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WO1993009137A1 - Inhibiteurs de l'agregation plaquettaire provoquee par le collagene - Google Patents

Inhibiteurs de l'agregation plaquettaire provoquee par le collagene Download PDF

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Publication number
WO1993009137A1
WO1993009137A1 PCT/US1991/007958 US9107958W WO9309137A1 WO 1993009137 A1 WO1993009137 A1 WO 1993009137A1 US 9107958 W US9107958 W US 9107958W WO 9309137 A1 WO9309137 A1 WO 9309137A1
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Prior art keywords
polypeptide
collagen
platelets
amino acid
monoclonal antibody
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PCT/US1991/007958
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English (en)
Inventor
J. Bryan Smith
Carol A. Dangelmaier
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Temple University-Of The Commonwealth System Of
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Priority to PCT/US1991/007958 priority Critical patent/WO1993009137A1/fr
Publication of WO1993009137A1 publication Critical patent/WO1993009137A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to inhibition of platelet aggregation, by novel polypeptides which interfere with the platelet's adhesion to fibrillar collagen.
  • Fibrillar collagen has been identified as the most thrombogenic macromolecular component of the blood vessel wall (Id.). Platelets which adhere to the thus-exposed collagen release the contents of their dense, nucleotide and amine storage granules, including adenosine diphosphate (ADP) (Zucker, Amer. J.
  • Substantially purified polypeptides corresponding to polypeptides obtainable from snake venoms are provided, which polypeptides inhibit adhesion of platelets to collagen.
  • Such platelet adhesion-inhibiting polypeptides (hereinafter collectively referred to as "antihesins") contain an antigenic determinant which is recognized by monoclonal antibody from hybridoma ATCC HB-10904.
  • antihesins contain an antigenic determinant which is recognized by monoclonal antibody from hybridoma ATCC HB-10904.
  • Recognized is meant that the monoclonal an tibody specifically binds to the antihesin antigenic determinant.
  • pharmaceutical compositions comprising one or more antihesins and a pharmaceutically acceptable carrier, useful for inhibiting platelet adhesion and aggregation.
  • the invention also relates to hybridomas, such as ATCC HB10904, which have been prepared providing cell lines producing monoclonal antibodies which recognize an antigenic determinant shared by the antihesins.
  • the hybridomas comprise cell hybrids formed by fusion of cells from a myeloma line and spleen cells from a donor previously immunized with an immunogen containing an antihesin, preferably substantially purified antihesin.
  • ATCC HB-10904 is one such hybridoma. It was deposited on October 17, 1991 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD.
  • the hybridomas may be cultured in vitro or in vivo to secrete antibodies.
  • the invention also relates to the monoclonal antibodies so produced.
  • the hybrid cell lines may be prepared by first immunizing a splenocyte donor with immunogen comprising antihesin.
  • the spleen cells are fused with myeloma cells in the presence of a fusion promotor.
  • the fused cells are diluted and cultured in separate wells in a medium which will not support the unfused myeloma cells.
  • the supernatant of each well is assayed for the presence of antibody to antihesin by an assay, such as an enzyme-linked immunosorbent assay ("ELISA").
  • ELISA enzyme-linked immunosorbent assay
  • the hybridomas are cultured in a suitable medium and the antibody is recovered from the supernatant.
  • the clones are transferred intraperitoneally into a suitable host, e.g. mice, and the resulting malignant ascites and/or serum containing the desired antibody are harvested.
  • the invention provides a method for purifying antihesins from a liquid, such as snake venom.
  • the liquid is contacted with an immobilized antibody, preferably a monoclonal antibody, which recognizes an antigenic determinant of antihesin to absorb that polypeptide from the liquid.
  • the antihesin is thereafter eluted from the immobilized monoclonal antibody.
  • lyophylized venom is dissolved in a solvent.
  • the venom is fractionated to separate the polypeptides contained therein.
  • the venom fractions are then assayed for activity in inhibiting the adhesion of platelets to collagen, or, alternatively, for polypeptide which is recognized by monoclonal antibody from hybridoma ATCC HB- 10904.
  • Polypeptide which inhibits platelet adhesion to platelets and/or is recognized by the aforesaid antibody is then purified from the active fractions.
  • the invention yet further provides a method for inhibiting adhesion of platelets to fibrillar collagen in a mammal comprising administering antihesin to the mammal.
  • a method of inhibiting collagen-induced aggregation of mammalian platelets comprising administering antihesin to a mammal to inhibit the occurrence of platelet aggregation in the bloodstream of the mammal.
  • antibody inclusive of both monoclonal and polyclonal antibodies, shall include not only the intact antibody, but also fragments thereof capable of binding antigen, including, but not limited to, Fab and F(ab) 2 fragments.
  • the expression “corresponds to” or “corresponding to” with regard to the relationship between a substantially purified polypeptide and the native snake polypeptide means that the purified polypeptide is identical to the native polypeptide, or is a synthetic or genetically engineered polypeptide or fragment of the native polypeptide, which has an amino acid sequence substantially the same as that of the native polypeptide, or at least sufficiently duplicative of the native sequence to retain the biological activity of the native polypeptide.
  • Fig. 1 shows the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%, non-reduced) of B. atrox antihesin at various stages of purification.
  • Lane 1 molecular weight standards; lane 2, crude B. atrox venom; lane 3, B. atrox venom after SEPHADEX G-100 column chromatography; lane 4, 50 kDa B. atrox antihesin after high performance liquid chromatography (HPLC).
  • Fig.2 shows the SDS-PAGE (12%, non-reduced) of c. atrox antihesin at various stages of purification.
  • Lane 1 molecular weight standards; lane 2, crude C. atrox venom; lane 3, C. atrox venom after SEPHADEX G-100 column chromatography; lane 4, C. atrox 13 kDa antihesin after HPLC; lane 5, C. atrox 50 kDa antihesin after HPLC.
  • Fig. 3 is a Western blot of crude snake venoms with ATCC HB-10904 monoclonal antibody following SDS-PAGE (12%, non-reduced). Lane 1, C. basiliscus (20 micrograms); lane 2, B. atrox (200 ng); lane 3, B. jararaca (200 ng); lane 4, A. halys blomohoffii (200 ng). Molecular weight standards are shown on the right.
  • Fig. 4 is a concentration-response curve for inhibition of platelet adhesion to collagen (25 micrograms/ml) by the purified antihesin polypeptide from B. atrox (SVP).
  • Figs. 5A through. 5E are a series of aggregometer tracings showing the effect of the purified antihesin from B. atrox (indicated by + sign) on aggregation in human citrated platelet-rich plasma induced by (5A) collagen (1 microgram/ml); (5B) ADP (5 micromolar); (5C) epinephrine (5 micromolar); (5D) platelet-activating factor (0.25 micromolar); and (5E) the thromboxane mimetic SQ 26655 (0.2 micromolar).
  • Adhesion of platelets to collagen at 37°C is a very rapid process, which is essentially complete by 1 minute.
  • the factor limiting the extent of adhesion is the amount of collagen to which the platelets are exposed.
  • the polypeptides of the present invention inhibit platelet aggregation in plasma by interfering with this initial step in hemostasis/thrombosis. This is in contrast to the so-called "disintegrin" class of antithrombotic agents which inhibit platelet aggregation by interfering with platelet binding to fibrinogen, a subsequent step in the pathway leading to thrombus formation.
  • the disintegrins which contain the tetrapeptide sequence Arg-Gly-Asp-Ser, bind to the fibrinogen receptor of platelet glycoprotein Ilb/IIIa, and thereby prevent build-up of platelet aggregates.
  • the disintegrins thus inhibit platelet aggregation induced by ADP, thrombin, epinephrine, and sodium arachidonate, as well as collagen.
  • the anti-adhesive polypeptides of the invention have no effect on platelet aggregation induced by platelet agonists other than collagen. They do not act on the fibrinogen receptor on platelet glycoprotein Ilb/IIIa. Rather, they bind to distinct receptors on the collagen molecule.
  • the antihesins inhibit platelet aggregation by preventing initial platelet attachment via collagen to the site of vascular injury.
  • Biological materials such as snake venoms may be screened for antihesins by an assay for platelet adhesion to collagen in the absence of platelet aggregation.
  • an assay for platelet adhesion to collagen in the absence of platelet aggregation.
  • suspensions of human platelets are incubated with or without the biological source material, in the presence of an ADP-removing system and antagonists to TxA 2 , platelet activating factor (PAF), serotonin, and fibrinogen, which comprise the platelet agonists responsible for aggregation.
  • PAF platelet activating factor
  • fibrinogen fibrinogen
  • the polypeptide may be isolated from the material in one of two ways.
  • the antihesin is purified by a combination of gel filtration and high performance liquid chromatography.
  • immobilized antibody which binds antihesin, preferably monoclonal antibody, is contacted with the biological material. Antihesin is absorbed from the material and thereafter eluted from the immobilized antibody.
  • the activity of the antihesins is such that they produce greater than 50% inhibition of platelet adhesion to collagen at an antihesin concentration of 100 ⁇ g/ml (about 2 ⁇ M, based upon an antihesin molecular weight of about 50kDa).
  • Antihesins have been isolated with greater than 95 weight percent purity based upon the presence of a single band upon Coomassie blue or silver stained SDS-PAGE.
  • substantially purified as the expression is used herein, is meant a purity of at least about 50 weight percent. While a purity level of 95 weight percent is generally utilized for clinical use, lower purity material may be satisfactory for in vitro applications.
  • the monoclonal antibodies which may be employed in immunoaffinity purification of antihesins recognize an antigenic determinant shared by such polypeptides, regardless of the source of the polypeptide.
  • Anti-antihesin antibodies may be prepared by immunizing an appropriate host with antihesin, purified as hereinafter described from the venom of any of the following species: Bothrops atrox, Bothrops jararaca, Bothrops mooqeni, Akistrodon halys blomhoffii, Crotalus atrox or Crotalus basiliscus.
  • mice are immunized with purified antihesin.
  • BALB/c AnSkh mice are preferred, although other strains may be used.
  • the immunization schedule and concentration of immunogen administered should be such as to produce useful quantities of suitably primed splenocytes.
  • mice Upon completion of the immunization regimen, more fully described below, the mice are sacrificed and their spleens removed. A suspension of splenocytes in a suitable medium is prepared. Approximately 2.5-5 ml of medium per spleen is sufficient. The protocols for in vitro suspension are well established.
  • the spleen cells are fused with mouse myeloma cells by means of a fusion promotor.
  • the preferred fusion promotor is polyethylene glycol (PEG), molecular weight 1,300-1,600.
  • PEG polyethylene glycol
  • the mouse myeloma cell line is preferably one of the drug-resistant types, to facilitate selection of hybrids.
  • the most frequently utilized class of myelomas are the 8-azaguanine-resistant cell lines, which are widely known and available. These cell lines lack the enzyme hypoxanthine guanine phosphoribosyl transferase. They do not therefore survive in "HAT"
  • myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
  • myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
  • myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
  • myeloma cells with different genetic deficiencies e.g., other enzyme deficiencies, drug sensitivities, etc.
  • the myeloma cell line should not itself produce any antibody, although in some circumstances secreting myeloma cell lines may be used.
  • fusion promotor is PEG of average molecular weight 1,300-1,600
  • other known fusion promotors may be used.
  • Fusion of cells may be carried out in an adherent monolayer, such as according to the method described by T. J. McKearn, "Fusion of Cells in an Adherent Monolayer” in Monoclonal Antibodies: Hybridomas: A New Dimension in Biological Analysis (Kennett, R. H., McKearn, T. J., and Bechtol, K.B., eds., Plenum Press, New York and London, 368-369, 1980). Other fusion techniques may be employed. A cell ratio of 2-5:1 spleen cells per myeloma cell may be used. This ratio may be varied depending on the source of spleen or myeloma cells.
  • a mixture of unfused myeloma cells, unfused spleen cells and fused cells are distributed for culturing in separate compartments (e.g., the wells of microtiter plates) in a selective medium in which the unfused myeloma cells will not survive. Distribution of the cells may be by resuspension in a volume of diluent which is statistically calculated to isolate a desired number of cells per compartment. See, for example, McKearn, "Cloning of Hybridoma Cell Lines by Limiting Dilution in Fluid Phase" in Monoclonal Antibodies, p. 374.
  • unfused 8-azaguanine-resistant myeloma cells When HAT is used as the medium, unfused 8-azaguanine-resistant myeloma cells will not grow. Unfused spleen cells will normally die after a few days, since they are non-malignant. Culturing proceeds for a time sufficient to allow their death. Fused cells continue to reproduce and grow in the selective medium.
  • the supernatant in each container or compartment having hybrid cell growth is screened and evaluated for the presence of antibody against antihesin.
  • Any suitable antibody-binding detection method may be used, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, etc.
  • monoclonal antibody to antihesin may be produced by in vitro culturing of the hybridomas or by in vivo peritoneal exudate in mice.
  • the first method will yield monoclonal antibody of higher purity.
  • the antibody is recovered from the supernatant essentially free of undesired immunoglobulin.
  • Antibody concentrations of 25-50 micrograms/ml are possible by this method.
  • growth media containing serum such as fetal calf serum
  • the subject hybridomas When concentrations of antibody larger than those obtained by in vitro culturing are required, the subject hybridomas may be injected into the peritoneal cavity of syngeneic or semisyngeneic mice. After a suitable period of incubation, the hybridomas cause formation of antibody-secreting tumors, which will produce 4-10 mg of antibody per ml of peritoneal exudate of the injected mouse. Since mice normally have antibodies in their blood and ascites, a contamination of about 5% from the host mouse is inevitable. Purification of ascites monoclonal antibody may remove these contaminants. The resultant antibody is of high titer.
  • the antihesins may be administered in any situation where inhibition of mammalian (inclusive of human) collagen-induced platelet aggregation is desired.
  • mammalian inclusive of human
  • collagen exposure and platelet adhesion plays a thrombotic role in patients with unstable angina and those recovering from myocardial infarction.
  • the antihesins are believed useful in inhibiting platelet aggregation in these conditions and to clear obstructed coronary arteries.
  • Platelet adhesion and aggregation is also a consequence of invasive medical techniques which cause epithelical damage and exposure of fibrillar collagen.
  • the polypeptides may find utility in surgery on peripheral arteries, coronary by-pass and other openheart surgical techniques, angioplasty, or other procedures where damage to the endothelial wall of blood vessels may result in collagen exposure, triggering platelet adhesion and aggregation.
  • Antihesins may be administered to patients undergoing these procedures to prevent platelet aggregation and formation of thrombii. The antihesins are thus believed useful in inhibiting thrombosis and reocclusion during and after such procedures. It is believed that the antihesins are particularly useful in preventing reocclusion after angioplasty.
  • the antihesins may be administered by any convenient route which will result in delivery to the blood stream in an amount effective for inhibiting platelet adhesion to exposed collagen.
  • the antihesins are most effectively administered parenterally, preferably intravenously or intraarterially.
  • parenterally preferably intravenously or intraarterially.
  • intravenous/intraarterial 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, having a buffered pH compatible with physiologic conditions.
  • Such intravenous delivery vehicles are known to those skilled in the art.
  • the most suitable vehicle is a sterile saline solution.
  • the antihesins may be administered in conjunction with other antithrombotic agents, such as tissue plasminogen activator or streptokinase in order to inhibit platelet aggregation.
  • tissue plasminogen activator or streptokinase
  • the amount of antihesin administered will depend on the individual clinical circumstances. For example, for clearance of obstructed coronary arteries, the antihesin may be given at a concentration ranging from about 1 to about 10 mg/ml in a solution further containing a clot lysing agent such as tissue plasminogen activator.
  • a clot lysing agent such as tissue plasminogen activator.
  • dosages may advantageously range from about 0.1 to 100 mg/kg. More or less antihesin may be administered as needed.
  • Bothrops atrox venom (Sigma Chemical Co., St. Louis, MO) was reconstituted in 0.9% NaCl at 100 mg/ml. Bothrops atrox is a South American viper, family Viperidae. subfamily Crotalinae. 0.5 ml of the aqueous solution was applied to a 2 cm ⁇ 45 cm SEPHADEX G-100 column and eluted with 0.9% NaCl at a flow rate of 6 ml per hour. Fractions of 2 ml were collected and examined spectroscopically for protein content at 280 nm. The fractions were also examined for ability to inhibit platelet adhesion to collagen according to the following adhesion assay which we have previously described (Smith, J.B. and Dangelmaier, C., Anal. Biochem. 187, 173-178 (1990)).
  • PRP obtained by centrifugation at 180gr for 20 min. at room temperature, was centrifuged at 800g for 15 min. at room temperature.
  • the platelet pellet was resuspended in 0.2 volume of autologous plasma and incubated for 1 hour with 1 microCi/ml [9,10- 3 H(N)]oleic acid (8.9 Ci/nmol) at 37oC. Following the incubation, the platelets were separated from unincorporated radiolabel by gel filtration on a SEPHAROSE 2B column using a calcium-free Tyrode's buffer containing 0.2% fatty acid-free bovine serum albumin (Sigma Chem. Co.) and 5 mM glucose.
  • the gel-filtered platelets were adjusted to 2.5 ⁇ 10 8 cells/ml and 1-ml samples were stirred at 800 rpm at 37oC for 1 min. before addition of the following (i) the fibrinogen/fibronectin inhibitor, Arg-Gly-Asp-Ser (220 micromolar); (ii) the TxA 2 receptor agonist, (SQ 29,548) [1S-[1 ⁇ a,2 ⁇ b(5Z)3 ⁇ b,4 ⁇ a]]-7-[3-[[2-[phenyl-amino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]- 5-heptenoic acid (6 micromolar), Bristol-Myers Squibb Co., Princeton, NJ); (iii) the ADP-removing system, creatine phosphate (20 mM) and creatine phosphokinase (50 U/ml); (iv) the platelet-activating factor antagonist, ginkgolide B (100 microm
  • the partially purified 50 kDa polypeptide was concentrated from approximately 15 ml to 3-5 ml using a CENTRICON PREP apparatus.
  • the concentrate was applied to a reverse phase HPLC column (semi-preparative, 1 ⁇ 25 cm, VYDAC C4).
  • the column was eluted over 60 min. using a linear gradient consisting of increasing amounts of acetonitrile in 0.1% trifluoroacetic acid. Elution of protein was followed by monitoring the absorbance at 220 nm. The peak of protein eluting at 70% acetonitrile in 0.1% trifluoroacetic acid was collected, lyophilized and reexamined for its activity in the adhesion assay.
  • the purification of the B. atrox polypeptide was monitored by SDS-PAGE, as shown in Fig. 1 (Lane 1, molecular weight standards; lane 2, crude venom; lane 3, venom after SEPHADEX G-100 column chromatography; lane 4, purified 50 kDa polypeptide after HPLC).
  • the purified polypeptide migrated as a single band with an apparent molecular weight of 50 kDa in both reduced and nonreduced gels, suggesting that the inhibitory protein is composed of a single polypeptide chain.
  • larger aggregates of the venom protein were visible, apparently reflecting disulfide interchange.
  • a partial amino acid sequence of the B. atrox antihesin was determined as follows.
  • the purified polypeptide was pyridethylated in 8M guanidine-HCl with dithiothriotol at pH 8.5, followed by HPLC on a VYDAC C4 column to remove salts.
  • the sample was then cleaved with CNBr in 5M guanidine-HCl/70% formic acid using an overnight incubation at room temperature.
  • the CNBr digest was washed, lyophilized and injected into HPLC. Of the four fragments obtained, a 13 kDa fragment was selected for limited N-terminal sequencing.
  • the amino acid composition of the B. atrox 50 kDa antihesin was determined by hydrolyzing samples in constant boiling 5.7 M HCl in vacuo at 107°C for 24-, 48- and 96-hour periods, followed by amino acid analysis on a Beckman Model 121 M amino acid analyzer using physiologic methodology.
  • the number of cysteine/half-cystine residues was determined as cysteic acid after hydrolysis with dimethylsulfoxide/HCl (Spencer and Wold, Anal. Biochem., 32, 185-190 (1969)). Tryptophan was determined after hydrolysis with mercaptoethanesulfonic acid (Penke et al., Anal. Biochem., 60, 45-50 (1974)).
  • the number of threonine and serine residues was extrapolated to zero time of hydrolysis.
  • the resulting amino acid composition, indicating a 452-residue polypeptide is set forth in Table 1, below. Examples 2-6
  • Crotalus Atrox Crotalus Basiliscus
  • Example 2 the venom of the following snakes was screened for the presence of antihesin.
  • Each venom obtained in lyophilized form from Sigma Chemical Co., St. Louis, MO
  • A. piscivorus leuostoma A. piscivorous piscivorous, A. rhodostoma, Austrelaps superba, Bitis arietans, Bothrops asper, B. jararaca, B. moogeni, B. neuwiedi, B. nummifer,
  • the amino acid composition of four of the 50 kDa snake venom antihesins is set forth in Table 1.
  • the values in parentheses are the numbers of each amino acid residue.
  • the total number of residues is believed to be accurate within about + 3 residues.
  • the residue numbers are based on a molecular weight of 50 kDa.
  • Venom was harvested from C. atrox glands (Biotoxins Inc., St. Cloud, FL) by standard techniques. The venom was fractionated by SEPHADEX G-100 chromatography and the fractions analyzed for antihesin activity according to Example 1. Partially purified 50 kDa and 13 kDa active polypeptides were further purified by reverse phase HPLC chromatography as in Exmaple 1. The purification was monitored by SDS-PAGE, as shown in Fig. 2 (Lane 1, molecular weight standards; lane 2, crude venom; lane 3, venom after SEPHADEX G-100 column chromatography; lane 4, 13 kDa antihesin after HPLC; lane 5, 50 kDa antihesin after HPLC).
  • the 13 kDa C. atrox antihesin unlike the 50 kDa polypeptides we have isolated, does not form aggregates after storage.
  • the 50 kDa antihesin was subjected to CNBr digestion and HPLC as in Example 1. In excess of ten cleavage fragments were obtained.
  • An 8 kDa fragment included the amino acid sequence, SEQUENCE ID NO:2, below.
  • a 13 kDa CNBr digest fragment included SEQUENCE ID NO: 3, wherein Xaa represents an undetermined amino acid.
  • the purified polypeptide from three of the South American vipers (B. atrox, B. iararaca and A. halys blomhoffii) exhibited a strikingly high degree of similarity in amino acid composition, while less similarity was found with C. basiliscus.
  • the South American venom proteins contained 13 methionine residues per molecule, while the protein from C. basiliscus contained only five.
  • Comparison of the 50 kDa anti-adhesive polypeptides with other proteins revealed that the snake venom molecules are unusual in the large number of cysteine residues they posses (an average of 30 residues per molecule, or 6.5 mol%).
  • the B. moogeni and C. atrox antihesins were not analyzed for amino acid composition.
  • the purified polypeptides inhibit platelet adhesion to collagen in a competitive fashion as exemplified by the B. atrox antihesin in Fig. 4.
  • the concentration required for 50% inhibition of platelet adhesion was approximately 10 microgram/ml, or 0.2 micromolar, assuming a molecular weight of 50 kDa. Increasing the preincubation time with the snake venom protein beyond one minute did not increase the extent of inhibition (data not shown).
  • the following experiment demonstrates that the antihesins function by binding to receptors on collagen, thereby blocking adhesion of platelets.
  • the antihesin inhibited adhesion of platelets to collagen in a concentration-related fashion, with amounts of antihesin from 10 to 100 ⁇ g per well. When collagen was treated with antihesin for only five minutes, the inhibitor was most effective in preventing adhesion of subsequently added platelets.
  • Fig. 5A collagen, 1 microgram/ml
  • 5B ADP, 5 micromolar
  • 5C epinephrine, 5 micromolar
  • 5D platelet-activating factor, 0.25 micromolar
  • 5E SQ 26655, 0.2 micromolar
  • the purified polypeptides inhibited both calcium mobilization and release of [ 14 C]serotonin in fura 2-loaded platelets stimulated by collagen in the presence of the feedback pathway antagonists (data not shown).
  • RIBI ADJUVANT SYSTEM (RIBI, Hamilton, MT) on day 0.
  • the latter consists of 0.5 mg monophosphoryl lipid A from S. minnesota, and 0.5 mg trehalose dimycolate from M. phlei lyophilized in 40 microliters of SQUALENE and 0.2 % TWEEN 80.
  • the adjuvant which enhances the immune response, was reconstituted in 1.0 ml sterile phosphate-buffered saline.
  • RIBI adjuvant and antigen were prepared. Immunizations were repeated at 3, 6, and 9 weeks. Ten days after each of these injections, blood was removed from the retro-orbital plexis of each mouse, and the mouse displaying the highest antibody titer was selected as the spleen donor. At week 12 the selected donor mouse was immunized by the intraperitoneal route with the same preparation previously used. Four days later the spleen of this mouse was aseptically removed and placed in a sterile plastic petri dish (15 ⁇ 60 mm) containing glucose/potassium/sodium solution (GKN).
  • GKN glucose/potassium/sodium solution
  • the latter consists of 137 mM NaCl, 110 mM glucose, 11 mM Na 2 HPO 4 , 5 mM NaH 2 PO 4 ⁇ H 2 O and 5 mM KCl.
  • the spleen was teased apart with sterile forceps and then transferred into a centrifuge tube which was placed on ice for two minutes to allow clumps to settle.
  • the cell-suspension was transferred into another centrifuge tube and spun for ten minutes at 1200 rpm. After discarding the supernatant, the cells were resuspended in 5-10 ml of 0.17 M NH 2 Cl (ice cold) and placed in ice for 5 minutes with occasional mixing in order to lyse red blood cells.
  • the cell suspension was gently underlain into 10 ml of a 1:1 dilution of GKN:normal serum and centrifuged at 1200 rpm for ten minutes. Fetal bovine serum (FCS) was used as the normal serum. The cells were then washed thrice in Dulbeco's Modified Eagle's Medium (DME, GIBCO, Grand Island, NY). The number and viability of the cells were then determined.
  • DME Dulbeco's Modified Eagle's Medium
  • SP2/O-Agl4 myeloma cells used in the hybridization procedure were washed in the same way as the unlysed splenocytes.
  • Fusion was carried out as follows. 1.5 ml of immune splenocytes and 1.5 ml of SP2/O-Agl4 cells were pipeted onto a concanavalin A-coated plate. The cell concentration of each cell type was adjusted so that the ratio of splenocytes to SP2/O-Agl4 cells was 2-3:1, with a total of 7-10 ⁇ 10 7 cells/plate. The plates were then incubated in 5% CO 2 at 37°C for 45-60 minutes to allow for attachment of the cells to concanavalin A. Fusion was performed by adding 1 ml of PEG 1500 (Boehringer Mannheim Biochemicals, Indianapolis, IN) dropwise to each plate. One minute after the addition of the first drop, the PEG solution was removed. The cells were then washed twice with 5 ml of DME. Following addition of 5 ml of DME plus 20% FCS, per plate, the cells were incubated overnight.
  • PEG 1500 Boehringer Mannheim Biochemicals, Indianapolis, IN
  • Antibody from ascites fluid prepared above may be purified by Protein-A affinity chromatography using a commercially available kit (AFFI-GEL PROTEIN-A, BioRad
  • the sub-class of the monoclonal antibody was determined with a mouse immunoglobulin subtype identification kit (Boehringer Mannheim). Monoclonal antibody 2C3-1 (ATCC HB-10904) was identified as sub-class IgG 1 , kappa light chain. The quality of the final antibody preparation was determined by SDS-PAGE according to a modified procedure of Laemmli, Nature 227, 680-685 (1970). Under non-reducing conditions, the antibody was observed to migrate as a single band of approximately 200 kDa. Upon reduction, the purified antibody resulted in bands at 50 kDa and 28 kDa, representing the heavy and light chains of the IgG immunoglobulins.
  • a combined Western blot/ELISA technique (Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350 (1979)) was performed using either crude snake venoms or purified snake venom proteins to confirm whether other venom proteins possess the epitope recognized by the monoclonal antibody directed against the 50 kDa antihesin from B. atrox.
  • the procedure in brief, is as follows. Purified snake venom proteins or crude snake venoms were subjected to SDS-PAGE using a 12% acrylamide running gel and a 4% acrylamide stacking gel, under non-reducing condition.
  • the SDS-PAGE gel was transferred to an electroeluation apparatus which electrically transferred the protein bands onto a poly vinylidene fluoride membrane (IMMOBILON, Millipore Corp., Bedford, MA). After transfer, the membrane was then blocked for 2 hours by incubation with Bovine Lacto Transfer Optimizer (BLOTTO) Johnson et al., Gene Anal. Tech. 1, 3 (1984)). The blocked membrane containing the proteins was then incubated for two hours at room temp, in BLOTTO containing 2 micrograms/ml of the purified mouse monoclonal antibody from ATCC HB-10904. The membrane was washed thrice with BLOTTO containing 0.1% TWEEN 20 detergent.
  • BLOTTO Bovine Lacto Transfer Optimizer
  • the membrane was then incubated in BLOTTO containing an alkaline phosphataseconjugated antimouse IgG polyclonal antibody (Sigma Chem. Co.) for two hours at room temperature.
  • BLOTTO alkaline phosphataseconjugated antimouse IgG polyclonal antibody
  • the membrane was then washed thrice with BLOTTO containing 0.1% TWEEN 20, and developed in an alkaline phosphate substrate comprising nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (both from Sigma), which left a colored precipitate where antigen-monoclonal antibody-(alkaline phosphatase-polyclonal antibody)-complex was present.
  • the results are shown in Fig. 3.
  • the monoclonal antibody raised against B. atrox antihesin recognized a single homogeneous protein of approximately 50 kDa in each of four crude venoms: Lane 1, C. basiliscus (20 micrograms); lane 2, B. atrox (200 ng); lane 3, B. jararaca (200 ng); lane 4, A. halys blomhoffii (200 ng).
  • Molecular weight standards are shown to the right in Fig. 3. Based upon the strength of the immunoblotting signal, it is believed that the monoclonal antibody binds antihesin with an affinity of about 10 8 mol -1 .
  • Monoclonal antibody against antihesin of one snake venom may be used to purify antihesin from the same or different snake venoms.
  • Example 10 describes the construction and operation of a representative immunoaffinity column for this purpose.
  • Monoclonal antibody such as ATCC HB-10904 is immobilized to form an immunoaffinity resin using commercially available agarose beads coupled to an activated hydroxysuccinimide ester (AFFIGEL 100, Bio-Rad Laboratories, Richmond, CA). Methods are well-established for coupling antibody to resins via hydroxysuccinimide esters. Covalent coupling of the antibody occurs through epsilon-amino groups of lysine in the protein. Coupling is performed using 2-20 mg/ml antibody in a phosphate or sodium bicarbonate buffer system, and is typically conducted overnight at 4°C. Residual reactive groups in the gel matrix are inactivated using 1 M ethanolamine-HCl, pH 8, for two hours at ambient temperature.
  • the resulting immunoaffinity resin is then washed with a storage buffer supplemented with 0.5% sodium azide and stored at 4°C until required.
  • the immunoaffinity resin is packed into a suitable column and the snake venom or other biological source material to be purified is loaded onto the column. Proteins possessing the epitope recognized by the monoclonal antibody are retained in the column and all other nonadherent proteins are eluted using a wash buffer composed of 10 mM phosphate, pH 6.8. Specifically bound proteins are eluted from the column using 100 mM glycine, pH 2.5, thereby regenerating the immunoaffinity matrix. The column resin is reequilibrated using the wash buffer. The column is then ready for another cycle of use.
  • antihesins Purification of the antihesins to chemical homogeneity has permitted partial amino acid sequencing. It is contemplated that the antihesins may be prepared through genetic engineering techniques, utilizing either partial or complete amino acid sequence information. It is thus understood that the scope of the invention is not iimited to polypeptides isolated by following the chromatographic procedures disclosed herein, but also includes antihesin polypeptides as they may be prepared by genetic engineering techniques.
  • antihesin may be obtained by recombination and cloning of the appropriate native gene obtained from venom producing cells.
  • a partial amino acid sequence such as the partial sequences disclosed herein for the Crotalus atrox and Bothrops atrox 50 kDa antihesins
  • an appropriate cDNA library may be prepared according to any of the known techniques for preparing such libraries, such as the techniques described in Chapter 8 of Molecular Cloning: A Laboratory Manual, Second Edition, 1989 (J. Sambrook, E.F. Fritsch and T. Maniatis, editors).
  • a cDNA library is prepared from polyA + mRNA using a snake venom gland.
  • the library is constructed using, for example, the insertion vector ⁇ ZAPII (Stratagene, La Jolla, CA) which is equipped with multiple cloning sites within plasmid sequences that can be excised in vivo and converted to a plasmid vector, Bluescript SK (M13-).
  • ⁇ ZAPII carries a polycloning site downstream from the E. coli lacZ promotor. See the map of ⁇ ZAP/R in Molecular Cloning, supra. at page 2.52.
  • ⁇ ZAPII is equivalent to ⁇ ZAP except that the Sam100 mutation has been removed to allow better growth of the bacteriophage, which, in turn, causes the plaques to become blue much sooner.
  • cDNAs up to 10kb in length may be inserted into the ⁇ ZAPII polycloning site and expressed in either infected bacteria or induced lysogens.
  • the Bluescript SK(M13-) plasmid carrying the cloned DNA is excised in the presence of f1 or M13 helper bacteriophages, e.g., f1 R408 (Russel et al., Gene 45:333 (1986)).
  • ⁇ ZAPII containing a cDNA library generated from snake venom gland genetic material is mixed and incubated with a plating bacteria, e.g. NM522, suitable for propagation of the ⁇ ZAPII bacteriophage, and grown on agar plates.
  • the plaques are transferred to nitrocellulose filters when they reach a diameter of approximately 1.5 mm.
  • the plaques are lysed, washed and fixed to the nitrocellulose filters and hybridized overnight at 42°C using appropriate 32 P-labeled probes for antihesin genes.
  • the probes may take the form of oligonucleotides synthesized on the basis of the least degenerate portions of CNBr cleavage fragments of the purified 13 kDa or 50 kDa antihesins.
  • the oligonucleotides are end-labeled to high specific activity with 32 P using T4 polynucleotide kinase and gamma 32 P-ATP.
  • the nitrocellulose filters are dried following hybridization with oligonucleotide probe, and then autoradiographed to identify positive clones.
  • Plaques containing positive clones are recovered from the agar plates and treated with chloroform to release the ⁇ -particles. Suitable host bacteria are coinfected with the release ⁇ -particles and a helper phage, e.g., R408. Following incubation, the mixture is heated to kill the bacteria and inactivate the parent ⁇ ZAPII, but not packaged Bluescript phage particles containing single stranded DNA (ssDNA) which are present in the supernatant following centrifugation. The ssDNA is isolated by standard methods well-known to those skilled in the art and analyzed by electrophoresis on agarose gels following EcoR1 digestion of the DNA to determine the size of the selected cloned inserts.
  • ssDNA single stranded DNA
  • ssDNA 32 P-labeled and used as hybridization probes under high stringency conditions in
  • Northern blot indicates transcript sizes similar to those of the 13 kDa and 50 kDa antihesins, respectively.
  • the mRNA identified by Northern blotting is recovered, solubilized and translated in vitro according to known techniques.
  • the translated protein is then analyzed by SDS-PAGE, and then
  • DNA from the final selected clone(s) is sequenced using the Sanger dideoxy-mediated chain termination method utilizing oligonucleotide primers according to standard published methodologies (Sambrook, et al. Molecular Cloning,

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Abstract

Des polypeptides isolés à partir de venin de serpent inhibent l'agrégation de plaquettes en inhibant l'adhésion desdites plaquettes au collagène. L'invention décrit des anticorps monoclonaux spécifiques aux polypeptides anti-adhésifs, qu'on peut utiliser afin de détecter et de purifier lesdits agents à partir de liquides.
PCT/US1991/007958 1991-10-28 1991-10-28 Inhibiteurs de l'agregation plaquettaire provoquee par le collagene WO1993009137A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103384A1 (fr) * 2003-05-20 2004-12-02 Toximed Gmbh Principe actif pharmaceutique contenant une toxine peptidique extraite du venin de serpents de la famille des crotales

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 28(21), issued 1989, ANDREWS et al., "Purification of Botrocetin From Bothrops Jararaca Venom Analysis of the Botrocetin-Medicated Interaction Between Vol Willebrand Factor and the Human Platelet Membrane Glycoprotein IB-IX Complex", pages 8317-8326. *
BIOCHEMISTRY, Volume 30, issued February 1991, FUJIMURA et al., "Isolation and Chemistry Characterization of Two Structurally and Functionally Distinct Forms of Botrocetin, the Platelet Coagglutinin Isolated From the Venom of Bothrops Jararaca", pages 1957-1964. *
J. BIOL. CHEM., Volume 264(20), issued 15 July 1989, SHANNON et al., "Amino Acid Sequence of a Crotalus Atrox Venom Metalloproteinase Which Cleaves Type IV Collagen and Gelatin", pages 11575-11583. *
THROMBOSIS RES., Volume 58, issued 1990, ZINGALI et al., "Bothrops Jararaca Snake Venom: Effects on Platelet Aggregation", pages 303-316. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103384A1 (fr) * 2003-05-20 2004-12-02 Toximed Gmbh Principe actif pharmaceutique contenant une toxine peptidique extraite du venin de serpents de la famille des crotales

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