EP1581551A2 - Antagonistes du récepteur de f11 (f11r) en tant qu'agents thérapeutiques - Google Patents
Antagonistes du récepteur de f11 (f11r) en tant qu'agents thérapeutiquesInfo
- Publication number
- EP1581551A2 EP1581551A2 EP03815213A EP03815213A EP1581551A2 EP 1581551 A2 EP1581551 A2 EP 1581551A2 EP 03815213 A EP03815213 A EP 03815213A EP 03815213 A EP03815213 A EP 03815213A EP 1581551 A2 EP1581551 A2 EP 1581551A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- peptide
- peptides
- present
- fllr
- 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.)
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Classifications
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- 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
- C07K14/70503—Immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- the present invention relates to protein and peptide chemistry, as well as crystallography and organic chemistry.
- the present invention is directed to a cell adhesion molecule (CAM) and fragments thereof, and more particularly to a CAM designated as the FI 1 receptor (FI IR), or a polypeptide fragment thereof.
- CAM cell adhesion molecule
- FI IR FI 1 receptor
- the present invention also relates to FI lR-antagonists and methods for t he prevention and treatment of thrombosis, heart attacks, stroke and other clinical disorders involving thrombus formation.
- the vasculature is recognized as a dynamic metabolic organ that exists under normal physiological conditions in an intact, undisturbed state (Karsan, et al. In: Hematology: Basic Principles and Practice, 3rd Ed. Hoffman, et al. (eds) 2000; pp. 1770 -82).
- Endothelial cells (EC) which line the exposed (luminal) surface of blood vessels, are normally not thrombogenic. That is, healthy EC do not attract nor bind circulating platelets (Cines, et al. Blood 1998, 91: 3527-61; May, et al. Thromb Haemost 1999, 82: 962-70).
- the physiological function of the endothelium is to facilitate blood flow by providing a highly thromboresistant surface to flowing blood that inhibits platelet adhesion and clotting (Cines, et al.).
- Cines platelet adhesion and clotting
- the nonthrombotic surface of EC can be transformed to a prothrombotic surface foil owing exposure to cytokines (May, et al.; Diquelou et al. Thromb Haemost 1995, 74: 778-83), resulting in procoagulant activity and a predisposition to thrombosis (May, et al; Dardik, et al. Br J Haemarol 2000, 109:512 -8; Andre, et al.
- cytokines induce alterations in EC which result in the adhesion of non-stimulated platelets.
- a novel adhesion protein of the Ig superfamily has been described with properties indicating a potential triggering role in the pathogenesis of in flammatory thrombosis.
- This protein was identified first on the surface of human platelets and called the FI 1 receptor (FUR; Komecki, et al. JBiol Chem 1990, 265: 10042-8; aik, et al. Biochem J 1995, 311: 155 -62), and then on the surface of murine endothelial and epithelial cells and called JAM (Martin-Padura, et al. J.CellBiol 1998, 142:117-27.).
- the human platelet Fll receptor is a surface glycoprotein duplex (32 and 35 kD at core protein: 29 kDa) member of the immunoglobulin superfamily.
- the FUR was first discovered as the target of a potent stimulatory monoclonal antibody, M.Ab.Fll, that induces platelet secretion followed by aggregation ( Komecki, et al.; ⁇ aik, et al.; Komecki, et al. JLab Clin. Med. 1988, 111:618-26; Wang et al. Biochem. J. 1995, 311: 401-6; Komecki, et al. In: Leucocyte Typing N. Schlossman, et al.
- Signal transduction mechanisms for platelet secretion and aggregation induced by M.Ab.Fl 1 following its initial binding to FI 1 include: crosslinking of the FI 1 to the Fc ⁇ RII (Naik, et al.), activation and translocation of specific PKC isozymes (Wang, et al.), phosphorylation of the FI IR through activation of PKC (Naik, et al.; Wang, et al.), phosphorylation of the FI IR following induction of platelet aggregation by the physiological agonists thrombin and collagen and by M.Ab.Fl 1 itself (Sobocka, et al. 1997; Sobocka; Sobocka, et al.
- the FUR participates in mechanisms underlying adhesion of platelets, endothelial cells, and epithelial cells (Martin-Padura, et al.; Sobocka, et al. 2000).
- the FI IR in addition to its role as a receptor that triggers signal transduction leading to secretion, the FI IR also serves as a CAM involved in platelet adhesion was supported by the high degree of sequence similarity found between the human platelet FI IR and an adhesion protein called Junctional Adhesion Molecule (JAM), a protein found in murine endothelial cells (Martin-Padura, et al).
- JAM Junctional Adhesion Molecule
- Comparison of the murine JAM sequence to the previously-reported sequences of human-platelet FI IR revealed over 70% homology of JAM to the N -temiinus (23 amino acids) of FI IR and to two digested products of FUR.
- both the human platelet FUR core protein and the murine JAM protein were found to contain a single transmembrane domain and two pairs of cysteine residues in their extracellular domains that allow formation of intermolecular disulfide bridges forming characteristic Ig-like folds.
- JAM the protein referred to as JAM (Martin-Padura, et al; Ozaki, et al. J. Immunol 1999, 163: 553 -7; Williams, et al. Mol Immunol. 1999, 36: 1175 -88; Liu, et al. J. Cell Science 2000, 113: 2363-74; Gupta, et al.
- the platelet antibody M.Ab.Fl 1 was found to recognize FUR molecules present at intercellular junctions of cultured human umbilical vein endothelial cells (Sobocka, et al., manuscript in preparation; Sobocka, et al. XVIII ISTH Congress, July, 2001, Paris, France, Abs# PI 902).
- Sobocka, et al. manuscript in preparation; Sobocka, et al. XVIII ISTH Congress, July, 2001, Paris, France, Abs# PI 902
- a recent study conducted by the inventors has determined that two domains of FI IR are critical for the induction of platelet aggregation by M.Ab.Fl 1 and the adhesion of platelets to M.Ab.Fl 1.
- FI IR the role of FI IR in physiological and pathophysiological processes involving the adhesion of platelets to endothelial cells has remained unknown.
- the inventors have now determined that the N-terminus of FI IR and the first Ig fold of FI IR contain protein sequences which are critical for the adhesion of platelets to endothelial cells, and that recombinant soluble FUR and FI IR-peptides block approximately 60% of the adhesion of platelets to cytokine-treated EC, demonstrating the involvement of the FI IR in platelet -endothelial cell interactions, which under pathological conditions result in thrombosis.
- the present invention provides the full length cDNA sequence of the FI 1 receptor (FI IR) (SEQ ID NO: 6) and the encoded FI IR amino acid sequence (SEQ ID NO: 7).
- the present invention also provides FI lR-antagonists including peptide antagonists.
- the present invention is directed to methods and compositions for treating FllR-mediated disorders such as thrombosis, heart attacks, stroke and all other clinical disorders involving thrombus formation.
- the present invention provides specific compositions containing at least one FI IR -antagonist peptide which inhibits, suppresses or causes the cessation of at least one FllR-mediated biological activity in a mammal, and preferably humans.
- Nucleic acid molecules coding for any of the above FUR -antagonist peptides of the present invention expression vectors which include any of such nucleic acid molecules, as well as related host cells containing such nucleotide sequences or vectors, are also contemplated by the present invention.
- Still another embodiment of the present invention is directed to antibodies raised against the FI lR-antagonist peptides of the present invention.
- the antibodies of the present invention are raised against those FI lR-antagonist peptides whose sequences coincide with the corresponding sequences of a mammalian FI IR or Junctional Adhesion Molecule (JAM) protein.
- the antibodies of the present invention can antagonize or neutralize the activity of FI IR.
- Both polyclonal antibodies and monoclonal antibodies are contemplated by the present invention. Examples of such antibodies include M. Ab.F 11.
- Figure 1 shows the 3-D structure of the external domain of the mature human platelet FllR.
- the two Ig-like folds of the human recombinant FllR protein (FllR) are shown as a backbone structure based on the template of the mouse JAM which shares about 70% homology to that of human FI IR.
- Figure 2 shows that potentiation of ADP-induced platelet aggregation by MAb.Fll is blocked by Fl lR peptides (SEQ ID NO: 1 and SEQ ID NO: 4).
- Figure 2A demonstrates strong potentiation of aggregation using subthreshold levels of M.Ab.Fl 1 (0.3 ⁇ g/ml) and ADP (0.5 ⁇ M). No aggregation with ADP or M.Ab.Fll alone.
- Figure 2B demonstrates that a subthreshold concentration of MAb.Fll (0.3 ⁇ g/ml) does not induce platelet aggregation. However, ADP (0.5 ⁇ M) plus M.Ab.Fll causes a strong aggregation.
- Figure 2C demonstrates inhibition of the potentiation of aggregation by FllR-peptide (SEQ ID NO: 1).
- SEQ ID NO: 1 50 ⁇ M preincubated with platelets for about 30 sec prior to the addition of M.Ab.Fll (0.3 ⁇ g ml) followed by the addition of ADP (0.5 ⁇ M) inhibited aggregation.
- Control aggregation in the absence of SEQ ID NO: 1.
- Figure 2D demonstrates inhibition of the potentiation of aggregation by FI IR -peptide (SEQ ID NO: 4). SEQ ID NO: 4 preincubated with platelets for about 42 sec.
- Figure 3 shows inhibition of M.Ab.Fl l -induced platelet aggregation by Fl lR peptides (Fig. 3A); Inhibition of M.Ab.Fl l (2.45 ⁇ g/ml)-induced platelet aggregation by 50 ⁇ M FllR peptide (SEQ ID NO: 1) and by 50 ⁇ M FllR peptide (SEQ ID NO: 4)
- Figure 3B Control:
- Fig. 3C shows lack of inhibition of M.Ab.Fll (2.45 ⁇ g/ml)-induced platelet aggregation by FI IR (SEQ ID NO: 2) (500 ⁇ M).
- Figures 4A-4D show that potentiation of collagen -inducted platelet aggregation by M.Ab.Fl 1 is inhibited by FI IR peptides (SEQ ID NOS: 1 and 4).
- Figure 5 shows inhibition of adhesion by FI IR peptides (SEQ ID NOS: 1 and
- Figure 6 provides the FI IR cDNA sequence (full length) (SEQ ID NO: 6).
- Figure 7 provides the FI IR amino acid sequence (SEQ ID NO: 7).
- the present invention is directed to FI IR- antagonists.
- FI lR-antagonist is meant a molecule that inhibits, suppresses or causes the cessation of at least one F 11R- mediated biological activity by, e.g., interfering with, blocking or otherwise preventing the interaction or binding of FI IR to its target, e.g. FI IR on another cell.
- novel FI lR-antagonist peptides derived from or corresponding to the FI IR have been isolated and synthesized.
- FI IR antagonistic properties including the ability to selectively bind to FI IR and inhibit FI IR-mediated biological activity which, for example, is associated with adhesion of platelets to endothelial cells in mammals.
- the peptides of the present invention preferably correspond to specific portions of the native human FI 1 receptor and include variations thereof, and therefore are non-immunogenic when administered to humans.
- the FI IR- antagonist peptides of the present invention substantially correspond to the amino acids of the N-terminus or first Ig domain of human FI IR.
- a preferred FI IR- antagonist peptide of the present invention is a sequence of the N-terminal peptide of the FI IR structure: SNTVHSSEPEVRIPENNPVKLSC (SEQ ID NO: 1).
- Another preferred FI IR- antagonist peptide of the present invention is a sequence of the first Ig fold of the FllR structure: KSVTREDTGTYTC (SEQ ID NO: 4).
- Homologs, analogs and fragments of these peptides are also contemplated by the present invention which maintain FI IR- antagonist activity in a mammal, particularly humans.
- Another aspect of the present invention provides methods of interfering with, blocking or otherwise preventing the interaction or binding of platelets to endothelial cells with FI IR by employing the FI lR-antagonists contemplated by the present invention.
- the present invention also provides compositions for the treatment of FI IR - mediated disorders such as thrombosis, heart attacks, stroke and all other clinical disorders involving thrombus formation, in animals, including humans and includes methods of treating such disorders.
- the compositions include at least one of the FI lR-antagonists, preferably at least one FI IR peptide antagonist according to the present invention, admixed with a pharmaceutically acceptable carrier.
- the protein FI IR serves a significant role in the adhesion of platelets to inflamed endothelial cells.
- the present invention has identified that the activity of FllR is critical for initiating the formation of platelet plaques in blood vessels and for the formation of thrombi.
- any agents, chemicals or drugs that inhibit the action of FI IR (named here col lectively: FI lR-antagonists) will serve as powerful inhibitors of thrombus development in the circulation.
- the present invention provides FI IR- antagonists as drugs useful for the prevention and treatment of thrombosis, heart attacks, strokes, iminunothrombocytoper-ia, posttransfusion purpura, acute and chronic immunothrombocytopenia, acquired disorders of platelet function, myeloproliferative disorders, uremia, liver disease, cardiopulmonary bypass, various types of thrombosis inflammatory thrombosis, peripheral vein thrombosis, coronary artery thrombosis and other arterial thrombosis, atherosclerosis, disorders of angiogenesis, cancer growth and metastasis, and all other human disorders that involve thrombus formation.
- One aspect of the invention is directed to methods of identifying a compound which prevents the adhesion of platelets to endothelial cells and inhibits platelet aggregation, also referred to as "FUR- antagonists.”
- compound is taken to include both organic compounds such as peptides, as well as inorganic compounds such as ion chelators.
- Antibodies e.g., polyclonal or monoclonal antibodies directed against FI IR, the Fab, Fab', F(ab') 2 fragments of such antibodies, as well as single- chain anti-FllR antibodies can also be considered as compounds useful in the present methods.
- Other preferred compounds include chemical compounds that can be derived from the knowledge of the sequence of the FllR, from each of the above sequences (i.e. SEQ ID NOS: 1-7) and from the combination of the sequences together. These include linear sequences, cyclic sequences, annealing of the peptides together (preferably SEQ ID NOS: 1 and 4), and any other possible derivations using standard peptide chemistry techniques. In one embodiment the present invention contemplates any compound whose structure is based on the interaction of peptides 1 and 4 (SEQ ID NOS. 1 and 4), which form the binding site of the mature human platelet FI IR.
- a "mimetic" or “peptidomimetic” of a compound 's functional site refers to a compound in which chemical structures of protein or peptide sequences necessary for functional activity of a compound's functional site have been replaced with other chemical structures that mimic the conformation of the functional site.
- An example of a peptidomimetic contemplated by the present invention includes a compound (e.g. a small organic molecule) including portions with residues which interact sterically with the binding site of the FI IR molecule.
- a “residue” refers to an amino acid incorporated in the peptide by an amide bond, for example.
- Approaches to designing peptide mimetics are known in the art. For example, see Farmer, P.S.
- the present invention contemplates all peptidomimetics which can be designed based on the knowledge of the three-dimensional stracture of the FI IR molecule of the invention including but not limited to all mimetic compounds which can be conventionally synthesized by an ordinarily skilled chemist to bind to, antagonize, act as an agonist, inhibit, promote, block, or otherwise functionally interact with the binding site of the FI IR as illustrated in Figure 1, for example.
- Most preferred compounds of the present methods ar e peptides which are made to resemble the monoclonal antibody FI 1 ("M.Ab.Fl 1") binding site on platelets.
- FI IR refers to a receptor protein on the surface of human platelets as a target for a stimulatory M.Ab.Fl 1.
- FI IR is also referred to as human ortholog of the murine protein called junctional adhesion molecule (JAM).
- JAM junctional adhesion molecule
- FI IR is depicted in Figure 1 as a backbone structure in its entirety including the extracellular, soluble domain, consisting of two Ig-folds, a transmembrance domain and a short cytoplasmic portion, on both human platelets and endothelial cells.
- FI IR antagonists and "FI IR antagonist peptides” further refers to any compound that can bind to the pocket formed by the N-terminal 23 amino acid region and 13 amino acid region in the first Ig fold. By such binding, the action of FI IR is inhibited, i.e. alignment of platelets and endothelial cells in FI IR-mediated trans -homophilic interaction through the steric pocket, as depicted in Figures 1- 2, is blocked so that platelet aggregation or thrombosis, heart attacks, strokes, and all other human disorders that involve thrombus formation, can be prevented or treated.
- Fl lR antagonist peptide is also meant a peptide that inhibits, suppresses or causes the cessation of at least one Fl lR mediated biological activity by e.g. interfering with or otherwise preventing the interaction or binding of platelets to endothelial cells and thereby inhibit platelet aggregation.
- two peptide sequences of the FI IR have been determined the sequences correspond to (the N -terminus SVTVHSSEPEVRIPENNPVKLSC (SEQ ID NO: 1), and the first Ig fold sequence KSVTREDTGTYTC (SEQ ID NO: 4).
- the peptide sequences of the present invention inhibit the adhesion of platelets to endothelial cells and inhibit platelet aggregation.
- peptide refers to a linear series of amino acid residues linked to one another by peptide bonds between the alpha -amino and carboxy groups of adjacent amino acid residues.
- synthetic peptide is intended to refer to a chemically derived chain of amino acid residues linked together by peptide bonds.
- synthetic peptide is also intended to refer to recombinantly produced peptides in accordance with the present invention.
- preferred FI IR antagonists include peptides (referred to herein as "FI IR antagomst peptides") and antibodies. Additionally, analogs, homologs and fragments of the novel peptides provided herein are included within the scope of the term " FI IR antagonist peptide”.
- homologs are meant the corresponding peptides from FI IR proteins of other mammalian species substantially homologous at the overall protein (i.e., mature protein) level to human FI IR, so long as such homologous peptides retain the FllR antagonist activity.
- an analog is meant peptides which differ by one or more amino acid alterations, which alterations, e.g., substitutions, additions or deletions of amino acid residues, do not abolish the FI IR antagonist properties of the relevant peptides.
- an analog may comprise a peptide having a substantially identical amino acid sequence to a peptide provided herein and in which one or more amino acid residues have been conservatively or non- conservatively substituted. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another.
- the present invention contemplates the substitution of one polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of one acidic resi due such as aspartic acid or glutamic acid for another is also contemplated.
- non-conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residues such as cyteine, glutamine, glutamic acid, lysine and/or a polar residue for a non -polar residue.
- a non-polar residue such as isoleucine, valine, leucine, alanine, methionine
- a polar (hydrophilic) residues such as cyteine, glutamine, glutamic acid, lysine and/or a polar residue for a non -polar residue.
- analogs of the subject peptides can contain an N- or C-terminal cysteine, by which, if desired, the peptide may be covalently attached to a carrier protein, e.g., albumin. Such attachment, it is believed, will minimize clearing of the peptide from the blood and also prevent proteolysis of the peptides.
- peptides containing D-amino acids in place of L-amino acids are also included in the term "conservative substitution.”
- the prese nee of such D-isomers can help minimize proteolytic activity and clearing of the peptide.
- fragment refers to any subject peptide having an amino acid sequence shorter than that of any peptide depicted in SEQ ID NOS: 1-5 and 7 and which fragment retains the FI IR-mediated antagonist activity of the subject peptides.
- the peptides of the present invention may be synthesized by a number of known techniques.
- the peptides may be prepared using the solid -phase synthetic technique initially described by Me ⁇ field, in J. Am. Chem. Soc. 85:2149-2154 (1963).
- Other peptide synthesis techniques may be found in M. Bodanszky, et al. Peptide Synthesis , John Wiley & Sons, 2d Ed., (1976) and other references readily available to those skilled in the art.
- a summary of polypeptide synthesis techniques can be found in J. Stuart and J.D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, 111., (1984).
- Peptides may also be synthesized by solution methods as described in The Proteins, Vol. II. 3d Ed., Neurath, H. et al., Eds., p. 105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the above- mentioned texts as well as in J.F.W. McOmie, Protective Groups in Organic Ch emistry, Plenum Press, New York, N.Y. (1973).
- the peptides of the present invention can also be prepared by chemical or enzymatic cleavage from larger portions of the FllR molecule or from the entire FllR molecule.
- the peptides of the present invention may also be prepared by recombinant DNA techniques (see e.g. Current Protocols in Molecular Cloning Ausubel et al., 1995, John Wiley & Sons, New York); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York; Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, New York (1994)).
- the skilled artisan understands that any of a wide variety of expression systems can be used to provide the recombinant p eptides of the present invention.
- the precise host cell used is not critical to the invention.
- the FllR antagonist peptides can be produced in a prokaryotic host (e.g. E. coli), or in a eukaryotic host (e.g., S. cerevisiae or mammalian cells, e.g. COS1, CHO, NIH3T3, and JEG3 cells, or in the cells of an arthropod, e.g. S.frugiperda).
- a prokaryotic host e.g. E. coli
- a eukaryotic host e.g., S. cerevisiae or mammalian cells, e.g. COS1, CHO, NIH3T3, and JEG3 cells, or in the cells of an arthropod, e.g. S.frugiperda.
- Such cells are available from e.g. the American Type Culture Collection, Manassas, NA.
- the method of transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection
- the present invention also contemplates a deoxyribonucleic acid (DNA) molecule or segment that defines a gene coding for, i.e., capable of expressing, a subject peptide or a subject cbimeric peptide from which a peptide of the present invention may be enzymatically or chemically cleaved.
- DNA deoxyribonucleic acid
- DNA molecules that encode peptides of the present invention can be synthesized by chemical techniques, for example, the phosphotriester method of Matteuccie, et al., J. Am. Chem. Soc. 103:3185(1981). Using a chemical DNA synthesis technique, desired modifications in the peptide sequence can be made by making substitutions for bases which code for the native amino acid sequence. Ribonucleic acid equivalents of the above described DNA molecules may also be used.
- a nucleic acid molecule comprising a vector capable of replication and expression of a DNA molecule defining coding sequence for a subject polypeptide or subject cbimeric polypeptide is also contemplated.
- the peptides of the present invention are chemically synthesized by conventional techniques such as the Merrifield solid phase technique.
- the method comprises the sequential addition of one or more amino acid residues to a growing peptide chain.
- a suitable, selectively removable protecting group is protected by a suitable, selectively removable protecting group.
- a different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
- a preferred method of solid phase synthesis entails attaching the protected or derivatized amino acid to an inert solid support through its unprotected carboxyl or ammo group.
- the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support.
- the protecting group of the amino carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth.
- any remaining terminal and side group protecting groups including the solid support are removed sequentially or concurrently to yield the final peptide.
- the lyophilized oligopeptides are resuspended in double distilled H O at 2 mg/ml as stock solutions and subsequently diluted in Ml 99 -HPS for experiments. Consistent with the observed properties of the peptides of the invention, the present peptides can be used to inhibit, suppress, or cause the cessation of at least one FllR- mediated biological activity.
- FI IR functions in the biochemical events associated with platelets aggregation and adhesion of platelets to endothelial cells. Accordingly, the present invention contemplates methods to block, interrupt or otherwise prevent the association of platelets to endothelial cells and thereby effectively treat or prevent FllR- cell associated disorders such as thrombosis, for example.
- FllR-mediated disorders such as, for example, thrombosis, heart attacks and strokes are FI lR-dependent therefore treatable w ith the FI IR antagonists, preferably FI IR antagonist peptides of the present invention.
- Other Fl lR related diseases are also contemplated by the present invention.
- one or more FI IR antagonists are included in pharmaceutical compositions.
- compositions containing the FI IR antagonist peptides of the present invention are administered intravenously to inhibit, suppress, or cause the cessation of at least one FI IR-mediated biological activity.
- the peptide compositions may be combined with other ingredients, such as carriers and/or adjuvants.
- the peptides may also be covalently attached to a protein carrier, such as albu min, so as to minimize clearing of the peptides.
- ingredients there are no limitations on the nature of the other ingredients, except that such ingredients must be pharmaceutically acceptable, efficacious for their intended administration and cannot degrade the activity of the active ingredients of the compositions.
- examples of other anti -inflammatory ingredients contemplated by the present invention include, but are not limited to anti-Fl IR antibodies, NSAIDS, steroids, or cyclosporin-A. When employed together with FllR antagonists, these agents may be employed in lesser dosages than when used alone.
- the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the ultimate solution form must be sterile and fluid.
- Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils.
- Sterilization can be accomplished by any art-recognized technique, including but not limited to, filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutano, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
- Production of sterile injectable solutions containi ng the subject peptides is accomplished by incorporated these compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization. To obtain a sterile powder, the above solutions are vacuum - dried or freeze-dried as necessary.
- the pharmaceutical compositions thereof containing an effective dose of the peptide can also contain an inert diluent, as assimilable edible carrier and the like, be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.
- the subject peptides are thus compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dose.
- the peptides should preferably be administered in an amount of at least about 50 mg per dose, more preferably in an amount up to about 500 mg to about 1 gram per dose. Since the peptide compositions of this invention will eventually be cleared from the bloodstream, re- administration of the compositions is indicated and preferred.
- the peptides can be administered in a manner compatible with the dosage formulation and in such amount as well be therapeutically effective.
- Systemic dosages depend on the age, weight and conditions of the patient and on the administration route. For example, a suitable dose for the administration to adult humans ranges from about lmg/kg of body weight about 10 mg per kilogram of body weight.
- a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents the like. The use of such media and agents are well-known in the art.
- the pharmaceutically acceptable carriers used in conjunction with the peptides of the present invention vary according to the mode of administration.
- the compositions may be formulated in any suitable carrier for oral liquid formulation such as suspensions, elixirs and solutions.
- Compositions for liquid oral dosage include any of the usual pharmaceutical media such as, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like.
- carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used.
- carriers such as liposomes and microemulsions may be used.
- the pharmaceutical compositions of the present invention are employed for the treatment of FI IR mediated pathological disorders.
- the present invention provides methods of treating an FI IR mediated disorder in a subject by administering a therapeutically effective amount of a pharmaceutical composition of the present invention.
- terapéuticaally effective amount means the dose required to treat an FI IR-mediated disorder.
- an FI IR-mediated disorder is meant a pathological disorder, the onset, progression or the persistence of the symptoms of which requires the participation of FI IR molecules.
- FI IR-mediated disorders contemplated by the present invention include thrombosis, heart attacks and strokes.
- treatment refers to effective inhibition, suppression or cessation of the FllR activity so as to prevent or delay the onset, retard the progression or ameliorate the symptoms of the disorder.
- subject refers to any mammalian subject. Preferably, the subject is a human.
- the present invention thus provides methods of interfering with, blocking or otherwise preventing the interaction or binding of platelets with endothelial cells by employing the FI IR antagonists contemplated by the present invention.
- the FI IR antagonist peptides of the present invention can be used to raise single-chain antibodies (S Ab) or humanized monoclonal antibodies useful in the invention.
- the peptides can be coupled to a carrier protein such as KLH as described in Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
- KLH- antagonist peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, donkeys and the like or preferably into rabbits.
- Antibodies may be purified by peptide antigen affinity chromatography.
- a single-chain antibody (SAb) is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
- Such single-chain antibody variable fragments Fvs can be fused to all or a portion of the constant domains of the heavy chain of an immunoglobulin molecule, if necessary.
- the use of sAb avoids the technical difficulties in the introduction of more than one gene construct into host cells.
- Single chain antibodies and methods for their production are known in the art. See, e.g., Bedzyk et al. (1990) J. Biol. Chem., 265:18615; Chaudhary et al. (1990) Proc. Natl. Acad. Sci., 87:9491; U.S. Patent No. 4,946,778 to Ladner et al.; and U.S. Patent No. 5,359,046 to Capon et al.
- Monoclonal antibodies can be prepared using FI IR antagonist peptides and standard hybridoma technology (see e.g. Kohler et al., (1975) Nature 256:495; Hammerling et al., (1981) In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York).
- monoclonal antibodies to FI IR antagonist peptides can be raised in Balb/C or other similar strains of mice by immunization with purified or partially purified preparations of FI IR antagonist peptides. The spleens of the mice can be removed, and their lymphocytes fused to a mouse myeloma cell line.
- a stable hybrid will be isolated that produces antibodies against FllR antagonist peptides.
- the monoclonal antibody can be examined for its ability to inhibit the biological activity of FI IR, e.g. platelet aggregation. Once produced, monoclonal antibodies are tested for specific FllR recognition by Western blot or immunoprecipitation analysis (by methods described in Ausubel et al., supra). Antibodies which antagonize FI lR/platelet aggregation are considered to be useful antagonists in the invention.
- the monoclonal antibodies of the present invention can be humanized to reduce the immunogenicity for use in humans.
- One approach is to make mouse-human chimeric antibodies having the original variable region of the murine mAb, joined to constant regions of a human immunoglobulin.
- Chimeric antibodies and methods for their production are known in the art. See, e.g., Cabilly et al., European Patent Application 125023 (pub lished Nov. 14, 1984); Taniguchi et al, European patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al, PCT Application WO 86/01533, (published Mar.
- DNA segments encoding the H and L chain antigen-binding regions of the murine mAb can be cloned from the inAb-producin g hybridoma cells, which can then be joined to DNA segments encoding C H and C L regions of a human immunoglobulin, respectively, to produce murine -human chimeric immunoglobulin -encoding genes.
- Monoclonal antibody M.Ab.Fll (IgGl isotype) was affinity - purified as described (Id.). Histidine antibody was obtained from InNitrogen (Carlsbad, CA).
- E. coli transformation, plasmid DNA isolation, restriction analysis, extraction of DNA from agarose gells and ligation of insert into pcDNA3.1/myc-His(+)C vector were carried out as described (Sambrook et al. Molecular- Cloning: A Laboratory Manuel 2 n Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989, inco ⁇ orated herein by reference). Plasmids were isolated from E. coli DH5 ⁇ (Life Technologies, Grand Island, NY) using Qiaprep columns (Qiagen, Valencia, CA). DNA restriction fragments were separated by agarose gel eletrophoresis and isolated with the QIAquick Gel extraction kit (Qiagen).
- PCR was performed using the Perkin Elmer Gene Amp 2400PCR System. DNA sequencing was performed by PCR-cycle sequencing using ABI PRISM Dye Terminator Cycle Sequencing Kit from Perkin Elmer (Foster City, CA) and the ABI Prism 377 DNA Squencer. Computer analysis of sequence data was performed with the Biology WorkBench, release 3.2.
- COS-7 cells were grown in DMEM/10%FBS (Cellgro Mediatech, Inc.) and 1% antimycotic (Life Technologies), at 37°C/5% CO 2 . Cells (about 50% confluency), plated in a 75 mm flask, were used for transfection one day later.
- the plasmid pcDNA3.1/F1 IR (10 ⁇ g) was transfected into cells using 30 ⁇ l of FuGENE -6 (Roche Diagnostics). Cells were maintained at 37°C/5% CO 2 in 7 ml complete medium. Cells were also treated with FuGENE -6 alone as controls.
- Total RNA was isolated (RNeasy Mini Kit, Qiagen) and used for subsequent RT -PCR.
- RNA (2 ⁇ g) was used for reverse transcription (Omniscript Reverse Transcriptase, Omniscript RT Kit, Qiagen). Half of the reaction mixture was used to amplify FI IR in a 35-cycle PCR using the specific FI IR primers as detailed previously (Sobocka, supra ). PCR cycling was as follows: 94°C for 5 min followed by 35 cycles of 94°C for 45 sec, 55°C for 45 sec, 72°C for 2 min and 94°C for 2 min. A 726-bp fragment was obtained only from pcDNA3.1/Fl IR transfected cells.
- Conditioned media collected 72 h posttransfection, were pooled and passed twice over a M.Ab.Fl 1-immunoaffinity column. After washing of the affinity column, the bound sFl IR was eluted by use of 50 mM diethylamine (pH 11.5), fractions were collected into 1 M Tris- HCI buffer (pH 8.0) and immediately dialysed against 10 mM Tris-HCI buffer (pH 7.4). The sFl IR solution was concentrated 5x by Centricon YM-10 (Bedford, MA), and stored frozen at -20°C.
- FI IR-peptides Five FI lRpeptides (95% pure) were synthesized (New England Peptides, Inc., Fitchburg, MA). The sequence of amino acids in these peptides and their location within the Fl lR molecule are shown in Table. Their mass was determined by MALDI-TOF DE mass spectrometry. TABLE 1
- PEPTIDE SEQUENCE
- amino acid numbers refer to the sequence of the mature platelet cell surface Fll receptor and of the recombinant protein, sFllR, which does not include the leader peptide sequence.
- ser-1 is the first amino acid that follows the 27 amino acid leader peptide sequence of the nascent protein (Sobocka et al. (2000) Blood 95:2600-2609).
- TBS/1%BSA for 1 h at 37°C, and washed with TBS/0.1 mM MnCl 2 /0.1 mM CaCl 2 .
- Isolated platelet suspensions (100 ⁇ l) (3 x 10 8 /ml) were added and plates were incubated at 37°C for
- FI IR recombinant FI IR protein
- COS-7 cells which contained only the extracellular portion (amino acids ser -1 to asn-208) of the mature FI IR molecule.
- the transcription of the recombinant sFl IR in COS -7 cells was determined by RT-PCR.
- a 726-base-pair fragment was detected only in pcDNA3.1/Fl IR transfected cells.
- the sFl IR protein was engineered to contain a Histidine tag sequence, and indeed, it was recognized by an anti - His antibody.
- sFl IR was purified from COS-7 cell media using M.Ab.Fl 1 immunoaffinity chromatography. The purified sFl IR was recognized by both the platelet stimulatory monoclonal antibody, MAb.Fl 1, and by a polyclonal FllR antibody, directed against the N -terminal amino acids ser-1 to cys-23. The results detailed above demonstrate that transfected COS-7 cells not only synthesize by also secrete sFl lR. EXAMPLE 3
- FI IR-peptides inhibit the potentiation of collagen-induced platelet aggregation.
- the effect of FI IR peptides on M.Ab.Fl 1 -induced potentiation of platelet aggregation induced by nonaggregating concentrations of collagen (0.5 ⁇ g/ml) also was examined. These results are shown in Fig. 4.
- Subthreshold concentrations of collagen and of M.Ab.Fl 1 used here w ere determined separately for each blood donor.
- Panel A of Fig. 4 demonstrates that the selected concentrations of each of the agonists were not able to induce platelet aggregation when added alone. However, the addition of collagen just after adding M.Ab.Fl 1 readily triggered a full -blown platelet aggregation.
- Peptide 4 (SEQ ID NO: 4), at 50 ⁇ M, produced very little (10%) inhibition compared to peptide 1 (SEQ ID NO: 1) at similar concentrations.
- 500 ⁇ M of peptide 4 (SEQ ID NO: 4) produced approximately 70% inhibition in the adhesion of platelets to MAb.Fl 1, similar to that observed with peptide 1 (SEQ ID NO: 1) at the same concentration.
- the addition of 500 ⁇ M (or higher) of peptides 2, 3 or 5 did not cause significant inhibition of platelet adhesion to M.Ab.Fl 1.
- FIG. 1 Three-dimensional structure of the recombinant human platelet FllR.
- a 3 - dimensional structural model of the human platelet FI IR highlighting the two domains that are occupied by sequences of the platelet inhibitory peptides 1 and 4, are depicted in Fig. 1. From this modeling it appears that the N -terminal portion of the molecule (containing the sequence of peptide 1 (SEQ ID NO: 1)), forms a loop around the 1 st Ig-fold (containing the sequence of peptide 4 (SEQ ID NO: 4)) to form an "active pocket" containing the sequences of both peptides 1 and 4, arranged in an anti-parallel orientation.
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Abstract
La présente invention concerne une molécule d'adhésion cellulaire (CAM), désignée en tant que récepteur de F11 (F11R), qui fait partie de la superfamille de l'immunoglobuline localisée à la surface des plaquettes humaines, et a pour rôle de promouvoir l'agrégation des plaquettes, la sécrétion, l'étalement des plaquettes et l'adhésion cellulaire. L'invention a également pour objet de l'ADNc de F11R cloné, de l'ADNc de F11R pleine longueur et des séquences d'acides aminés, ainsi que des antagonistes de F11R et des procédés pour prévenir et traiter la thrombose, l'infarctus du myocarde, l'accident vasculaire cérébral et autres troubles cliniques liés à la formation de thrombus.
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US43866903P | 2003-01-03 | 2003-01-03 | |
US438669P | 2003-01-03 | ||
PCT/US2003/039890 WO2004063327A2 (fr) | 2003-01-03 | 2003-12-16 | Antagonistes du recepteur de f11 (f11r) en tant qu'agents therapeutiques |
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US20050265992A1 (en) | 2003-01-03 | 2005-12-01 | The Research Foundation Of State University Of New York | F11 receptor (F11R) antagonists as therapeutic agents |
US8557957B2 (en) | 2003-01-03 | 2013-10-15 | Elizabeth Kornecki | Methods of treating disorders by administration of F11 receptor antagonists |
WO2020256795A1 (fr) * | 2019-06-19 | 2020-12-24 | The Research Foundation For The State University Of New York | Procédés de traitement de l'hyperplasie néointimale au moyen d'inhibiteurs de f11r/jam-a |
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US5665701A (en) * | 1994-11-17 | 1997-09-09 | The Research Foundation Of State University Of New York | Platelet membrane glycoprotein F11 and polypeptide fragments thereof |
WO1999002561A1 (fr) * | 1997-07-10 | 1999-01-21 | Smithkline Beecham Corporation | Antigene f11 humain, constituant un recepteur de surface cellulaire implique dans l'agregation plaquettaire |
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- 2003-12-16 JP JP2004566554A patent/JP2006516255A/ja active Pending
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US5665701A (en) * | 1994-11-17 | 1997-09-09 | The Research Foundation Of State University Of New York | Platelet membrane glycoprotein F11 and polypeptide fragments thereof |
WO1999002561A1 (fr) * | 1997-07-10 | 1999-01-21 | Smithkline Beecham Corporation | Antigene f11 humain, constituant un recepteur de surface cellulaire implique dans l'agregation plaquettaire |
Non-Patent Citations (3)
Title |
---|
BABINSKA ANNA ET AL: "Two regions of the human platelet F11-receptor (F11R) are critical for platelet aggregation, potentiation and adhesion" THROMBOSIS AND HAEMOSTASIS, vol. 87, no. 4, April 2002 (2002-04), pages 712-721, XP002456220 ISSN: 0340-6245 * |
See also references of WO2004063327A2 * |
SOBOCKA MALGORZATA B ET AL: "Cloning of the human platelet F11 receptor: A cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation" BLOOD, vol. 95, no. 8, 15 April 2000 (2000-04-15), pages 2600-2609, XP002456219 ISSN: 0006-4971 * |
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JP2006516255A (ja) | 2006-06-29 |
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