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WO1999013898A9 - Ec-3 en tant qu'inhibiteur des integrines alpha4 beta1 et alpha4 beta7 - Google Patents

Ec-3 en tant qu'inhibiteur des integrines alpha4 beta1 et alpha4 beta7

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Publication number
WO1999013898A9
WO1999013898A9 PCT/US1998/016719 US9816719W WO9913898A9 WO 1999013898 A9 WO1999013898 A9 WO 1999013898A9 US 9816719 W US9816719 W US 9816719W WO 9913898 A9 WO9913898 A9 WO 9913898A9
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WO
WIPO (PCT)
Prior art keywords
peptide
cells
integrin
protein
substantially purified
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Application number
PCT/US1998/016719
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English (en)
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WO1999013898A1 (fr
Inventor
Stefan Niewiarowski
Cezary Marcinkiewicz
Original Assignee
Stefan Niewiarowski
Cezary Marcinkiewicz
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 Stefan Niewiarowski, Cezary Marcinkiewicz filed Critical Stefan Niewiarowski
Priority to US09/485,323 priority Critical patent/US6818617B1/en
Publication of WO1999013898A1 publication Critical patent/WO1999013898A1/fr
Publication of WO1999013898A9 publication Critical patent/WO1999013898A9/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention generally relates to methods and compositions for modulating cell adhesion and for inhibiting the interaction between integrins and their ligands.
  • the invention relates to peptides that selectively inhibit ⁇ 4 integrins.
  • Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound and ⁇ subunits. In humans there are at least 15 different and eight different ⁇ subunits, and these can combine to form proteins with diverse biological activities and ligand specificities.
  • Integrins play important roles in many diverse biological processes including platelet aggregation, tissue repair, angiogenesis, bone destruction, tumor invasion, inflammation, and immune reactions.
  • the integrins are, therefore, important targets for therapeutic intervention in human disease.
  • Integrin ⁇ llb ⁇ 3 glycoprotein Ilb/IIIa complex
  • Integrin ⁇ v ⁇ 3 is predominantly expressed on endothelial cells and plays an important role in angiogenesis. Integrin v ⁇ 3 is also expressed on osteoclasts and participates in bone destruction.
  • Integrin ⁇ 5 ⁇ l is widely distributed on a variety of cells; it plays a critical role in cell adhesion to extracellular matrix as well as in the formation of tissues and organs during embryonic development. All three integrins ⁇ llb ⁇ 3, ⁇ v ⁇ 3 and ⁇ 5 ⁇ l recognize RGD sequences in the adhesive ligands.
  • integrin inhibitors As antithrombotic agents. Peptides and peptidomimetics that block the adhesion of GPIIb/IIIa to fibrinogen can prolong bleeding times and prevent thrombotic occlusion in vivo.
  • One group of naturally occurring peptides that inhibit platelet aggregation by interfering with fibrinogen binding to GPIIb/IIIa has been called the "disintegrins. "
  • the disintegrins are a family of low molecular weight cysteine-rich peptides that have been isolated from the venom of various snakes (reviewed in Niewiarowski et al. , Seminars in Hematology 31(4):289-300 (1994)). Most disintegrins described to date contain an RGD motif. RGD is a recognition site for many integrins, and disintegrins inhibit fibrinogen binding to GPIIb/IIIa, as well as the binding of other ligands to RGD-dependant integrins on the surface of cells . Peptides modeled on the structure of disintegrins have potential clinical applications in the prevention and treatment of coronary thrombosis, stroke, and other vascular diseases.
  • Trigramin The first disintegrin described in the literature, trigramin, was identified and characterized on the basis of its ability to block platelet aggregation and inhibit fibrinogen binding to ⁇ llb ⁇ 3. Trigramin contains 72 amino acids including 12 cysteines, is linked by S-S bonds, and contains an RGD sequence. Subsequently several other RGD containing viper venom disintegrins of similar size were isolated.
  • echistatin A 49 amino acid disintegrin, called echistatin, has been isolated from the venom of Echis carinatus (Gan et al. , J. Biol. Chem. 263:19827-32 (1988)). Like other disintegrins, echistatin contains an RGD sequence and inhibits GPIIb/IIIa binding of fibrinogen. Echistatin has been called a "promiscuous disintegrin" because it blocks ⁇ llb ⁇ 3, ⁇ v ⁇ 3 and ⁇ 5 ⁇ l with similar potency.
  • the disintegrin eristostatin originally described as a potent inhibitor of ⁇ llb ⁇ 3, also inhibits human melanoma cell (MV3) metastases in immune deficient mice. It has been suggested that this effect is mediated by altering the function of an ⁇ 4 integrin expressed on MV3 cells (Danen et al, Exp. Cell Res. 238:188-196 (1998)).
  • the ⁇ 4 integrins are expressed on leukocytes and lymphoid cells, and play a major role in inflammation and auto-immune diseases.
  • the ⁇ 4 ⁇ l integrin (which has also been called VLA-4, very late activation antigen 4) mediates cell adhesion to vascular cell adhesion molecule- 1 (VCAM-1), an adhesive molecule belonging to the IgG superfamily which is expressed on endothelial cells at sites of inflammation.
  • VCAM-1 vascular cell adhesion molecule- 1
  • the integrin ⁇ 4 ⁇ l also binds to alternatively spliced variants of fibronectin containing connecting segment 1 (CS-1).
  • the 4 ⁇ 7 integrin binds to the gut homing receptor mucosa addressin cell adhesion molecule- 1 (MadCAM-1) and to a lesser extent to CS-1 and VCAM-1.
  • McCAM-1 gut homing receptor mucosa addressin cell adhesion molecule- 1
  • Cytokine activated leukocytes express 4 ⁇ l and ⁇ 4 ⁇ 7 integrins.
  • VCAM-1 or MadCAM-1 which are also up- regulated by cytokines
  • endothelium mediates capillary infiltration by leukocytes, which can lead to tissue and organ destruction.
  • Selectins and ⁇ 2 integrins also contribute to this process.
  • Altevost et al. reported that the 4 subunit itself is a ligand for ⁇ 4 ⁇ l and ⁇ 4 ⁇ 7 integrins (J. Exp. Med. 182:345-55 (1995)), suggesting that ⁇ 4 integrins may play a role in leukocyte communication during the immune response.
  • Activation and up-regulation of ⁇ 4 ⁇ l or ⁇ 4 ⁇ 7 on lymphocytes or macrophages is believed to play a significant role in the progression of many disease states, including insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, organ rejection, and restenosis of arteries after surgery or angioplasty.
  • the 4 integrins are therefore targets for therapeutic intervention in a variety of inflammatory, auto-immune, and other diseases.
  • anti-oc4 monoclonal antibodies There are several animal models of inflammatory and autoimmune diseases in which endothelial infiltration by lymphocytes and organ destruction are blocked by anti-oc4 monoclonal antibodies.
  • anti- 4 antibody inhibits lymphocyte infiltration of Langerhans islets in NOD mice, thus preventing development of spontaneous insulin dependent diabetes (Yang et al. , Proc. Natl. Acad. Sci. USA 91: 12604-08 (1994)).
  • An ⁇ i- ⁇ 4 monoclonal antibodies have also shown in vivo efficacy in animal models of asthma (Abraham et al. , J. Clin. Invest. 93:776 (1994)), multiple sclerosis (Yednock et al.
  • Synthetic inhibitors that have been reported include cyclic RGD peptides and short peptides based on the sequences of MadCAM-1, VCAM-1, and CS-1. These peptides are typically active in vitro at the micromolar level.
  • Cyclic RGDS (SEQ ID NO: 15) peptides have been reported as strong inhibitors of ⁇ 4 ⁇ l and ⁇ 4 ⁇ 7 integrins (Cardarelli et al., J. Biol. Chem. 269: 18668-73 (1994); Yang et al. Eur. J. Immunol. 28:995-1004 (1998)).
  • the present invention relates to the discovery of the EC-3 protein, a heterodimeric disintegrin that is an extremely potent antagonist of 4 integrins.
  • EC-3 proteins and peptides inhibit adhesion of cells expressing ⁇ 4 integrins in an RGD-independent manner.
  • the invention provides a substantially purified EC-3 protein, isolated from E. carinatus venom and characterized by: (a) an apparent molecular weight of about 14,762 Da, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 40% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.
  • the invention also provides a substantially purified EC-3A peptide, isolated from EC-3 protein which has been reduced and alkylated, and characterized by: (a) a molecular mass of about 8478 Da in its ethylpyridylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 42% acetonitrile; and (c) the ability to inhibit adhesion of K562 cells to fibronectin.
  • the invention further provides a substantially purified EC-3B peptide, isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, and characterized by: (a) a molecular mass of about 7950 Da in its carboxymethylated form, as determined by electrospray ionization mass spectrometry ; (b) elution from a C- 18 HPLC column at about 46 % acetonitrile ; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.
  • One preferred embodiment of the invention is a substantially purified EC-3 A peptide comprising the sequence SEQ ID NO: 19, or a biologically active fragment or derivative thereof.
  • a more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO:2, or a biologically active fragment or derivative thereof.
  • Another preferred embodiment of the invention is a substantially purified EC-3B peptide comprising the sequence SEQ ID NO: 20, or a biologically active fragment or derivative thereof.
  • a more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO: 3, or a biologically active fragment or derivative thereof.
  • EC-3 protein comprising two subunits, wherein one subunit comprises the sequence SEQ ID NO: 19 or a biologically active fragment or derivative thereof and one subunit comprises the sequence SEQ ID NO: 20 or a biologically active fragment or derivative thereof.
  • the invention is also directed to a biologically active peptide fragment of EC-3B having the sequence X-Y-Met-Leu-Asp-Z, where X is H or a blocking group, Y is zero or more amino acids, and Z is OH or zero or more amino acids.
  • the biologically active peptide is from about 3 to about 20 amino acids.
  • the peptide has the sequence SEQ ID NO: 16.
  • the peptide has the sequence SEQ ID NO: 14.
  • Another aspect of the invention is a substantially purified nucleic acid encoding a protein or peptide according to the invention.
  • One embodiment of the invention is a vector comprising a nucleic acid encoding a protein or peptide according to the invention.
  • Another embodiment of the invention is a recombinant cell comprising a nucleic acid encoding a protein or peptide according to the invention.
  • the invention further provides an antibody which specifically binds to a protein or peptide according to the invention.
  • the antibody may be a monoclonal antibody or a polyclonal antibody or an antibody fragment that is capable of binding antigen.
  • One aspect of the invention is a hybridoma that produces a monoclonal antibody which specifically binds to a protein or peptide according to the invention.
  • Another aspect of the invention is a substantially purified echistatin polypeptide in which the Arg-Gly-Asp residues at positions 24-26 are replaced by Met-Leu-Asp, or a biologically active fragment or derivative thereof.
  • the invention further provides a method of isolating a peptide that binds to an integrin of interest from venom comprising: (a) dissolving venom in a solvent, (b) centrifuging the dissolved venom to remove high molecular weight proteins, (c) fractionating the supernatant from step (b), (d) immobilizing the fractions from step (c) on a solid support, (e) adding detectably labeled cells which express the integrin of interest to the immobilized fractions, (f) detecting the number of cells bound to each immobilized fraction, and (g) isolating peptide from those fractions which showed enhanced cell binding in step (f).
  • compositions comprising a pharmaceutically acceptable carrier and a protein, peptide, or nucleic acid according to the invention.
  • the invention encompasses a method of inhibiting the binding of an 4 integrin to VCAM-1 comprising contacting a cell that expresses the ⁇ 4 integrin with an effective amount of a protein or peptide according to the invention.
  • the integrin is ⁇ 4 ⁇ l or ⁇ 4 ⁇ 7.
  • the invention also encompasses a method of inhibiting the binding of ⁇ 4 ⁇ 7 integrin to MadCAM-1 comprising contacting a cell that expresses ⁇ 4 ⁇ 7 with an effective amount of a protein or peptide according to the invention.
  • the invention further encompasses a method of inhibiting the binding of an 4 integrin to CS-1 comprising contacting a cell that expresses the ⁇ 4 integrin with an effective amount of a protein or peptide according to the invention.
  • One preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an ⁇ 4 integrin and VCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
  • Another preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an ⁇ 4 integrin and MadCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
  • a further preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an 4 integrin and CS-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
  • the invention is also directed to the use of a protein, peptide, or nucleic acid according to the invention for the preparation of a medicament for inhibiting the interaction between cells expressing an 4 integrin and its ligands.
  • Figure 1 shows the HPLC fractionation of E. carinatus venom on a reverse phase C-18 HPLC column eluted with an acetonitrile gradient (dashed line). Fractions containing the disintegrin echistatin ( ⁇ C1), the echicetin protein (which binds to GPIb/IX receptors on platelets, EC 10) and EC3 are indicated.
  • Figure 2 shows the effect of selected fractions obtained from Echis carinatus venom on adhesion of Jurkat cells to immobilized VCAM-1 (Fig. 2 A) and K562 cells to immobilized fibronectin (Fig. 2B).
  • Figure 3 shows the elution profile of reduced and alkylated EC-3 from a reverse phase HPLC column eluted with an acetonitrile gradient (dashed line). Fractions containing EC3A and EC3B are indicated.
  • Figure 4 shows the amino acid sequences of EC-3 subunits EC-3 A (SEQ ID NOs: 19 and 2; Fig. 4A) and EC-3B (SEQ ID Nos: 20 and 3; Fig. 4B), which were established by N-terminal sequencing of reduced and ethylpyridylethylated subunits and by sequencing of peptides isolated from digests with endoproteinase Lys-C. EC-3B was also digested by CNBr and two peptides were isolated.
  • Figure 5 shows a comparison of the amino acid sequence of EC3 A (SEQ ID NO:2), EC3B (SEQ ID NO:3), with two short disintegrins eristostatin (SEQ ID NO:8) and echistatin (SEQ ID NO:9), with two medium size disintegrins flavoridin (SEQ ID NO: 10) and kistrin (SEQ ID NO: 11), and with a disintegrin domain of Le3 (SEQ ID NO: 7), a metallproteinase from Vipera lebetina venom. Boxes show the positions of conserved cysteine residues.
  • the typical active site of the disintegrins (RGD) as well as the corresponding position in EC-3A(VGD), EC-3B (MLD), and Le3 (VGD) are underlined.
  • Figure 6 shows the effect of EC-3 on Jurkat cell adhesion to immobilized VCAM-1 in the presence (filled circles) or absence (open circles) of l mM Mn + + .
  • Figure 7 shows the effect of EC-3 on CHO ( ⁇ 4 + , ⁇ 5-) cell adhesion to immobilized VCAM-1.
  • Figure 8 shows the effect of EC3 on the adhesion of CHO cells transfected with different integrins. Adhesion experiments were performed using
  • Open circles show the effect of EC-3 on the adhesion of CHO cells transfected with 4 to immobilized VCAM-1; closed circles: CHO cells transfected with cdlb ⁇ 3 to fibrinogen; open triangles: CHO cells transfected with ⁇ v ⁇ 3 to vitronectin; and closed triangles: non-transfected CHO cells expressing hamster ⁇ 5 ⁇ l to fibronectin. Error bars represent S.D. from three experiments.
  • Figure 9 shows the effect of RGD peptides and HP2/1 on Jurkat cell-EC-3 interactions.
  • Figure 9 A shows the effect of GRGDSP (SEQ ID NO: 12), GRGESP (SEQ ID NO: 13) and HP 2/1 monoclonal antibody on the adhesion of Jurkat cells to immobilized EC-3.
  • Figure 9B shows the effect of EC- 3 on the binding HP 2/1 monoclonal antibody to cells expressing ⁇ 4 ⁇ l integrin. Error bars represent the S.D. from three experiments.
  • Figure 10 shows the adhesion of CHO cells transfected with 4 and with ⁇ 4 inactive G190A mutant to immobilized EC-3 and to immobilized HP2/1 antibody.
  • the number of adhered cells is shown for: (a) CHO cells transfected with wild-type ⁇ 4, (b) CHO cells transfected with G190A cc4, (c) control CHO-K1 cells.
  • the results with EC-3 are shown as open bars, and the results with HP2/1 are shown as closed bars. Error bars represent S.D. from three experiments.
  • Figure 11 shows the effect of reduced and ethylpyridylethylated EC3A (open circles) and EC3B (closed circles) on adhesion of Jurkat cells to immobilized VCAM-1 (Fig. 11 A) and K562 cells to immobilized fibronectin (Fig. 1 IB). Error bars indicate S.D. from three experiments.
  • Fig. 12 shows the effect of EC-3B derived peptides on Jurkat cell adhesion to immobilized VCAM-1.
  • the inhibitory effect of MLDG peptide (SEQ ID NO: 14) is shown by open circles, RGDS peptide (SEQ ID NO: 15) by closed circles, cyclic *CKRAMLDGLNDYC* (SEQ ID NO: 16) by open triangles and *CKRAMLAGLNDYC* (SEQ ID NO: 17) by closed triangles.
  • Error bars indicate S.D. from three experiments.
  • the present invention relates to the discovery of a novel protein, EC-3, which is a potent inhibitor of ⁇ 4 ⁇ l and ⁇ 4 ⁇ 7 integrins.
  • the EC-3 protein was purified from Echis carinatus suchoreki viper venom.
  • the EC-3 inhibitory effect is not RGD dependent but is competed by HP 2/4 and HP 2/1 monoclonal antibodies, which recognize epitopes on ⁇ 4.
  • EC-3 comprises two types of subunits, EC-3A and EC-3B, which are covalently linked.
  • the EC-3A and EC-3B subunits show a high degree of homology with other viper venom disintegrins, including the alignment of conserved cysteines.
  • the conserved RGD motif of integrins is replaced, however, by VGD in EC-3 A and by MLD in EC-3B.
  • BSA bovine serum albumin
  • CHO cells are Chinese hamster ovary cells.
  • CMFDA 5-chloromethylfluorescein diacetate
  • CS-1 is connecting segment 1 of fibronectin.
  • DBA is direct binding assay.
  • EC-3 protein (also called EC3 protein) is a heterodimeric protein comprising an EC-3A (also called EC3A) subunit and an EC-3B (also called EC3B) subunit.
  • EC-3 peptide (also called EC3 peptide) as used herein encompasses the EC-3 protein as well as the EC-3A and EC-3B subunits, and fragments thereof.
  • ECM extracellular matrix
  • ep is ethylpyridylethylated.
  • the ethylpyridylethylated forms of EC-3A and EC-3B are designated “epEC3A” and “epEC3B.
  • HBSS Hanks' balanced salt solution.
  • HPLC high performance liquid chromatography.
  • Immunoglobulin is immunoglobulin.
  • IC 50 is the concentration of a biologically active agent such as a peptide, which inhibits 50% of the activity obtained in the absence of the agent.
  • Mc is monoclonal antibody.
  • McCAM-1 is mucosal addressin cell adhesion molecule-1, a member of the IgG protein superfamily having a mucin component. MadCAM-1 is a cellular ligand for the ⁇ 4 ⁇ 7 integrin.
  • MLD is the one letter designation of the amino acid sequence Met-Leu-Asp, which occurs in the EC-3 protein.
  • NOTE mouse is a Non Obese Diabetic mouse.
  • PBS phosphate buffered saline solution
  • Arg-Gly-Asp is a recognition site for many disintegrins.
  • a related recognition site is "KGD" which is the one letter designation for Lys-Gly-Asp.
  • TFA is trifluoroacetic acid.
  • VCAM-1 is vascular cell adhesion molecule-1 , a member of the IgG protein superfamily which is a cellular ligand for the 4 ⁇ l and ⁇ 4 ⁇ 7 integrins.
  • VLA-4" is very late activation antigen 4.
  • a "peptide” is a compound comprised of amino acid residues covalently linked by peptide bonds.
  • Amino acids have the following general structure:
  • Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxy lie (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • Peptides comprising a large number of amino acids are sometimes called "poly peptides" .
  • the EC-3A and EC-3B subunits of EC-3 are peptides.
  • a "protein” is a polypeptide which plays a structural or functional role in a biological system. Proteins comprise one or more peptides. EC-3 is a protein.
  • Fibrinogen is a blood plasma glycoprotein, which is involved in platelet aggregation and fibrin formation.
  • Integrins are a family of heterodimeric cell surface proteins which mediate adhesion between cells and between cells and extracellular matrix proteins.
  • Disintegrins are a family of peptides isolated from snake venoms which inhibit the binding of various ligands to integrins.
  • Echistatin is a 49 amino acid disintegrin isolated from Echis carinatus venom.
  • Homology means similarity of sequence reflecting a common evolutionary origin. Peptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or
  • nucleic acids are said to have homology if a substantial number of their nucleotides are identical.
  • Substantial amino acid sequence homology means an amino acid sequence homology greater than about 30 percent, preferably greater than about 60% , more preferably greater than about 80% , and most preferably greater than about 90 percent.
  • Substantially purified peptide or substantially purified protein means a peptide or protein which is substantially free of those compounds that are normally associated therewith in its natural state (e.g. , other proteins or peptides, nucleic acids, carbohydrates, lipids).
  • substantially purified is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
  • a " biologically active fragment " of an EC-3 peptide is a fragment derived from an EC-3 peptide which retains at least one biological activity of the EC-3 peptide.
  • a "biologically active derivative" of an EC-3 peptide is any analogue, variant, derivative, or mutant which is derived from an EC-3 peptide, which has substantial amino acid sequence homology with the EC-3 peptide, and which retains at least one biological property of the EC-3 peptide.
  • Different variants of EC-3 peptides may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for EC-3 peptides, or may involve differential splicing or post-translational modification. The skilled artisan can produce derivatives having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • These derivatives may include, inter alia: (a) derivatives in which one or more amino acid residues are substituted with conservative or non- conservative amino acids, (b) derivatives in which one or more amino acids are added to an EC-3 peptide, (c) derivatives in which one or more of the amino acids includes a substituent group, and (d) derivatives in which the EC-3 peptide is fused with another peptide such as serum albumin.
  • the techniques for obtaining these derivatives including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • Biologically active fragments and biologically active derivatives of EC-3 peptides are intended to be included within the scope of this invention.
  • blocking group is any group capable of blocking the N- terminal amino group of a peptide.
  • a preferred blocking group is an alkyl group; a most preferred blocking group is an acetyl group.
  • nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence.
  • isolated nucleic acid means a nucleic acid which is substantially free of those compounds that are normally associated therewith in its natural state. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
  • a nucleic acid which hybridizes at high stringency means that the hybridized nucleic acids are able to withstand a washing under high stringency conditions.
  • An example of high stringency washing conditions for DNA-DNA hybrids is 0.1X SSC, 0.5 % SDS at 68°C. Other conditions of high stringency washing are known to persons having ordinary skill in the art.
  • regulatory region means a nucleic acid sequence which regulates the expression of a nucleic acid.
  • a regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid
  • a homologous region may include sequences of a different origin (responsible for expressing different proteins or even synthetic proteins).
  • the sequences can be sequences of eukaryotic or viral genes or derived sequences which stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner.
  • Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences which direct the polypeptide into the secretory pathways of the target cell, and promoters.
  • a regulatory region from a "heterologous source” is a regulatory region which is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.
  • “Operatively linked to a regulatory region” means that the peptide or polypeptide coding region is connected to transcriptional and translational regulatory sequences in such a way as to permit polypeptide expression when the appropriate molecules (such as activator proteins and polymerases) are present in a cell or cell free system.
  • a “vector” is any means for the transfer of a nucleic acid according to the invention into a host cell.
  • the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a prokaryotic or eukaryotic cell in vitro, ex vivo, or in vivo.
  • Non-viral vectors include plasmids, liposomes , electrically charged lipids (cy tof ectins) , DN A-protein complexes , and biopolymers.
  • Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors.
  • a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring , and monitoring nucleic acid transfer results (transfer to which tissues , duration of expression, etc.).
  • a “recombinant cell” is a cell which contains a nucleic acid which is not naturally present in the cell.
  • “Recombinant cell” includes higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells, and archaebacterial cells.
  • “Antibody” as used herein includes monoclonal and polyclonal antibodies as well as fragments capable of binding antigen, including but not limited to Fab and F(ab) 2 fragments.
  • “Pharmaceutically acceptable carrier” includes diluents and fillers which are pharmaceutically acceptable for methods of administration, may be sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents.
  • the particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.
  • the present invention provides substantially purified EC-3 peptides, including the EC-3 protein, the EC-3A and EC-3B peptides, and biologically active fragments and derivatives of EC-3, EC-3A, and EC-3B.
  • the invention also provides methods of isolation and peptides isolated from other venoms having biological activity similar to EC-3.
  • the peptides of the present invention may be recombinant peptides, natural peptides, or synthetic peptides. Each peptide is characterized by a reproducible single molecular weight and/or multiple set of molecular weights, chromatographic response and elution profile, amino acid composition and sequence, and biological activity.
  • the peptides of the present invention may be isolated from natural sources, such as viper venom, using the methods disclosed herein.
  • the peptides of the invention may also be chemically synthesized, using, for example, solid phase synthesis methods.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • N-protecting groups e.g. , the carbobenzyloxy group of the t- butyloxycarbonyl group
  • various coupling reagents e. g. , dicyclohexylcarbodiimide or carbonyldimidazole, various active esters, e.g. , esters of N-hydroxyphthalimide or N-hydroxy-succinimide
  • various cleavage reagents e.g.
  • Crude peptides may be purified using preparative HPLC. The amino terminus may be blocked according, for example, to the methods described by Yang et al. (FEBS Lett. 272:61-64 (1990)). Peptide synthesis includes both manual and automated techniques employing commercially available peptide synthesizers. Fragments and derivatives of EC-3 peptides may be prepared by chemical synthesis and biological activity can be tested using the methods disclosed herein.
  • the peptides of the invention may be prepared utilizing recombinant DNA technology, which comprises combining a nucleic acid encoding the peptide thereof in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered.
  • the peptides of the present invention may be used in the form of a pharmaceutically acceptable salt.
  • suitable acids which are capable of forming salts with the peptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, gly colic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.
  • Suitable bases capable of forming salts with the peptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. , triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e. g. , ethanolamine, diethanolamine and the like).
  • inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
  • organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. , triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e. g. , ethanolamine, diethanolamine and the like).
  • the present invention provides substantially purified nucleic acids which encode peptides according to the invention.
  • EC-3A and EC-3B amino acid sequences disclosed herein one of skill in the art can isolate or prepare a gene that encodes the peptide of interest. Synthetic genes may be synthesized directly on a DNA synthesizer, or may be synthesized as complementary oligonucleotides which are ligated together to form the synthetic gene. Alternatively, the native gene encoding the peptides of the invention may be isolated from genomic or cDNA libraries. As an example, based upon the amino acid sequences disclosed herein, one of skill in the art can prepare suitable oligonucleotide probes and polymerase chain reaction (PCR) primers, which can be used to screen a cDNA library prepared from E. carinatus venom glands. Positive clones are purified and sequenced to confirm their identity.
  • PCR polymerase chain reaction
  • the nucleic acids encoding ⁇ C-3 peptides may be operatively linked to one or more regulatory regions. Regulatory regions include promoters, poly adenylation signals, translation initiation signals (Kozak regions), termination codons, peptide cleavage sites, and enhancers.
  • the regulatory sequences used must be functional within the cells of the vertebrate to be immunized. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art.
  • Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters.
  • the promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are lad, lacZ, T3, T7, lambda P r . P r and tip promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g.
  • tissue-specific promoters e.g. actin promoter in smooth muscle cells
  • promoters which respond to a stimulus e.g. steroid hormone receptor, retinoic acid receptor
  • tetracycline-regulated transcriptional modulators cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela, and MLP promoters.
  • Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and 5,385,839, the entire disclosures of which are incorporated herein by reference.
  • poly adenylation signals examples include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • Fragments and derivatives of EC-3 peptides may be prepared using recombinant DNA technology. The biological activity of the fragments and derivatives can be assayed using the methods disclosed herein.
  • the present invention provides antibodies against EC-3 peptides. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of an Fab expression library.
  • Polyclonal antibodies may be generated against the intact protein or peptide, or against a fragment, derivative, or epitope of the protein or peptide.
  • Antibodies may be obtained following the administration of the protein, polypeptide, fragment, derivative, or epitope to an animal, using the techniques and procedures known in the art.
  • Monoclonal antibodies may be prepared using the method of
  • a peptide of the present invention is used to immunize spleen cells of Balb/C mice.
  • the immunized spleen cells are fused with myeloma cells.
  • Fused cells containing spleen and myeloma cell characteristics are isolated by growth in HAT medium, a medium which kills both parental cells, but allows the fused products to survive and grow.
  • Antibodies may be used to purify the peptides according to the invention, using immunoaffinity techniques which are well known by those of skill in the art.
  • the present invention provides a method for isolating peptides that bind to an integrin of interest from the venom of snakes or other organisms. This method comprises the steps of: (a) dissolving venom in a solvent,
  • step (c) fractionating the supernatant from step (b), (d) immobilizing the fractions from step (c) on a solid support,
  • step (g) isolating peptide from those fractions which showed enhanced cell binding in step (f).
  • This method may be used to isolate peptides from the venom of species including but not limited to vipers and other snakes including Agkistrodon acutus, Agkistrodon halys blomhoffi, Agkistrodon contortrix mokasen, Bitis arietans, Bitis caudalis, Bitis gabonica, Bitis g. rhinoceros, Bothrops asper, Bothrops alternata, Bothrops atrox, Bothrops cotiara, Bothrops jararaca, Bothrops newiedi, Bothrops medusa, Bothrops schlegli, Cerastes cerastes, Cerastes vipera, Crotalus adamanteus, C. atrox, C. basilicus, C.
  • vipers and other snakes including Agkistrodon acutus, Agkistrodon halys blomhoffi, Agkistrodon contortrix mokasen, Bitis arietans, Bitis caudalis, Bitis gabonica
  • the present invention provides methods of inhibiting the binding of 4 ⁇ l or ⁇ 4 ⁇ 7 integrins to a ligand.
  • a preferred embodiment is a method of inhibiting the interaction between cells expressing ⁇ 4 ⁇ l and/or ⁇ 4 ⁇ 7 integrins and a ligand, in a patient in need of such treatment, comprising administration of a therapeutically effective amount of a composition comprising one or more peptides according to the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • a composition comprising one or more peptides according to the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • compositions include physiologically tolerable or acceptable diluents, excipients, solvents, adjuvants, or vehicles, for parenteral injection, for intranasal or sublingual delivery, for oral administration, for rectal or topical administration or the like.
  • the compositions are preferably sterile and nonpyrogenic.
  • suitable carriers include but are not limited to water, saline, dextrose, mannitol, lactose, or other sugars, lecithin, albumin, sodium glutamate cystein hydrochloride, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyehtylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used.
  • auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like).
  • absorption enhancing or delaying agents such as liposomes, aluminum monostearate, or gelatin
  • the compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • compositions may be administered by any convenient route which will result in delivery to the blood stream in an amount effective for inhibiting ⁇ 4 ⁇ l- and ⁇ 4 ⁇ 7-mediated adhesion, including orally, rectally, parenterally (intravenously, intramuscularly, intraarterially, or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol.
  • the compositions can also be delivered through a catheter for local delivery at a target site, or via a biodegradable polymer.
  • the compositions may also be complexed to ligands, or antibodies, for targeted delivery of the compositions.
  • compositions are most effectively administered parenterally, preferably intravenously.
  • intravenous administration they may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances, such as sodium chloride, glycine, and the like, having a buffered pH compatible with physiologic conditions.
  • physiologically compatible substances such as sodium chloride, glycine, and the like
  • the vehicle is a sterile saline solution. If the peptides are sufficiently small (e.g. , less than about 8-10 amino acids) other preferred routes of administration are intranasal, sublingual, and the like.
  • the compositions according to the invention can be administered in any circumstance in which inhibition of 4 ⁇ l and ⁇ 4 ⁇ 7 integrin function is desirable.
  • Disease states which may be treated include but are not limited to diabetes, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, autoimmune disorders, transplant rejection, and restenosis of the arteries following surgery or angioplasty. Because the ⁇ 4 integrins are expressed on various cancer cells, including leukemia, melanomas, lymphomas, and sarcomas, inhibitors of ⁇ 4 binding can also be useful in the treatment of some forms of cancer.
  • the amount of peptide administered depends upon the degree of integrin inhibition that is desired. Those skilled in the art will derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient. Typically, dosages are between about 0.001 mg/kg and about 100 mg/kg body weight. In some embodiments dosages are between about 0.01 mg/kg and about 10 mg/kg body weight. In some embodiments dosages are between about 0.05 mg/kg and about 5 mg/kg body weight. Examples The following examples illustrate the invention. These examples are illustrative only, and do not limit the scope of the invention.
  • HP2/1 anti- 4 subunit of VLA-4) and SAM-1 (anti- ⁇ 5 subunit of VLA-5) were purchased from Immunotech, Inc. (Westbrook, ME).
  • HP2/4 was received as a gift from Dr. Francisco Sanchez- Madrid (Madrid, Spain). The biological effects of HP2/1 and HP2/4 were identical.
  • Highly purified human fibrinogen was gift from Dr. A. Budzynski (Temple University, Philadelphia PA)
  • recombinant human VCAM-1 was a gift from Dr. M.
  • Renz Genentech, San Francisco, CA
  • human vitronectin and fibronectin were purchased from Calbiochem (La Jolla, CA) and Sigma (St. Louis, MO), respectively.
  • GRGDSP and GRGESP peptides were purchased from Bachem (Torrance, CA).
  • RGDS was purchased from Sigma.
  • Highly purified MLDG peptide was kindly provided by Dr. Z. Huang (Thomas Jefferson University, Philadelphia, PA) .
  • Echistatin was isolated from Echis carinatus suchoreki venom as described by McLane et al. (Biochem. J. 301:429-436 (1994).
  • Fluorescein isothiocynate (FITC)-conjugated goat anti-mouse IgG for flow cytometry was purchased from Jackson Immune Research (West Grove, PA).
  • A5 and VNRC3 cells CHO cells transfected with human ⁇ llb ⁇ 3 and ⁇ v ⁇ 3 integrins, respectively (O'Toole etal. , Cell. Regul. 1:883-893 (1990)), were kindly provided by Dr. M. Ginsberg (Scripps Research Institute, La Jolla, CA).
  • CHO cells with deleted ⁇ 5 integrin (B2 cells) were kindly provided by Dr. R. Juliano (University of North Carolina, Chapel Hill, NC).
  • CHO cells transfected with human 4 and its G190A mutant Karlina et al, Biochem. J. 305:945-951 (1995)
  • B2 cells transfected with human 4 4B2 were kindly provided by dr.
  • JY cells expressing ⁇ 4 ⁇ 7 were a gift of Dr. S. Burakoff (Dana-Farber Cancer Institute, Boston MA)
  • K562 cells transfected with ⁇ 6 integrin were a gift of Dr. A. Sonnenberg (Netherlands Cancer Institute, Amsterdam, Holland).
  • Jurkat cells, K562 cells, and non transfected CHO-K1 cells were purchased from ATCC (Rockville , MD) .
  • CMFDA 5-chloromethylfluorescein diacetate
  • Unbound label was removed by washing with the same buffer. Labeled cells (1 x 10 5 per sample) were added to the well in the presence or absence of inhibitors and incubated at 37°C for 30 min. Unbound cells were removed by aspiration, the wells were washed and bound cells were lysed by adding 0.5% Triton X-100. In parallel the standard curve was prepared in the same plate from known concentrations of labelled cells. The plate was read using a Cytofluor 2350 fluorescence plate reader (Millipore, Bedford, MA) at 485 nm EX(excitation) filter and 530 nm (emmision) filter.
  • DBA direct binding assay
  • VCAM-1 Ig alkaline phosphatase conjugate was performed using Jurkat ( ⁇ 4 ⁇ l expressing) and JY ( ⁇ 4 ⁇ 7 expressing) cells according to procedure described in Lobb et al, Cell Adhes. Commun. 3:385-398 (1995)).
  • Lyophilized Echis carinatus suchoreki venom obtained from Latoxan Serpentarium, Rosans 05150 France
  • 0.1 % trifluoroacetic acid 10 mg/300 ⁇ X
  • the solution was centrifuged for 5 minutes at 5000 rpm to remove the insoluble proteins, and the pellet was discarded.
  • the supernatant was applied to a C-18 HPLC column (250 x 10 mm, Vydac TPRP equilibrated in 0.1 % TFA. The column was eluted with an acetonitrile gradient (0-80% over 45 min.) at a flow rate of 2.0 ml/min.
  • ECl As shown in Figure 1 , the venom separated into 17 main fractions, which were designated “ECl " through "EC 17.
  • EC 10 was identified as echicetin (Peng et al., Blood 81:2321-28 (1993)).
  • EC-3 eluted at approximately 40% acetonitrile.
  • each eluted fraction was lyophilized, then dissolved in water.
  • the protein concentration in each fraction was estimated using the BCA assay
  • Jurkat cells were labeled by incubation with 12.5 ⁇ M CMFDA (5- chloromethylfluorescein iacetate) in HBSS (Hanks' balanced salt solution) buffer containing 1 % BSA (bovine serum albumin) for 15 minutes at 37 °C. Cells were washed three times to remove excess CMFDA.
  • CMFDA chloromethylfluorescein iacetate
  • the labeled Jurkat cells were added to the microtiter plate (1 x 10 5 cells per well), and the plate was incubated for 30 minutes at 37 °C. After incubation, unbound cells were removed by aspiration and the wells were washed three times with HBSS/BSA buffer.
  • Bound cells were lysed by the addition of 0.5 % Triton X-100, and fluorescence was measured in a Cytofluor 2350 fluorescence plate reader (Millipore) using a 485 nm EX (excitation) filter and a 530 nm EM (emission) filter.
  • the cell adhesion assay (as described in Example 1) was used to study the inhibitory effect of the eluted fractions on Jurkat cell adhesion to immobilized VCAM-1 and on K562 cell adhesion to fibronectin.
  • Recombinant VCAM-1 (0.5 ⁇ g/well) or fibronectin (0.5 ⁇ g/well) were immobilized overnight at 4°C on a 96-well plate (Falcon) in PBS buffer. After blocking, the CMFDA- labeled cells were added to each well in the presence of 5 ⁇ g per sample of protein from selected fractions. The results are shown in Fig. 2.
  • EXAMPLE 3 Purification of EC-3
  • the venom fraction eluting at approx. 40% and designated EC3 was purified to homogeneity by two steps of reverse phase HPLC using a C-18 column.
  • proteins from crude venom were separated as in Example 2.
  • the column was eluted with an acetonitrile linear gradient 0-80% over 45 minutes, and the EC 3 fraction eluting at approximately 40% of acetonitrile was collected, lyophilized, and dissolved in water.
  • the recovery of protein after the first step was 7 mg per 1 g of dried venom.
  • EC-3 The reduction and alkylation of EC-3 were performed according to a procedure used for trigramin (Huang et al. , J. Biol. Chem. 262: 16157- 63 (1987)). Briefly, 100 ⁇ g of EC-3 was incubated in 200 ⁇ l of 6 M guanidine hydrochloride, 4 mM EDTA, 0.1 M Tris-HCl, pH 8.5, 3.2 mM dithiothreitol, and 2 ⁇ l of vinyl pyridine. The reaction mixture was incubated for two hours at room temperature in the dark, then applied to a C-18 HPLC column and eluted with an acetonitrile gradient (0-80% over 45 min.). In some experiments EC-3 subunits were reduced and carboxymethylated with iodoacetate acid followed by HPLC separation.
  • N-terminal sequence of native EC-3 electroblotted onto PVDF membrane (Madsudaira, J. Biol. Chem. 262: 10035-10038 (1987)) and residues 1-40 of epEC3A and epEC3B were determined by N-terminal sequence analysis using an Applied Biosystem Precise instrument.
  • EC- 3A and EC-3B were deduced from Edman degradation of overlapping peptides obtained by digestion with endoproteinase Lys-C (Boehringer Mainhem) (2 mg/ml protein in 100 mM ammonium bicarbonate, pH 8.3, for 18 h at 37°C using an enzyme : substrate ratio of 1 : 100 (w/w)) and CNBr (10 mg/ml protein and 100 mg/ml CNBr in 70% formic acid for 6 h under N 2 atmosphere and in the dark).
  • endoproteinase Lys-C Boehringer Mainhem
  • CNBr 10 mg/ml protein and 100 mg/ml CNBr in 70% formic acid for 6 h under N 2 atmosphere and in the dark.
  • Immobilon-P revealed a single amino acid sequence : NSVHPXXDPV(K/T)XEPREGEHXISGP (SEQ ID NO: 1).
  • Both, EC3A and EC3B are cysteine-rich proteins of 67 amino acids and display heterogeneity at several positions, indicating the existence of isomorphs (isomorphs are given as SEQ ID NOs: 19 and 20; the predicted major isomorphs are given as SEQ ID NOs: 2 and 3).
  • the isotope-averaged molecular masses calculated for the reduced EC3A isomorphs (1-67: N33 137 G64 E66), (1-67: R33, V37, G64, E66), and (1-66: N33, 137, D64, D66) are 7412 Da, 7440, Da and 7341 Da, corresponding to major and minor ions of reduced EC-3 mass spectrum.
  • the major EC3A isomorph might be that with molecular weight 7412, which yields a mass of 8476 after reduction and ethylpyridylation.
  • reduced EC3B isomorphs (1-67: Tl 1 , K40, S55), (1-67: Kl 1 , R40, T55), and (1-67: Til, R40, T55) have calculated masses, 7370 Da, 7439 Da, and 7412 Da, respectively.
  • the major EC3B isomorph i.e. the one that would have a molecular mass of 7950 after reduction and carboxymethylation, is EC3B 7370 Da.
  • EC3A and EC3B isomorphs could combine into a number of dimers.
  • EC3A-EC3B heterodimers may represent the major species, however, because i) homodimers would not yield separated subunits displaying the distinct biological activities demonstrated for HPLC purified A and B fractions, and ii) a mixture of EC3A and EC3B homodimers would display a more complex HPLC separation profile.
  • EC3A and EC3B show a high degree of sequence similarity between themselves and with eristostatin (Gould et al. , Proc. Soc. Exp. Biol. Med.
  • Recombinant human VCAM-1 (0.5 ⁇ g/well) was immobilized in the wells of an ELISA plate overnight in PBS buffer. The plate was blocked using HBSS buffer containing 1 % BSA. CMFDA-labelled Jurkat cells (1 x 10 5 cells per sample) were added to the wells in the presence or absence of EC-3 in HBSS buffer containing 1 % BSA. The plate was incubated for 30 minutes at 37°C. Unbound cells were removed by aspiration and the wells were washed with HBSS buffer. The bound cells were lysed with 0.5% Triton X-100, and fluorescence was measured. Percent inhibition was calculated by comparing the fluorescence obtained for adhered cells in the absence (0% inhibition) and presence of EC-3. Figure 6 shows the percent inhibition with increasing concentrations of EC-3 , in the presence (filled circles) or absence (open circles) of l mM Mn + + .
  • EC-3 inhibits the adhesion of Chinese hamster ovary cells, which are ⁇ 5 deficient (B2 cells) and have been transfected with human ⁇ 4 integrin, to VCAM-1.
  • Chinese hamster ovary cells transfected with human ⁇ 4 integrin were provided by Dr. Y. Takada (Scripps Research Institute, La Jolla, CA).
  • Recombinant human VCAM-1 (0.5 ⁇ g/well) was immobilized in the wells of an ELISA plate overnight in PBS buffer. The plate was blocked using HBSS buffer containing 1 % BSA.
  • CMFDA-labelled 4B2 cells (1 x 10 5 cells per sample) were added to the wells in the presence or absence of EC-3 in HBSS buffer containing 1 % BSA. The plate was incubated for 1 hour at 37 °C. Unbound cells were removed by aspiration and the wells were washed with HBSS buffer. The bound cells were lysed with 0.5 % Triton X-100, and fluorescence was measured. Figure 7 shows the percent inhibition with increasing concentrations of EC-3.
  • VNRC3 (CHO ⁇ v ⁇ 3+) ctv ⁇ 3 Vn CA SO >10 4
  • Fg fibrinogen
  • Fn fibronectin
  • Vn vitronectin
  • CS-1 conective segment 1 of fibronectin
  • Lm laminin
  • PA platelet aggregation
  • CA cell adhesion
  • DBA direct binding assay
  • ND not determined
  • EC3 was a highly potent inhibitor of the interaction of both anchorage- dependent and anchorage- independent cells expressing ⁇ 4 ⁇ 1 with either VCAM- 1 or the CS-1 fragment of fibronectin, while echistatin showed no detectable activity at 10 ⁇ M.
  • EC3 inhibited to the same extent adhesion of A2 (CHO ⁇ 4+ ⁇ 5 +) cells and ⁇ 4B2 (CHO 4 + 5-) cells to immobilized VCAM-1, confirming direct inhibition of 4 ⁇ l integrin.
  • Figure 9A shows the effect of GRGDSP (SEQ ID NO: 12), GRGESP (SEQ ID NO: 13) and HP 2/1 monoclonal antibody on the adhesion of Jurkat cells to immobilized EC3.
  • the adhesion study was performed using CMFDA-labeled Jurkat cells in the absence or presence 1 mM GRGDSP or GRGESP, or 10 ⁇ g/ml HP 2/1. Competition between EC3 and HP2/1 was also confirmed in cell suspension using FACS analysis.
  • Fig. 9B shows inhibition of HP2/1 binding to ⁇ 4B2 cells. The experiment was performed using flow cytometry.
  • Jurkat cells open bars or B2-CHO cells (CHO cells with deleted ⁇ 5 transfected with 4) (closed bars) were incubated with 10 ⁇ g/ml HP 2/1 in the absence (a) or presence 60 nM EC3 (b) for 30 min at room temperature. After washing, 10 ⁇ g/ml of FITC-conjugated goat anti-mouse IgG was added and samples were incubated for another 30 min at room temperature. The samples were fixed by addition of 1 % paraformaldehyde before measurement of fluorescence intensity. No inhibitory effect was observed in Jurkat cells, which express both 5 ⁇ l and ⁇ 4 ⁇ l.
  • Figure 10 shows the adhesion of CHO cells transfected with ⁇ 4 and with 4 inactive G190A mutant to immobilized EC3 and to immobilized HP2/1 antibody.
  • EC3 (2 ⁇ g/well) and HP2/1 (1 ⁇ g/well) were immobilized on the 96-well plate.
  • the CHO cells were labeled with CMFDA and adhesion was performed as described above.
  • the inactive G190A ⁇ 4 mutant did not interact with EC3, since adhesion of CHO transfected with this mutant to immobilized EC3 was identical with adhesion of non transfected cells. Only A2 cells expressing wild type ⁇ 4 adhered to immobilized HP2/1.
  • Blood from a healthy donor was collected into a syringe containing sodium citrate as an anticoagulant.
  • the anticoagulated blood was centrifuged at 400g for 17 minutes, and platelet rich plasma (PRP) was removed by aspiration.
  • PRP platelet rich plasma
  • the aggregation of platelets in PRP in the presence of 30 ⁇ M ADP was measured in the absence and presence of echistatin and EC-3, using an aggregation meter (Sinco).
  • Echistatin and EC-3 inhibited ADP induced platelet aggregation with an IC 50 of 130 nM and 1,000 nM, respectively.
  • EXAMPLE 6 Biological Activity of EC-3 Peptides
  • RGDX motif in monomeric disintegrins is replaced by MLDG (SEQ ID NO: 14) in EC-3B. Both RGDX and MLDG therefore appear to represent integrin binding sites.
  • An RGDS peptide (SEQ ID NO: 15) which is a known inhibitor of several ⁇ l and ⁇ 3 integrins, was inactive at this concentration.
  • Removal of the side-chain protecting groups and cleavage of the assembled peptide from the resin were achieved by treating with trifluoroacetic acid in the presence of ethane dithiol, crystalline phenol, thioanisole and tri-isopropyl silane as scavengers.
  • the peptides were purified using HPLC on C18 column with a water/acetonitrile gradient, containing 0.1 % TFA.
  • the amino acid composition of the peptides was determined by the Pico Tag method, and the molecular mass of the peptides was confirmed using Mass spectrometry.
  • Additional peptides comprising fragments and derivatives of EC-3 such as Met Leu Asp Gly Leu (SEQ ID NO: 18), are synthesized and inhibitory activity is measured using assays such as those described in Example 5.
  • a modified echistatin polypeptide is produced.
  • the Arg- Gly-Asp residues at positions 24-26 are replaced by Met-Leu-Asp.
  • the biological activity of the modified echistatin, as well as fragments and derivatives thereof, is determined.
  • Lymphocyte reticulation and accumulation at a site of inflammation is a multistep process which involves interaction between lymphocyte adhesion molecules and their endothelial ligands.
  • Non Obese Diabetic mice develop an age related lymphocyte infiltration of pancreatic islets and salivary glands, which resembles human type I diabetes and Sjogren's syndrome.
  • Studies on the adhesion molecules involved in lymphocyte invasion of pancreas and salivary glands in NOD mice have suggested that the integrins ⁇ 4 ⁇ l and ⁇ 4 ⁇ 7 are important for infiltration of Langerhans islets in the pancreas.
  • the following experiment showed that EC-3, which selectively inhibits ⁇ 4 integrins, can block lymphocyte invasion of pancreatic parenchyma in NOD mice.
  • Echistatin which inhibits v ⁇ 3- and ⁇ 5 ⁇ l -dependent interactions, but not ⁇ 4-dependent interactions, was inactive in this experiment.
  • mice from 1 to 5 weeks of age were purchased from
  • mice were divided into 3 groups, and each group was treated with PBS, echistatin, or EC-3 for nine weeks.
  • the PBS, echistatin, and EC-3 were administered intraperitoneally in a total volume of 0.5 ml three times per week.
  • the treatment groups were: (a) PBS (control); (b) 1 ⁇ g/mouse of echistatin; and (c) 3 ⁇ g/mouse of EC-3.
  • the 3 ⁇ g/mouse EC-3 dose is the molar equivalent of the 1 ⁇ g/mouse echistatin dose, since the molecular weight of EC-3 is approximately three times that of echistatin.
  • the animals were sacrificed by exposure to CO 2 .
  • pancreas was removed, frozen, and stored at -70 °C until the histological analysis.
  • histological analysis five ⁇ m sections of frozen tissue were fixed with acetone.
  • the sections were stained with hematoxylin/eosin, and observed by light microscopy.

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Abstract

Identification, purification et caractérisation d'une nouvelle désintégrine hétérodimère, EC-3, tirée du venin de la vipère Echis carinatus. EC-3 inhibe les intégrines α-4 d'une manière indépendante de RGD. La présente invention concerne en outre des procédés d'utilisation de EC-3, ou d'un fragment ou dérivé biologiquement actif de ladite substance, pour inhiber l'interaction entre des cellules exprimant les intégrines α4 et des ligands cellulaires.
PCT/US1998/016719 1997-08-15 1998-08-13 Ec-3 en tant qu'inhibiteur des integrines alpha4 beta1 et alpha4 beta7 WO1999013898A1 (fr)

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