[0065] PAR1 is comprised of 425 amino acids, with a 99-amino acid N-terminal exodomain and a 51 amino acid C-terminal tail. The N-terminus of PAR-1 comprises two thrombin recognition sites, the cleavage site at arginine 41 (,..LDPR41 / SFLLRN...), and the hirudin-like sequence at lysine 51 (K51YEPF). Protease activated receptors are highly expressed on platelets, endothelial cells, fibroblasts, immune cells, myocytes, neurons, and tissues that line the gastrointestinal tract.

[0066] In an embodiment PAR-1 comprises the following sequence: SEQ ID NO 41.

[0067] MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPRSFLLRNPNDKYEPFWEDEEKN ESGLTEYRLVSINKSSPLQKQLPAFISEDASGYLTSSWLTLFVPSVYTGVFWSLPLNIMAIVVFI LKMKVKKPAVVYMLHLATADVLFVSVLPFKISYYFSGSDWQFGSELCRFVTAAFYCNMYASIL LMTVISIDRFLAVVYPMQSLSWRTLGRASFTCLAIWALAIAGVVPLLLKEQTIQVPGLNITTCHD VLNETLLEGYYAYYFSAFSAVFFFVPLIISTVCYVSIIRCLSSSAVANRSKKSRALFLSAAVFCIFII CFGPTNVLLIAHYSFLSHTSTTEAAYFAYLLCVCVSSISCCIDPLIYYYASSECQRYVYSILCCKE SSDPSSYNSSGQLMASKMDTCSSNLNNSIYKKLLT

[0068] Uniprot accession number P25116.

[0069] In an embodiment the term PAR-1 refers to any functional fragment of PAR-1. In an embodiment the term PAR-1 refers to variants and isoforms of PAR-1 , in particular variants and isoforms of human PAR-1 . In an embodiment the term PAR-1 may refer to the N-terminal peptide of PAR-1 i.e., SEQ ID NO. 42. In an embodiment PAR-1 comprises SEQ ID NO. 42.

[0070] SEQ ID NO.42

[0071] ARTRARRPESKATNATLDPRSFLLRNPNDKYEPFWEDEEKNESGLTEYRLVSINKSSPL

[0072] The term “anti-PAR-1 antibody”, “anti-PAR-1 antigen binding fragment”, “anti-PAR-1 antigen binding molecule”, “PAR-1 antibody”, “PAR-1 antigen binding fragment”, “PAR-1 binding molecule / fragment / agent / protein / moiety” all refer to molecules capable of binding PAR-1. The antibodies or antigen binding fragment of the present invention specifically bind PAR-1. In an embodiment the antibodies or antigen binding fragment thereof of the present invention specifically bind human PAR-1.

[0073] “Specifically binds", "specific binding" or "selective binding" means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody or antigen binding fragment thereof to bind to a specific antigenic determinant can be measured either through any suitable techniques for example enzyme- linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays. The binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.

[0074] The antibody or antigen-binding fragment thereof described herein, "which binds" or is “capable of binding” the antigen of interest, e.g. PAR-1 , binds the antigen with sufficient affinity such that the antibody or antigen-binding fragment thereof is useful as a therapeutic or diagnostic agent in targeting PAR-1 as described herein.

[0075] The antibody or antigen binding fragment of the invention may bind to a specific epitope of PAR-1 . The antibody or antigen binding fragment may bind to the extracellular portion of PAR- 1. The antibody or antigen binding fragment may bind to a portion of the PAR-1 protein which comprises the cleavage site, the cleavage site of PAR-1 is found within the N-terminal peptide i.e., SEQ ID NO. 42. In an embodiment the antibody or antigen binding fragment of the invention may bind to an epitope within the N-terminal peptide (SEQ ID NO. 42). In an embodiment the antibody or antigen binding fragment of the invention may bind to an epitope comprising one or more amino acids of SEQ ID NO. 42.

[0076] The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[0077] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antigen-binding fragment thereof specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes. The antibody or antigen binding fragment of the present invention binds to at least one epitope of PAR-1 .

[0078] Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antigen-binding fragment thereof (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides are tested for reactivity with a given antibody or antigen-binding fragment thereof. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. Suitable competition assays are mentioned elsewhere herein and also shown in the examples. In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g, site-directed mutagenesis mapping). The terms “polypeptide(s)” and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.

[0079] The terms “binding molecule” "antibody" “antigen binding fragment” or “antigen binding molecule” as used herein refers to an immunoglobulin protein that is capable of binding an antigen target of interest, i.e., PAR-1. In particular, the term "antibody" as used herein broadly refers to any polypeptide comprising complementarity determining regions (CDRs) that confer specific binding affinity of the polypeptide for an antigen. The term antibody may refer to any molecule comprising and immunoglobulin heavy chain or a portion thereof, comprising complementarity determining regions. The term antibody as used herein encompasses polyclonal and monoclonal antibody preparations. The term “antibody” as used herein encompasses binding molecules with different antibody formats.

[0080] Antibodies can be modified in various ways, therefore the term "antibody", “antigen binding fragment”, and “antigen binding molecule” should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain such as the binding molecules described herein. The terms “antibody", “antigen binding fragment”, and “antigen binding molecule” also extend to different antibody formats, such as formats containing an scFV, VH domain, VHH domain, nanobody, Fab, Fab', F(ab')2, F(ab')3, Fabc, Fd, (scFv)2, Fv, scFv-Fc, heavy chain only antibody, diabody, tetrabody, triabody, minibody, or antibody mimetic protein as in specific aspects of the invention. The term antibody also refers to full-length antibodies for example including polyclonal and monoclonal antibodies.

[0081] A "full-length antibody” refers to a molecule comprising two heavy chains and two light chains. In a full length antibody, the two heavy chains may be linked via one or more disulphide bond and each heavy chain may be linked to one light chain via one or more disulphide bond. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR, or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR, or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0082] Antibodies may include the kappa (K) and lambda (A) light chains and the alpha (IgA), gamma (lgG1 , lgG2, lgG3, lgG4), delta (IgD), epsilon (IgE) and mu (IgM) heavy chains, or their equivalents in other species. Full-length immunoglobulin “light chains” (usually of about 25 kDa or usually about 214 amino acids long) consist of a variable region of approximately 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full- length immunoglobulin “heavy chains” (usually of about 50 kDa or 446 amino acids long), likewise consist of a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g. gamma (of about 330 amino acids).

[0083] Light or heavy chain variable regions are generally composed of a “framework” region (FR) interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and CDRs have been precisely defined. The sequences of the framework regions of different light and heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0084] Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3. Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.

[0085] "Framework" or "FR" refers to variable domain residues other than hypervariable region (UVR) residues. The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0086] In one embodiment, the antibody is comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.

[0087] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. In one embodiment, the antibody is of the lgG1 type.

[0088] Different definitions of the CDRs are commonly in use. The method described by Kabat is the most commonly used and CDRs are based on sequence variability (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1 -113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme (Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005)). According to the IMGT numbering scheme, a CDR is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG). Another numbering system is the Honnegger numbering scheme (AHo). AHo defines conserved residues (C23, W43, C106, G140) and gaps on specific positions (#27-28, #36, #63, #123). The CDR1 has a “two span bridge” conformation created by a conserved hydrophobic residue at position #31 which is deeply inserted into the structure and therefore divides the loop into two distinct parts. The AHo scheme describes two gap regions located onto these two parts, one located in the first part and the other one located in the second part. The AHo system can be advantageous as the numbering system is based on structural alignments and therefore better allows for antibody 3-dimensional structural features. The AHo system is used herein unless otherwise stated. Where embodiments refer to a CDR comprising a specific sequence this refers to a CDR as defined by any numbering system.

[0089] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which generally retain the specificity of the original antibody. Such techniques may involve introducing the CDRs into a different immunoglobulin framework, or grafting variable regions onto a different immunoglobulin constant regions. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0090] The antibody or antigen-binding fragment thereof may be chimeric, human or humanised. A “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody. Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody therapies, as such molecules would elicit little or no immune response in the human subject. In an embodiment the antibody or antigen binding fragment is human or humanised.

[0091] As discussed above the term “antibody” or “antigen binding fragment” as used herein specifically includes antibody fragments / antigen-binding fragments / portions thereof unless stated otherwise. The antibody fragment / antigen-binding fragments may be selected from any fragment capable of binding the antigen or antigenic fragment of interest. Exemplary antibody fragments include, but are not limited to, Fab, Fab1, F(ab')2, F(ab')3, Fabc, Fv, single chain Fv (scFv), (scFv)2, Fv, scFv-Fc, bis-scFv heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, heavy chain only antibody, single domain antibody, diabody, tetrabody, triabody, maxibody, minibody, intrabody, antibody mimetic protein or a polypeptide that contains at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Thus, the antibody fragment / antigen-binding fragment may comprise or consist of any of these fragments.

[0092] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and / or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[0093] A "Fab molecule" refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin. In certain embodiments the Fab light chain and Fab heavy chain in the Fab construct are linked by a polypeptide sequence to yield a single chain Fab (scFab).

[0094] The "Fab fragment" of an antibody (also referred to as fragment antigen binding) contains the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab’)2 differ from Fab fragments in that they are bivalent binding molecules comprising two Fab molecules linked via disulphide between the heavy hinge region.

[0095] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties, e.g., antigen binding polypeptide construct, is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single- chain Fab molecule. Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv). In certain other embodiments, one of the antigen binding moieties is a single-chain Fv molecule (scFv).

[0096] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. “Heavy chain only antibody” refers to a molecule comprising one or more immunoglobulin heavy chain or a portion thereof and lacking a light chain. The antigen binding region of heavy chain only antibodies comprises CDR 1 , 2, and 3 of the heavy chain. Heavy chain only antibodies are found in sharks and camelids such as dromedaries, camels, llamas and alpacas, as such the heavy chain only antibodies may be derived from a shark or camelid antibody.

[0097] “Single domain antibody” refers to a binding molecule comprising a single heavy chain variable domain. A “single domain antibody”, as used herein, therefore does not comprise any other parts of a full antibody, but the antigen binding VH domain only; e.g. it only includes the heavy chain variable domain and does not comprise constant heavy chain domains and does not comprise a light chain. A single variable heavy chain domain antibody is capable of specific binding to an antigen in the absence of light chain or other antibody fragments. The single heavy chain variable domain may be derived from a full-length antibody such as an IgG antibody (a VH domain) or a heavy chain only antibody (a VHH domain).

[0098] The term "Fc" or "Fc domain" or "Fc region" or "Fc construct" herein is used to define a C- terminal region of an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions.

[0099] "Fc region", as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences.

[0100] The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "Fc polypeptide" herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence. The antibody may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody is in one embodiment the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g the CH2 domain of human lgG1. The sequences of human IgG domains are known in the art.

[0101] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. In one embodiment, the hinge region, or part thereof, is a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, is an lgG1 hinge region, or part thereof.

[0102] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise a CH3 domain. The sequence of the CH3 domain is not particularly limited. In one embodiment, the CH3 domain is a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[0103] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human lgG1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. A binding molecule, antibody or antigen binding fragment of the invention may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0104] The Fc includes two Fc polypeptides each having a CH3 domain for dimerization. The N- terminal end of each Fc polypeptide is linked to the C-terminus of one of the antigen binding polypeptide constructs with or without a linker.

[0105] The antibody or antigen binding fragment thereof of the invention may comprise a human IgG Fc with or without effector function. The antibody or antigen binding fragment thereof of the invention may comprise a human IgG Fc which has been engineered for enhanced characteristics which are described herein.

[0106] Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRlla, FcyRllb and FcyRllc) and CD16 (FcyRllla and FcyRlllb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRI 11 are low to intermediate affinity (micromolar range KD).

[0107] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0108] "Complement dependent cytotoxicity" and "CDC" refer to the lysing of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.

[0109] "Antibody-dependent cellular phagocytosis and "ADCP" refer to the destruction of target cells via monocyte or macrophage-mediated phagocytosis. In antibody ADCC, FcyRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function various for IgG subtypes. Although this is dependent on the allotype and specific FcyR in simple terms ADCC effector function is high for human IgG 1 and lgG3, and low for lgG2 and lgG4.

[0110] FcyRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions

[0111] The, antibody or antigen binding fragment thereof of the invention of the invention may have an Fc region with effector function, with enhanced effector function or with reduced effector function. In particular, antibody or antigen binding fragment thereof may include a modified Fc region.

[0112] The potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such ADCC and ADCP. A number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D / A330L / I332E (“3M”), F243L or G236A. Alternatively, enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, FcyRs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity. Afucosylated (non- fucosylated) antibodies, exhibit greatly enhanced ADCC activity through increased binding to FcyRllla.

[0113] Activation of ADCC and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred. Due to their lack of effector functions, lgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, lgG4 molecules can exchange half molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous lgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of lgG4 antibodies with a reduced propensity for Fab-arm exchange. Fc engineering approaches have been used to determine the key interaction sites for the lgG1 Fc domain with Fey receptors and C1 q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors, such as FcyRI, FcyRlla, FcyRllb, FcyRIII and / or to complement. CH2 domains of human IgG domains normally bind to Fey receptors and complement, decreased binding to Fey receptors is expected to decrease ADCC and decreased binding to complement is expected to decrease CDC activity of the antibody molecule.

[0114] Mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors and / or complement are known in the art. An antibody molecule of the invention may comprise an Fc with modifications K322A / L234A / L235A or L234F / L235E / P331S (“TM”), which almost completely abolish FcyR and C1q binding. An antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be decreased by mutation of Pro329 (position according to EU numbering), e.g., to either P329A or P329G. The antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA- PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering). Additionally or alternatively, an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297.

[0115] Modification of glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction may confer a loss of binding to FcRs; a loss of binding to FcRs has been observed in N297 point mutations. An antibody molecule of the invention may comprise an Fc with an N297A, N297G or N297Q mutation. An antibody molecule of the invention with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor or following the expression of Fc domains in bacteria.

[0116] IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant, conferred an approximately 2- fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant (YTE”), gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy.

[0117] In one embodiment, the Fc is an lgG1 Fc construct, and lgG2 Fc construct, an lgG3 Fc construct, or an lgG4 Fc construct.

[0118] In one embodiment the antibody or antigen binding fragment comprises a binding affinity in the region of 0.1 nM to 100 nM, 0.1 nM to 90 nM, 0.1 nM to 80 nM, 0.1 nM to 70 nM, 0.1 nM to 60 nM, 0.1 nM to 50 nM, 0.1 nM to 40 nM, 0.1 nM to 35 nM, 1 nM to 100 nM, 1 nM to 90 nM, 1 nM to 80 nM, 1 nM to 70 nM, 1 nM to 60 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 35 nM,2 nM to 100 nM, 2 nM to 90 nM, 2 nM to 80 nM, 2 nM to 70 nM, 2 nM to 60 nM, 2 nM to 50 nM, 2 nM to 40 nM, 2 nM to 35 nM,3 nM to 100 nM, 3 nM to 90 nM, 3 nM to 80 nM, 3 nM to 70 nM, 3 nM to 60 nM, 3 nM to 50 nM, 3 nM to 40 nM, 3 nM to 35 nM, 4 nM to 100 nM, 4 nM to 90 nM, 4 nM to 80 nM, 4 nM to 70 nM, 4 nM to 60 nM, 4 nM to 50 nM, 4 nM to 40 nM, or 4 nM to 35 nM. In one embodiment the binding affinity is in the range of 4.8 to 36nM.

[0119] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g, antibody or antigen -binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).

[0120] Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff / kon, whereas KA is calculated from the quotient of kon / koff. Kon refers to the association rate constant of, e.g, an antibody or antigenbinding fragment thereof to an antigen, and koff refers to the dissociation of, e.g, an antibody or antigen-binding fragment thereof from an antigen. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA. the antibody of antigen binding fragment has an IC50 in the range 0.1 nM to 100 nM, 0.1 nM to 90 nM, 0.1 nM to 80 nM, 0.1 nM to 70 nM, 0.1 nM to 60 nM, 0.1 nM to 50 nM, 0.1 nM to 40 nM, 0.1 nM to 35 nM, 1 nM to 100 nM, 1 nM to 90 nM, 1 nM to 80 nM, 1 nM to 70 nM, 1 nM to 60 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 35 nM,2 nM to 100 nM, 2 nM to 90 nM, 2 nM to

[0121] 80 nM, 2 nM to 70 nM, 2 nM to 60 nM, 2 nM to 50 nM, 2 nM to 40 nM, 2 nM to 35 nM,3 nM to

[0122] 100 nM, 3 nM to 90 nM, 3 nM to 80 nM, 3 nM to 70 nM, 3 nM to 60 nM, 3 nM to 50 nM, 3 nM to

[0123] 40 nM, 3 nM to 35 nM, 4 nM to 100 nM, 4 nM to 90 nM, 4 nM to 80 nM, 4 nM to 70 nM, 4 nM to

[0124] 60 nM, 4 nM to 50 nM, 4 nM to 40 nM, or 4 nM to 35 nM, In one embodiment the binding affinity is in the range of 4.8 to 36nM.

[0125] The term IC50 refers to the half-maximal inhibitory concentration i.e. the concentration of the antibody or antigen binding fragment needed to inhibit a biological process or response by 50%. The IC50 may be determined by any suitable assay for example a whole cell viability assay. The IC50 may be determined by inhibition of inositol-1 -phosphate as set out in Example 6.

[0126] In an embodiment the antibody of antigen binding fragment is capable of inhibiting or reducing cell proliferation. The antibody or antigen binding fragment of the invention may be capable of targeting PAR-1 which plays a role in cellular proliferation, by inhibiting PAR-1 the antibody of antigen binding fragment is capable of reducing cell proliferation.

[0127] In an embodiment the antibody of antigen binding fragment is capable of inhibiting or reducing P-arrestin recruitment, p-arrestin recruitment may be inhibited or reduced by approximately 1 to 100%, 5 to 100%, 10 to 100%, 15 to 100%, 20 to 100%, 25 to 100%, 30 to 100%, 35 to 100%, 40 to 100%, 45 to 100%, 50 to 100%, 55 to 100%, 60 to 100%, 65 to 100%, 70 to 100%, 75 to 100%, 80 to 100%, 85 to 100%, 5 to 95%, 5 to 95%, 10 to 95%, 15 to 95%, 20 to 95%, 25 to 95%, 30 to 95%, 35 to 95%, 40 to 95%, 45 to 95%, 50 to 95%, 55 to 95%, 60 to 95%, 65 to 95%, 70 to 95%, 75 to 95%, 80 to 95%, 85 to 95%. Preferably p-arrestin recruitment may be inhibited or reduced by more than 60%, more than 70%, more than 80% or more than 90%. p-arrestin recruitment may be determined by any suitable method for example the method set out in Example 7

[0128] In an embodiment the antibody of antigen binding fragment is capable of inhibiting or reducing platelet activation. In an embodiment the antibody of antigen binding fragment is capable of inhibiting or reducing platelet aggregation. In an embodiment the antibody of antigen binding fragment is capable of inhibiting or reducing thrombin-related platelet aggregation. Platelet activation may be inhibited or reduced by approximately 1 to 100%, 5 to 100%, 10 to 100%, 15 to 100%, 20 to 100%, 25 to 100%, 30 to 100%, 35 to 100%, 1 to 90%, 5 to 90%, 10 to 90%, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%, 1 to 80%, 5 to 80%, 10 to 80%, 15 to 80%, 20 to 80%, 25 to 80%, 30 to 80%, 35 to 80%, 1 to 75%, 5 to 75%, 10 to 75%, 15 to 75%, 20 to 75%, 25 to 75%, 30 to 75%, 35 to 75%, 1 to 70%, 5 to 70%, 10 to 70%, 15 to 100%, 20 to 70%, 25 to 70%, 30 to 70%, 35 to 70%, 1 to 65%, 5 to 65%, 10 to 65%, 15 to 65%, 20 to

[0129] 65%, 25 to 65%, 30 to 65%, 35 to 65%, 40 to 100%, 45 to 100%, 50 to 100%, 55 to 100%, 60 to 100%, 40 to 99%, 45 to 99%, 50 to 99%, 55 to 99%, 60 to 99%, 40 to 98%, 45 to 98%, 50 to 98%, 55 to 98%, 60 to 98%, 40 to 95%, 45 to 95%, 50 to 95%, 55 to 95%, 60 to 95%, 40 to

[0130] 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 40 to 85%, 45 to 85%, 50 to 85%, 55 to

[0131] 85%, 60 to 85%, 40 to 80%, 45 to 80%, 50 to 80%, 55 to 80%, or 60 to 80%. Platelet aggregation may be inhibited or reduced by approximately 1 to 100%, 5 to 100%, 10 to 100%, 15 to 100%, 20 to 100%, 25 to 100%, 30 to 100%, 35 to 100%, 1 to 90%, 5 to 90%, 10 to 90%, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%, 1 to 80%, 5 to 80%, 10 to 80%, 15 to 80%, 20 to 80%, 25 to 80%, 30 to 80%, 35 to 80%, 1 to 75%, 5 to 75%, 10 to 75%, 15 to 75%, 20 to 75%, 25 to 75%, 30 to 75%, 35 to 75%, 1 to 70%, 5 to 70%, 10 to 70%, 15 to 100%, 20 to 70%, 25 to 70%, 30 to 70%, 35 to 70%, 1 to 65%, 5 to 65%, 10 to 65%, 15 to 65%, 20 to

[0132] 65%, 25 to 65%, 30 to 65%, 35 to 65%, 40 to 100%, 45 to 100%, 50 to 100%, 55 to 100%, 60 to 100%, 40 to 99%, 45 to 99%, 50 to 99%, 55 to 99%, 60 to 99%, 40 to 98%, 45 to 98%, 50 to 98%, 55 to 98%, 60 to 98%, 40 to 95%, 45 to 95%, 50 to 95%, 55 to 95%, 60 to 95%, 40 to

[0133] 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 40 to 85%, 45 to 85%, 50 to 85%, 55 to

[0134] 85%, 60 to 85%, 40 to 80%, 45 to 80%, 50 to 80%, 55 to 80%, or 60 to 80%. Thrombin-related platelet aggregation may be inhibited or reduced by approximately 1 to 100%, 5 to 100%, 10 to 100%, 15 to 100%, 20 to 100%, 25 to 100%, 30 to 100%, 35 to 100%, 1 to 90%, 5 to 90%, 10 to 90%, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%, 1 to 80%, 5 to 80%, 10 to

[0135] 80%, 15 to 80%, 20 to 80%, 25 to 80%, 30 to 80%, 35 to 80%, 1 to 75%, 5 to 75%, 10 to 75%,

[0136] 15 to 75%, 20 to 75%, 25 to 75%, 30 to 75%, 35 to 75%, 1 to 70%, 5 to 70%, 10 to 70%, 15 to

[0137] 70%, 20 to 70%, 25 to 70%, 30 to 70%, 35 to 70%, 1 to 65%, 5 to 65%, 10 to 65%, 15 to 65%,

[0138] 20 to 65%, 25 to 65%, 30 to 65%, 35 to 65%, 40 to 100%, 45 to 100%, 50 to 100%, 55 to 100%, 60 to 100%, 40 to 99%, 45 to 99%, 50 to 99%, 55 to 99%, 60 to 99%, 40 to 98%, 45 to

[0139] 98%, 50 to 98%, 55 to 98%, 60 to 98%, 40 to 95%, 45 to 95%, 50 to 95%, 55 to 95%, 60 to

[0140] 95%, 40 to 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 40 to 85%, 45 to 85%, 50 to

[0141] 85%, 55 to 85%, 60 to 85%, 40 to 80%, 45 to 80%, 50 to 80%, 55 to 80%, or 60 to 80%. In some embodiments the antibody or antigen binding fragment of the invention are advantageously capable of achieving a significant level, for example 60 to 98%, of platelet aggregation inhibition even when used at a lower concentration, as demonstrated in Example 9. The antibody or antigen binding fragments of the invention have been shown to effectively reduce platelet aggregation at various concentrations including lower concentrations as shown in Example 9. In particular some of the antibodies or antigen binding fragments thereof may achieve a higher level of platelet aggregation inhibition when used at a lower concentration. In some embodiments where the antibody or antigen binding fragment thereof is used at a lower concentration platelet aggregation may be inhibited 40 to 100%, 45 to 100%, 50 to 100%, 55 to 100%, 60 to 100%, 40 to 99%, 45 to 99%, 50 to 99%, 55 to 99%, 60 to 99%, 40 to 98%, 45 to 98%, 50 to 98%, 55 to 98%, 60 to 98%, 40 to 95%, 45 to 95%, 50 to 95%, 55 to 95%, 60 to 95%, 40 to 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 40 to 85%, 45 to 85%, 50 to 85%, 55 to 85%, 60 to 85%, 40 to 80%, 45 to 80%, 50 to 80%, 55 to 80%, or 60 to 80%, in a preferred embodiment platelet aggregation may be inhibited 60 to 100%.

[0142] The antibody or antigen binding fragment of the invention may comprise modifications for example derivatives of the antibody or antigen binding fragment are encompassed. A "derivative" of an antibody contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

[0143] In some embodiments the antibody or fragment thereof is linked to a further moiety. The further moiety may provide a certain function to the molecule for example the further moiety is selected from a further binding molecule, a therapeutic moiety, a half-life extending moiety, and / or a label. The further moiety that may serve to prolong the half-life of the molecule. The further moiety may comprise a protein, for example an antibody, or part thereof that binds a serum albumin, e.g., dog or cat serum albumin. Other modifications that prolong half-life are also known and include, for example, modification by PEG or by incorporation in a liposome. The therapeutic moiety may be any molecule with therapeutic utility, in particular the therapeutic molecule may be a drug, for example a chemotherapeutic drug, a tubulin inhibitor, a DNA damage compound, a PROTAC, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, a carmaphycin, an RNA inhibitor, an immunomodulatory drug. The label may be any suitable label for example a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.

[0144] The present invention also relates to a binding molecule comprising the antibody or antigen binding fragment as described herein. The binding molecule may be a molecule comprising a binding domain in combination with additional domains, for example a chimeric antigen receptor. A chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signalling domain, the antigen binding domain may comprise the antibody or antigen binding fragment as described herein.

[0145] The binding molecule may be a multispecific binding molecule. The multispecific binding molecule may comprise one or more binding domains wherein one of the binding domains comprises the antibody or antigen binding fragment as described herein. For example the antibody or antigen binding fragment of the invention may be a building block in a multispecific binding molecule, for example a bispecific or trispecific molecule. In the multispecific molecule the antibody or antigen binding fragment may be linked to one or more further binding domains wherein the further binding domain(s) binds to a different target antigen. The further binding domain may be a therapeutic moiety for example a antibody or antigen binding domain or antibody mimetic. The binding domains may be linked in any suitable manner known in the art. For example a linker may be used to link binding domains. Suitable linkers include linkers that include G and / or S residues, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n" is generally a number between 1 and 10.

[0146] An aspect of the invention also relates to an immunoconjugate comprising the antibody or antigen binding fragment of the invention, conjugated to a therapeutic moiety, a half-life extending moiety, and / or a label. The therapeutic moiety, a half-life extending moiety, and / or a label may be any molecules as described herein.

[0147] Pharmaceutical Composition

[0148] An aspect of the invention relates to a pharmaceutical composition comprising the antibody or antigen binding fragment of the invention, or the immunoconjugate of the invention and a pharmaceutically acceptable excipient, diluent and or carrier.

[0149] The pharmaceutical composition of the present invention can be administered by any convenient route, including parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Compositions can take the form of one or more dosage units.

[0150] The composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

[0151] The amount of the pharmaceutical composition of the present invention that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0152] The compositions of the invention comprise an effective amount of a binding molecule of the present invention such that a suitable dosage will be obtained. The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

[0153] Typically, this amount is at least about 0.01% of a binding molecule of the present invention by weight of the composition.

[0154] Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present invention.

[0155] For intravenous administration, the composition can comprise from typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the subject’s body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the subject’s body weight. The present compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0156] The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a binding molecule of the present invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

[0157] The antibody, antigen binding fragment or pharmaceutical composition of the invention can be co-administered with other therapeutics, for example anti-cancer agents.

[0158] Exemplary combinations with other agents

[0159] The antibody, antigen binding fragment or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, or antibody mimetic protein.

[0160] In one embodiment, the antibody, antigen binding fragment or pharmaceutical composition described herein is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of the antibody, antigen binding fragment or pharmaceutical composition described herein and an anti-cancer therapy.

[0161] The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens, including EGFR antagonists), an antiinflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the antibody, antigen binding fragment or pharmaceutical composition is used in combination with surgery. In a specific embodiment of the present invention, the antibody, antigen binding fragment or pharmaceutical composition described herein is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

[0162] In some embodiments, he antibody, antigen binding fragment or pharmaceutical composition described herein may be administered with two or more therapeutic agents. In some embodiments, the binding agents of the invention may be administered with two or more therapeutic agents.

[0163] The antibody, antigen binding fragment or pharmaceutical composition as described herein may be administered at the same time or at a different time as the other therapy or therapeutic compound or therapy, e.g., simultaneously, separately or sequentially.

[0164] Therapy

[0165] The antibody or antigen binding fragment of the invention is capable of targeting PAR-1 and modulate PAR-1 signalling as such they have therapeutic utility.

[0166] An aspect of the invention relates to the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition thereof for use in therapy.

[0167] An aspect of the invention relates to a method of treatment or therapy comprising administering a therapeutically effective amount of the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition according to the invention to a subject in need thereof.

[0168] An aspect of the invention relates to the use of the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition of the invention in the manufacture of a medicament for use in therapy.

[0169] The antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition may be useful in the treatment or prevention of a thrombin-mediated or PAR-1 mediated disease.

[0170] The antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition may be useful in the treatment or prevention of one or more of the following diseases or conditions; cancer, inflammatory conditions, sepsis, multiple sclerosis, acute lung injury (ALI), cardiovascular disease, autoimmune disease.

[0171] In one embodiment the disease is an inflammatory condition which refers to pathological states resulting in inflammation or an autoimmune disease. Suitable examples of inflammatory conditions that may be treated by the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition described herein include but are not limited to: inflammation of the gastrointestinal tract, such as inflammatory bowel disease, ulcerative colitis, Crohn’s disease, irritable bowel syndrome; inflammatory respiratory diseases, such as chronic obstructive pulmonary disease (COPD), pneumonitis, emphysema, bronchitis, asthma; inflammatory skin diseases, such as atopic dermatitis (atopy), allergic dermatitis, pruritus, psoriasis, scleroderma, or eczema. Suitable examples of autoimmune conditions that may be treated by the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition described herein includes but is not limited to: rheumatoid arthritis, Sjorgen's syndrome, vasculitis, lupus, sarcoidosis, scleroderma.

[0172] In one embodiment the disease is a cancer, which refers to pathological states resulting for uncontrolled growth of abnormal cells. Examples of cancer that may be treated by the antibody, antigen binding fragment, immunoconjugate, or pharmaceutical composition described herein include but are not limited to: bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, endometrial cancer, cancer of the esophagus, cancer of the small intestine, liver cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, thymoma, urothelial carcinoma, prostate cancer, leukaemia, acute myeloid leukaemia, glioblastoma, glioma, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

[0173] In one embodiment, post-treatment, the subject has at least 5 days, at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days disease progression-free.

[0174] In one embodiment, the number of days of survival, the number of disease-free days, or the number of disease-progression free days is at least 1 month, at least 2 months, or at least 3 months, or at least 4 months, e.g. at least 5 months, such as at least 6 months.

[0175] In one embodiment, the number of days of survival, the number of disease-free days, or the number of disease-progression free days is at least 9 months, or at least one year. The invention provides methods of treating or preventing thrombin-mediated or PAR-1 mediated diseases or disorders in a mammal, e.g., a human, comprising administering an effective amount of an antibody or fragment of the present invention to the human in need thereof.

[0176] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0177] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. By way of example only, a subject includes, human.

[0178] As will be appreciated by the skilled person, the therapeutically effective amount of the antibody or antigen binding fragment thereof or pharmaceutical composition as described herein, will necessarily vary depending on the subject to be treated, the route of administration and the nature and severity of the disease to be treated. The antibody or antigen binding fragments of the invention have been shown to effectively reduce platelet aggregation at various concentrations including lower concentrations as shown in Example 9. In particular some of the antibodies or antigen binding fragments thereof may achieve a higher level of platelet aggregation inhibition when used at a lower concentration. In some embodiments the therapeutically effective dose may be a lower dose.

[0179] The term “therapeutically effective amount” refers to that amount of the antibody or antigen binding fragment thereof or pharmaceutical composition being administered which will relieve to some extent one or more of the symptoms of the disorder being treated, or result in inhibition of the progress or at least partial reversal of the condition. The effective amount of antibody or antigen-binding fragment thereof of the invention, is an amount that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration. The term “effective amount” of a composition, as used herein, is intended to denote a non-lethal but sufficient amount of the composition to provide the desired effect. For example, in order to elicit a favourable response in a subject when treating a disorder or infection, the effective amount is the one which eliminates or diminishes the symptoms associated with the disorder. An effective amount may be determined by one of ordinary skill in the art, using routine experimentation.

[0180] In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0181] Methods

[0182] The invention relates to antibodies and antigen binding fragments which target PAR-1 as such the invention also relates to methods of using said antibodies or antigen binding fragments.

[0183] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of detecting PAR-1 in a biological sample comprising contacting said biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0184] The invention relates to a method for reducing platelet aggregation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0185] The invention relates to a method for reducing platelet activation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0186] The invention relates to a method for reducing cell proliferation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0187] The invention relates to a method for blocking thrombin induced Beta-arrestin 1 recruitment comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to the invention.

[0188] The biological sample may be a biological tissue sample, a biological fluid. A biological fluid may be, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.

[0189] The methods of the invention may be performed in vitro, ex vivo or in vivo. The methods may be performed on a biological sample that has been obtained from a subject or patient. The methods of the invention may comprise a step of obtaining a biological sample. The methods of the invention may comprise a step of providing a biological sample. The methods may be performed on a subject or patient.

[0190] Reduction of platelet activation, cell proliferation or blocking of thrombin induced Beta-arrestin 1 recruitment, can be assessed by using suitable in vitro or in vivo assays.

[0191] Nucleic Acids

[0192] The invention also relates to a nucleic acid construct comprising at least one nucleic acid molecule wherein the nucleic acid molecule encodes the antibody, antigen binding fragment, binding molecule or immunoconjugate of the invention. A nucleic acid construct is an artificially constructed portion of nucleic acid that can be used to introduce exogenous nucleic acid into a host. The nucleic acid construct may be any suitable construct, for example a plasmid, vector, artificial chromosome, bacteriophage vector, cosmid, fosmid, transcription or expression cassette.

[0193] An expression vector can be, for example, a plasmid, such as pBR322, pUC, or Co1 E1 , or an adenovirus vector, such as an adenovirus Type 2 vector or Type 5 vector. Vectors suitable for use in the present invention include, for example, bacterial vectors, mammalian vectors, viral vectors (such as retroviral, adenoviral, adeno-associated viral, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors) and baculovirus-derived vectors for use in insect cells. Polynucleotides in such vectors are preferably operably linked to a promoter, which is selected based on, e.g., the cell type in which expression is sought.

[0194] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. The invention includes host cells containing polynucleotides encoding an antibody of the invention (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, or a fragment or variant thereof), operably linked to a heterologous promoter. For the expression of entire antibody molecules, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule. In one embodiment, the expression vector is one which enables heterologous expression e.g., expression in a host organism from different species.

[0195] The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid construct as described herein, i.e., nucleic acid molecules encoding antibodies of the invention. Host cells useful in the present invention are prokaryotic, yeast, or higher eukaryotic cells and include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0196] Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). Examples of suitable mammalian host cells include but are not limited to: Chinese hamster ovary (CHO) cells, such as ExpiCHO cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS); human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Examples of suitable insect host cells include but are not limited to: insect cell lines e.g., Spodoptera frugiperda or Trichoplusia ni. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.

[0197] Prokaryotes are useful as host cells in the present invention include gram negative or grampositive organisms such as E. coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas, and Streptomyces. These examples are illustrative rather than limiting, other examples of host cells include plant cells and amphibian cells. In one embodiment, a method of making an anti- anti-PAR1 antibody as described herein is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the antibody and isolating the antibody.

[0198] The nucleic acid or nucleic acid construct may be introduced to the host cell in any suitable manner, in particular transformation may be used. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.

[0199] Kits

[0200] An aspect of the invention relates to a kit comprising the antibody, antigen binding fragment, binding molecule, immunoconjugate or pharmaceutical composition described herein and optionally instructions for use.

[0201] The kit may contain materials useful for the treatment of the disorders described above is provided. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the antibody, antigen binding fragment, binding molecule, immunoconjugate or pharmaceutical composition of the invention which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or fragment thereof. The label or package insert indicates that the composition is used for treating the condition of choice, such as an inflammatory condition, autoimmune disease or cancer. The kit may further contain a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0202] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0203] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0204] "and / or" where used herein 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.

[0205] The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0206] The term “consisting of’ or “consists of’ means including the components specified but excluding other components. Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0207] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0208] It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

[0209] The invention is further defined in the following numbered embodiments:

[0210] 1. An antibody or antigen binding fragment thereof, that binds to Proteinase-Activated Receptor-1 (PAR-1) comprising ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 10 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 11 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 14 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 15 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 18 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 19 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and cii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 22 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 23 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 26 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 27 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 30 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 31 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or ei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 34 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 35 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and eii). a variable light chain (VL) CDR1 comprising SEQ ID NO. 37 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 38 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 39 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or

[0211] 2. The antibody or antigen binding fragment thereof according to embodiment 1 , comprising i) a VH comprising SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and ii) a VL comprising SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

[0212] 3. The antibody or antigen binding fragment thereof according to claim 1 or 2, i) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2,

[0213] 3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and ii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

[0214] 4. The antibody or antigen binding fragment thereof, according to any preceding embodiment ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 , a VH CDR2 comprising SEQ ID NO. 2, a VH CDR3 comprising SEQ ID NO. 3, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5, VL CDR2 comprising SEQ ID NO. 6, VL CDR3 comprising SEQ ID NO. 7, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9, a VH CDR2 comprising SEQ ID NO. 10, a VH CDR3 comprising SEQ ID NO. 11 , and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13, VL CDR2 comprising SEQ ID NO. 14, VL CDR3 comprising SEQ ID NO. 15, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17, a VH CDR2 comprising SEQ ID NO. 18, a VH CDR3 comprising SEQ ID NO. 19, and cii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 , VL CDR2 comprising SEQ ID NO. 22, VL CDR3 comprising SEQ ID NO. 23, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25, a VH CDR2 comprising SEQ ID NO. 26, a VH CDR3 comprising SEQ ID NO. 27, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29, VL CDR2 comprising SEQ ID NO. 30, VL CDR3 comprising SEQ ID NO. 31 , or ei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33, a VH CDR2 comprising SEQ ID NO. 34, a VH CDR3 comprising SEQ ID NO. 35, and eii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 37, VL CDR2 comprising SEQ ID NO. 38, VL CDR3 comprising SEQ ID NO. 39.

[0215] 5. The antibody or antigen binding fragment thereof according to any preceding embodiment, comprising ai) a VH comprising SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and aii) a VL comprising SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or bi) a VH comprising SEQ ID NO. 12 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and bii) a VL comprising SEQ ID NO. 16 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ci) a VH comprising SEQ ID NO. 20 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and cii) a VL comprising SEQ ID NO. 24 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or di) a VH comprising SEQ ID NO. 28 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and dii) a VL comprising SEQ ID NO. 32 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ei) a VH comprising SEQ ID NO. 36 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and eii) a VL comprising SEQ ID NO. 40 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

[0216] 6. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen fragment thereof binds to human PAR1 .

[0217] 7. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody of antigen binding fragment is capable of reducing platelet activation, preferably reducing thrombin-related platelet aggregation.

[0218] 8. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody of antigen binding fragment has an IC50 in the range 1.0 to

[0219] 50 nM. 9. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody of antigen binding fragment is capable of inhibiting arrestin recruitment by 5 to 100%.

[0220] 10. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody of antigen binding fragment is capable of inhibiting platelet aggregation, preferably reducing thrombin-related platelet aggregation, by 25 to 100%.

[0221] 11 . The antibody or antigen binding fragment according to a preceding embodiment wherein said fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, or a polypeptide that contains at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

[0222] 12. The antibody or antigen binding fragment according to a preceding embodiment wherein said antibody or fragment thereof is linked to a further moiety.

[0223] 13. The antibody or antigen binding fragment according to embodiment 12, wherein the further moiety is selected from a further binding molecule, a therapeutic moiety, a half life extending moiety, and / or a label.

[0224] 14. An immunoconjugate comprising the antibody or antigen binding fragment according to any of embodiments 1 to 13, conjugated to a therapeutic moiety, a half-life extending moiety, and / or a label.

[0225] 15. An isolated nucleic acid encoding the antibody or antigen binding fragment according to any of embodiments 1 to 13.

[0226] 16. A vector comprising the nucleic acid according to embodiment 15.

[0227] 17. A host cell comprising the nucleic acid according to embodiment 15 the vector according to embodiment 16.

[0228] 18. The host cell of embodiment 17 wherein the host cell is a bacterial, yeast, viral or mammalian cell.

[0229] 19. A pharmaceutical composition comprising the antibody or antigen binding fragment according to any of embodiments 1 to 13, or the immunoconjugate of claim 14 and a pharmaceutically acceptable excipient, diluent and or carrier.

[0230] 20. The antibody of embodiments 1 to 13, the immunoconjugate of embodiment 14 or the pharmaceutical composition according to embodiment 15, for use in therapy.

[0231] 21. The antibody of embodiments 1 to 13, the immunoconjugate of embodiment 14 or the pharmaceutical composition according to embodiment 15, for use in treatment of a thrombin- mediated disease or a PAR-1 -mediated disease. 22. A method of treating a thrombin-mediated disease or a PAR-1 -mediated disease, comprising administering a therapeutically effective amount of antibody of embodiments 1 to 13, the immunoconjugate of embodiment 14 or the pharmaceutical composition according to embodiment 15.

[0232] 23. The antibody of embodiments 1 to 13, for use in the manufacture of a medicament for the treatment of a thrombin-mediated disease or a PAR-1 -mediated disease.

[0233] 24. The antibody, immunoconjugate, pharmaceutical for use according to embodiment 21 , the method according to embodiment 22 or the use according to embodiment 23, wherein the PAR-1 -mediated disease is selected from cancer, inflammatory conditions, sepsis, multiple sclerosis, acute lung injury (ALI), cardiovascular disease, autoimmune disease

[0234] 25. The antibody, immunoconjugate, pharmaceutical for use according to embodiment 24, the method according to embodiment 24 or the use according to embodiment 24, wherein the cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, endometrial cancer, cancer of the esophagus, cancer of the small intestine, liver cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, thymoma, urothelial carcinoma, prostate cancer, leukaemia, acute myeloid leukaemia, glioblastoma, glioma, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

[0235] 26. The antibody, immunoconjugate, pharmaceutical for use according to embodiment 21 , the method according to embodiment 22 or the use according to embodiment 19, wherein said antibody or antigen binding fragment is administered in combination with a further therapy.

[0236] 27. The antibody, immunoconjugate, pharmaceutical for use, the method, the use according to embodiment 26, wherein the further therapy is selected from an anti-cancer therapy, an anti-inflammatory, or a cardiovascular disease therapy.

[0237] 28. A binding molecule comprising the antibody or antigen binding fragment according to any of embodiments 1 to 13.

[0238] 29. A multispecific binding molecule comprising the antibody or antigen binding fragment according to any of embodiments 1 to 13. 30. An in vitro, ex vivo or in vivo method of detecting PAR-1 in a biological sample comprising contacting said biological sample with the antibody or antigen binding fragment thereof according to any of embodiments 1 to 13.

[0239] 31 . A method for reducing platelet activation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of embodiments 1 to 13.

[0240] 32. A method for reducing cell proliferation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of embodiments 1 to 13.

[0241] 33. A method for blocking thrombin induced Beta-arrestin 1 recruitment comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of embodiments 1 to 13.

[0242] 34. A method for reducing platelet aggregation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of embodiments 1 to 13.

[0243] 35. A kit comprising the antibody or antigen binding fragment according to any of embodiments 1 to 13 and optionally instructions for use.

[0244] EXAMPLES

[0245] Example 1 - Antibody library design

[0246] A naive antibody library was computationally designed to match the properties of natural human antibody repertoire, within limits of synthesis technology. We selected a human germline pairing that was known to express and otherwise perform well. We analysed the subset of the human immune repertoire using that germline and matched the properties of our library to best represent its properties - including the fact that different-length CDRs have different sequence patterns. Our analysis resolved that CDRL1 and CDRL2 should be left as germline but diversity inserted into the remaining 4 CDRs; this decision was partly influenced by synthesis considerations. This library was designed to cover as broad a swathe of the natural repertoire as possible.

[0247] Example 2 - Library construction and expression

[0248] A phagemid library was constructed based on the antibody library design set out in Example 1 . A total of 1 ,5pg of DNA was electroporated into E.coli TG1 cells for storage of the phagemid library. After electroporation, transformed cells were recovered in 1 mL of SOC medium, then incubated at 37°C for 1 hour for recovery and initial expression of ampicillin resistance. From the recovered cell suspension, 900 uL was spread onto TYE agar containing ampicillin (100 pg / mL) and glucose (1% w / v), then grown overnight at 37°C; the resultant mixed culture was recovered in 2xYT containing ampicillin (100 pg / mL), glucose (1% w / v), and glycerol (15% w / v) and stored at -80°C for later use. The remaining 100 uL of the recovered cell suspension was used to determine the diversity of the library. In brief, a six-step 10-fold serial dilution was prepared, with 100 uL of each dilution used to inoculate further TYE agar plates that were incubated overnight at 37°C. Colony counts on the titration plates revealed the library diversity to be 108.

[0249] Example 3 - Panning phage:scFv library against biotinylated hPAR1 peptide

[0250] Panning of the scFV library, developed by Antiverse and displayed on the M13 phage capsid, was performed against a biotinylated hPAR1 peptide. Three rounds of panning were performed. Briefly, library phage was blocked in a solution of phosphate-buffered saline (PBS) containing milk proteins (3% w / v), before biotinylated hPAR1 peptide was added and mixtures were incubated to allow scFv:hPAR1 binding interactions to occur. Magnetic beads coated in streptavidin that were also blocked in PBS containing milk proteins (3% w / v) were later added to the blocked phage:scFv-hPAR1 mixtures. The mixtures were further incubated to allow capture of the phage:scFv-hPAR1 -biotin complexes onto the streptavidin-coated beads. Unbound phage was removed by several washes in PBS, before any phage that remained bound to the beads was eluted by trypsin digestion.

[0251] Example 4 - Screening phage:scFv library panning round 3 output hPAR1 binders

[0252] Isolated clones from TG1s that had been infected to contain round 3 output phage were hand- picked into 96-well plates, and the corresponding phage:scFv clones were produced. These supernatants were used directly to assess the binding potency of the monoclonal phage:scFv for the same biotinylated hPAR1 peptide was assayed by ELISA.

[0253] ELISA:

[0254] In brief, Maxisorp 96-well plates were coated with avidin before capture of the biotinylated hPAR1 peptide. To control for nonspecific binding potency of the monoclonal phage:scFv, wells coated with a cocktail of biotinylated irrelevant peptides were assayed in parallel. Bacterial supernatant containing monoclonal phage:scFv were added to plates and incubated to allow phage:scFv-hPAR1 binding to occur. After incubation, wells were washed three times in PBS containing Tween 20 (0.1% v / v), then two times in PBS, before the addition of an HRP- conjugated anti-M13 antibody. After further incubation, unbound anti-M13-HRP was removed by repeat of the washing steps, before the HRP substrate TMB was added. The colorimetric reaction was allowed to develop before the OD of wells were read at 450 nm (with 630 nm subtraction) using a plate reader. All OD values were adjusted by subtracting the background signal from a blank well that received no phage:scFv. A cut-off for ‘negative’ and ‘positive’ binder signals were determined by scrutinizing a histogram of all ‘blank-subtracted’ signals that clearly displayed two distinct populations - a relatively discrete group of low signal values that represented the minimally heterogeneous negatives, and a more heterogeneous group with larger signal values that were deemed to be positives. Example 5 - Antibody-PAR1 receptor binding assay

[0255] 17 clones were selected based on: ELISA data, abundance in pans 2 and 3 (based on NGS counts), and sequence clustering. The 5 candidates were selected from those 17 based on functional readouts, five candidate antibodies (AV-002, AV-004, AV-005, AV-013, AV-015), were further evaluated in a PAR-1 receptor binding assay.

[0256] Interaction of the candidate antibodies with the PAR1 receptor was quantified by flow cytometry, testing the antibodies on cells transfected with and over-expressing the human PAR1 receptor (CHO-PAR1). The antibodies were also tested on cells not transfected with the receptor as a negative control (CHO).

[0257] Stably PAR1 -transfected cells were obtained as follows. T-Rex-CHO cells were grown overnight such that they were approximately 70% confluent at the time of transfection. Cells were then transfected with a mammalian expression vector plasmid that contained tetracycline-inducible human PAR1 receptor (pcDNA5 / TO:hPAR1) using the lipofectamine reagent kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Transfected cells were cultured in the presence of hygromycin in order to select for positive transfectants. Monoclonal cell lines were derived from the resultant polyclonal culture by limiting dilution and further culture to expand from single cells. Both polyclonal and monoclonal cell lines were validated by flow cytometry using a commercially available anti-PAR1 antibody (clone ATAP- 2). Overexpression of PAR1 was induced by the addition of tetracycline. Cells were seeded to flasks and grown overnight such that they were 70% confluent at the time of induction. Tetracycline was added to culture media at a final concentration of 5 pg / ml. Cells were harvested after a further 24 hours of incubation and used for staining and flow cytometry analysis.

[0258] The flow cytometry analysis was performed as follows: Growth medium was removed from cells prior to washing with excess phosphate-buffered saline (PBS). Cells were then harvested using PBS with 1 mM EDTA. Harvested cells were diluted in excess ice-cold PBS, before being pelleted by centrifugation at 300 x g for 5 minutes at 4°C; cells were washed in ice-cold PBS two times. The washed cells were resuspended in 5 ml ice-cold PBS before the cell count and cell viability were determined using the Countess 3 cell counter (Thermo Fisher Scientific). Cell suspensions were diluted with further ice-cold PBS to achieve a final cell density of 1 x 106cells / ml. Cell staining was performed in a 96-well polypropylene plate loaded with 100 pl per well cell suspension (105cells per well). Cells were first pelleted in the wells by centrifugation at 300 x g at 4°C for 5 minutes, before being re-suspended in 50 pl of the different primary antibody suspensions prepared in PBS + bovine serum albumin (BSA, 0.5% w / v). The primary antibodies tested were AV-002, AV-004, AV-005, AV-013, AV-015, and the negative control IgGlK. Antibodies were tested at 10 nM, 1 nM, 0.1 nM and 0.01 nM concentration. Staining with primary antibody occurred during incubation at 4°C for 30 minutes. Cell-antibody suspensions were then diluted by the addition of 150 pl ice-cold PBS, before cells were pelleted away from unbound antibody by centrifugation at 300 x g at 4°C for 5 minutes. The washing of cells in 150 pl ice-cold PBS was repeated once more. Washed cells were then re-suspended in 50 pl of a suspension of fluorescein isothiocyanate (FITC)- conjugated secondary / reporter antibody prepared in PBS + BSA (0.5% w / v). For detection of the candidate human antibodies being tested, rabbit anti-human IgG-FITC (Invitrogen) was used. Staining with the secondary / reporter antibodies occurred during incubation at 4°C for 30 minutes. Stained cell suspensions were then diluted by the addition of 150 pl ice-cold PBS, before cells were pelleted away from unbound antibody by centrifugation at 200 x g at 4°C for 5 minutes. The washing of cells in 150 pl ice-cold PBS was repeated twice more. Washed cells were resuspended in 100 pl fresh PBS prior to analysis by flow cytometry on the CytoFlex platform (Beckman Dickenson). Recorded events were scrutinized under FSC-H vs FSC-A for gating on singlets, then by SSC-A vs FSC-A for gating on live cells. Finally, events were scrutinized by FSC-A vs FITC-A for any indication of positive staining. The threshold for - / + FITC staining was set such that between 0.5-1 % of cells stained with secondary antibody only were deemed positive. Antibody binding was quantified as the mean fluorescence intensity.

[0259] Example 6 - Inhibition of PAR1 -stimulated cellular signalling, measured using inositol-1- phosphate

[0260] Upon activation by thrombin, the PAR1 receptor stimulates intracellular signalling, primarily via the Gq G-protein pathway. This can be measured by quantifying the levels of inositol-1- phosphate (IPi), which is generated via Gq-mediated activation of phospholipase C. For these experiments, a human PAR1 receptor tagged with yellow fluorescent protein at the PAR1 intracellular C-terminus was used (PAR1-YFP). This construct was used to enable expression of the receptor to be easily verified during development of the IPi assay. The intracellular tagging is unlikely to affect thrombin or antibody activity since these agents are targeted to the extracellular region of the receptor.

[0261] Cells transiently overexpressing PAR1-YFP were prepared as follows: HEK293T cells were plated in T25 tissue culture flasks at 100,000 cells / cm2and grown overnight. The following day, the cells were transfected using FuGENE HD (Promega E2311) according to the manufacturer’s instructions with 7.8 pg / flask of PAR1-YFP DNA, using a 3:1 FuGENE HD:DNA ratio. After 18 hours, the cells were washed with PBS then lifted from the flask using enzyme free cell dissociation buffer (Thermo Fisher 13150-016) for 10 minutes at 37°C in 5% CO2. Serum-free Dulbecco's modified Eagle’s medium (DMEM) was then added and the cells collected by centrifugation. The cells were then resuspended in IPi assay buffer supplied with the IP-One HTRF assay kit (Rewity 62IPAPEC), plus 0.1 % bovine serum albumin (BSA).

[0262] The IPi assay was performed as follows: Antibodies were added to a 384 well Optiplate (Perkin Elmer 6007299) in 14 pl IPi assay buffer + 0.1 % BSA. 7 pl cell suspension was added (30,000 cells per well) and the cells and antibody were incubated for 30 minutes at 37°C. Thrombin (Abeam AB62452) was then applied in a volume of 7 pl assay buffer + 0.1 % BSA, at a final assay concentration of 0.9 nM (a concentration equivalent to the ECso / go concentration for stimulating I Pi production, based on the thrombin EC50 range of 0.09 - 0.20 nM measured in concentration-response experiments). Following a further 60 minute incubation at 37°C, I Pi was quantified using the IP-One HTRF assay kit (Rewity 62IPAPEC) according to the manufacturer’s instructions, using a PHERAstar reader (BMG Labtech) to quantify TR-FRET. The TR-FRET ratios (665 nm 1620 nm emission following 337nm excitation) were interpolated using an IP1 standard curve to derive the assay concentration of IP1 in nM for each data point. Antibody concentration inhibition curves were performed in technical duplicate and fitted by 3 parameter non-linear regression (Hill slope equal to one) to derive IC50, as the concentration of antibody that inhibited the thrombin IP1 response by 50 %.

[0263] Table 1. IC50 values of PAR1 antibodies for inhibition of thrombin-stimulated IP1 generation by the PAR1 receptor. Data values are mean ± SEM from three experiments (AV-002, AV-004, AV-005, AV-013) or two experiments (AV-015). The thrombin concentration was 0.9 nM.

[0264] Example 7 - Inhibition of PAR1 -stimulated arrestin recruitment p-arrestin recruitment to PAR1 is a complementary method to the IP1 assay for measuring PAR1 activation. Upon activation by thrombin, p-arrestin interacts with the PAR1 receptor, which can result in blockade of G-protein activation, and also propagation of p-arrestin- dependent signalling. PAR1 -p-arrestin interaction was measured using bioluminescence resonance energy transfer (BRET). The human PAR1 receptor was tagged at the intracellular C-terminus with YFP and p-arrestin1 with the light-generating protein Renilla luciferase (Rluc). When the arrestin interacts with the receptor, the light generated by the luciferase on p- arrestinl is transmitted to the fluorescent acceptor of YFP on the PAR1 receptor, causing a fluorescence emission of the YFP that can be detected in a plate reader. This process is called bioluminescence resonance energy transfer (BRET). Cells transiently-expressing PAR1-YFP and Rluc-p-arrestin1 were prepared as follows: HEK293 cells were seeded at 4.5 x 105cells per well of a 6-well plate, grown overnight, and then transfected with RLuc-p-arrestin1 (100 ng) together with PAR1-YFP (400 ng), with polyethylenimine (PEI) diluted in Opti-MEM. The next day, cells were pooled and re-seeded into 32 wells of a poly-D-lysine coated 96-well plate at 3 x 104cells per well and grown overnight.

[0265] The arrestin recruitment assay was performed as follows: Cells were washed twice with PBS and then incubated for 1 hour at room temperature with 35 pl of a 1 :1 equal mixture of DMEM without phenol red (Gibco, #31053-028) and PBS. After 1 hour, cells were incubated with 5 pl PAR1 antibodies (final concentrations 1.0 uM) for 10 minutes at room temperature. 5 pl of RLuc substrate coelenterazine H (5 pM final concentration) (Biotium, #10111-1) was added for 3 minutes followed by the addition of 5 pl of thrombin (1 nM final concentration). BRET measurements were taken 2 minutes later at 37°C. Each well was read for 3 sec and wells were measured every 1 .27 minutes for 30 minutes. All BRET measurements were performed using a Berthold TriStar LB941 multimode plate reader running MicroWIN 2010 software (Berthold Biotechnologies) using two filter settings: 480 nm for Rluc and 530 nm for YFP. The BRET signal was calculated as the emission at 530 nm divided by the emission at 480 nm. The BRET signals were normalized to basal BRET ratios and expressed as the percent over basal. Two technical replicates were used for each condition and the experiment was performed three times.

[0266] Table 2. Arrestin recruitment % inhibition values for PAR1 antibodies. The AUC for the time course of arrestin recruitment stimulated by thrombin (Figure 3) was calculated, and the AUC in the presence of the antibodies expressed as a percentage inhibition of the AUC in the absence of antibody. The antibody concentration was 1 pM and thrombin concentration was 1 nM. Data values are the mean ± SEM from three experiments.

[0267] Example 8 - Inhibition of human platelet aggregation This assay reports activity of the antibodies in a physiologically-meaningful context. In clinical use, efficacy of PAR1 antibodies for treating cardiovascular disease will involve inhibition of platelet aggregation. Thrombin activates the PAR1 receptor on platelets resulting in a signalling cascade that results in platelet aggregation. This activity can be measured in isolated human platelets. Aggregation is measured as a decrease of light absorbance, quantified in a cuvette using an aggregometer.

[0268] In this assay the lead antibodies were compared a commercially available anti-PAR1 antibody (clone ATAP-2). ATAP-2 is a murine IgG 1 K monoclonal PAR-1 antibody.

[0269] The human platelet suspension was prepared as follows: Following an institutionally approved protocol, human blood was obtained from healthy donors who gave their informed consent according to the Declaration of Helsinki. Blood (five parts) collected in one part acid-citrate- dextrose (ACD, 71 mM citric acid, 85 mM sodium citrate, 111 mM dextrose, pH 4.5) was centrifuged at 180 g for 15 minutes to prepare platelet-rich plasma. A platelet pellet was then obtained by centrifuging platelet-rich plasma at 800 g for 15 minutes in the presence of 15% w / w of ACD. The pellet was washed once by resuspension and centrifugation in modified Tyrode buffer, pH 6.5 (135 mM NaCI, 2.9 mM KCI, 1 mM sodium phosphate monobasic, 10 mM HEPES, 2.5 mM dextrose), and finally resuspended in the same buffer but at pH 7.4.

[0270] The aggregation assay was performed as follows: Aliquots of washed platelet suspension were incubated with antibody (130 nM final concentration) or vehicle control for 14 minutes at 37° C in a volume of 250 pl and an additional minute in the presence of 1 mM CaCh under stirring. Platelet aggregation was initiated with thrombin (0.5 nM final concentration) and monitored for 4 minutes on a digital aggregometer (Chrono-log). Light absorbance was measured every 0.5 seconds. Baseline light transmission was measured for 1.5 minutes before application of thrombin.

[0271] Platelet aggregation was quantified as the AUC of light absorbance below the baseline level before application of thrombin. This was done for the no-antibody vehicle control and for the antibody-treated sample. The AUC for the antibody-treated sample was then expressed as a percentage inhibition of the AUC for the no-antibody vehicle control.

[0272] Table 3. Platelet aggregation % inhibition values for PAR1 antibodies. The AUC of the absorbance decrease, reflecting the degree of platelet aggregation, was quantified for the noantibody vehicle control and for the antibody-treated sample (Figure 4). Then the AUC for the antibody-treated sample was expressed as a percentage inhibition of the AUC for the noantibody vehicle control. The antibody concentration was 130 nM and the thrombin concentration was 0.5 nM. Data values are the mean ± SEM from three experiments.

[0273] Example 9 - Inhibition of human platelet aggregation at three antibody concentrations

[0274] This assay reports activity of the antibodies in a physiologically-meaningful context. In clinical use, efficacy of PAR1 antibodies for treating cardiovascular disease will involve inhibition of platelet aggregation. Thrombin activates the PAR1 receptor on platelets resulting in a signalling cascade that results in platelet aggregation. This activity can be measured in isolated human platelets. Aggregation is measured as a decrease of light absorbance, quantified in a cuvette using an aggregometer.

[0275] In this assay the lead antibodies were compared a commercially available anti-PAR1 antibody (clone ATAP-2). ATAP-2 is a murine IgG 1 K monoclonal PAR-1 antibody. Antibodies were tested at three concentrations, 27 nM, 130 nM and 670 nM.

[0276] The human platelet suspension was prepared as follows: Following an institutionally approved protocol, human blood was obtained from healthy donors who gave their informed consent according to the Declaration of Helsinki. Blood (five parts) collected in one part acid-citrate- dextrose (ACD, 71 mM citric acid, 85 mM sodium citrate, 111 mM dextrose, pH 4.5) was centrifuged at 180 g for 15 minutes to prepare platelet-rich plasma. A platelet pellet was then obtained by centrifuging platelet-rich plasma at 800 g for 15 minutes in the presence of 15% w / w of ACD. The pellet was washed once by resuspension and centrifugation in modified Tyrode buffer, pH 6.5 (135 mM NaCI, 2.9 mM KCI, 1 mM sodium phosphate monobasic, 10 mM HEPES, 2.5 mM dextrose), and finally resuspended in the same buffer but at pH 7.4.

[0277] The aggregation assay was performed as follows: Aliquots of washed platelet suspension were incubated with antibody (27, 130 or 670 nM final concentration) or vehicle control for 14 minutes at 37° C in a volume of 250 pl and an additional minute in the presence of 1 mM CaCI2 under stirring. Platelet aggregation was initiated with thrombin (0.5 nM final concentration) and monitored for 4 minutes on a digital aggregometer (Chrono-log). Light absorbance was measured every 0.5 seconds. Baseline light transmission was measured for 1.5 minutes before application of thrombin. Platelet aggregation was quantified as the AUC of light absorbance below the baseline level before application of thrombin. This was done for the no-antibody vehicle control and for the antibody-treated sample. The AUC for the antibody-treated sample was then expressed as a percentage inhibition of the AUC for the no-antibody vehicle control. The results shown in Figure 5 demonstrate that the antibodies almost completely inhibit aggregation and are similarly active at the low concentration of 27 nM, intermediate concentration of 130 nM, and high concentration of 670 nM. The extent of inhibition is similar for the human antibodies, for ATAP-2, and for a high concentration of the small molecule PAR1 inhibitor vorapaxar (170 nM). Further the results shown in Figure 6 show a similar degree of inhibition is achieved when the antibodies are used at the three concentrations tested; that the degree of inhibition is strong (from 60 - 98% inhibition); and that the inhibition is similar to that of a high concentration of the small molecule PAR1 inhibitor vorapaxar (83%, dashed horizontal line, 170 nM concentration).

[0278] Table 4. Human platelet aggregation % inhibition AUC values for PAR1 antibodies at three concentrations. The AUC of the absorbance decrease, reflecting the degree of platelet aggregation, was quantified for the no-antibody vehicle control and for the antibody-treated sample (Figure 5). Then the AUC for the antibody-treated sample was expressed as a percentage inhibition of the AUC for the no-antibody vehicle control. The thrombin concentration was 0.5 nM. Data values are the mean ± SEM from three to five experiments.

[0279]

[0280] Table 5. Sequences

[0281]

[0282] References

[0283] Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11023-7. doi: 10.1073 / pnas.96.20.11023. PMID: 10500117; PMCID: PMC34235. Zhang C, Srinivasan Y, Arlow DH, Fung JJ, Palmer D, Zheng Y, Green HF, Pandey A, Dror RO, Shaw DE, Weis Wl, Coughlin SR, Kobilka BK. High-resolution crystal structure of human protease-activated receptor 1. Nature. 2012 Dec 20;492(7429):387-92. doi: 10.1038 / naturel 1701. Epub 2012 Dec 9. PMID: 23222541 ; PMCID: PMC3531875. Soh UJ, Dores MR, Chen B, Trejo J. Signal transduction by protease-activated receptors. Br J Pharmacol. 2010 May;160(2):191-203. doi: 10.1111 / j.1476-5381 ,2010.00705.x. PMID: 20423334; PMCID: PMC2874842.

[0284] Flaumenhaft R, De Ceunynck K. Targeting PARI : Now What? Trends Pharmacol Sci. 2017 Aug;38(8):701-716. doi: 10.1016 / j.tips.2017.05.001. Epub 2017 May 27. PMID: 28558960;

[0285] PMCID: PMC5580498.

[0286] Tricoci P, Huang Z, Held C, Moliterno DJ, Armstrong PW, Van de Werf F, White HD, Aylward PE, Wallentin L, Chen E, Lokhnygina Y, Pei J, Leonardi S, Rorick TL, Kilian AM, Jennings LH, Ambrosio G, Bode C, Cequier A, Cornel JH, Diaz R, Erkan A, Huber K, Hudson MP, Jiang L, Jukema JW, Lewis BS, Lincoff AM, Montalescot G, Nicolau JC, Ogawa H, Pfisterer M, Prieto JC, Ruzyllo W, Sinnaeve PR, Storey RF, Valgimigli M, Whellan DJ, Widimsky P, Strony J, Harrington RA, Mahaffey KW; TRACER Investigators. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes. N Engl J Med. 2012 Jan 5;366(1):20-33. doi: 10.1056 / NEJMoa1109719. Epub 2011 Nov 13. PMID: 22077816.

Claims

CLAIMS1. An antibody or antigen binding fragment thereof, that binds to Proteinase-Activated Receptor-1 (PAR-1) comprising ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 10 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 11 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 14 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 15 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 18 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 19 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and cii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 22 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 23 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 26 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 27 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 30 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 31 or a sequence comprising 80%, 90% or 95% sequence identity thereto, orei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR2 comprising SEQ ID NO. 34 or a sequence comprising 80%, 90% or 95% sequence identity thereto, a VH CDR3 comprising SEQ ID NO. 35 or a sequence comprising 80%, 90% or 95% sequence identity thereto, and eii). a variable light chain (VL) CDR1 comprising SEQ ID NO. 37 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR2 comprising SEQ ID NO. 38 or a sequence comprising 80%, 90% or 95% sequence identity thereto, VL CDR3 comprising SEQ ID NO. 39 or a sequence comprising 80%, 90% or 95% sequence identity thereto, or2. The antibody or antigen binding fragment thereof according to claim 1 , comprising i) a VH comprising SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and ii) a VL comprising SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

3. The antibody or antigen binding fragment thereof according to claim 1 or 2, comprising i) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 or a sequence comprising 1 , 2,3, or 4 amino acid substitutions, a VH CDR2 comprising SEQ ID NO. 2 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, a VH CDR3 comprising SEQ ID NO. 3 or a sequence comprising 1 , 2, 3, 4, 5 or 6 amino acid substitutions, and ii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR2 comprising SEQ ID NO. 6 or a sequence comprising 1 , 2, 3 or 4 amino acid substitutions, VL CDR3 comprising SEQ ID NO. 7 or a sequence comprising 1 , 2, 3, 4, 5, or 6 amino acid substitutions.

4. The antibody or antigen binding fragment thereof, according to any preceding claim, comprising ai) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 1 , a VH CDR2 comprising SEQ ID NO. 2, a VH CDR3 comprising SEQ ID NO. 3, and aii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 5, VL CDR2 comprising SEQ ID NO. 6, VL CDR3 comprising SEQ ID NO. 7, or bi) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 9, a VH CDR2 comprising SEQ ID NO. 10, a VH CDR3 comprising SEQ ID NO. 11 , and bii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 13, VL CDR2 comprising SEQ ID NO. 14, VL CDR3 comprising SEQ ID NO. 15, or ci) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 17, a VH CDR2 comprising SEQ ID NO. 18, a VH CDR3 comprising SEQ ID NO. 19, andcii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 21 , VL CDR2 comprising SEQ ID NO. 22, VL CDR3 comprising SEQ ID NO. 23, or di) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 25, a VH CDR2 comprising SEQ ID NO. 26, a VH CDR3 comprising SEQ ID NO. 27, and dii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 29, VL CDR2 comprising SEQ ID NO. 30, VL CDR3 comprising SEQ ID NO. 31 , or ei) a variable heavy chain (VH) CDR1 comprising SEQ ID NO. 33, a VH CDR2 comprising SEQ ID NO. 34, a VH CDR3 comprising SEQ ID NO. 35, and eii) a variable light chain (VL) CDR1 comprising SEQ ID NO. 37, VL CDR2 comprising SEQ ID NO. 38, VL CDR3 comprising SEQ ID NO. 39.

5. The antibody or antigen binding fragment thereof according to any preceding claim, comprising ai) a VH comprising SEQ ID NO. 4 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and aii) a VL comprising SEQ ID NO. 8 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or bi) a VH comprising SEQ ID NO. 12 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and bii) a VL comprising SEQ ID NO. 16 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ci) a VH comprising SEQ ID NO. 20 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and cii) a VL comprising SEQ ID NO. 24 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or di) a VH comprising SEQ ID NO. 28 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and dii) a VL comprising SEQ ID NO. 32 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto, or ei) a VH comprising SEQ ID NO. 36 or a sequence comprising 75%, 80%, 85%, 90% or 95% sequence identity thereto, and eii) a VL comprising SEQ ID NO. 40 or a sequence comprising 80%, 85%, 90% or 95% sequence identity thereto.

6. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or antigen fragment thereof binds to human PAR1 .

7. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody of antigen binding fragment is capable of reducing platelet activation, preferably reducing thrombin-related platelet aggregation.

8. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody of antigen binding fragment has an IC50 in the range 1 .0 to 50 nM.

9. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody of antigen binding fragment is capable of inhibiting arrestin recruitment by 5 to 100%.

10. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody of antigen binding fragment is capable of inhibiting platelet aggregation, preferably reducing thrombin-related platelet aggregation, by 25 to 100%.11 . The antibody or antigen binding fragment according to any preceding claim wherein said fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, or a polypeptide that contains at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

12. The antibody or antigen binding fragment according to any preceding claim wherein said antibody or fragment thereof is linked to a further moiety.

13. The antibody or antigen binding fragment according to claim 12, wherein the further moiety is selected from a further binding molecule, a therapeutic moiety, a half life extending moiety, and / or a label.

14. An immunoconjugate comprising the antibody or antigen binding fragment according to any of claims 1 to 13, conjugated to a therapeutic moiety, a half-life extending moiety, and / or a label.

15. An isolated nucleic acid encoding the antibody or antigen binding fragment according to any of claims 1 to 13.

16. A vector comprising the nucleic acid according to claim 15.

17. A host cell comprising the nucleic acid according to claim 15 the vector according to claim 16.

18. The host cell of claim 17 wherein the host cell is a bacterial, yeast, viral or mammalian cell.

19. A pharmaceutical composition comprising the antibody or antigen binding fragment according to any of claims 1 to 13, or the immunoconjugate of claim 14 and a pharmaceutically acceptable excipient, diluent and or carrier.

20. The antibody of claims 1 to 13, the immunoconjugate of claim 14 or the pharmaceutical composition according to claim 15, for use in therapy.

21. The antibody of claims 1 to 13, the immunoconjugate of claim 14 or the pharmaceutical composition according to claim 15, for use in treatment of a thrombin-mediated disease or a PAR-1 -mediated disease.

22. A method of treating a thrombin-mediated disease or a PAR-1 -mediated disease, comprising administering a therapeutically effective amount of antibody of claims 1 to 13, the immunoconjugate of claim 14 or the pharmaceutical composition according to claim 15.

23. The antibody of claims 1 to 13, for use in the manufacture of a medicament for the treatment of a thrombin-mediated disease or a PAR-1 -mediated disease.

24. The antibody, immunoconjugate, pharmaceutical for use according to claim 21 , the method according to claim 22 or the use according to claim 23, wherein the PAR-1 -mediated disease is selected from cancer, inflammatory conditions, sepsis, multiple sclerosis, acute lung injury (ALI), cardiovascular disease, autoimmune disease25. The antibody, immunoconjugate, pharmaceutical for use according to claim 24, the method according to claim 24 or the use according to claim 24, wherein the cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, endometrial cancer, cancer of the esophagus, cancer of the small intestine, liver cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, thymoma, urothelial carcinoma, prostate cancer, leukaemia, acute myeloid leukaemia, glioblastoma, glioma, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

26. The antibody, immunoconjugate, pharmaceutical for use according to claim 21 , the method according to claim 22 or the use according to claim 23, wherein said antibody or antigen binding fragment is administered in combination with a further therapy.

27. The antibody, immunoconjugate, pharmaceutical for use, the method, the use according to claim 26, wherein the further therapy is selected from an anti-cancer therapy, an anti-inflammatory, or a cardiovascular disease therapy.

28. A binding molecule comprising the antibody or antigen binding fragment according to any of claims 1 to 13.

29. A multispecific binding molecule comprising the antibody or antigen binding fragment according to any of claims 1 to 13.

30. An in vitro, ex vivo or in vivo method of detecting PAR-1 in a biological sample comprising contacting said biological sample with the antibody or antigen binding fragment thereof according to any of claims 1 to 13.31 . A method for reducing platelet activation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of claims 1 to 13.

32. A method for reducing cell proliferation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of claims 1 to 13.

33. A method for blocking thrombin induced Beta-arrestin 1 recruitment comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of claims 1 to 13.

34. A method for reducing platelet aggregation comprising contacting a biological sample with the antibody or antigen binding fragment thereof according to any of claims 1 to 13.

35. A kit comprising the antibody or antigen binding fragment according to any of claims 1 to 13 and optionally instructions for use.

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