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EP1943277A2 - Agents antithrombiques - Google Patents

Agents antithrombiques

Info

Publication number
EP1943277A2
EP1943277A2 EP06763512A EP06763512A EP1943277A2 EP 1943277 A2 EP1943277 A2 EP 1943277A2 EP 06763512 A EP06763512 A EP 06763512A EP 06763512 A EP06763512 A EP 06763512A EP 1943277 A2 EP1943277 A2 EP 1943277A2
Authority
EP
European Patent Office
Prior art keywords
agent
antibody
seq
hgp
gpvi
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06763512A
Other languages
German (de)
English (en)
Inventor
Götz Münch
Andreas Bültmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Trigen GmbH
Original Assignee
Trigen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trigen GmbH filed Critical Trigen GmbH
Publication of EP1943277A2 publication Critical patent/EP1943277A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present disclosure relates to agents which bind to glycoprotein VI (GPVI) protein or sequences thereof. More particularly, though without limitation, the invention concerns agents which bind one or more epitopes of human GPVI. The invention also relates to certain epitopes of human GPVI, methods of identifying or screening agents which bind to the epitopes. The invention also relates to the use of agents for treatment or prevention of diseases arising from processes of blood platelet aggregation, as well as other subject matter
  • Collagen is the major extracellular matrix protein present in the subendothelium of blood vessels. Collagen binds directly to platelets via specific platelet receptors such as integrin, collagen receptor, glycoprotein IV and GPVI.
  • WO 01/16321, WO 01/00810 and WO 00/68377 disclose a DNA and protein sequence of the human GPVI receptor.
  • WO 03/05020 discloses specific binding members directed against human GPVI and specific inhibitors of collagen-induced platelet aggregation.
  • Antibodies of the single chain format and in particular single chain antibodies with a particular sequence are also disclosed.
  • EP 1224942 and EP 1228768 disclose a monoclonal anti-GPVI murine antibody JAQl, which specifically binds to mouse GPVI, for the treatment of thrombotic disease. JAQl antibody induces irreversible internalization of the GPVI receptor on mouse platelets. This mechanism has only been observed in mice and cannot be used in a patient.
  • WO 03/008454 and WO 01/00810 disclose polypeptides, proteins and fusion proteins of GPVI as a pharmaceutical composition. There is some suggestion of antibodies and single chain variable fragments (scFv) against GPVI.
  • Acute coronary or carotid syndromes are a major cause of death in Western societies. Even in the case of initial survival of such a cardiovascular event, many patients suffer from life- threatening complications such as intravascular thrombosis leading to further myocardial infarction or stroke.
  • the dislodging of the atherosclerotic plaque initiates a cascade of events culminating in arterial thrombosis and ischemia of the downstream tissue, precipitating diseases such as myocardial infarction or ischemic stroke.
  • the first response to vascular injury is adhesion of circulating platelets to exposed subendothelial matrix proteins, which triggers subsequent platelet aggregation.
  • fibrillar collagen is considered the most thrombogenic constituent, as it acts as a strong activator of platelets and supports platelet adhesion both in vitro and in vivo (see Baumgartner, H. R. (1977). Thromb. Haemost 37, 1-16; Clemetson, K. 1, Clemetson, J. M. (2001). Thromb. Haemost. 86, 189-197; Massberg, S., Gawaz, M. Eta/. J.Exp.Med. 197, 41-49).
  • the platelet membrane proteins which have been reported to be putative collagen receptors, may be divided into those which interact indirectly with collagen through collagen-bound von
  • Willebrand factor including GPIb and the integrin GPIIb,IIIa, and those which interact directly with collagen including GPVI, the integrin ⁇ 2 ⁇ i, and CD36 (reviewed in Clemetson, K. J.,
  • GPVI has been identified as the major platelet collagen receptor (Moroi, M., eta/. (1989). J C/in.Invest 84, 1440-1445).
  • GPVI is a 60-65 kDa type I transmembrane glycoprotein, which belongs to the immunoglobulin superfamily (Clemetson, J. M., et a/ (1999) J Biol.Chem. 274, 29019-29024; Jandrot-Perrus, M., Busfield, et a/ (2000). Blood 96, 1798-1807; Gibbins, J. M., et al (1997). FEBS Lett. 413, 255- 259; Zheng, Y. M., etal '(2001). J Biol.Chem. 276, 12999-13006; Suzuki-Inoue, K., Tulasne, D., et a/.
  • Platelets deficient in GPVI show loss of collagen-induced adhesion and aggregation in vitro (Sugiyama, T., et a/ (1987) Blood 69, 1712-1720).
  • function blocking anti-GPVI monoclonal antibodies attenuate ex vivo platelet aggregation in response to collagen and collagen-related peptide (CRP), which mimics collagen triple helix (Sugiyama, T., Ishibashi, T., Okuma, M. (1993) IntJ.Hematol. 58, 99-104; Schulte, V., et a/ (2001) J Biol. Chem. 276, 364- 368).
  • GPIIb/IIIa antagonists have no influence on the release mechanism of platelet or even enhance pro-inflammatory responses such CD 4OL or P-Selectin expression (for review see Bhatt DL and Topol EJ. (2003) Nature Reviews Drug Discovery ⁇ ; 2: 15- 28).
  • Antibodies directed against GPVI have been reported to induce platelet activation (Schulte, V., Snell, D., et a/. (2001). J Biol.Chem. 276, 364-368) and immuno-thrombocytopenia, hampering their use in the clinical setting.
  • PCT Application No. PCT/EP2004/013779 filed 3 December 2004 and claiming priority from European patent application 03027772.7 filed 3 December 2003 discloses an anti-GPVI monoclonal antibody, hGP 5C4 and fragments thereof, including a Fab fragment.
  • This antibody fragment is an inhibitory antibody which binds to GPVI and does not activate platelets.
  • Monoclonal antibody hGP 5C4 Fab prevents platelet adhesion and aggregation.
  • monoclonal antibody 5C4 Fab binds to GPVI and prevents binding of GPVI to collagen, but also does not induce significant platelet activation.
  • Antibodies disclosed by WO 01/00810 lead to massive induction of platelet aggregation, which is, however, an absolute contraindication for the use of such antibodies for the treatment of patients for acute and chronic vascular diseases.
  • Antibody 5C4 and its fragments, notably the antibody fragment hGP 5C4 Fab have marked inhibitory effects on the main physiological functions of platelets induced by collagen stimulation.
  • the stimulation of collagen-mediated physiological activation parameters PAC-I and CD 62P- Selectin was completely prevented by hGP 5C4 Fab.
  • Other putative anti-GPVI antibodies had no significant inhibitory effect on PAC-I and CD 62 P.
  • other putative anti-GPVI antibodies presented with a well known problem for inhibitors: Despite specific binding to GPVI some antibodies like 14E11 and 4C9 activated PAC-I and CD 62P even in the absence of agonists. This is a common problem for the development of inhibitory antibodies.
  • the antibody fragment hGP 5C4 Fab did not show any intrinsic GPVI activity.
  • hGP 5C4 Fab potently inhibited human platelet aggregation ex vivo without any intrinsic activity.
  • hGP 5C4 and hGP 5C4 Fab fragment were highly selective for collagen-mediated effects.
  • the inhibitory antibody had no effects on ADP-mediated activation of PAC-I and CD 62P.
  • hGP 5C4 Fab had no effect on TRAP- and ADP-mediated aggregation and ATP release of human platelets ex vivo.
  • hGP 5C4 Fab is a highly selective inhibitor of arterial thrombosis with no effects on venous thrombosis and hemostasis. Experiments have shown that bleeding time of human blood was not prolonged in the PFA-IOO device.
  • hGP 5C4 Fab circumvents an almost inherent problem of anti-platelet drugs (Quinn MJ; et a/ 2002: Platelet Glycoprotein Ilb/IIIa inhibitors - Recognition of a two- edged sword. Circulation 106: 379-385; Bhatt DL & Topol FJ; (2003): Nat Rev Drug Discovl: 15- 28).
  • hGP 5C4 Fab shows highly potent and selective inhibition of platelet activation with no prolongation of bleeding time.
  • hGP 5C4 Fab shows potent and highly selective inhibition of release of transmitter substances such as ATP from intracellular storage vesicles of human platelets. This is a crucial parameter for the platelet-endothelium interaction promoting atherosclerosis.
  • the antibody fragment hGP 5C4 Fab is a GPVI inhibitor selectively inhibiting the activated branch of GPVI mediated effects without significant bleeding complications.
  • the antibody fragment hGP 5C4 Fab can be used for the treatment of atherosclerotic complications caused by unstable atherosclerotic plaques with plaque rupture or endothelial lesion. Therefore, the antibody fragment hGP 5C4 Fab serve as therapeutic inhibitors for collagen-mediated GPVI activation without affecting the intrinsic activity of the GPVI receptor with the relevant signalling system. Moreover, these inhibitors can be used for the prevention and treatment of atherosclerosis.
  • agents to hGP 5C4 which are inhibitory agents for GPVI, in particular, agents which do not activate platelets when binding to a GPVI receptor on the surface of platelets. It is further considered useful to provide an agent which may be used to prevent or treat cardiovascular diseases for example but not limited to chronic atherosclerotic disease and intra-vascular thrombosis. It is also desirable to provide alternative agents which do not significantly activate a GPVI receptor, and to provide an inhibitory agent which prevents or substantially reduces the release mechanism of platelets and the expression of pro-inflammatory responses from platelets.
  • the present invention provides an agent that binds to GPVI or a sequence thereof. In an embodiment, it provides an agent which binds immunoglobulin like C2 domain 1 (Dl) of GPVI.
  • Dl immunoglobulin like C2 domain 1
  • One class of agents binds specifically to GPVI. In one embodiment, the agent specifically binds to Dl of GPVI. In that the agent specifically binds to domain 1 of GPVI it does not bind significantly to immunoglobulin domain 2 of GPVI.
  • the junction between Dl and D2 appears to be located in the region of amino acid residues 113/114 as shown in Figure 18.
  • these are generally insignificant compared to the binding to Dl.
  • the inventors believe that the binding of agents to Dl and not to D2 may avoid any cross-linking of D2 and the FcR- ⁇ chain.
  • the invention provides an agent that binds to a ligand, the ligand consisting of one or a combination of:
  • the agent binds to ligand (a) only and not ligand (b) or ligand (c).
  • the agent binds to ligand (a) and ligand (b) but does not bind to ligand (c).
  • the agent binds to ligand (a) and ligand (c) and does not bind to ligand (b).
  • the agent binds to ligand (b) and ligand (c) and does not bind to ligand (a).
  • the agent binds to ligand (a), ligand (b) and ligand (c).
  • the binding of the agent is optionally binding with an affinity of greater than 10 "7 M, 10 "8 M, 10 “9 M, 10 "10 M, 10 "11 M or 10 "12 M.
  • the binding may be specific for the ligand or non-specific, although in some instances there is a degree of lower affinity non-specific binding to certain other ligands unrelated to GP VI.
  • Peptide moieties (a), (b) and (c) independently of one another may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. They may have 5 to 13, 5 to 11 or 5 to 9 residues e.g. 13 amino acid residues, 11 amino acid residues or 9 residues. Also, within the scope of the invention are peptide moieties (a), (b) and (c) having (independently of one another) 5, 6, 7, 8, 10, 12, 14 or 15 amino acid residues. Larger numbers of amino acid residues for peptide moieties of (a), (b) and (c) are possible including 17, 18, 19, 20, 25 or 30 residues.
  • the peptide moiety (a) may be a sequence starting at position 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 of the sequence of Figure 18.
  • the peptide moiety (a) may be selected from one of the following sequences of contiguous amino acids of a human GPVI as shown in Figure 18:
  • the peptide moiety (a) may be selected from one of the following sequences of contiguous amino acids of human GP VI as shown in Figure 18:
  • the aforementioned portions of the GPVI sequence do not include the leader sequence (though the numbering given starts at residue M of the leader). It is believed that the signal sequence is cleaved from the native GPVI protein before expression on the platelet cell surface.
  • the peptide moiety (a) may be selected from one of the following amino acid sequences (see Table 2 for peptide SEQ ID. NOS):
  • the ligand includes a peptide moiety (a) which has amino acid residue 27 as part of the contiguous sequence of said peptide moiety.
  • the agent may bind at amino acid residue 27 comprised in the ligand. Whilst this will usually be lysine, conservative subsitution of this residue is possible meaning, for example, that amino acid residue 27 may be a basic amino acid other than lysine e.g. arginine.
  • the invention includes agents which bind to a sequence of contiguous amino acids, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, such as 5 to 13 amino acids, of the sequence of Figure 18, which sequence of contiguous amino acids includes lysine 27.
  • a sequence of contiguous amino acids e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, such as 5 to 13 amino acids, of the sequence of Figure 18, which sequence of contiguous amino acids includes lysine 27.
  • Peptide moiety (b) may be selected from the contiguous sequence of amino acids from position 75 to position 85 of human GPVI as shown in Figure 18.
  • the number of contiguous amino acids forming peptide moiety (b) may be 5, 6, 7, 8, 9 or 10 and the starting position for selecting the contiguous sequences of less than 10 residues may be selected so that all possible contiguous length variants are made available.
  • peptide moiety (b) may be selected from one of the following amino acid sequences:
  • the peptide moiety (c) may be selected from the contiguous sequence of amino acids from position 111 to position 119 of human GP VI as shown in Figure 18.
  • the number of contiguous amino acids may be 5, 6, 7, 8 or 9 and the starting position of the GP VI sequence for selecting sequences less than maximum number of residues may be performed so that all possible contiguous length segments are made available.
  • Peptide moiety (c) may be selected from one of the following amino acid sequences:
  • the ligand which the agent of the invention binds to consists of one or a combination of (a), (b) and (c) below:
  • SEQ ID. No. 136 KPSLQ
  • SEQ ID. No. 146 SLSAQ.
  • polypeptide (a), (b) and (c) comprising the peptide motifs as described above do not include the entirety of D2.
  • the amino acid sequence of the ligand to which the agent binds may be modified by one or more changes in sequence which do not eliminate the underlying biological function and utility of the agents as described herein. Modifications may include substitution of individual amino acids with other naturally occurring or non-naturally occurring amino acids, as described in more detail later on.
  • the agents of the invention may be, for example, an antibody or fragment thereof, e.g. a Fab. fragment.
  • Preferred antibodies and fragments are Fab fragments or scFv.
  • Other agents which bind the ligand as defined herein are encompassed within the present invention.
  • a "humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical, e.g.
  • humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the described products, methods and uses do not include the subject matter of the monoclonal antibody described in PCT Application No. PCT/EP2004/013779 filed on 3 December 2004 or EP patent application No. 0302777.2 filed on 3 December 2003.
  • a monoclonal antibody hGP 5C4 and fragments thereof e.g. Fab fragment, as well as humanised hGP 5C4 and fragments are excluded from the invention.
  • the invention does provide agents which are a Fab 1 , a (Fab')2, Fv or dsFv fragment of hGP 5C4.
  • agents as described herein which bind to platelet-bound GPVI include agents as described herein which bind to platelet-bound GPVI. Included are agents which do not significantly activate platelets.
  • the invention provides an isolated nucleic acid comprising a nucleic acid sequence, which sequence encodes an agent described herein which is an antibody, a fusion protein, a peptide or a protein.
  • the agents of the present invention if comprising a peptide sequence, for example an antibody, a fusion protein, a peptide or a protein, may be encoded by a nucleic acid sequence.
  • the present invention includes any nucleic acid sequence which encodes an agent as defined herein.
  • the present invention also includes a nucleic acid sequence which encodes the agent of the invention but which differs from the wild-type nucleic acid as a result of the degeneracy of the genetic code.
  • the present invention also includes nucleic acids that share at least 90% homology with a nucleic acid sequence which encodes an agent of the present invention.
  • the nucleic acid may have 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98% or 99% homology to a nucleic acid which encodes an antibody or fragment thereof of the present invention.
  • nucleic acid molecule which hybridises under stringent conditions to a nucleic acid molecule which encodes an agent of the present invention, when said agent is an antibody or fragment thereof or a fusion protein.
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et a/., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following have been found as exemplary for hybridization conditions but without limitation:
  • Hybridization 5x SSC at 65 0 C for 16 hours
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours
  • Hybridization 6x SSC at RT to 55 0 C for 16-20 hours
  • the invention provides an expression vector comprising a nucleic acid as described above and associated regulatory sequences necessary for expression of a protein or polypeptide in a host cell.
  • regulatory sequences include promoters, termination sequences and enhancers, for example.
  • the invention provides a host cell comprising a nucleic acid or a vector as described above. Such host cells are transfected or transformed so that they contain the nucleic acid or vector in such a way that they are effective in expressing the desired polypeptide/protein when cultured in appropriate media under the necessary growth conditions.
  • the host cell is selected from a HeLa cell and a CHO (Chinese Hamster Ovary) cell.
  • the host cells to be used are not particularly circumscribed so as long as they can be transfected by a vector to be used and can express the DNA of the present invention.
  • bacteria such as Escherichia coli, yeast such as Saccharomyces cerevisiae, and an animal cell such as a COS cell, a CHO cell, etc.
  • prokaryotic host cells appropriate for use with this invention include E coli.
  • eukaryotic host cells include avian, insect, plant, and animal cells such as C0S7, HeLa, and CHO cells.
  • an agent of the invention for example a fusion protein, antibody or antibody fragment having hGP 5C4 Fab activity or function can be produced in a cell or a culture medium. Then, by collecting the produced antibody (or antibody fragment), the antibody of the first aspect of the present invention can be obtained.
  • the obtained antibody or protein can be isolated and purified by appropriately combining methods, for example, centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion-exchange chromatography, or gel-filtration chromatography.
  • the cells can be cultured in a suitable medium, and spent medium can be used as an antibody source.
  • matrix-coated channels or beads and cell cocultures may be included to enhance growth of antibody-producing cells.
  • the method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse.
  • the mammal is optionally primed for ascites production by prior administration of a suitable composition, for example, Pristane.
  • Antibodies of the invention may also be obtained by employing routine recombinant methods such as described in Sambrook et a/. (1989) supra.
  • nucleic acid sequences of the invention can be cloned into a suitable expression vector (which contains control sequences for transcription, such as a promoter).
  • the expression vector is in turn introduced into a host cell.
  • the host cell is grown under suitable conditions such that the polynucleotide is transcribed and translated into a protein.
  • Heavy and light chains of antibodies of the invention may be produced separately, and then combined by disulfide bond rearrangement.
  • vectors with separate polynucleotides encoding each chain of an antibody of the invention, or a vector with a single polynucleotide encoding both chains as separate transcripts may be transfected into a single host cell which may then produce and assemble the entire molecule.
  • the host cell is a higher eukaryotic cell that can provide the normal carbohydrate complement of the molecule.
  • the fusion protein or antibody is thus produced in the host cell can be purified using standard techniques in the art.
  • the method of isolation may depend on the immunoglobulin isotype.
  • Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.
  • the agent of the invention is purified by using Protein G-Sepharose columns.
  • the agents of this invention can be made by any suitable procedure, including by recombinant methods or by chemical synthesis. Peptides which are produced may then be separated from each other by techniques known in the art, including but not limited to gel filtration chromatography, gel electrophoresis, and reverse-phase HPLC.
  • the polypeptide is at least partially purified from other cellular constituents.
  • the polypeptide is at least about 50% pure, as a weight percent of total protein. More preferably, the protein is at least about 50-75% pure.
  • the polypeptide is preferably at least about 80% pure.
  • the invention also provides a ligand consisting of one or more of: (a) a peptide moiety of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues including a sequence of contiguous amino acid residues selected from the sequence of contiguous amino acids from position 15 to position 39 of human GP VI protein as shown in Figure 18;
  • the ligand has a peptide sequence selected from the following peptide sequences: a) LGRVPAQSGPLPK (SEQ ID. NO 8) b) RVPAQSGPLPKPS (SEQ ID. NO 9) C) PAQSGPLPKPSLQ (SEQ ID. NO 10) d) QSGPLPKPSLQAL (SEQ ID. No 11) e) GPLPKPSLQALPS (SEQ ID. Nol2) f) LPKPSLQALPSSL (SEQ ID. No 13) g) KPSLQALPSSLVP (SEQ ID. No 14) h) LFIPAMKRSLAGR (SEQ ID. No 37) i) IPAMKRSLAGRYR (SEQ ID. No 38)
  • An artificial homolog of the GP VI protein may be produced by synthetic or recombinant means.
  • Such a homolog would comprise one or more of (a), (b) or (c) described above.
  • the epitopes (a), (b) or (c) above could be combined in pairs, or all three together, plus a suitable framework in order to provide a GP VI homolog which could be used a ligand for binding studies, screening of binding agents, e.g. antibodies, or for generating antibodies by immunising an animal.
  • Such homologs comprising the three epitopes (a), (b) and (c) would not include the native full length GP VI protein.
  • ligands in accordance with another aspect of the invention include a polypeptide comprising a peptide motif selected from:
  • SEQ ID. No. 136 KPSLQ
  • polypeptide is not full-length GPVI.
  • polypeptide (a), (b) and (c) comprising the peptide motifs as described above preferably do not include the entirety of D2.
  • the ligands of the invention may include any of the further features as hereinbefore described.
  • agents of the invention can be chemically synthesized using information provided in this disclosure, in conjunction with standard methods of protein synthesis.
  • a suitable method is the solid-phase Merrifield technique.
  • Automated peptide synthesizers are commercially available, such as those manufactured by Applied Biosystems, Inc. (Foster City, Calif.).
  • the ligand is a peptide and may be a fusion protein.
  • a preferred fusion protein partner for the ligand is PR-15, as shown in Figure 10.
  • the invention provides an array of ligands as hereinbefore described.
  • the array is provided on a solid substrate which in preferred arrangements is a flat substrate in the form of a membrane or sheet, e.g. a chip.
  • ligands which differ from one another in the peptide moieties (a), (b) and/or (c) which they contain.
  • Such a population of ligands may be used to screen for additional binding agents of the invention.
  • the ligands are preferably tagged with labels which may represent which of moieties (a), (b) or (c) (or combination thereof) are provided in the individual ligand.
  • the invention provides a humanized antibody comprising complementarity determining regions that bind, e.g. specifically binds, a ligand as described herein, and a human framework region, or a conservative substitution thereof of 1, 2, 3, 4, or 5 residues of the complementarity determining regions or the framework regions, wherein the antibody retains the binding affinity to the ligand as described herein.
  • the humanized antibody may comprise the complementarity determining regions of hGP 5C4 antibody or a conservative substitution thereof of 1, 2, 3, 4, or 5 residues of the complementarity determining regions or the framework regions hGP 5C4. In other embodiments, the humanized antibody does not include the CDRs of hGP 5C4.
  • the humanized antibody preferably has a binding affinity which is greater for Dl than D2 of human GPVI.
  • the binding affinity of the antibody for Dl may be greater than 10 "6 M, preferably greater than 10 "7 M, 10 "8 M, 10 "9 M, 10 "10 M, 10 "11 M, 10 "12 M.
  • the invention provides an agent, a ligand or a humanised antibody as hereinbefore described for use as a pharmaceutical.
  • a pharmaceutical formulation comprising an agent, a ligand or a humanised antibody as hereinbefore described.
  • the formulation may contain at least one additional pharmaceutically acceptable component, e.g. an excipient, diluent or carrier.
  • the formulation is intended for parenteral administration.
  • the ligands disclosed herein to which the described agents, e.g. antibodies, bind have potential application as antidotes or reversal agents for the described agents, for example, for hGP 5C4, hGP 5C4 Fab fragments and humanized versions thereof.
  • a ligand described herein for the manufacture of a medicament for therapeutically neutralising (i.e. reducing or substantially destroying the activity of) an anti- GPVI agent, i.e. an agent which binds to GPVI.
  • the agent may be one described herein, e.g. hGP 5C4, humanised hGP 5C4 or fragments (e.g. a Fab fragment) thereof.
  • a a ligand described herein for the manufacture of a medicament for treating bleeding resulting from the administration of an anti-GPVI agent.
  • a pharmaceutical formulation comprising a ligand described herein; in embodiments the formulation is a composition comprising the ligand and a pharmaceutically acceptable diluent, carrier or excipient.
  • the described ligands for use as a pharmaceutical may be an intravenous formulation.
  • the invention also provides a method of neutralising (i.e. reducing or substantially destroying the activity of) an anti-GPVI agent comprising contacting said agent with a ligand of the disclosure.
  • said agent which has been administered to a patient and the the method comprises administering an effective amount of the ligand to the patient.
  • a method for treating bleeding resulting from the administration of an anti-GPVI agent to a subject comprising administering an effective amount of a ligand of the disclosure to the subject.
  • the ligand is administered intravenously.
  • the invention also includes a method of making an antibody comprising immunising an animal with a ligand as hereinbefore described.
  • the invention provides a method of humanising antibodies comprising:
  • the invention provides a method of identifying an agent for binding to GP VI comprising contacting a candidate agent with any of the ligands of the invention as hereinbefore described.
  • the binding assay can be carried out in a variety of formats, whether solid or liquid phase.
  • a labelled ligand may be employed.
  • the method can be used to screen libraries of potential GPVI binding agents. The screening can take advantage of protein array technology or it can rely on traditional binding assays where bound and free labelled ligand are separated and measured across a range of test agents and concentrations of test agents and/ or ligand.
  • the invention provides for the use of ligands as hereinbefore described in a binding assay for identifying an agent capable of binding GPVI, preferably an agent capable of inhibiting platelet aggregation, more preferably an agent capable of inhibiting platelet aggregation by collagen or collagen related peptides (CRP).
  • an agent capable of binding GPVI preferably an agent capable of inhibiting platelet aggregation, more preferably an agent capable of inhibiting platelet aggregation by collagen or collagen related peptides (CRP).
  • the invention provides a method for inhibiting platelet aggregation in a subject, comprising administering to a subject a therapeutically effective amount of an agent as hereinbefore described, thereby inhibiting platelet aggregation.
  • the agent is an antibody or fragment thereof.
  • the invention provides a method for inhibiting platelet aggregation, comprising contacting platelets with an effective amount of the agent or an antibody as hereinbefore described, thereby inhibiting platelet aggregation.
  • the platelets may be in vitro or they may be in vivo.
  • the invention provides a method for treating a disease or disorder selected from therapeutic or prophylactic cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides comprising administering an agent of the invention, to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • the treatment may be therapeutic and/or prophylactic.
  • an agent of the invention for the manufacture of a medicament to treat or prevent of a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • the agents of the invention have a reduced tendency to cause unwanted bleeding in patients and medical uses of the agents described herein are useful in the treatment of certain patient groups such as those suffering from identifiable bleeding disorders for example thrombocytopenia purpura.
  • agents which bind to a GPVI receptor on platelets and not activate the platelets are provided. Also provided are agents which have little or no effects on ADP-mediated activation of PAC-I and CD 62P. Additionally, agents are provided which have little or no effect on TRAP- and ADP-mediated platelet aggregation and ATP release of human platelets ex vivo. As a consequence, the agents should be a highly selective inhibitor of arterial thrombosis with reduced or no effects on venous thrombosis and hemostasis. Further provided are inhibitors which do not activate platelets and should have reduced or no effect in the induction of immuno- thrombocytopenia. The inhibitor of the present invention may also inhibit the release of proinflammatory substances by platelets and therefore inhibit pro-inflammatory responses.
  • the agent is not a full length antibody; it may be an antibody fragment or humanized antibody fragment for example a Fab or a single chain variable fragment e.g. humanized in either case.
  • the present invention includes agents which advantageously circumvent an almost inherent problem of anti-platelet drugs (Quinn MJ et a/ (2002): Circulation 106: 379-385; Bhatt DL & Topol FJ; (2003): Nat Rev Drug Discov 2: 15-28) i.e. agents which are used to block platelet interactions can also trigger pro-inflammatory platelet responses which can potentially lead to fatality.
  • the agents of the invention at least in some embodiments are advantageously highly potent and selective inhibititors of platelet activation and show little prolongation of bleeding time.
  • the invention includes agents which can advantageously also be drugs for use in the treatment of acute vascular syndromes like acute coronary syndromes or ischemic stroke because they avoid or reduce unwanted and potentially fatal side effects like intra cranial hemorrhage or other bleeding complications.
  • the agents of the invention advantageously are also potent and highly selective inhibitors of release of transmitter substances such as ATP from intracellular storage vesicles of human platelets. Without being bound by any particular theory, this may be important for platelet-endothelium interactions which promote atherosclerosis.
  • Agents of the invention are advantageously used as drugs for the treatment and prevention of atherosclerosis. The agents also solve the problem of treatment of atherosclerosis by inhibition of platelet secretion.
  • the invention provides GPVI inhibitors, which in embodiments are selective GPVI inhibitors.
  • the invention further includes medical devices which are coated or impregnated with an anti- GPVI agent described herein, for example implants and prostheses, particularly cardiovascular implants and prostheses, of which may be mentioned arterial prostheses, venous prostheses, vascular grafts, vascular stents, vascular catheters, prosthetic valves, ventricular assist devices, anuloplasty rings, prostheses for aortic aneurysms and vena cava filters; haemodialysis and other apheresis machines and parts and fittings therefor; extracorporeal blood circuit equipment, for example cardiopulmonary bypass machines and parts and fittings therefor.
  • implants and prostheses particularly cardiovascular implants and prostheses, of which may be mentioned arterial prostheses, venous prostheses, vascular grafts, vascular stents, vascular catheters, prosthetic valves, ventricular assist devices, anuloplasty rings, prostheses for aortic aneurysms and vena cava filters; haemodialysis and other
  • the extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount. Included in the scope of protection therefore are packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species.
  • hGP 5C4 specifically detected purified Fc-GPVI-nt fusion protein and GPVI receptor on platelets, but not the control Fc protein, (b) The generation of Fab fragments of the IgG hGP 5C4 was verified in a SDS gel with Coomassie staining after digestion with an ImmunoPure Fab Kit (Pierce Biotechnology, Inc., Rockford, IL, USA).
  • Figu re 3 The binding of different antibodies to stable GPVI-expressing CHO cells was measured by FACS. The binding of the specific antibodies to control CHO cells served as control. After incubation with the primary antibodies CHO cells were incubated with anti-IgG rat antibodies labelled with PE. Specific PE fluorescence was determined in a Becton Dickenson FACScalibur device.
  • Figu re 4 The binding of different antibodies to human platelets was determined by FACS. Platelets were washed and platelet rich plasma was prepared as described in Material and Methods. After incubation with the primary antibodies, human platelets were incubated with anti- IgG rat antibodies labeled with peroxidase (PE). Specific PE fluorescence was determined in a Becton Dickenson FACScalibur device. The means ⁇ SEM are shown.
  • Figu re 5 The ability of different anti-GPVI antibodies to inhibit collagen-mediated platelet activation was measured for different activation markers by FACS.
  • Specific PAC-I fluorescence was determined in a Becton Dickenson FACScalibur device.
  • the means ⁇ SEM are shown, (b) Pre-incubation of human platelets with hGP 5C4 Fab (20 ⁇ g/ml) inhibited CD 62 P activation by collagen type I (10 ⁇ g/ml), whereas other antibodies could not prevent collagen-mediated CD 62P activation. 4C9 (20 mg/ml) or 14E11 (20 ⁇ g/ml) had even stimulating effects on human platelets.
  • the means ⁇ SEM are shown.
  • FIG. 6 The specificity of hGP 5C4 for collagen-mediated processes was investigated by ADP- and thrombin induced human platelet activation in FACS.
  • the CD 62 P expression was determined in a Becton Dickenson FACScalibur device with specific antibodies as described in Material and Methods.
  • hGP 5C4 had no influence on ADP-mediated CD 62 P activation in human platelets.
  • the TRAP-mediated activation of CD62P was also tested (bottom).
  • hGP 5C4 had no effect on TRAP (25 ⁇ M)-mediated CD62P activation in human platelets.
  • the means ⁇ SEM are summarized, (b) Human platelets were incubated with 0.5 ⁇ g/ml to 5 ⁇ g/ml hGP 5C4 Fab and stimulated with ADP (20 ⁇ M; top) or TRAP (25 ⁇ M; bottom). PAC-I fluorescence was measured in a Becton Dickenson FACScalibur. hGP 5C4 had no influence on ADP- mediated or TRAP-mediated PAC-I activation.
  • the means ⁇ SEM are shown, (c) Collagen (lO ⁇ g/ml), ADP (20 ⁇ M) and TRAP-mediated (25 ⁇ M) platelet activation and the effect of hGP 5C4 Fab on CD63 activation was investigated. hGP 5C4 inhibited collagen-mediated CD63 activation, whereas ADP- and TRAP- mediated CD63 activation was unaffected.
  • the means ⁇ SEM are ssummarized.
  • FIG. 7 The inhibition of collagen-mediated aggregation and ATP release by hGP 5C4 was tested in human platelets. Increasing concentrations of hGP 5C4 Fab (0.1 ⁇ g/ml to 2 ⁇ g/ml) were incubated with human platelets and collagen-induced (3 ⁇ g/ml) aggregation and ATP release was measured simultaneously ex vivo in an aggregometer. (a) The aggregation is expressed relative to an internal standard [in %] (see Examples for description). From 0.25 ⁇ g/ml hGP 5C4 Fab concentration the collagen-mediated aggregation of human platelets was almost completely abolished, (b) In simultaneous experiments, ATP release was measured form these platelets. Collagen induced marked release of ATP from intracellular stores. hGP 5C4 potently inhibited this collagen-induced ATP release. The ATP release is given in % of control release. The means ⁇ SEM are shown.
  • hGP 5C4Fab The specificity of hGP 5C4Fab for collagen-mediated inhibition of human platelet aggregation and ATP release was tested, (a) Different agonists (collagen 2 ⁇ g/ml, TRAP 10 mmol/l, and ADP 5 ⁇ mol/l) were used to induce aggregation ex vivo in an aggregometer [in % of internal standard](see Examples for description). hGP 5C4 Fab (1 ⁇ g/ml and 2 ⁇ g/ml) almost completely abolished the collagen-mediated aggregation of human platelets.
  • TRAP- and ADP- mediated aggregation was largely unaffected by substantially higher doses of hGP 5C4 Fab (2 ⁇ g/ml and 6 ⁇ g/ml).
  • ATP release was measured simultaneously given in pmol ATP/I.
  • hGP 5C4 (1 ⁇ g/ml and 2 ⁇ g/ml) inhibited collagen-mediated ATP release.
  • Substantially higher concentrations of hGP 5C4 Fab (2 ⁇ g/ml and 6 ⁇ g/ml) had no effect on thrombin/TRAP-mediated ATP release.
  • the highest dose of hGP 5C4 Fab (6 ⁇ g/ml) also inhibited ADP-mediated ATP release.
  • the means ⁇ SEM are shown.
  • hGP 5C4 Fab (1 ⁇ g/ml and 5 ⁇ g/ml) did not show any prolongation of bleeding time.
  • Figu re 1 1 a Amino acid sequence of monoclonal antibody hGP 5C4 heavy chain variable domain ( ⁇ 2a)
  • Figure 1 2a Amino acid sequence of monoclonal antibody 5C4 light chain variable domain (K)
  • Figu re 14 Intrinsic activating effect of various anti-GPVI antibodies on platelet aggregation (in the absense of an agonist)
  • Figu re 1 7 Epitope mapping studies using a control monoclonal antibody anti-rat HRP to screen a a human Glycoprotein VI peptide library.
  • Figu re 1 8 Amino acid sequence of a human Glycoprotein VI.
  • Figu re 1 9 Nucleic acid sequence encoding a human Glycoprotein VI.
  • the "Immunological activity" of hGP 5C4 Fab refers to any of the following activities: ability to bind human glycoprotein VI; ability to specifically bind human glycoprotein VI; ability to inhibit the binding of human glycoprotein VI to collagen in a specific manner; lack of activation of platelets or induction of immuno-thrombocytopenia; inhibition of the release mechanism of platelets and the expression of pro-inflammatory responses.
  • hGP 5C4 Fab “activity” or “function” refers to any of the immunological activities of hGP 5C4 Fab, or to any other biological activity ascribed to hGP 5C4 Fab, including the role of hGP 5C4 or hGP 5C4 Fab in the prevention or treatment of acute or chronic cardiovascular disease associated with intraarterial and/or intravenous thrombosis.
  • a specific aspect of hGP 5C4 Fab "activity” or “function” relates to the binding of human glycoprotein VI exposed on the surface of platelets thereby functionally neutralizing the glycoprotein VI mediated activation of the platelets by collagen. This activity or function is differs from the activity or function of an antibody which binds human glycoprotein VI and thereby activates the platelet.
  • the antibody fragment hGP 5C4 Fab has inhibitory effects on the main physiological functions of platelets induced by collagen stimulation.
  • the stimulation of collagen-mediated physiological activation parameters PAC-I and CD 62P-Selectin is prevented by hGP 5C4 Fab.
  • Anti-GPVI antibodies other than hGP 5C4 present with a well known problem for inhibitors: despite specific binding to GPVI some antibodies activate PAC-I and CD 62P even in the absence of agonists which can lead to platelet activation. Additionally, hGP 5C4 Fab inhibits human platelet aggregation ex vivo without any intrinsic activity.
  • hGP 5C4 Fab is selective for collagen-mediated effects.
  • the inhibitory antibody had no effects on ADP-mediated activation of PAC-I and CD 62P.
  • hGP 5C4 Fab had no effect on TRAP- and ADP-mediated aggregation and ATP release of human platelets ex vivo.
  • hGP 5C4 Fab is a highly selective inhibitor of arterial thrombosis with no effects on venous thrombosis and hemostasis. Experiments have shown that bleeding time of human blood was not prolonged in the PFA-100 device.
  • hGP 5C4 Fab circumvents an almost inherent problem of anti-platelet drugs (Quinn MJ; Plow EF; Topol EEJ. 2002: Platelet Glycoprotein Ilb/IIIa inhibitors - Recognition of a two-edged sword. Circulation 106: 379-385; Bhatt DL & Topol EEJ; 2003: Scientific and therapeutic advances in antiplatelet therapy. Nat Rev Drug Discov 2: 15-28).
  • hGP 5C4 Fab shows highly potent and selective inhibition of platelet activation with no prolongation of bleeding time.
  • hGP 5C4 is an ideal drug for the treatment of acute vascular syndromes like acute coronary syndromes or ischemic stroke without unwanted and potentially fatal side effects like intra cranial hemorrhage or other bleeding complications.
  • hGP 5C4 Fab shows potent and highly selective inhibition of release of transmitter substances such as ATP from intracellular storage vesicles of human platelets. As this is a crucial parameter for the platelet-endothelium interaction promoting atherosclerosis, the hGP 5C4 Fab could be an ideal drug for the treatment and prevention of atherosclerosis.
  • the antibody hGP 5C4 is a GPVI inhibitor selectively inhibiting the activated branch of GPVI mediated effects without significant bleeding complications.
  • the antibody fragment hGP 5C4 Fab can be used for the treatment of atherosclerotic complications caused by unstable atherosclerotic plaques with plaque rupture or endothelial lesion. Therefore, the antibody fragment hGP 5C4 Fab serve as therapeutic inhibitors for collagen-mediated GPVI activation without affecting the intrinsic activity of the GPVI receptor with the relevant signalling system. Moreover, these inhibitors can be used for the prevention and treatment of atherosclerosis.
  • hGP 5C4 Fab fragment in the specification further includes other hGP 5C4 fragments for example scFv or F(ab) 2 fragments.
  • Antibody f ragment (f ragment w it h specif ic ant igen binding) : Various fragments of antibodies have been defined, including Fab, (Fab ⁇ 2 , Fv, dsFV and single-chain Fv (scFv).
  • antibody fragments are defined as follows: (1) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain or equivalently by genetic engineering; (2) Fab 1 , the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab 1 fragments are obtained per antibody molecule; (3) (Fab') 2 , the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalently by genetic engineering; (4) F(Ab') 2 , a dimer of two FAb 1 fragments held together by disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; dsFV, which is the variable region of the light chain and the variable region of the heavy chain linked by disulfide bonds and (6) single chain antibody
  • Cell line/ Cell cultu re A "cell line” or “cell culture” denotes higher eukaryotic cells gown or maintained in vitro. It is understood that the progeny of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. "Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • An "isolated" polynucleotide or polypeptide is one that is substantially free of the materials with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of the materials with which it is associated in nature.
  • CDR Com plementarity-determ ining region
  • the CDRs are three hypervariable regions within each of the variable light (VL) and variable heavy (VH) regions of an antibody molecule that form the antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Proceeding from the N-terminus of a heavy or light chain, these complementarity-determining regions are denoted as "CDRl”, “CDR2,” and “CDR3,” respectively.
  • CDRs are involved in antigen-antibody binding, and the CDR3 comprises a unique region specific for antigen-antibody binding.
  • An antigen-binding site therefore, may include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region.
  • Alteration of a single amino acid within a CDR region can alter the affinity of an antibody for a specific antigen (see Abbas et al., Cellular and Molecular Immunology, 4th ed. 143-5, 2000).
  • the locations of the CDRs have been precisely defined, e.g., by Kabat et al., Sequences of Proteins of Immunologic Interest, U.S. Department of Health and Human Services, 1983.
  • the light and heavy chains of an Ig each have three CDRs, designated L-CDRl, L-CDR2, L-CDR3 and H-CDRl, H-CDR2, H-CDR3, respectively.
  • the CDRs of the light chain are bounded by the residues at positions 24 and 34 (L-CDRl), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded by the residues at positions 31 and 35b (H-CDRl), 50 and 65 (H-CDR2), 95 and 102 (H- CDR3), using the numbering convention delineated by Kabat eta/., (1991) Sequences of Proteins of Immunological Interest, 5 th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda (NIH Publication No. 91-3242).
  • Alignment may be done by hand, or by computer using commonly accepted computer programs; an example of such a program is the Align 2 program discussed in this description. Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences.
  • the light and heavy chains each typically have two cysteines which have the same residue numbers; in VL domain the two cysteines are typically at residue numbers 23 and 88, and in the VH domain the two cysteine residues are typically numbered 22 and 92.
  • Framework residues generally, but not always, have approximately the same number of residues, however the CDRs will vary in size.
  • residues lOOabcde For example, in the case of a CDR from a candidate sequence which is longer than the CDR in the sequence in Kabat to which it is aligned, typically suffixes are added to the residue number to indicate the insertion of additional residues (see, e.g. residues lOOabcde in fig. 5).
  • residues lOOabcde For candidate sequences which, for example, align with a Kabat sequence for residues 34 and 36 but have no residue between them to align with residue 35, the number 35 is simply not assigned to a residue.
  • CDR and FR residues are also determined according to a structural definition (as in Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987). Where these two methods result in slightly different identifications of a CDR, the structural definition is preferred, but the residues identified by the sequence definition method are considered important FR residues for determination of which framework residues to import into a consensus sequence.
  • Constant Region The portion of the antibody molecule which confers effector functions.
  • the variant antibodies of use can include constant regions derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma or mu.
  • Heavy chains of various subclasses are responsible for different effector functions.
  • the light chain constant region can be of the kappa or lambda type.
  • Epitope The site on an antigen recognized by an agent as determined by the specificity of the amino acid sequence. Two agents are said to bind to the same epitope if each competitively inhibits (blocks) binding of the other to the antigen as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495-1502, 1990). Alternatively, two antibodies have the same epitope if most amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are said to have overlapping epitopes if each partially inhibits binding of the other to the antigen, and/or if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Framework region Relatively conserved sequences flanking the three highly divergent complementarity-determining regions (CDRs) within the variable regions of the heavy and light chains of an antibody.
  • CDRs complementarity-determining regions
  • the variable region of an antibody heavy or light chain consists of a FR and three CDRs.
  • Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the variable region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs.
  • the framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.
  • the numbering of the residues in the light chain and heavy chain framework regions follows the numbering convention delineated by Kabat et al., (1991, supra).
  • a "human” framework region is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.
  • I m m u nogenicity A measure of the ability of a targeting protein, a therapeutic moiety or an agent to elicit an immune response (humoral or cellular) when administered to a subject.
  • Ig Immunoglobulin molecules and immunologically active portions of Ig molecules, for instance, molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
  • a naturally occurring antibody for example, IgG
  • IgG includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • the two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond.
  • Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.
  • Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length.
  • Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus.
  • Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.
  • the basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions.
  • Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. Sd.
  • Each chain contains distinct sequence domains.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHl, CH2 and CH3, collectively referred to as CH).
  • the variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors.
  • An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • CDR's complementarity determining regions
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
  • the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
  • a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715, which is herein incorporated by reference.
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
  • a humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr (see U.S. Patent No. 5,585,089, which is incorporated herein by reference).
  • Humanized immunoglobulins can be constructed by means of genetic engineering, e.g., see U.S. Patent No. 5,225,539 and U.S. Patent No. 5,585,089, which are herein incorporated by reference.
  • a human antibody is an antibody wherein the light and heavy chain genes are of human origin.
  • Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., Dower et al., PCT Publication No. WO91/17271; McCafferty et al., PCT Publication No. WO92/001047; and Winter, PCT Publication No.
  • Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No. WO91/10741, which are herein incorporated by reference).
  • Antibodies may also be obtained using phage display technology.
  • Phase display technology is known in the art for example Marks et al J. MoI. Biol. 222: 581-597 and Ckackson et al, Nature 352: 624-628, both incorporated herein by reference.
  • Phage display technology can also be used to increase the affinity of an antibody.
  • the antibody sequence is diversified, a phage antibody library is constructed, and a higher affinity binders are selected on antigen (see for example Marks et al Bio/ Technology 10:779-783, Barbas et al Proc. Natl. Acad. Sci USA 91:3809-3813 and Schier et al J. MoI. Biol. 263: 551-567, all incorporated herein by reference.
  • Aptamer The agent of the present invention may also be an aptamer.
  • Aptamers have been defined as artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and re-amplification.
  • RNA aptamers are nucleic acid molecules with affinities for specific target molecules. They have been likened to antibodies because of their ligand binding properties. They may be considered as useful agents for a variety of reasons. Specifically, they are soluble in a wide variety of solution conditions and concentrations, and their binding specificities are largely undisturbed by reagents such as detergents and other mild denaturants. Moreover, they are relatively cheap to isolate and produce. They may also readily be modified to generate species with improved properties.. Extensive studies show that nucleic acids are largely non-toxic and non-immunogenic and aptamers have already found clinical application. Furthermore, it is known how to modulate the activities of aptamers in biological samples by the production of inactive dsRNA molecules in the presence of complementary RNA single strands (Rusconi eta/., 2002).
  • RNAs synthesised chemically based on L-ribose sugars will bind the natural target, that is to say the mirror image of the selection target. This process is conveniently referred to as reflection- selection or mirror selection and the L-ribose species produced are significantly more stable in biological environments because they are less susceptible to normal enzymatic cleavage, i.e.they are nuclease resistant.
  • I m m u noreactivity A measure of the ability of an agent, sometimes an antibody, to recognize and bind to a specific antigen.
  • Specifically binds refers to the ability of individual agents or antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an agent, for example but not limited to a antibody molecule, and the antigen.
  • the antigen is glycoprotein VI (GPVI). Binding specificity is typically determined from the reference point of the ability of the agent to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • An antibody that specifically binds to a particular epitope is referred to as a "specific antibody.”
  • specificity may be determined by means of a binding assay such as ELISA employing a panel of antigens, e. g. as disclosed herein with reference to Table 2 .
  • a binding assay such as ELISA employing a panel of antigens, e. g. as disclosed herein with reference to Table 2 .
  • An agent according to the present invention may recognise GPVI on cells of the platelet/megakaryocyte lineage, and not other human blood cells, in particular granulocytes, lymphocytes and erythrocytes. Reactivity of a specific binding member according to the invention with human platelets may be abolished by competition with recombinant GPVI.
  • Monoclonal ant ibody An antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • a monoclonal antibody is produced by a specific hybridoma cell, or a progeny of the hybridoma cell propaged in culture.
  • a hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • nucleic acid is a polymeric form of nucleotides of any length, which contain deoxyribonudeotides, ribonucleotides, and analogs in any combination. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown.
  • the term "nucleic acid” includes double-, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a nucleic acid encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form.
  • Specific examples of nucleic acid sequences of the variable domains that characterize the invention are the nucleic acid sequences given in SEQ ID No. 9 and SEQ ID No. 11 of the heavy chain and light chain of hGP 5C4, respectively.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component.
  • Amino acid substitutions can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region.
  • Amino acid substitutions are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide.
  • Groups of functionally related amino acids within which conservative substitutions may be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan.
  • Polypeptides of this invention may be in glycosylated or unglycosylated form, may be modified post-translationally (e.g., acetylation, and phosphorylation) or may be modified synthetically (e.g., the attachment of a labeling group).
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the platelet receptor GPVI mediates both adhesion and signaling responses to collagen in a receptor density-dependent fashion. J Biol.Chem. 277, 3011-3019) aid under (patho-)physiological conditions in vivo (Massberg, S., Gawaz, M., Gr ⁇ ner, S., Schulte, V., Konrad, I., Zohlnh ⁇ fer, D., Heinzmann, U., Nieswandt, B. (2003) A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J.Exp.Med. 197, 41-49). This identifies the inhibition of GPVI as a promising strategy to prevent platelet recruitment and arterial thrombosis in patients with advanced atherosclerosis.
  • Variable region also variable dom ain or V dom ain
  • the regions are composed of polypeptide chains containing four relatively invariant "framework regions” (FRs) and three highly variant “hypervariable regions” (HVs). Because the HVs constitute the binding site for antigen(s) and determine specificity by forming a surface complementarity to the antigen, they are more commonly termed the “complementarity-determining regions,” or CDRs, and are denoted CDRl, CDR2, and CDR3.
  • the framework regions surround the CDRs. Proceeding from the N-terminus of a heavy or light chain, the order of regions is: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.
  • the term "variable region” is intended to encompass a complete set of four framework regions and three complementarity-determining regions. Thus, a sequence encoding a "variable region” would provide the sequence of a complete set of four framework regions and three complementarity-determining regions.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and
  • F(ab') 2 fragments a bivalent fragment comprising two linked Fab fragments
  • scFv single chain Fv molecules
  • a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site
  • Fv,scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and
  • VL domains (Y. Reiter etal, Nature Biotech, 14, 1239-1245,1996).
  • Minibodies comprising ascFv joined to a CH3 domain may also be made (S. Hu etal, Cancer Res., 56, 3055-3061,1996).
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4,446-449 (1993) ), e. g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti- idiotypic reaction.
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (W094/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against GPVI, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al, Protein Eng., 9,616-621,1996). or substance having an antibody antigen-binding domain with the required specificity.
  • this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • amino-group protecting moiety refers to any group used to derivatise an amino group, especially an N-terminal amino group of a peptide or amino acid. Such groups include, without limitation, alkyl, acyl, alkoxycarbonyl, aminocarbonyl, and sulfonyl moieties. However, the term “amino-group protecting moiety” is not intended to be limited to those particular protecting groups that are commonly employed in organic synthesis, nor is it intended to be limited to groups that are readily cleavable.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • GPVI inhibitor refers to a product which, within the scope of sound pharmacological judgement, is potentially or actually pharmaceutically useful as an inhibitor of GPVI, and includes reference to substance which comprises a pharmaceutically active species and is described, promoted or authorised as a GPVI inhibitor. Such GPVI inhibitors may be selective, that is they are regarded, within the scope of sound pharmacological judgement, as selective towards GPVI in contrast to other receptors or targets; the term “selective GPVI inhibitor” includes reference to substance which comprises a pharmaceutically active species and is described, promoted or authorised as a selective GPVI inhibitor.
  • agents which bind to GPVI and more specifically, to a ligand of the disclosure.
  • the agent will have the same or similar immunological activity as hGP 5C4 Fab.
  • the invention includes all variant forms of the described GPVI binding agents which retain GPVI binding function, in particular clinically useful function, especially ability to inhibit GPVI-collagen binding and not to significantly activate platelets to promote aggregation.
  • proteins and other poly (amino acids) may be derivatised as by glycosylation, for example, to modify their properties.
  • Other modifications included within the disclosure include without limitation attachment of natural or synthetic polymers (e.g. a polyethylene glycol or dextran) albumin affinity tags (see for example Bioorg Med Chem Lett. 2002 Oct 21; 12(20): 2883-6).
  • peptides containing one or more d-amino acids are also included.
  • Table 2 shows peptides consisting of contiguous sequences of GPVI.
  • the binding affinity of peptides 9 to 14 may be compared to a comparative peptide.
  • the comparative peptide may contain between five and fifteen contiguous amino acid residues of human GPVI ( Figure 18), excluding amino acid residues 15 to 39 of Figure 18.
  • the comparative peptide may contain amino acid residues 1 to 14 or 40 onwards of Figure 18.
  • the agent of the present invention binds to one or more than one or a combination of peptide sequences selected from SEQ ID Nos. 9, 10 11, 12, 13 and 14 with greater affinity than any comparative peptide of human GPVI e.g. at least twice as strongly as any comparative peptide sequence of a human GPVI.
  • the strength of the binding of an agent to a peptide sequence of GPVI may be analysed using chemiluminescence and quantified by measuring signal intensity. Thus, the strength of binding of an agent to a peptide may be indicated by a signahnoise ratio.
  • the agent of the present invention binds to one or more than one or a combination of peptide sequences selected from SEQ ID Nos.
  • the agent of the present invention binds to one or more than one or a combination of peptide sequences selected from SEQ ID Nos. 11 and 12 at least twice as strongly as any comparative peptide sequence of a human GPVI.
  • the agent of the invention binds to peptide sequence SEQ ID No. 9. with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • the agent of the invention binds to peptide sequence SEQ ID No. 10 with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • agent of the invention binds to peptide sequence SEQ ID No. 11 with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • agent of the invention binds to peptide sequence SEQ ID No. 12 with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • the invention also includes embodiments in which the agent of the invention binds to peptide sequence SEQ ID No. 13 with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • the invention further includes embodiments in which the agent of the invention binds to peptide sequence SEQ ID No. 14 with greater affinity than it does to a peptide having a sequence of any of SEQ ID NOS 1-8 and 15-131.
  • Agents of the invention may bind with greater affinity to each of the peptides of sequences SEQ ID Nos. 9, 10 11, 12, 13 and 14 affinity than it does to any of the peptides having a sequence of SEQ ID NOS 1-8 and 15-131.
  • agents which bind with greater affinity to a particular peptide or peptides than to other specified peptide(s) may have an affinity two, five, 10, fifteen or twenty times greater for the particular peptide(s) than for any of the specified peptide(s).
  • the binding affinity of an agent to a peptide may be analysed using chemiluminescence and measured, at least relative to the binding affinity to other peptides, by measuring signalintensity.
  • analysis of agent binding to the peptides may be carried out by PepSpotTM analysis (see: Molecular Basis for the Binding Promiscuity of an Anti-p24 (HIV-I) Monoclonal Antibody, Kramer et al., Cell Vol. 91 (1997), p. 799-809, Antigen sequence and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis, Reineke et al., Curr. Top. Microbiol. Immunol. Vol. 243 (1999), p. 23-36 ; Coherent Membrane Supports for Parallel Microsynthesis and Screening of Bioactive Peptides, Wenschuh et al., Biopolymers Vol. 55 (2000), p.
  • the agent binds to peptide sequence SEQ ID No. 11 at least five times as strongly as any comparative peptide sequence of human GPVI. In an embodiment, the agent binds to peptide sequence SEQ ID No. 11 at least ten times as strongly as any comparative peptide sequence of human GPVI.
  • the strength of the binding of an agent to a peptide sequence of GPVI may be analysed using chemiluminescence and quantified by measuring signal intensity. Thus, the strength of binding of an agent to a peptide may be indicated by a signal: noise ratio.
  • the agent binds to peptide sequence SEQ ID No. 11 at least fifteen times as strongly as it binds to any comparative peptide sequence of human GPVI. In one embodiment, the agent binds to peptide sequence SEQ ID No. 11 at least twenty times as strongly as it binds to any comparative peptide sequence of human GPVI.
  • the strength of the binding of an agent to a peptide sequence of GPVI may be analysed using chemiluminescence and quantified by measuring signal intensity. Thus, the strength of binding of an agent to a peptide may be indicated by a signaknoise ratio and quantified by Light Units (LU).
  • the agent binds to one of or both of peptide sequence SEQ ID No. 37 and 38 at least twice as strongly as any peptide sequence in Table 2 other than SEQ ID NOS 9 to 14, 37, 38, 54, 55, and 56. In one embodiment, the agent binds to one of or both of peptide sequence SEQ ID No. 37 and 38 at least twice as strongly as any comparative peptide sequence of human GPVI, wherein the comparative peptide sequence contains between five and fifteen contiguous amino acid residues.
  • the agent binds to one or more than one or a combination of peptide sequence SEQ ID. NOS 54, 55, and 56 at least one and a half times as strongly as any comparative peptide sequence. In one embodiment, the agent binds to one of or a combination of peptide sequences SEQ ID No. 54, 55 and 56 at least one and a half times as strongly as any comparative peptide sequence of human GPVI, wherein the comparative peptide sequence contains between five and fifteen contiguous amino acid residues.
  • the agent binds to one or more than one or a combination of peptide sequences selected from SEQ ID Nos. 9, 10 11, 12, 13 and 14 and also to peptides SEQ ID No. 37 and 38 at least twice as strongly as to any comparative peptide disclosed in Table 2 excluding SEQ ID NOS 9, 10 11, 12, 13, 14, 37, 38, 54, 55 and 56.
  • the agent binds to one or more than one or a combination of peptide sequences selected from SEQ ID Nos. 9, 10 11, 12, 13 and 14 and also to peptides SEQ ID No. 37 and 38 at least twice as strongly as to any comparative peptide disclosed in Table 2 and also to one of or a combination of peptide sequences SEQ ID No. 54, 55 and 56 at least twice as strongly as to any comparative peptide disclosed in Table 2, excluding SEQ ID NOS 9, 10 11, 12, 13, 14, 37, 38, 54, 55 and 56 .
  • the agent binds to one or both of peptides sequences SEQ ID No.
  • the agent binds to one or more than one or a combination of peptide sequences SEQ ID NOS. 9, 10 11, 12, 13 and 14 at least twice as strongly as to any comparative peptide disclosed in Table 2 and also to one of or a combination of peptide sequences SEQ ID No. 54, 55 and 56 at least twice as strongly as to any comparative peptide sequence disclosed in
  • the agents of the invention bind with greater affinity to one or more of peptides 8, 9, 10 11, 12, 13 or 14 of Table 2 than to any comparative peptide sequence listed in Table 2.
  • the agent binds to peptides 9, 10 11, 12, 13 and 14 with a binding affinity of within 5% of the binding affinity of hGP 5C4 for each of peptides 9, 10 11, 12, 13 and 14.
  • the agent binds to peptides 9, 10 11, 12, 13 or 14 with a binding affinity of within 10% of the binding affinity of hGP 5C4 for each of peptides 9, 10 11, 12, 13 and 14. Particularly, these are provided agents which bind to peptide 11 with an affinity described previously in this paragraph.
  • Binding affinity of an agent for example an antibody or fusion protein may be measured using for example BIACORE systems.
  • the agents bind to one or more than one or a combination of peptide sequences selected from SEQ ID. No. 9, 10, 11, 12, 13 and 14 (shown in Table 2) such that the signal: noise ratio produced by chemiluminescence analysis is at least twice the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the agent of the invention binds to one or more or a combination of peptides sequences ID Nos. 9, 10, 11, 12, 13 or 14 such that the signal: noise ratio produced by chemiluminescence analysis is at least twice the signal produced as background.
  • the agents bind to one or more than one or a combination of peptide sequences selected from SEQ ID. No. 11, 12 and 13 (shown in Table 2) such that the signal: noise ratio produced by chemiluminescence analysis is at least three times the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the agents bind to one or more than one or a combination of peptide sequences selected from SEQ ID. No. 11 (shown in Table 2) such that the signal: noise ratio produced by chemiluminescence analysis is at least five times the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the agent of the present invention binds to at least one of or a combination of peptide sequences selected from SEQ ID. No 37 and 38 with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least twice the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the invention provides agents which bind to at least one of or a combination of peptide sequences selected from SEQ ID N0S.8, 54, 55 and 56 with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least one and a half times the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the invention provides agents which bind to at least one of or a combination of peptide sequences selected from SEQ ID NOS 9, 10, 11, 12, 13 and 14 and also with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least twice the signal produced as background and wherein the agent also binds to SEQ ID NOS 8, 54, 55 and 56 with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least one and a half times the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the invention provides agents which bind to at least one of or a combination of peptide sequences selected from SEQ ID NOS 9, 10, 11, 12, 13 and 14 and also with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least twice the signal produced as background and wherein the agent also binds to SEQ ID NOS 37 and 37 with an affinity such that the signahnoise ratio produced by chemiluminescence analysis is at least twice the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the invention provides agents which bind to at least one of or a combination of peptide sequences selected from SEQ ID NOS 37 and 38 and also with an affinity such that the signaknoise ratio produced by chemiluminescence analysis is at least twice the signal produced as background and wherein the agent also binds to SEQ ID NOS 54, 55 and 56 with an affinity such that the signal: noise ratio produced by chemiluminescence analysis is at least one and a half times the signal produced as background.
  • the binding affinity of the agent in this embodiment is quantified in Light Units (LU).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutical acceptable carrier and an effective amount, e. g. a therapeutically effective amount, including a prophylactically effective amount, of one or more products of the invention.
  • a method for preparing a hybridoma cell-line producing monoclonal antibodies comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one peptide moiety (a) described herein, e.g. one which includes lysine 27 for example, polypeptide having the amino acid sequence as represented in SEQ ID NOS 8 to 14, e.g.
  • the said immunocompetent mammal is a mouse.
  • said immunocompetent mammal is a rat.
  • a method of identifying agents which bind to the ligands as hereinbefore described comprising using competition assays.
  • competition between binding members or agents may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one agent which can be detected in the presence of other untagged agent (s), to enable identification of agents which bind the same epitope or linear peptide sequence or an overlapping epitope or linear peptide sequence.
  • the present invention provides a method of obtaining one or more agents able to bind an epitope of GPVI, the method including bringing into contact a library of agents according to the invention and the GPVI epitope or linear peptide sequence, and selecting one or more agents of the library able to bind the epitope or linear peptide sequence.
  • the GPVI epitope or linear peptide epitope is in particular a peptide moiety (a) as described herein e.g. one which binds to lysine 27 e.g. one represented by SEQ ID NOS 8-14 e.g. SEQ ID No 11.
  • the library may be displayed on the surface of bacteriophage particles, each particle containing nucleic acid encoding the antibody VH variable domain displayed on its surface, and optionally also a displayed VL domain if present.
  • nucleic acid may be taken from a bacteriophage particle displaying a said selected agent.
  • nucleic acid may be used in subsequent production of an agent or an antibody VH variable domain (optionally an antibody VL variable domain) by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage particle displaying a said selected agent.
  • An antibody VH variable domain with the amino acid sequence of an antibody VH variable domain of a said selected specific binding member may be provided in isolated form, as may an agent comprising such a VH domain.
  • an agent comprising such a VH domain there is ability to bind human GPVI may be further tested, also ability to compete with hGP 5C4.
  • an agent to bind human glycoprotein VI may be tested by immunoassay. Any form of direct binding assay is suitable.
  • the human glycoprotein VI or alternatively the agent is labeled. Suitable labels include radioisotopes such as 125 I, enzymes such as peroxidase, fluorescent labels such as fluorescein, and chemiluminescent labels.
  • the other binding partner is insolubilized (for example, by coating onto a microtiter plate) to facilitate washing. After combining the labeled component with the insolubilized component, the solid phase is washed and the amount of bound label is determined.
  • the agent is titered for its ability to decrease the binding of for example hGP 5C4 Fab to human glycoprotein VI, or human glycoprotein VI to subendothelial collagen. Either of the binding pairs in the reaction to be inhibited is labeled, while the other is typically insolubilized in order to facilitate washing. Agents with the characteristics of hGP 5C4 Fab will proportionately decrease the amount of label attached to the solid phase, compared with control polypeptides. Any other characterization may be carried out as specifically described in the examples.
  • the present invention provides a method of inhibiting platelet-collagen interaction in a mammal, which method comprises acutely or chronically administering to a mammal in need of inhibition of platelet-collagen interaction a therapeutically effective amount, including a prophylactically effective amount, of one or more products of the invention.
  • Included in the invention is a method of screening a plurality of compounds by an assay which utilises at least one ligand, (poly)peptide or agent of the present disclosure to determine whether a compound binds to GPVI, in particular at an epitope identified herein, or to an epitope identified herein.
  • Also provided by the present invention is a process for preparing a pharmaceutical composition for treating thrombotic disorders, comprising:
  • the binding affinity may be measured by measuring the IC50 values of the compounds.
  • step (a) may comprise obtaining IC50 value for each compound.
  • the selected compound has an IC50 of less than 500 nM, e.g. less than 100 nM, less than 10 nm, less than 1 nM or less than 0.1 nM.
  • the predetermined binding affinity may be selected accordingly. In any event, the predetermined binding affinity is suitably one which, within the scope of sound pharmacological judgement, is potentially or actually useful for a therapeutic inhibitor of GPVI.
  • the screening methods described above may further include the step of providing the plurality of compounds, e.g. at least 100 compounds, at least 100 compounds or at least 10,000 compounds.
  • the at least one target is at least one ligand or (poly)peptide of the disclosure which binds to antibody hGP 5C4 or includes an amino acid sequence comprising at least a portion of an epitope to the antibody.
  • the at least one target is at least one agent of the disclosure which binds to a said ligand or (poly)peptide, e.g. which binds to antibody hGP 5C4.
  • Also provided by the present invention is a process for preparing a pharmaceutical composition for treating thrombotic disorders comprising:
  • the present invention also provides use of a compound as identified by the methods described above in a method to treat and/or prevent thrombotic disorders.
  • GPVI antagonists which bind to a ligand, epitope or (poly)peptide of the disclosure, for example with a binding affinity which, within the scope of sound pharmacological judgement, is potentially or actually useful for a therapeutic inhibitor of GPVI.
  • Exemplary agonist have an IC50 of less than 100OnM, more particularly of less than 500 nM, e.g. less than 100 nM, less than 10 nm, less than 1 nM or less than 0.1 nM.
  • the antagonist may be an antibody (including an antibody fragment or a molecule comprising an antigen-binding region of an antibody) or an aptamer.
  • agents which bind to an epitope of, or linear peptide sequence comprised within, human Glycoprotein VI can be used in the treatment of any disease, state or condition involving the interaction of platelet bound GPVI and collagen and subsequent activation of the platelets.
  • Active lesions are characterized by the unmasking of subendothelial matrix collagens and platelet activation.
  • the occurrence of such lesions can be investigated e.g. by intravascular ultrasound or thermography (e.g., Fayed and Fuster, Clinical imaging of the high-risk or vulnerable atherosclerotic plaque. Circulation 2001; 89:305-316) or nuclear resonance imaging (Helft et al., Progression and Regression of Atherosclerotic Lesions. Circulation 2002; 105:993-998).
  • the dimeric form of the Fc-GPVI-nt fusion protein serves as and ideal diagnostic tool for the identification of endothelial lesions in patients (EP 03/05929).
  • Such lesions are highly probable in patients with acute coronary or carotid syndromes, and the risk of the reoccurrence of acute clinical complications such as myocardial infarction or stroke is very high, decreasing progressively with increasing time distance from the primary event.
  • the present invention provides a method of treating a patient suffering from an acute coronary or carotid syndrome for avoiding intravascular thrombosis. Moreover, based on the present invention, it is possible to treat patients being at risk of intravascular thrombosis due to the rupture of complex arteriosclerotic plaques. The rupture also unmasks the subendothelial collagen matrix. As a consequence of intraarterial thrombus formation, the perfusion of vital organs is blocked with the above described important and life threatening clinical syndromes.
  • aspects of the invention provide methods of treatment comprising, administration of an agent as provided, pharmaceutical compositions comprising such a agent, and use of such an agent in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the agent with a pharmaceutically acceptable excipient.
  • Clinical indications in which an agent which binds to an epitope of GPVI may be used to provide therapeutic benefit include any condition in which collagen recognition by GPVI has pathological consequences, for example in cardiovascular conditions such as thrombosis, including for example arterial thrombosis occurring in blood vessel wall disease (e. g. coronary artery thrombosis, which causes myocardial infarction). Similar thrombotic processes may occur in other serious conditions at diverse anatomical locations, for instance in the cerebral vasculature, leading to stroke, or in the peripheral extremities. In the latter case for instance, patients with intermittent claudication may be treated. Agent-mediated blockade of GPVI may be used and be beneficial during therapeutic procedures which induce damage to the blood vessel wall, for instance vascular surgery.
  • thrombosis including for example arterial thrombosis occurring in blood vessel wall disease (e. g. coronary artery thrombosis, which causes myocardial infarction). Similar thrombotic processes may occur in other serious conditions at diverse anatomic
  • EExamples of vascular surgery may include, but are not limited to, coronary artery bypass grafting, balloon angioplasty and stenting.
  • circulating platelets may be exposed to collagens where they may contribute to local thrombotic effects and to the inflammatory processes which ensue.
  • An example of the latter occurs in hepatitis where the hepatic circulation is compromised by the disease.
  • diseases of generalised platelet activation such as thrombocytopenic purpura and haemolytic uraemic syndrome and other clinical conditions with disseminated intravascular coagulation may be ameliorated.
  • multi-organ damage because of arterial insufficiency in patients with homozygous sickle disease may be beneficially affected by inhibiting the activation of platelets via GPVI.
  • kidney damage by platelet and fibrin disposition on the glomerular membrane and other conditions such as micro-angiopathic vasculitides may be treated by agent- mediated GPVI blockade.
  • Anti-GPVI treatment in accordance with the present invention may be used to provide clear benefit for patients with cardiovascular disease, especially those who have undergone corrective vessel surgery or angioplasties with or without stenting.
  • Anti-GPVI treatment may be given by injection (e. g. intravenously) or by local delivery methods (e.g. pre- coating of stents or other indwelling devices).
  • Anti-GPVI may be delivered by gene-mediated technologies.
  • Alternative formulation strategies may provide preparations suitable for oral or suppository route. The route of administration may be determined by the physicochemical characteristics of the treatment, by special considerations for the disease, to optimise efficacy or to minimise side-effects.
  • the agents of the inventions may be used to treat and/or protect against a variety of disorders, including, for example, seizures, transient ischemic shock, strokes, focal ischemia originating from thrombus or cerebral hemorrhage,global ischemia originating from cardiac arrest, trauma, neonatal palsy, hypovolemic shock, and hyperglycemia and associated neuropathies.
  • the present inventive method includes the administration to an animal, such as a mammal, particularly a human, in need of the inhibition of platelet activation of an effective amount, e. g., a therapeutical effective amount, of one or more of the aforementioned present inventive agents, alone or in combination with one or more other pharmaceutically active agents.
  • the agents of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation,
  • the agents may be administered in the form of pharmaceutical preparations in a pharmaceutical acceptable dosage form.
  • the compositions may be administered at varying doses.
  • an agent of the invention is to be administered to an individual, it is particularly at least 80% pure, more preferably it is at least 90% pure, even more preferably it is at least 95% pure and free of pyrogens and other contaminants.
  • the percent purity is calculated as a weight percent of the total protein content of the preparation, and does not include constituents which are deliberately added to the composition after the agent is purified.
  • the present invention provides a method of treatment of a human or animal body in need of treatment or prevention of acute or chronic vascular diseases associated with intraarterial and/or intravenous thrombosis, which comprises administration to a human or animal of a pharmaceutically effective amount of an inhibitor or agent of the invention.
  • the dosage will usually be in the range of from 0.1 to 100 mg/patient/ day.
  • the agents are used as lyophilised powders solubilised in PBS/succrose/manitol-buffer prior to parenteral administration.
  • a human or animal body in need of treatment or prevention of acute or chronic vascular diseases associated with intraarterial and/or intravenous thrombosis is characterized by active lesions due to unmasking of subendothelial matrix collagens and platelet activation. The occurrence of such lesions can be investigated e.g. by intravascular ultrasound or thermography (e.g., Fayed and Fuster, Clinical imaging of the high-risk or vulnerable atherosclerotic plaque.
  • Such lesions are highly probable in patients with acute coronary or carotid syndromes, and the risk of the reoccurrence of acute clinical complications such as myocardial infarction or stroke is very high, decreasing progressively with increasing time distance from the primary event.
  • the most preferred routes of administration are injection and infusion, especially intravenous administration.
  • the compounds of the invention may be combined and/or co-administered with any antithrombotic agent, such as the antiplatelet agents acetylsalicylic acid,ticlopidine, dopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists.
  • any antithrombotic agent such as the antiplatelet agents acetylsalicylic acid,ticlopidine, dopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists.
  • antithrombotic agent such as the antiplatelet agents acetylsalicylic acid,ticlopidine, dopidogrel, thromboxane receptor and/or synthetase inhibitor
  • the agents of the invention may be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
  • tissue plasminogen activator naturally, recombinant or modified
  • streptokinase urokinase
  • prourokinase prourokinase
  • anisoylated plasminogen-streptokinase activator complex APSAC
  • animal salivary gland plasminogen activators and the like
  • the pharmaceutical compounds of the invention may be administered orally or parenterally ("parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion of which intavenous is most preferred) to a host to obtain a desired effect, for example protection against thrombosis.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion of which intavenous is most preferred
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion of which intavenous is most preferred
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion of which intavenous is most preferred
  • the compounds may be administered alone or as compositions
  • Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active agent (s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration.
  • the selected dosage level will depend upon the activity of the particular agent, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the agent at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the products of this invention are administered prophylactically.
  • Another aspect of this invention is directed to methods for treating cardiovascular diseases comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating arteriosclerosis comprising administering to a mammal a therapeutical effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating arrhythmia comprising administering to a mammal a therapeutical effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating angina pectoris comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating cardiac hypertrophy comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating renal diseases comprising administering to a mammal a therapeutical effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating diabetic complications comprising administering to a mammal a therapeutical effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating restenosis comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating organ hypertrophies or hyperplasias comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • Another aspect of this invention is directed to methods for treating septic shock and other inflammatory diseases (septicemia, endotoxcemia) comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • septic shock and other inflammatory diseases septicemia, endotoxcemia
  • Another aspect of this invention is directed to methods for treating cerebro ischemic disorders comprising administering to a mammal a therapeutically effective amount of a product of the invention.
  • the present invention further provides a method of modulating GPVI activity comprising administering an effective amount of an agent of the present invention.
  • a method for treating therapeutic or prophylactic a disease or disorder selected from therapeutic or prophylactic cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides comprising administering an agent of the disclosure, to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • the agents included in the invention may be used for the manufacture of a medicament to treat or prevent of a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • the compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation,
  • the compounds may be administered in the form of pharmaceutical preparations.
  • the compositions may be administered at varying doses.
  • the products of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the inhibitors of thrombin or other coagulation enzymes (e.g. Factor IXa or X), antiplatelet agents acetylsalicylic acid, tidopidine, dopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P 2 T) antagonists.
  • thrombin or other coagulation enzymes e.g. Factor IXa or X
  • antiplatelet agents acetylsalicylic acid, tidopidine, dopidogrel, thromboxane receptor and/or synthetase inhibitors
  • fibrinogen receptor antagonists e.g., prostacyclin mimetics and phosphodiesterase inhibitors
  • the GPVI inhibitors of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
  • tissue plasminogen activator naturally, recombinant or modified
  • streptokinase urokinase
  • prourokinase prourokinase
  • anisoylated plasminogen-streptokinase activator complex APSAC
  • animal salivary gland plasminogen activators and the like
  • the products of the disclosure may be combined and/or co-administered with any cardiovascular treatment agent.
  • cardiovascular treatment agents available in commercial use, in clinical evaluation and in pre-clinical development, which could be selected for use with a product of the disclosure for the prevention of cardiovascular disorders by combination drug therapy.
  • Such agent can be one or more agents selected from, but not limited to several major categories, namely, a lipid-lowering drug, including an IBAT (ileal Na + /bile acid cotransporter) inhibitor, a fibrate, niacin, a statin, a CEETP (cholesteryl ester transfer protein) inhibitor, and a bile acid sequestrant, an anti-oxidant, including vitamin E and probucol, a Ilb/IIIa antagonist (e.g. abciximab, eptifibatide, tirofiban), an aldosterone inhibitor (e.g. spirolactone and epoxymexrenone), an adenosine A2 receptor antagonist (e.g.
  • losartan an adenosine A3 receptor agonist, a beta-blocker, acetylsalicylic acid, a loop diuretic, an angiotensin receptor blocker and an ACE (angiotensin converting enzyme) inhibitor.
  • the products of the disclosure may be combined and/or co-administered with a cardioprotectant, for example an adenosine Al or A3 receptor agonist.
  • a cardioprotectant for example an adenosine Al or A3 receptor agonist.
  • a method for treating a cardiovascular disease in a patient that comprises treating the patient with a product of the disclosure and an NSAID, e.g., a COX-2 inhibitor. Accordingly, the products of the disclosure may be combined and/or co-administered with an NSAID.
  • an NSAID e.g., a COX-2 inhibitor
  • Actual dosage levels of active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration (referred to herein as a "therapeutically effective amount").
  • the selected dosage level will depend upon the activity of the particular compound, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • a parenteral formulation including a product as described herein.
  • the formulation may consist of the product alone or it may contain additional components, in particular the product may be in combination with a pharmaceutically acceptable diluent, excipient or carrier, for example a tonicity agent for the purpose of making the formulation substantially isotonic with the body of the subject to receive the formulation, e.g. with human plasma.
  • a pharmaceutically acceptable diluent, excipient or carrier for example a tonicity agent for the purpose of making the formulation substantially isotonic with the body of the subject to receive the formulation, e.g. with human plasma.
  • the formulation may be in ready-to-use form or in a form requiring reconstitution prior to administration.
  • Parenteral preparations can be administered by one or more routes, such as intravenous, subcutaneous, intradermal and infusion; a particular example is intravenous.
  • a formulation disclosed herein may be administered using a syringe, injector, plunger for solid formulations, pump, or any other device recognized in the art for parenteral administration.
  • Liquid dosage forms for parenteral administration may include solutions, suspensions, liposome formulations, or emulsions in oily or aqueous vehicles.
  • the liquid dosage forms may contain other compounds.
  • Tonicity agents for the purpose of making the formulations substantially isotonic with the subject's body, e.g.
  • cellulose derivatives e.g. methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose
  • gelatin and/or acacia may optionally be added to the parenteral formulation.
  • Solid dosage forms for parenteral administration may encompass solid and semi-solid forms and may include pellets, powders, granules, patches, and gels.
  • the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier.
  • the disclosed products may be presented as solids in finely divided solid form, for example they may be milled or micronised.
  • the formulations may also include antioxidants and/or preservatives.
  • antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid.
  • Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.
  • the parenteral formulations may be prepared as large volume parenterals (LVPs), e.g. larger than 100 ml, more particularly about 250 ml, of a liquid formulation of the active compound.
  • LVPs are infusion bags.
  • the parenteral formulations may alternatively be prepared as small volume parenterals (SVPs), e.g. about 100 ml or less of a liquid formulation of the active compound.
  • SVPs are vials with solution, vials for reconstitution, prefilled syringes for injection and dual chamber syringe devices.
  • the active compound or compounds can be present at varying concentrations, with a carrier acceptable for parenteral preparations making up the remainder.
  • the carrier is water, particularly pyrogen free water, or is aqueous based.
  • the carrier for such parenteral preparations is an aqueous solution comprising a tonicity agent, for example a sodium chloride solution.
  • aqueous based is meant that formulation comprises a solvent which consists of water or of water and water-miscible organic solvent or solvents; as well as containing a product of disclosure in dissolved form, the solvent may have dissolved therein one or more other substances, for example an antioxidant and/or an isotonicity agent.
  • organic cosolvents may be mentioned those water-miscible solvents commonly used in the art, for example propyleneglycol, polyethyleneglycol 300, polyethyleneglycol 400 and ethanol.
  • organic co-solvents are only used in cases where the active agent is not sufficiently soluble in water for a therapeutically effective amount to be provided in a single dosage form.
  • the solubility of the active compound in the present formulations may be such that the turbidity of the formulation is lower than 50 NTU, e.g. lower than 20 NTU such as lower than 10 NTU.
  • parenteral formulations are administered at or near physiological pH. It is believed that administration in a formulation at a high pH (i.e., greater than 8) or at a low pH (i.e., less than 5) is undesirable. In particular, it is contemplated that the formulations would most desirably be administered at a pH of between 6.0 and 7.0 such as a pH of 6.5.
  • the pH values mentioned in this paragraph are not critical, however, and formulations may fall outside them.
  • the parenteral formulation may be purged of air when being packaged.
  • the parenteral formulation may be packaged in a sterile container, e.g. vial, as a solution, suspension, gel, emulsion, solid or a powder.
  • a sterile container e.g. vial
  • Such formulations may be stored either in ready-to-use form or in a form requiring reconstitution prior to administration.
  • Parenteral formulations according to the disclosure may be packaged in containers.
  • Containers may be chosen which are made of material which is non-reactive or substantially non-reactive with the parenteral formulation.
  • Glass containers or plastics containers e.g. plastics infusion bags, may be used.
  • a concern of container systems is the protection they afford a solution against UV degradation. If desired, amber glass employing iron oxide or an opaque cover fitted over the container may afford the appropriate UV protection.
  • Plastics containers such as plastics infusion bags are advantageous in that they are relatively light weight and non-breakable and thus more easily stored. This is particularly the case for Large Volume parenterals.
  • the intravenous preparations may be prepared by combining the active product or products with the carrier. After the formulation is mixed, it may be sterilized, for example using known methods. Once the formulation has been sterilized, it is ready to be administered or packaged, particularly in dark packaging (e.g. bottles or plastics packaging), for storage. It is envisaged, however, that the disclosed products might not be stored in solution but as dry solids, particularly a finely divided form such as, for example, a lyophilisate, in order to prolong shelf life; this would of course apply to other parenteral formulations, not only intravenous ones.
  • the intravenous preparations may take the form of large volume parenterals or of small volume parenterals, as described above.
  • kits for producing a single-dose administration unit.
  • the products may each contain both a first container having the active compound (optionally combined with additives, for example antioxidant, preservative and, in some instances, tonicity agent) and a second container having the carrier/diluent (for example water, optionally containing one or more additives, for example tonicity agent).
  • additives for example antioxidant, preservative and, in some instances, tonicity agent
  • second container having the carrier/diluent (for example water, optionally containing one or more additives, for example tonicity agent).
  • additives for example antioxidant, preservative and, in some instances, tonicity agent
  • the carrier/diluent for example water, optionally containing one or more additives, for example tonicity agent
  • Such dual chamber syringes or binary syringes will have in one chamber a dry preparation including or consisting of the active compound and in another chamber a suitable carrier or diluent such as described herein.
  • the two chambers are joined in such a way that the solid and the liquid mix to form the final solution.
  • formulations disclosed herein comprises subcutaneous or intradermal formulations (for example formulations for injection) in which the active product (or active agent combination) is formulated into a parenteral preparation that can be injected subcutaneously or intradermally.
  • the formulation for administration will comprise the active product and a liquid carrier.
  • the carrier utilized in a parenteral preparation that will be injected subcutaneously or intradermally may be an aqueous carrier (for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent) or a nonaqueous carrier (again one or more additives may be incorporated).
  • a non-aqueous carrier for such parenteral preparations may be mentioned highly purified olive oil.
  • the active compound and the carrier are typically combined, for example in a mixer. After the formulation is mixed, it is preferably sterilized, such as with U.V. radiation. Once the formulation has been sterilized, it is ready to be injected or packaged for storage. It is envisaged, however, that the disclosed products will not be stored in liquid formulation but as dry solids, in order to prolong shelf life.
  • the active product may suitably be formulated together with one or more polymers that are gradually eroded or degraded when in use, e.g. silicone polymers, ethylene vinylacetate, polyethylene or polypropylene.
  • Transdermal formulations may be prepared in the form of matrices or membranes, or as fluid or viscous formulations in oil or hydrogels or as a compressed powder pellet.
  • an adhesive which is compatible with the skin may be included, such as polyacrylate, a silicone adhesive or polyisobutylene, as well as a foil made of, e.g., polyethylene, polypropylene, ethylene vinylacetate, polyvinylchloride, polyvinylidene chloride or polyester, and a removable protective foil made from, e.g., polyester or paper coated with silicone or a fluoropolymer.
  • water or organic solvents or mixtures thereof may be used.
  • Transdermal gels may furthermore contain one or more suitable gelling agents or thickeners such as silicone, tragacanth, starch or starch derivatives, cellulose or cellulose derivatives or polyacrylic acids or derivatives thereof.
  • Transdermal formulations may also suitably contain one or more substances that enhance absorption though the skin, such as bile salts or derivatives thereof and/or phospholipids.
  • Transdermal formulations may be prepared according to a method disclosed in, e.g., B W Barry, "Dermatological Formulations, Percutaneous Absorption", Marcel Dekker Inc., New York-Basel, 1983, or Y W Chien, "Transdermal Controlled Systemic Medications", Marcel Dekker Inc., New York-Basel, 1987.
  • the pharmaceutical products of the invention may be administered orally or parenterally ("parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.) to a host to obtain an protease-inhibitory effect.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
  • a pharmaceutical composition including a described product, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • compositions of this invention for parenteral injection or infusion suitably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • solvents co-solvents or additives for parenteral, e.g. intravenous, or other formulations may be mentioned: • Acids, e.g. with pH greater than 1.8
  • Cremophor EL e.g. up to 25% in water
  • Tween surfactants e.g. up to 2%
  • microsphere-based delivery systems composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
  • PLG poly-DL-lactide-co-glycolide
  • OEDs lipophilic carbohydrate excipients
  • Intravenous and other parenteral compositions may be provided as ready-to-use solutions or as lyophilisates or dry powders for reconstitution prior to administration.
  • Parenteral and other compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents.
  • adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example.
  • Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example aluminum monostearate and gelatin) which delay absorption.
  • the absorption of the drug in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are suitably made by forming microencapsule matrices of the drug in biodegradable polymers, for example polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. EExamples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate;
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.
  • oral formulations contain a dissolution aid.
  • the dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable.
  • EExamples include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, poly
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion.
  • coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion.
  • opacifying agents include polymeric substances and waxes.
  • the active compounds may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • the active compounds may be in finely divided form, for example it may be micronised.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, is
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p 33 et seq.
  • Dosage forms for topical administration of a compound of this invention include powders, sprays, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required.
  • Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • the present invention provides inter alia antibody hGP 5C4 and its fragments, as well as humanised versions thereof such as humanised Fab fragments, for example, in a formulation as described above.
  • Exemplary formulations are parenteral formulations as described above. To be mentioned, therefore, are the following formulations containing as active agent hGP 5C4, humanized hGP 5C4 or a Fab or other fragment, optionally in combination with one or more other active agents:
  • a liquid dosage form for parenteral administration for example a solution, suspension, liposome formulation, or emulsion in oily or aqueous vehicles.
  • the liquid dosage forms may contain e.g. tonicity agents (for the purpose of making the formulations substantially isotonic with the subject's body, e.g. with human plasma) such as, for instance, sodium chloride, sodium sulfate, dextrose, mannitol and/or glycerol may be optionally added to the parenteral formulation.
  • a pharmaceutically acceptable buffer may be added to control pH.
  • Thickening or viscosity agents for instance well known cellulose derivatives (e.g. methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropyl- methylcellulose), gelatin and/or acacia, may optionally be added to the parenteral formulation.
  • LVPs are infusion bags.
  • SVPs are vials with solution, vials for reconstitution, prefilled syringes for injection and dual chamber syringe devices.
  • Solid dosage forms for parenteral administration may encompass solid and semi-solid forms and may include pellets, powders, granules, patches, and gels.
  • Subcutaneous or intradermal formulations for example formulations for injection
  • the active product or active agent combination
  • the formulation for administration will comprise the active product and a liquid carrier.
  • a parenteral preparation having an aqueous carrier for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent, for example solutions, dispersions, suspensions or emulsions.
  • aqueous carrier for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent
  • solutions, dispersions, suspensions or emulsions for example solutions, dispersions, suspensions or emulsions.
  • a parenteral formulation having a nonaqueous carrier for example solutions, dispersions, suspensions or emulsions.
  • Pharmaceutically acceptable non-aqueous carriers can be fully saturated, or partially or fully unsaturated. EExamples of non-aqueous carriers include, but are not limited to:
  • Vegetable oils such as cottonseed oil, corn oil, sesame oil, soybean oil, olive oil, fractionated coconut oils, peanut oil, sunflower oil, safflower oil, almond oil, avocado oil, palm oil, palm kernel oil, babassu oil, beechnut oil, linseed oil, rape oil, and the like), mineral oils, synthetic oils, and combinations thereof.
  • Fully saturated non-aqueous carriers examples of which include, but are not limited to, medium to large chain fatty acids (e.g. capric acid and/or caprylic acid) and particularly esters thereof (such as fatty acid triglycerides with a chain length of about 6C to about 24C); mixtures of fatty acids are split from the natural oil (for example coconut oil palm kernel oil, babassu oil, or the like) and are refined. In some embodiments, about 8C to about 12C fatty acid medium chain triglycerides are useful.
  • medium to large chain fatty acids e.g. capric acid and/or caprylic acid
  • esters thereof such as fatty acid triglycerides with a chain length of about 6C to about 24C
  • mixtures of fatty acids are split from the natural oil (for example coconut oil palm kernel oil, babassu oil, or the like) and are refined.
  • about 8C to about 12C fatty acid medium chain triglycerides are useful.
  • non-aqueous carriers include, but are not limited to, saturated coconut oil (which typically includes a mixture of lauric, myristic, palmitic, capric and capric acids), including those sold under the Miglyo trademark from HuIs and bearing trade designations 810, 812, 829, and 840). Also noted are the NeoBee products sold by Drew Chemicals. Isopropyl myristate is another example of a non-aqueous carrier.
  • Synthetic oils examples of which include triglycerides, and propylene glycol diesters of saturated or unsaturated fatty acids having from 6 to 24 carbon atoms such as, for example hexanoic acid, octanoic (caprylic), nonanoic (pelargonic), decanoic (capric), undecanoic, lauric, tridecanoic, tetradecanoic (myristic), pentadecanoic, hexadecanoic (palmitic), heptadecanoic, octadecanoic (stearic), nonadecanoic, heptadecanoic, eicosanoic, heneicosanoic, docosanoic, and lignoceric acids, and the like.
  • hexanoic acid octanoic (caprylic), nonanoic (pelargonic), decanoic (capric), undecanoic, lauric, tri
  • Unsaturated carboxylic acids examples of which include oleic, linoleic, and linolenic acids, and the like.
  • non-oil for example polyethylene glycol.
  • non-aqueous carrier can comprise the mono-, di-, and triglyceryl esters of fatty acids or mixed glycerides and/or propylene glycol diesters wherein at least one molecule of glycerol has been esterified with fatty acids of varying carbon atom length 11.
  • the injectable formulation may be in an aqueous carrier or a non-aqueous carrier.
  • Formulations comprising as aqueous and nonaqueous carriers, diluents, solvents or vehicles water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, for example), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, for example
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • the features of this paragraph may be applied to the formulations of any of preceding paragraphs 1, 2, 3, 4, 5, 6, 8, 9, 10 and 11.
  • Formulations comprising an acid, e.g. formulations with pH greater than 1.8, for example at least 2, e.g. at least 3, as in the case of formulations having a pH of at least 4. Often acidic formulations have a pH of at least 5.
  • Formulations comprising a base, e.g. formulations with pH of less than 14 for example less than 13, e.g. no more than 12, as in the case of formulations having a pH of no more than 11.
  • a base e.g. formulations with pH of less than 14 for example less than 13, e.g. no more than 12, as in the case of formulations having a pH of no more than 11.
  • acidic formulations have a pH of no more than 10, particularly no more than 9.
  • the pH is no more than 8.
  • Cremophor EL aqueous carrier
  • Cremophor EL is also known asPolyoxyl 35 Castor Oil.
  • Cremophor EL is a non-ionic solubilizer and emulsifier obtained by causing ethylene oxide to react with castor oil of German Pharmacopoeia (DAB 8) quality in a molar ratio of 35 moles to 1 mole. Cremophor EL forms clear solutions in water.
  • Formulations comprising dextrose, e.g. up to 5%, in an aqueous solvent, for example water or saline.
  • Formulations comprising ethanol, e.g. up to 15% and optionally up to 5%, in an aqueous solvent, for example water or saline.
  • Formulations comprising glycerol, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising sorbitol e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising phosphate buffer.
  • Formulations comprising polyethylene glycol, e.g. PEG 300 or 400, neat or e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising propylene glycol or a propylene glycol derivative, for example propylene glycol alginate, neat or or e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising a sugar, e.g. lactose, sucrose or glucose, whether as a solid or in solution, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • a sugar e.g. lactose, sucrose or glucose
  • an alcoholic or aqueous solvent for example water or saline.
  • Formulations comprising an antioxidant, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising an amino acid additive, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising a lipid, e.g. a phospholipid.
  • Formulations comprising a polyoxyethylenesorbitan ester surfactant, e.g. Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan monopalmitate), Tween 60 (polyoxyethylenesorbitan monostearate), Tween 80 (polyoxyethylenesorbitan monooleate ) or Tween 85 (polyoxyethylenesorbitan trioleate).
  • a polyoxyethylenesorbitan ester surfactant e.g. Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan monopalmitate), Tween 60 (polyoxyethylenesorbitan monostearate), Tween 80 (polyoxyethylenesorbitan monooleate ) or Tween 85 (polyoxyethylenesorbitan trioleate).
  • Formulations comprising a poloxamer (poly(oxyethylene)-poly(oxypropylene) block copolymer).
  • Formulations comprising a Solutol, for example Solutol HS 15 (Polyethylene glycol-15- hydroxystearate), e.g. up to 30% in water
  • Formulations comprising a microsphere-based delivery system, e.g. composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
  • PLG poly-DL-lactide-co-glycolide
  • Formulations comprising lipophilic carbohydrate excipients, termed oligosaccharide ester derivatives (OEDs), which have been used to modify pharmacokinetic profiles of drugs (SoliDoseTM technology, Elan Pharmaceuticals).
  • Intravenous and other parenteral compositions provided as ready-to-use solutions, suspensions, liposome formulations, or emulsions in oily or aqueous vehicles
  • Intravenous and other parenteral compositions provided as lyophilisates or dry powders for reconstitution prior to administration.
  • transdermal formulation for example in the form of matrices or membranes, or as fluid or viscous formulations in oil or hydrogels or as a compressed powder pellet. See above for further details.
  • Injectable depot forms e.g. comprising microencapsule matrices of the drug in biodegradable polymers, for example a polylactide-polyglycolide, poly(orthoester) and poly(anhydride), or comprising the drug entrapped in liposomes or microemulsions which are compatible with body tissues.
  • biodegradable polymers for example a polylactide-polyglycolide, poly(orthoester) and poly(anhydride
  • Formulations comprising a biodegradable polymer, e.g. one mentioned previously.
  • the compounds of the invention are orally active, have rapid onset of activity and low toxicity.
  • the compounds of the invention have the advantage that they may be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or that they may have other useful pharmacological properties over, compounds known in the prior art.
  • the invention includes a method for the peri-interventional prevention of restenosis and/or thrombosis comprising administering to a subject an effective amount of an agent of the disclosure, e.g. hGP 5C4, humanised hGP 5C4 or a fragment of either.
  • the invention also includes a method for the long term or chroncic prevention of atherosclerosis and/or arterial thrombosis, comprising administering to a subject an effective amount of an agent of the disclosure, e.g. hGP 5C4, humanised hGP 5C4 or a fragment of either.
  • Lou/C rats were immunized with human dimeric Fc-GPVI-nt fusion protein having the amino acid sequence as shown in Figure 10.
  • Screening of hybridoma supernatants was performed in a solid-phase immunoassay using dimeric Fc-GPVI-nt or Fc lacking the GPVI domain. Screening identified the supernatant of hybridoma different antibodies to bind specifically to dimeric Fc-GPVI-nt but not to Fc lacking the external GPVI domain.
  • the immunoglobulin type was determined with rat Ig class (anti-IgM) and IgG subdass-specific mouse mAbs.
  • the monoclonal antibodies were purified using Protein G-Sepharose columns.
  • hGP 5C4 Antibody specificity of hGP 5C4 was verified by immunoblotting against dimericFc-GPVI-nt and control Fc. hGP 5C4 monoclonal antibody detected recombinant dimeric Fc-GPVI-nt but not control Fc (see figu re. 1 a, top). Furthermore, hGP 5C4 binds specifically to the surface of human platelets (see figu re 1 a, bottom).
  • the method of this example may be modified by using as an immunogen another ligand disclosed herein e.g. a peptide moiety (a) in particular one which includes lysine 27 for example are represented by SEQ ID NOS 9 to 14
  • Fab-fragments of monoclonal IgG antibodies Complete IgG antibodies were digested to generate Fab-fragments of anti-GPVI antibodies with ImmunoPure Fab Kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer's instructions. Accordingly, IgG molecules were digested into Fab fragments and Fc fragments by using immobilized papain. After digestion, the fragments were purified on an immobilized Protein A column. Detailed instructions allow for flexibility in the protocol for hard to digest antibodies. The success of Fab-fragment generation was tested by comparing molecular size of both antibody formats in SDS gels and staining with Coomassie blue (see f igu re 1 b).
  • the extra-cellular domain of human GPVI was cloned and fused to the human immunoglobin Fc-domain.
  • the Fc was amplified from a human heart cDNA library (Clonetech, Palo Alto, CA) by PCR using the forward primer 5'-cgcggggcggccgcgagt- ccaaatcttgtgacaaaac-3' and the reverse primer 5'-gcgggaagctttcatttacccggagacagggag-3'.
  • the PCR reaction was performed at 58 0 C annealing temperature and 20 cycles with the EExpand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).
  • the PCR fragment was cloned in the plasmid pADTrack CMV with Notl/Hindlll and the sequence was checked by sequencing (MediGenomix, Martinsried, Germany).
  • RNA from cultured megakaryocytes was isolated (RNeasy Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer's protocol and reverse transcription was performed (Omniscript RT Kit; Qiagen) with 2 ⁇ g RNA at 37 0 C overnight. 100 ng of the reaction was used as a template in PCR amplification of the hGPVI with the primer 5'-gcggggagatctaccaccatgtctccatccccgacc-3' and 5'- cgcggggcggccgcgttgcccttggtgtagtac-3'.
  • the PCR reaction was performed at 54 0 C annealing temperature and 24 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).
  • the PCR fragment was cloned in the plasmid pADTrack CMV Fc with Bglll/Notl and the sequence was checked by sequencing.
  • the human Fc-GPVI-nt was amplified from the plasmid pADTrackCMV human Fc-GPVI-nt by PCR using the forward primer 5'- gcgggggctagcaccaccatgtctccatccccgac -3' and the reverse primer 5'- cgcgggggatcctcatttacccggagacagggag -3'.
  • the PCR reaction was performed at 58 0 C annealing temperature and 24 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).
  • PCR fragment was cloned in the plasmid pREP4 (Invitrogen, Carlsbad, CA) with Nhel/BamHI and the sequence of the resulting plasmid pREP4 human Fc-GPVI-nt was checked by sequencing (MediGenomix, Martinsried, Germany).
  • CHO Kl cells DSMZ, Braunschweig, Germany
  • effectene transfection reagent Qiagen, Hilden, Germany
  • the Flp-in system (Invitrogen, Düsseldorf, Germany) was used to generate a stable GPVI expressing GPVI-CHO-FIp-In cell line.
  • full length human GPVI was cloned into the pcDNA5/FRT vector.
  • 0.9 ⁇ g of GPVI-pcDNA5/FRT were co-transfected with 10 ⁇ g of pOG44, encoding the FIp recombinase, into the CHO-FIp-On cells using LipofectAMINE tm together with the Plus 1 " 1 reagent (Invitrogen).
  • Transfected cells were selected in medium containing 0.1 mg/ml hygromycin B. Expression of human GPVI was confirmed by FACS analysis using anti-GPVI monoclonal antibodies.
  • Non-transfected CHO-FIp-In cells served as controls.
  • Fc-GPVI-nt protein and Fc control fully human fusion protein purification.
  • the culture supernatant of Fc-GPVI-nt CHO cells was collected, then applied onto two sequential tangential flow filtration devices using hollow fibre modules at a perfusion/recirculation rate of 10 I /min.
  • a first purification was achieved via a filter from MembraPure, Bodenheim, # 5421534 (Typ Minikross, PEF, 0.5 ⁇ m pore width; 4000 ca m area). Then, the filtrate was concentrated 10- fold via a second module (MembraPure # 5422532; 50 kDa pore width; 3900 cm 2 ).
  • the Fc-GPVI- nt fusion protein was centrifuged (3800 g, 30 min, 4 0 C) filtrated (0.45 ⁇ m) and precipitated by addition of 1 vol. ammonium sulphate (761 g/l) and stirred overnight at 4 0 C.
  • the proteins were pelleted by centrifugation (3000 g, 30 min, 4 0 C), dissolved in 0.1 Vol. PBS and dialysed in PBS overnight at 4 0 C.
  • BenzonaseTM (Merck, cat. no 101695) is added at a concentration of 5 ng/ml to the concentrated and filtrated supernatant to brake down chromosomal DNA into oligonucleotides.
  • the protein solution was clarified by centrifugation (3000 g, 30 min, 4 0 C) and loaded on a protein A column MabSelect (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Some column volumes (cv) of elution buffer, 100 mM Na-citrate pH 3.2- 4.2 are run into the column mainly to be sure that there is no residual protein left from previous runs in the column. The sample is applied at a flow rate of 5 ml/min corresponding to a linear flow of 150 cm/h.
  • the plates were washed with 250 ⁇ l/well PBS/0.05 % Tween 20 (PBST) twice and blocked with 250 ⁇ l/well Roti-Block (Roth, Düsseldorf, Germany) over night.
  • the plates were washed with 250 ⁇ l/well PBST twice, then Fc-GPVI-nt in PBST was added and the plate was incubated for 1 hr at room temperature.
  • Fc-GPVI-nt (20 ⁇ g/ml) was pre-incubated for 10 min with different Fab fragments of antibodies e.g. hGP 5C4 Fab (20 ⁇ g/ml) to determine inhibition of collagen - GPVI interaction.
  • GPVI-expressing CHO cells were generated as described above. Human citrate blood was collected from volunteers Platelet rich plasma (PRP) was generated after centrifugation and washing procedures (PBS 1 x; pH 7.2) with 2000 rpm at 4 0 C and resuspension. GPVI-expressing CHO or control CHO cells were incubated with different antibodies where appropriate. Similarly, human platelets where incubated with different antibodies. Thereafter, secondary anti rat IgG antibodies labelled with peroxidase (Immunotech) were added. FACS measurement was performed and specific mean peroxidase fluorescence was counted with a Becton Dickenson FACScalibur device.
  • FACS measurement for stimulation of different activation markers on platelets Human citrate blood was collected from volunteers. PRP was generated as described and diluted in staining buffer (Ix PBS (w/o Ca 2+ and Mg + ) with 0,1% sodium azide and 2% fetal bovine serum ( FBS ), 2 mM CaCI 2 ) was incubated with bovine collagen type 1 (0; 2; 5 and 10 ⁇ g/ml;
  • PAC-I activation in human platelets was determined in presence of 4C9 Fab and hGP 5C4 Fab.
  • hGP 5C4 Fab had no intrinsic activity for PAC-I expression in unstimulated human platelets, whereas 4C9 Fab leads to a small activation of PAC-I (see figu re 5a) .
  • Stimulation with bovine collagen (10 ⁇ g/ml) activated PAC- 1, which could be abolished by hGP 5C4 (20 ⁇ g/ml) but not by 4C9 Fab.
  • Collagen typically activates human platelets and leads to increased surface expression of CD 62- P.
  • Different anti-GPVI antibodies were tested and 4C4 Fab and hGP 5C4 Fab inhibited the collagen-mediated CD 62P activation (please see f igu re 5b).
  • 4C9 had a potent activating effect on CD 62P in human platelets.
  • hGP 5C4 inhibits collagen-mediated platelet activation without inducing intrinsic platelet activity.
  • Platelet aggregation and A TP release Platelet aggregation and A TP release. Platelet aggregation ex vivo and in vitro was evaluated by optical aggregometry in citrated blood samples at 37° C using a two-channel Chronolog aggregometer (Nobis, Germany). PRP was prepared as described and the final platelet count was adjusted to 2 x 10 8 platelets/ml by Thyrodes-HEPES buffer (2.5 mmol/l HEPES, 150 mmol/l NaCI, 12 mmol/l NaHCO 3 , 2,5 mmol/l KCI, 1 mmol/l MgCI 2 , 2 mmol/l CaCI 2 , 5,5 mmol D-Glucose, 1 mg/ml BSA, pH 7.4).
  • Thyrodes-HEPES buffer 2.5 mmol/l HEPES, 150 mmol/l NaCI, 12 mmol/l NaHCO 3 , 2,5 mmol/l KCI, 1 mmol
  • Chrono-Lume #395 (Chrono-Log Corporation) was added for ATP measurement.
  • PRP was incubated with various antibodies in different concentrations as indicated. Thereafter, agonists were added to the platelets, pipetted into the aggregometer and aggregation was started under defined stirring conditions. Aggregation was determined by change of light transmission due to coagulating platelets and normalised to an internal standard. ATP release is determined at the characteristic wavelength of Chrono-Lume for ATP and normalised to an internal standard according to the manufacturer's instructions. Inhibition of human platelet aggregation and A TP release by hGP 5C4 Fab.
  • hGP 5C4 Fab potently inhibited human platelet aggregation [in % of internal standard] ex vivo with an IC50 value of 1,2 x 10 ⁇ 7 g/ml (see f igu re 7a) .
  • hGP 5C4 inhibited collagen-mediated ATP release but had no effect on thrombin/TRAP-mediated ATP release even at higher concentrations (2 ⁇ g/ml and 6 ⁇ g/ml). ADP-mediated ATP release, however, was also inhibited by the maximal hGP 5C4 Fab concentrations (6 ⁇ g/ml) (see figu re 8b). This also underlines the specificity of inhibition of collagen-mediated platelet aggregation of hGP 5C4 Fab. Moreover, the release of potent mediator substances from intracellular stores was also potently and selectively inhibited by hGP 5C4.
  • In vitro bleeding time was determined with a PFA-100 device (Dade-Behring). 800 ⁇ l of human whole blood was injected in the PFA-100 device. Bleeding time was measured with ADP/collagen and epinephrine/collagen coated measuring cells according to the manufacturer's instructions.
  • hGP 5C4 Fab (1 ⁇ g/ml and 5 ⁇ g/ml) did not show any prolongation of bleeding time, whereas the IgG format of hGP 5C4 prolonged bleeding time in low concentrations (1 ⁇ g/ml).
  • 4C9 markedly prolonged bleeding time both as Fab and as IgG antibody (Please see f igu re 9b) .
  • GPVI as an antigen for hGP 5C4 was investigated.
  • Recombinant GPVI protein as dimeric fusion protein of the extracellular domain of the GPVI receptor and the Fc part of a human IgGl as a linker was generated as described.
  • Soluble GPVI protein was secreted from Fc- GPVI-nt expressing CHO cells as dimer and purified.
  • the identity of Fc-GPVI-nt was tested in a SDS gel with Coomassie stain for proteins and with an antibody directed against the Fc part of Fc-GPVI-nt (see f igu re 1 a, top).
  • hGP 5C4 recognised the Fc-GPVI-nt specifically as purified protein in a Western blot.
  • hGP 5C4 Under reducing conditions hGP 5C4 recognized a protein with approximately 80 kDalton (see figu re 1 a). A specific protein was also recognised from platelet lysates with approximately 65 kDalton. There was no cross reaction to the control protein Fc- control. Generation of Fab fragments of the IgG antibody hGP 5C4 was tested with SDS-PAGE and Coomassie staining (see f igu re 1 b) .
  • hGP 5C4 showed strong binding to native platelets with weaker binding of 4C4 and 4C9. 14El 1 and CD3 did not show increased binding to platelets compared to the control antibody (see f igure 4). This further supports the concept that hGP 5C4 shows specific binding to native GPVI on the surface of either GPVI-expressing CHO cells or native human platelets.
  • a peptide library (peptide scan format 13/11, 131 peptides) was scanned using the antibody hGP 5C4. All N-termini were acetylated.
  • the peptide library comprised the extracellular domain of human Glycoprotein VI, including the signal sequence. Since the fusion protein, PR-15 was used, peptide SEQ ID No. 131 contains terminal amino acids GGRE which do not occur in the native GPVI protein.
  • the peptides were synthesized on a cellulose membrane in a stepwise manner resulting in a defined arrangement (peptide array) and are covalently bound to the cellulose membrane. Binding assays were performed directly on the peptide array. In general an antigen peptide array is incubated with blocking buffer for several hours to reduce non-specific binding of proteins or antibodies. It follows the incubation with a primary (antigen peptide-binding) antibody in blocking buffer and an incubation with a horseradish peroxidase (HRP)-labelled secondary antibody, which binds selectively the primary antibody. Alternatively a HRP-labelled primary antibody in blocking buffer can be used.
  • HRP horseradish peroxidase
  • T(Tween)-TBS-buffer washing directly after the incubation of the antigen peptide array with the secondary protein or antibody or the HRP-labelled primary antibody followed by the first chemiluminescence experiment is made to get an first overview which antigen peptides do bind the primary antibody.
  • Several buffer washing steps follow (T-TBS- and TBS-buffer) to reduce false positve binding (unspecific antibody binding to the cellulose membrane itself)- After these washing steps the final chemiluminescence analysis is performed. The data were analysed with an imaging system showing the signal intensity (Light units, LU) as single measurements for each peptide.
  • incubations using the secondary antibody have to be performed in the absence of the primary antibody as control incubation.
  • the primary antibody was hGP 5C4.
  • the secondary antibody was anti-rat HRP antibody and was used as control.
  • the binding data for the antigen peptide scan is listed in tables 1 and 2 and shown in figu re
  • RVPAQSGPLPKPS SEQ. ID. No 9
  • PVTLRCQGPPGVD 76 (SEQ. ID. No 22)
  • DQAVLFIPAMKRS 186 (SEQ. ID. No 35) 36 AVLFIPAMKRSLA 125 (SEQ. ID. No 36) 37 LFIPAMKRSLAGR ICO (SEQ. ID. No 37) 38 IPAMKRSLAGRYR !0>1 (SEQ. ID. No 38) 39 AMKRSLAGRYRCS 134 (SEQ. ID. No 39) 40 KRSLAGRYRCSYQ 139 (SEQ. ID. No 40) 41 SLAGRYRCSYQNG 8 (SEQ. ID. No 41) 42 AGRYRCSYQNGSL 41 (SEQ. ID. No 42) 43 RYRCSYQNGSLWS 64 (SEQ. ID.
  • RWYRASFPIITVT 184 (SEQ. ID. No 80)
  • ELVVTGTSVTPSR 114 (SEQ. ID. No 100)
  • VVTGTSVTPSRLP 211 (SEQ. ID. No 101)
  • VTPSRLPTEPPSS 91 (SEQ. ID. No 104)
  • EATAELTVSFTNK 135 (SEQ. ID. No 113)
  • TSPKESDSPAGPA 25 (SEQ. ID. No 125)
  • PAGPARQYYTKGN 139 (SEQ. ID. No 129)

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Abstract

L'invention concerne des agents qui se lient à une protéine glycoprotéine VI (GPVI) ou à des séquences de celle-ci. L'invention concerne, plus particulièrement mais sans limitation, des agents qui se lient avec un ou plusieurs épitope(s) de GPVI humaine. L'invention concerne également certains épitopes de GPVI humaine, des méthodes permettant d'identifier ou de cribler des agents qui se lient à des épitopes. L'invention concerne enfin l'utilisation d'agents permettant de traiter ou de prévenir des maladies induites par des processus d'agrégation de plaquettes sanguines.
EP06763512A 2005-06-07 2006-06-05 Agents antithrombiques Withdrawn EP1943277A2 (fr)

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EP2000802A4 (fr) 2006-03-31 2009-04-15 Mochida Pharm Co Ltd Nouveau marqueur de l'activation plaquettaire et son procédé de détermination
EP2336188A1 (fr) * 2009-12-18 2011-06-22 Sanofi-Aventis Nouveaux anticorps antagonistes et leurs fragments Fab contre le GPVI et leurs utilisations
EP2397495A1 (fr) * 2010-06-21 2011-12-21 Sanofi Nouveaux anticorps antagonistes et leurs fragments Fab contre le GPVI et leurs utilisations
MA33930B1 (fr) * 2009-12-18 2013-01-02 Sanofi Sa Nouveaux anticorps antagonistes contre gpvi et leurs fragments fab et utilisations de ceux-ci
AU2016301969B2 (en) * 2015-08-05 2022-10-06 Acticor Biotech Novel anti-human GPVI antibodies and uses thereof
SG11201906530TA (en) * 2017-02-03 2019-08-27 Acticor Biotech Inhibition of platelet aggregation using anti-human gpvi antibodies
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US7291714B1 (en) * 1999-06-30 2007-11-06 Millennium Pharmaceuticals, Inc. Glycoprotein VI and uses thereof
WO2001016321A1 (fr) * 1999-09-01 2001-03-08 Otsuka Pharmaceutical Co., Ltd. Adn et sequences proteiniques de glycoproteine vi (gpvi) de membranes plaquettaires et utilisations de ceux-ci
EP1369128A1 (fr) * 2002-06-07 2003-12-10 Procorde GmbH Inhibiteurs de glycoprotéine VI et leur utilisation thérapeutique
EP1538165A1 (fr) * 2003-12-03 2005-06-08 Procorde GmbH Inhibiteurs de la glycoprotéine VI construits à partir de l'anticorps monoclonal hgp 5c4

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