WO1993022340A1 - Modulation des interactions entre cd36 et la thrombospondine - Google Patents

Modulation des interactions entre cd36 et la thrombospondine Download PDF

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Publication number
WO1993022340A1
WO1993022340A1 PCT/US1993/003748 US9303748W WO9322340A1 WO 1993022340 A1 WO1993022340 A1 WO 1993022340A1 US 9303748 W US9303748 W US 9303748W WO 9322340 A1 WO9322340 A1 WO 9322340A1
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polypeptide
thrombospondin
tsp
ligand
binding
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PCT/US1993/003748
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English (en)
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Russell John Howard
Lawrence Lit-King Leung
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Schering Corporation
The Board Of Trustees Of The Leland Stanford Junior University
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Priority to JP5519355A priority Critical patent/JPH07503024A/ja
Priority to EP93909610A priority patent/EP0640100A1/fr
Publication of WO1993022340A1 publication Critical patent/WO1993022340A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This application relates to thrombospondin and the receptor therefor, known as CD36, GPIIIb or GPIV, and to ligands and methods which can be used to either augment or inhibit interactions between thrombospondin and the receptor.
  • This application also relates to platelets and various cells bearing thrombospondin receptors.
  • Thrombospondin is a multi-domain adhesive macromolecule that interacts with a number of proteins, including fibrinogen, fibronectin and collagen [Leung et al, J. Clin. Invest. 70:542 (1982); Lahav et al, Cell 31 :253 (1982)].
  • TSP is a major platelet ⁇ granule protein and is released and expressed on activated platelet surfaces [Phillips et al, J. Biol Chem.255 :U629 (1980)]. By interacting with fibrinogen, TSP serves to reinforce platelet-platelet cohesion and to stabilize platelet aggregates [Leung, /. Clin. Invest. 74:1764 (1984)].
  • TSP is also synthesized by a variety of cell types and is incorporated into cell matrices, where it may modulate cell adhesion, mobility and growth. TSP also supports adhesion of Plasmodium falciparum-intected erythrocytes and has been implicated in the pathogenesis of cerebral malaria [Roberts et al, Nature 318:64 (1985); Howard et al, Blood 74:2603 (1989)].
  • CD36 also known as glycoprotein nib (GPIIIb) or glycoprotein IV (GPIV)
  • GPIIIb glycoprotein nib
  • GPIV glycoprotein IV
  • CD36 is an integral membrane protein present on platelets, endothelial cells, monocytes, erythroid precursors, epithelial cells and some tumor lines [Knowles et al, J. Immunol 252:2170 (1984); Asch et al., J. Clin. Invest. 79:1054 (1987); Kieffer et al, Biochem. J. 262:835 (1989); Greenwalt et al, Biochemistry 29:7054 (1990); Catimel et al, Blood 77:2649 (1991)].
  • GPIIIb glycoprotein nib
  • GPIV glycoprotein IV
  • CD36 is one of the cell surface receptors for TSP, and the CD36-TSP interaction plays a role in mediating platelet- platelet and platelet-monocyte cell adhesion [Asch et al, supra; McGregor et al, J. Biol. Chem.264:501 (1989); Silverstein et al, J. Clin. Invest. 84:546 (1989)], as well as cell attachment to matrix. In such interactions, TSP acts as a bridge.
  • TSP A substance that inhibits new blood vessel formation (an angiogenesis inhibitor) has been found to be a fragment or an isoform of TSP [Good et al, Proc. Natl. Acad. Sci. USA 87:6624 (1990)]. TSP, or a particular isoform of it, may therefore play a role in regulating cell growth by this mechanism.
  • the receptor site for this TSP isoform has not been defined but may be CD36.
  • CD36 can also function as a receptor for the adhesion of Plasmodium falciparum-mfected erythrocytes [Barnwell et al, J. Immunol. 755:3494 (1985); Ockenhouse et al, Science 243:1469 (1989)]. This interaction is mediated by a CD36 recognition protein which has recently been identified on parasitized cells.
  • CD36 also reacts with OKM5, an anti-CD36 monoclonal antibody which has been reported to inhibit the cell adhesive functions of CD36, including the in vitro binding of Plasmodium falciparum-inf ected erythrocytes to monocytes, endothelial cells and C32 melanoma cells (Barnwell et al, supra), suggesting that the OKM5 epitope on CD36 is functionally important.
  • OKM5 an anti-CD36 monoclonal antibody which has been reported to inhibit the cell adhesive functions of CD36, including the in vitro binding of Plasmodium falciparum-inf ected erythrocytes to monocytes, endothelial cells and C32 melanoma cells (Barnwell et al, supra), suggesting that the OKM5 epitope on CD36 is functionally important.
  • CD36 also binds a serum protein called platelet agglutinating protein p37, which is found in the sera of thrombotic thrombocytopenic purpura patients. Platelet agglutination protein p37 causes platelet agglutination through binding to membrane CD36 [Lian et al, Thrombosis and Haemostasis 65:102 (1991)].
  • the present invention fulfills this need by providing ligands which selectively bind to a region of thrombospondin that specifically binds to a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
  • This invention further provides ligands which selectively bind to a region of thrombospondin, the presence of which region is induced by the binding to thrombospondin of a polypeptide having an amino acid sequence defined by SEQ ID NO: L
  • This invention still further provides methods for augmenting thrombospondin-mediated effects comprising contacting thrombospondin in the presence of platelets or cells bearing receptors for thrombospondin with an effective amount of a ligand which selectively binds to a region of thrombospondin that specifically binds to a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
  • This invention still further provides methods for inhibiting thrombospondin-mediated effects comprising contacting thrombospondin in the presence of platelets or cells bearing receptors for thrombospondin with an effective amount of a ligand which selectively binds to a region of thrombospondin, the presence of which region is induced by the binding to thrombospondin of a polypeptide having an amino acid sequence defined by SEQ ED NO: 1.
  • Pharmaceutical compositions comprising one or more of the ligands and a pharmaceutically acceptable carrier are also provided by this invention.
  • Fig. 1 is a schematic representation of a proposed model of CD36-thrombospondin interaction.
  • Fig. 2 is a graphical representation of the effects of polypeptides P93-110 and P139-155 on the binding of CD36 to thrombospondin.
  • Fig. 3 shows platelet aggregation tracings for human platelets treated with various polypeptides plus ADP (A) or collagen (B) and (C).
  • Fig. 4 shows platelet aggregation tracings for human platelets treated with collagen plus control buffer, antibodies from hybridoma subclone 7AIh, or various amounts of antibodies from subclone 7AIe.
  • Fig. 5 is a graphical representation of the binding of 125 I-P93-110 to thrombospondin, alone and in the presence of various polypeptides.
  • the term "ligand” means a molecule which specifically binds to human TSP.
  • the ligands of the invention are antibodies or linear polypeptides, although cyclic peptides and non-peptide organic compounds that bind to TSP and exhibit the activities described below are also contemplated by this invention.
  • region of thrombospondin is defined herein to mean a localized sequence of amino acids on the surface of human TSP to which a ligand can bind.
  • polypeptide and “peptide” are used interchangeably herein and are intended to mean the same thing.
  • the term "specifically binds" is defined in the context of this invention to mean the binding of a ligand to human TSP which is mediated by noncovalent short-range interactions, including but not limited to hydrophobic, ionic, hydrogen bonding and van der Waals interactions. Such interactions are determined by specific amino acid sequences and/or organic compound functional groups.
  • the term "selectively binds to a region of thrombospondin” means that the binding of a ligand to human TSP is both specific and localized to a distinct region of the TSP. This definition specifically excludes molecules such as CD36, the binding of which is specific but not selective because they bind to TSP at more than one region.
  • receptor for thrombospondin is defined to mean an amino acid sequence on a platelet or cell which specifically binds to human TSP.
  • receptor is known as CD36, GPmb or GPIV.
  • Ligands are provided by this invention which can either augment or inhibit TSP-mediated effects.
  • the ligands of this invention which augment TSP-mediated effects are exemplified by a polypeptide designated P139-155 having an amino acid sequence defined by SEQ ID NO: 1.
  • This polypeptide represents an epitope on CD36 which is specifically recognized by monoclonal antibody OKM5. Since OKM5 is known to inhibit TSP-CD36 interactions, it would be expected that the polypeptide, by binding to TSP, would do so as well. Yet, surprisingly, the binding of P139-155 to TSP has the opposite effect — it actually augments the interaction between TSP and its receptor.
  • the inhibitory ligands of the invention are exemplified by a polypeptide designated P93-110 having an amino acid sequence defined by SEQ ID NO: 2.
  • This polypeptide binds to a site on TSP which is distinct from the site to which P139-155 binds.
  • the binding of P139-155 to TSP induces the appearance of the site to which P93-110 binds, thereby greatly increasing the binding of the latter polypeptide.
  • FIG. 1 A proposed model of the interactions between TSP and CD36 based upon the observed activities of the exemplary polypeptide ligands of the invention is shown in Fig. 1, although whether this model is correct or not is not essential to this invention.
  • Panel A of Fig. 1 shows that CD36 is believed to contain two regions which can bind to TSP. The binding of one of these regions (represented by polypeptide PI 39-155) occurs first, with later binding to TSP by the second region of CD36 (represented by polypeptide P93-110).
  • P93-110 binds, however, prevents CD36 from binding at that site (Panel C). Because the affinity of binding of CD36 to the other ⁇
  • TSP site is believed to be much lower, the net effect is inhibition of the interaction between TSP and CD36.
  • polypeptide ligands having amino acid sequences defined by SEQ ID NOs:l and 2 are preferred for use in modulating the interactions between TSP and its receptors, those skilled in the art will appreciate that polypeptides for use in this invention might also contain more or fewer amino acid residues based on the known sequence of CD36, as long as function is not substantially impaired. They may also contain residues not normally present in the sequence of CD36, as shown in the Example below.
  • the ligands of the invention encompass more than polypeptides and include other molecules such as antibodies as well.
  • a monoclonal antibody described below exhibited the same effects on TSP-CD36 interactions as did polypeptide P93-110.
  • This antibody is characterized by about 5 fold greater binding to human TSP to which has been bound a polypeptide having an amino acid sequence defined by SEQ ID NO: 1, compared to TSP to which such a polypeptide has not been bound.
  • This binding differential provides a convenient basis for screening hybridomas producing other antibodies having a similar specificity for binding TSP.
  • the ligands of this invention may be useful for modulating TSP-CD36 interactions in any process in which such interactions are known or can be shown to occur.
  • Plasmodium falciparum-infected erythrocyte adhesion to microvascular endothelial cells is believed to be mediated by infected cell recognition of CD36.
  • CD36 on tumor cells mediates adhesion to TSP [Asch et al, J. Biol. Chem.
  • the ligands of the invention which inhibit TSP-CD36 interactions may be used to block or reverse such adhesion. There is also evidence that TSP-CD36 interactions play a role in stabilizing platelet aggregates [Asch et al, J. Clin. Invest. 79:1054 (1987); McGregor et al, J. Biol. Chem. 264:501 (1989)].
  • the ligands of the invention which inhibit TSP-CD36 interactions may be thus also be useful for reducing or preventing platelet aggregation. Such use of these ligands provides a novel approach to the design of anti-platelet and anti-thrombotic agents.
  • anti-platelet agents are functionally quite potent, they also have the potential to produce undesired bleeding, as evidenced by a marked prolongation of the bleeding time (Yasuda et al, supra; Shebuski et al, supra).
  • polypeptide ligands of this invention which augment TSP-CD36 interactions may be useful for enhancing platelet aggregation in situations where such aggregation is desirable. Such a situation arise in the treatment of patients with functional disorders or immune-mediated thrombocytopenia who cannot tolerate conventional platelet transfusions.
  • DDAVP desmopressin
  • polypeptide ligands which augment TSP-CD36 interactions in the case of platelets do not act by themselves but instead enhance ADP- and collagen-induced platelet aggregation in platelet-rich plasma.
  • the polypeptide ligands of the invention are synthesized by a suitable method such as by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • the polypeptides are preferably prepared by solid phase peptide synthesis as described by Merrifield, /. Am. Chem. Soc. 85:2149 (1963).
  • the synthesis is carried out with amino acids that are protected at the alpha-amino terminus.
  • Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides.
  • the alpha-amino protecting group is selectively removed to allow subsequent reaction to take place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove the side-chain protecting groups.
  • the alpha-amino protecting groups are those known to be useful in the art of stepwise polypeptide synthesis. Included are acyl type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane type protecting groups [e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc)], aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) , and alkyl type protecting groups (e.g., benzyl, triphenylmethyl).
  • acyl type protecting groups e.g., formyl, trifluoroacetyl, acetyl
  • aromatic urethane type protecting groups e.g., benzyloxycarbonyl (
  • the preferred protecting group is Boc.
  • the side-chain protecting groups for Tyr include tetrahydropyranyl, tert. -butyl, trityl, benzyl, Cbz, 4-Br-Cbz and 2,6-dichlorobenzyl.
  • the preferred side-chain protecting group for Tyr is 2,6-dichlorobenzyl.
  • the side-chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl.
  • the preferred side-chain protecting group for Asp is cyclohexyl.
  • the side-chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz.
  • the preferred protecting group for Thr and Ser is benzyl.
  • the side-chain protecting groups for Arg include nitro, Tos, Cbz, adamantyloxycarbonyl and Boc.
  • the preferred protecting group for Arg is Tos.
  • the side-chain amino group of Lys may be protected with Cbz, 2-Cl-Cbz, Tos or Boc.
  • the 2-Cl-Cbz group is the preferred protecting group for Lys.
  • the side-chain protecting groups selected must remain intact during coupling and not be removed during the deprotection of the amino-terminus protecting group or during coupling conditions.
  • the side-chain protecting groups must also be removable upon the completion of synthesis, using reaction conditions that will not alter the finished polypeptide.
  • Solid phase synthesis is usually carried out from the carboxy-terminus by coupling the alpha-amino protected (side-chain protected) amino acid to a suitable solid support.
  • An ester linkage is formed when the attachment is made to a chloromethyl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C-terminus.
  • a benzhydrylamine or p-methylbenz- hydrylamine resin is used, an amide bond is formed and the resulting polypeptide will have a carboxamide group at the C-terminus.
  • These resins are commercially available, and their preparation has described by Stewart et al, "Solid Phase Peptide Synthesis” (2nd Edition), Pierce Chemical Co., Rockford, IL., 1984.
  • DCC dicyclohexylcarbodiimide
  • N,N'- diisopropylcarbodiimide N,N'- diisopropylcarbodiimide
  • carbonyldiimidazole carbonyldiimidazole
  • Dimethylsulfide is added to the TFA after the introduction of methionine (Met) to suppress possible S-alkylation. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the required order to obtain the desired sequence.
  • activating agents can be used for the coupling reactions including DCC, N,N'-diisopropyl- carbodiimide, benzotriazol-l-yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate (BOP) and DCC- hydroxybenzotriazole (HOBt).
  • BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate
  • HOBt DCC- hydroxybenzotriazole
  • the polypeptide-resin is cleaved with a reagent such as liquid HF for 1-2 hours at 0°C, which cleaves the polypeptide from the resin and removes all side-chain protecting groups.
  • a scavenger such as anisole is usually used with the liquid HF to prevent cations formed during the cleavage fom alkylating the amino acid residues present in the polypeptide.
  • the polypeptide-resin may be deprotected with TFA/dithioethane prior to cleavage if desired.
  • Recombinant DNA methodology dan also be used to prepare the polypeptides.
  • the known genetic code tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences.
  • oligonucleotides can be inserted into an appropriate vector and expressed in a compatible host organism.
  • polypeptides of the invention can be purified using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution or other known methods.
  • the antibody ligands of the invention which inhibit TSP-CD36 interactions can be prepared against TSP using methods described below. Although polyclonal antiserum could in principle be used, monoclonal antibodies are preferred.
  • Antibodies can be produced by immunizing a host animal such as a rabbit, rat, goat, sheep, mouse, etc. with TSP. Preferably, one or more booster injections are given after the initial injection, to increase the antibody titer. Blood is then drawn from the animal and serum is prepared and screened by standard methods such as enzyme-linked immunosorbent assay (ELISA) using TSP as the antigen.
  • ELISA enzyme-linked immunosorbent assay
  • the immunogenicity of the TSP used for immunization can be increased by combination with an adjuvant and/or by conversion to a larger form prior to immunization.
  • Suitable adjuvants for the vaccination of animals include but are not limited to Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as 'aluminum hydroxide, aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecyl-ammonium bromide, N,N-dioctadecyl-N',N'- bis(2-hydroxymethyl) propanediamine, methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides such as muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.
  • the polypeptides could
  • Monoclonal antibodies can be prepared using standard methods, e.g., as described by Kohler et al. [Nature 256:495 (1975); Eur. J. Immunol 6:511 (1976)]. Essentially, an animal is immunized as described above to produce antibody-secreting somatic cells. These cells are then removed from the immunized animal for fusion to myeloma cells.
  • Somatic cells with the potential to produce antibodies are suitable for fusion with a myeloma cell line.
  • These somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals.
  • rat spleen cells are used, in part because these cells produce a relatively high percentage of stable fusions with mouse myeloma lines. It would be possible, however, to use human, mouse, rabbit, sheep, goat or other cells instead.
  • myeloma cell lines have been developed from lymphocytic tumors for use in hyridoma-producing fusion procedures [Kohler and Milstein, Eur. J. Immunol 6:511 (1976); Shulman et al, Nature 276:269 (1978); Volk et al, J. Virol 42:220 (1982)]. These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas from unfused and similarly indefinitely self- propagating myeloma cells. Usually, this is accomplished by using myelomas with enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of hybridomas.
  • the purpose of using monoclonal techniques is to obtain fused hybrid cell lines with unlimited life spans that produce the desired single antibody under the genetic control of the somatic cell component of the hybridoma.
  • myeloma cell lines incapable of producing endogenous light or heavy immunoglobulin chains are used.
  • a third reason for selection of these cell lines is for their suitability and efficiency for fusion.
  • myeloma cell lines may be used for the production of fused cell hybrids, including, e.g., P3X63-Ag8, P3X63-AG8.653, P3/NSl-Ag4-l (NS-1), Sp2/0-Agl4 and S194/5.XXO.Bu.l.
  • the P3X63-Ag8 and NS-1 cell lines have been described by Kohler and Milstein [Eur. J. Immunol 6:511 (1976)].
  • Shulman et al [Nature 276:269 (1978)] developed the Sp2/0- Agl4 myeloma line.
  • the S 194/5.XXO.Bu.l line was reported by Trowbridge [J. Exp. Med. 148:313 (1979)].
  • Methods for generating hybrids of antibody- producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells with myeloma cells in a 10:1 proportion (although the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described by Kohler and Milstein, supra, Gefter et al. [Somatic Cell Genet. 3:231 (1977)], and Volk et al. (J. Virol 42:220 (1982)]. The fusion-promoting agents used by those investigators were Sendai virus and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • fusion procedures produce viable hybrids at very low frequency (e.g., when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly every 1 x 10-5 spleen cells). It is essential to have a means of selecting the fused cell hybrids from the remaining unfused cells, particularly the unfused myeloma cells. A means of detecting the desired antibody-producing hybridomas among other resulting fused cell hybrids is also necessary.
  • the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the unfused myeloma cells, which normally would go on dividing indefinitely.
  • the somatic cells used in the fusion do not maintain long-term viability in in vitro culture and hence do not pose a problem.
  • myeloma cells lacking hypoxanthine phosphoribosyl transferase HPRT-negative
  • Selection against these cells is made in hypoxanthine/aminopterin/thymidine (HAT) medium, a medium in which the fused cell hybrids survive due to the HPRT-positive genotype of the spleen cells.
  • HAT hypoxanthine/aminopterin/thymidine
  • the use of myeloma cells with different genetic deficiencies (drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.
  • each cell line may be propagated in either of two standard ways.
  • a suspension of the hybridoma cells can be injected into a histocompatible animal. The injected animal will then develop tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines may be propagated in vitro in laboratory culture vessels.
  • the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation, and subsequently purified.
  • Hybridomas producing monoclonal antibodies that can inhibit TSP-CD36 interactions are preferably identified by a two-stage screening process.
  • hybridoma clones producing antibodies against TSP generally are identified by, e.g., ELISA, radioimmunoassay or gel electrophoresis/Western blotting, using TSP as the antigen.
  • clones thus identified are further analyzed for increased binding affinity for TSP in the presence of polypeptide P139-155 or another ligand that binds to TSP at the same site.
  • prior binding of polypeptide P139-155 to TSP causes induction of the site to which the inhibitory antibody ligands of the invention bind and about a five-fold increase in binding of the antibodies.
  • Such increased binding of antibodies to TSP in the presence of the polypeptide can readily be detected and quantified, e.g., as described in the Example below.
  • the CDRs from a rodent monoclonal antibody can be grafted onto a human antibody, thereby "humanizing" the rodent antibody [Riechmann et al, Nature 332:323 (1988)]. More particularly, the CDRs can be grafted into a human antibody variable region with or without human constant regions.
  • Such methodology has been used, e.g., to humanize a mouse monoclonal antibody against the p55 (Tac) subunit of the human interleukin-2 receptor [Queen et al, Proc. Natl. Acad. Sci. USA 56:10029 (1989)].
  • human monoclonal antibodies can be prepared using the antigen disclosed herein and methods described by Banchereau et al. [Science 251 :10 (1991)].
  • the present invention also encompasses antibody binding fragments such as Fab, F(ab')2, Fv fragments, etc.
  • antibody binding fragments such as Fab, F(ab')2, Fv fragments, etc.
  • the use and generation of fragments of antibodies is well known, e.g., Fab fragments [Tijssen, Practice and Theory of Enzyme Immunoassay s (Elsevier, Amsterdam, 1985)], Fv fragments [Hochman et al, Biochemistry 72:1130 (1973); Sharon et al, Biochemistry 15:1591 (1976); Ehrlich et al, U.S. Patent No.
  • Human TSP for use in producing the hybridomas and monoclonal antibodies of the invention can be prepared and purified by known methods, e.g., as described in the Example below.
  • Ligands other than polypeptides and antibodies or antibody fragments can be identified using the methods disclosed herein.
  • other ligands that are functionally equivalent to polypeptide PI 39- 155 can be identified because they too, by binding to the same region on TSP, will augment TSP-CD36 interactions and enhance the binding of polypeptide P93-110 to TSP.
  • other ligands that are functionally equivalent to polypeptide P93-110 and the antibodies produced by hybridoma clone 7AIe can be identified because their binding to TSP will also be enhanced by prior binding of polypeptide P139-155, and they will inhibit TSP-CD36 interactions.
  • the ligands of the invention can be administered to a human patient requiring modulation of the interaction of TSP with platelets or cells bearing TSP receptors either directly in a buffered physiological solution or in the form of a pharmaceutical composition.
  • Pharmaceutical compositions can be prepared which contain effective amounts of one or more of the ligands and a physiologically acceptable carrier.
  • Such carriers are well known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, PA).
  • Administration may be by any suitable method, e.g., by parenteral administration, including intravenous, intraarterial, intraperitoneal, intramuscular and subcutaneous administration. Administration may be by bolus injection, continuous infusion, sustained release from implanted delivery systems [Urquhart et ⁇ l., Ann. Rev. Pharmacol. Toxicol. 24: 199 (1984)] or by other known methods.
  • the polypeptide and antibody ligands will be administered at a dosage of from about 1 ⁇ g/kilogram body weight/day to about 50 . mg/kilogram body weight/day, with a preferred dosage of from about 1 mg/kilogram body weight/day to about 10 mg/kilogram body weight/day.
  • Determination of the proper dosage of a ligand for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages that are less than optimum. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
  • the amount and frequency of administration of the ligands of the invention and the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician, taking into account such factors as age, condition and size of the patient and severity of the condition(s) being treated.
  • polypeptide ligands having amino acid sequences defined by SEQ ID NO: 1 and SEQ ID NO: 2 will for convenience be referred to below as polypeptides P139-155 and P93-110, respectively. These polypeptides, together with eight others . having amino acid sequences defined by SEQ ID NOs: 3-10, had sequences based upon subsequences of CD36.
  • polypeptides were prepared by solid phase synthesis using an Applied Biosystems Model 430 peptide synthesizer and t-boc chemistry. Cleavage of the peptides from the resin was performed with anhydrous HF at -5°C in the presence of 10% anisole. The peptides were precipitated with ether, dissolved in 0.25 M acetic acid and lyophilized.
  • a cysteine and a tyrosine residue were added to each peptide if the CD36 subsequence upon which the peptide was based did not contain these residues, to allow the peptides to be conjugated to carrier proteins or iodinated if desired.
  • the locations of these residues within the peptides to which they were added were as follows: i Residue (Number'.
  • synthesized peptides comprised about 40% of the extracellular domain of CD36.
  • CD36 was purified from Triton X-114-solubilized human platelet membrane extracts by FPLC anion exchange chromatography essentially as described by McGregor et al [J. Biol Chem. 264:501 (1989)] except that wheat germ agglutinin affinity chromatography was performed instead of FPLC gel filtration as the final purification step, as described by Tandon et al [J. Biol. Chem.264:1510 (1989)].
  • TSP was purified from thrombin-induced human platelet releasates, essentially as described by Leung et al. [J. Clin. Invest. 70:542 (1982)] and Clezardin et al [Eur. J. Biochem. 154:95 (1986)].
  • a three-month old female Lewis rat was immunized intraperitoneally (Lp.) with a 0.25 ml solution of 100 purified human TSP, emulsified with an equal volume of complete Freund's adjuvant.
  • the rat was boosted i.p. with 50 ⁇ g of TSP emulsified with incomplete Freund's adjuvant. This booster was repeated three weeks later.
  • the rat was boosted again.
  • the rat was sacrificed, blood was collected, and the spleen was removed for fusion.
  • Spleen cells were fused with mouse myeloma cells, P3X63-Ag8.653 (ATCC CRL 1580), in a 1:1 ratio using polyethylene glycol (PEG).
  • the cell suspension (3.5 x 10 cells/ml in HAT medium) was distributed into 40 96- well plates.
  • HAT medium Six days after plating the fusion products, the HAT medium was replaced with HT medium. Hybridoma supernatants were tested in a primary screen for their ability to bind to human TSP two, three and four weeks after fusion. Cells in positive wells were then expanded, and the medium was replaced with RPMI medium containing penicillin/streptomycin (Pen-Strep) and 10% fetal calf serum (FCS).
  • Pen-Strep penicillin/streptomycin
  • FCS fetal calf serum
  • the primary screen was a standard ELISA in which plates having U-shaped wells were coated overnight at 4°C with 50 ⁇ l of a 10 ⁇ g/ml solution of TSP in IX TBS (Tris-buffered saline; 20 mM Tris, 0.15 M NaCl, pH 7.4) with 2% CaCl.
  • the plates were washed twice with TBS containing 2% and 0.05% TWEEN 20® (polyoxyethylenesorbitan monolaurate), after which 100 ⁇ l of hybridoma supernatant were added to each well and the plates were incubated at room temperature for two hours.
  • the wells were washed twice as described above, and 50 ⁇ l of a 1:1,000 dilution of alkaline phosphatase-conjugated goat anti-rat IgG in TBS/CaCl were added to each well and incubated for one hour at room temperature.
  • the wells were again washed twice, and 50 ⁇ l of BIORAD alkaline phosphatase substrate solution (a buffered solution of p-nitrophenyl phosphate) were added to each well. After a 15 minute incubation at room temperature, the plates were read at 405 nm. About 200 hybridoma cell lines testing positive in the primary screen were thus identified. The positive hybridomas were then evaluated in a secondary ELISA screen.
  • Cells were selected from the secondary screen which secreted antibodies that bound at least about 4 times more to the TSP in the presence of polypeptide P139-155 than in the absence of the polypeptide. Three hybridoma cell lines were thus identified, one of which was designated 7A.
  • hybridoma cell lines testing positive in the secondary screen were then subcloned by limiting dilution. This was accomplished by seeding a solution containing 360 cells/well in RPMI/10% FCS/Pen-Strep into the top row of a microtiter plate, and then carrying out serial 2-fold dilutions down the plate. The bottom row would have been predicted to have 0.25 cell/well on average, as a result of this procedure. After two weeks, wells containing single colonies (as determined by visual inspection at 4X and 10X) were expanded. As a result of such cloning of hybridoma cell line 7A, three clones were isolated, two of which were designated 7AIe and 7AIh.
  • TSP, CD36 and the polypeptides having amino acid sequences defined by SEQ ID NOs: 1, 2 and 5 were radiolabeled with 1251 using the IODOBEAD® method (Pierce Chemical Co.), essentially as described by McGregor et al. [J. Biol. Chem. 264:501 (1989)].
  • the labeled peptides were separated from free 125 I by SEPHADEX G-25® gel filtration chromatography (1.3 x 25 cm) and eluted with phosphate buffered saline (PBS) at a flow rate of 0.5 ml/minute.
  • PBS phosphate buffered saline
  • 125I-CD36 or 1251-P139-155 (1 x 106 . cpm/sample) was incubated with 3 ⁇ g/sample of antibody OKM5 (Ortho Pharmaceuticals) or a control monoclonal antibody against an unrelated antigen (designated antibody 1F5) for 2 hours at room temperature in the presence or absence of 10 ⁇ g/sample of the various synthetic polypeptides.
  • OKM5 Ortho Pharmaceuticals
  • antibody 1F5 a control monoclonal antibody against an unrelated antigen
  • Protein A SEPHAROSE® (15 ⁇ l/sample) that had been pre-washed in PBS with 0.5% bovine serum albumin (BSA) was added, and after 1 hour of incubation at room temperature, the SEPHAROSE ® was washed three times with Tris-Tween buffer [20 mM Tris-HCl, 0.05% TWEEN 20®, pH 7.4] and the bound immune complexes were eluted by boiling with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [Laemmli, Nature 227 :680 (1970)].
  • Tris-Tween buffer 20 mM Tris-HCl, 0.05% TWEEN 20®, pH 7.4
  • SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
  • the eluted immune complexes were analyzed by
  • SDS-PAGE (Laemmli, supra) and autoradiography, with equal amounts of counts per sample loaded in the gels. 7.5% SDS-PAGE gels were used except for analysis of the labeled polypeptide, where 20% gels were used. The bands in the gels were located by staining with Coomassie blue.
  • OKM5 did in fact directly immunoadsorb the labeled P139-155 polypeptide but not other polypeptides, thereby ruling out an indirect, conformational effect on CD36. This specific immunoadsorption was inhibited by excess unlabeled P139-155.
  • polypeptide PI 39- 155 was coated onto microtiter wells at a concentration of 10 ⁇ g/ml in bicarbonate coating buffer (15 mM Na2C ⁇ 3, 34.8 mM NaHCO ⁇ , pH 9.5) by incubating overnight at 4°C. The wells were washed with Tris-Tween buffer, and nonspecific sites were blocked with Tris-Tween/0.5% BSA. TSP (1, 2, 4 and 8 ⁇ g/ml) was added in Tris-Tween buffer and incubated for 3 hours at 37°C.
  • TSP TSP to polypeptide P139-155
  • binding of TSP to polypeptide P139-155 was monitored by the sequential addition of anti-TSP antibody P10 35 (10 ⁇ g/ml) followed by goat anti-mouse IgG conjugated with alkaline phosphatase (1 :7,500) and enzyme substrate. Absorbance at 405 nm was measured in a TITERTEK® plate reader.
  • increasing amounts of soluble P139-155 were added during a binding assay carried out as described above. It was thereby found that the binding of TSP to immobilized polypeptide PI 39-155 was inhibited 90% by the addition of 80 ⁇ g/ml soluble P139-155.
  • polypeptide P139-155 represents part of the CD36 molecule involved in TSP-CD36 binding. Augmentation and Inhibition of the Binding of CD36 to Thrombospondin
  • polypeptide P139-155 appears to represent the part of the CD36 molecule involved in TSP-CD36 binding, it would be expected that by binding to TSP, the polypeptide would block the site at which TSP binds to CD36 and thereby inhibit the binding of the two proteins. Yet the data of Fig. 2 (upper curve) show just the opposite effect. Polypeptide P139-155 instead augmented or enhanced the binding of CD36 to TSP.
  • polypeptide P93-110 (Fig. 2, lower curve), which also specifically binds to TSP but has no effect on the immunoprecipitation of CD-36 by antibody OKM5, did inhibit the binding of 125 I-CD36 to immobilized TSP.
  • sequence Ser-Val-Thr-Cys-GLy has been identified as a cell adhesive motif in TSP having homology to the maralia circumsporozoite protein.
  • SEQ ID NO: 14 a polypeptide having an amino acid sequence corresponding to that of residues 487 to 498 of the known sequence of TSP which contained the motif sequence was synthesized as described above, together with two polypeptides containing the motif amino acid residues but in scrambled order.
  • the amino acid sequences of these polypeptides are defined in the Sequence Listing by SEQ ID NOs: 11 (correct sequence) and 12 and 13 (scrambled sequences).
  • Microtiter wells were coated with one of the polypeptides as described above, at a concentration of 10 ⁇ g/ml.
  • An unrelated control antibody or antibodies from subclone 7AIe or 7AD ⁇ were then added at a concentration of 8 ⁇ g/ml to the wells at 37°C, and the plate was incubated for 3 hours. Following the incubation, the plate was washed to remove unbound antibodies, and the bound antibodies were detected with goat anti-rat IgG conjugated with alkaline phosphatase. The results represented the mean of two experiments, each done in triplicate.
  • Hybridoma clones 7AIe and 7AIh were deposited April 29, 1992 with the American Type Culture
  • Cys Tyr lie Asn Lys Ser Lys Ser Ser Met Phe Gin Val Arg Thr Leu 1 5 10 15 Arg Glu Leu
  • Val lie Asp Gly Ser lie Cys Arg Gly Thr Thr Val 1 ' 5 10

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Abstract

L'invention décrit des ligands se fixant spécifiquement à la thrombospondine chez l'homme. Elle décrit également des procédés utilisant les ligands pour moduler les interactions de la thrombospondine avec des plaquettes et des cellules porteuses de récepteurs de la thrombospondine.
PCT/US1993/003748 1992-04-30 1993-04-28 Modulation des interactions entre cd36 et la thrombospondine WO1993022340A1 (fr)

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JP5519355A JPH07503024A (ja) 1992-04-30 1993-04-28 トロンボスポンジン−ｃｄ３６相互作用の変調
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578342A2 (fr) * 1992-05-14 1994-01-12 W.R. Grace & Co.-Conn. Purification et caractérisation d'un récepteur d'adhésion de cellules tumorales reconnaissant la séquence CSVTCG
WO1996033218A1 (fr) * 1995-04-21 1996-10-24 Allelix Biopharmaceuticals Inc. Peptides anti-hemorragiques
WO1996036349A1 (fr) * 1995-05-17 1996-11-21 Manitoba Cancer Treatment And Research Foundation ACTIVATION POST-TRANSLATIVE DU FACTEUR DE CROISSANCE TRANSFORMANT BETA1 (TGFβ1) FAISANT INTERVENIR LE RECEPTEUR CD36 DU TSP-1
EP0778014A1 (fr) 1995-07-07 1997-06-11 Jerome Douglas Muchin Dilatateur nasale
EP0941723A1 (fr) 1998-03-13 1999-09-15 Charles E. Lundy, Jr. Dilatateur nasal non-lineaire
CN106279362A (zh) * 2015-06-23 2017-01-04 首都医科大学 Arg-Leu-Val-Cys-Val,其合成,药理活性和应用
CN106317191A (zh) * 2015-06-23 2017-01-11 首都医科大学 Thr-Val-Gly-Cys-Ser,其合成,活性和应用
EP3117804A1 (fr) 2015-07-13 2017-01-18 Avataneo, Elisa Dispositif de dilatation nasale

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0443404A1 (fr) * 1990-02-22 1991-08-28 W.R. Grace & Co.-Conn. Fragments peptidiques et analogues de thrombospondine
WO1992001049A2 (fr) * 1990-07-13 1992-01-23 The General Hospital Corporation Antigene de surface de cellule cd53 et son utilisation
EP0478101A2 (fr) * 1990-09-24 1992-04-01 W.R. Grace & Co.-Conn. Peptides ayant une activité de type thrombospondine et leur utilisation thérapeutique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0443404A1 (fr) * 1990-02-22 1991-08-28 W.R. Grace & Co.-Conn. Fragments peptidiques et analogues de thrombospondine
WO1992001049A2 (fr) * 1990-07-13 1992-01-23 The General Hospital Corporation Antigene de surface de cellule cd53 et son utilisation
EP0478101A2 (fr) * 1990-09-24 1992-04-01 W.R. Grace & Co.-Conn. Peptides ayant une activité de type thrombospondine et leur utilisation thérapeutique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 182, no. 3, 14 February 1992, DULUTH, MINNESOTA US pages 1208 - 1217 ASCH AS;SILBIGER S;HEIMER E;NACHMAN RL; 'Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.' See the abstract *
CELL vol. 58, 4 July 1989, CAMBRIDGE, NA US pages 95 - 101 OQUENDO, P. ET AL.; 'CD36 directly mediates cytoadherence of plasmodium falciparum parasitized erythrocytes.' *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 266, no. 3, 1991, BALTIMORE US pages 1740 - 1745 ASCH AS;TEPLER J;SILBIGER S;NACHMAN RL; 'Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.' *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 267, no. 25, 5 September 1992, BALTIMORE US pages 18244 - 18250 LEUNG LL;LI WX;MCGREGOR JL;ALBRECHT G;HOWARD RJ; 'CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.' *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578342A2 (fr) * 1992-05-14 1994-01-12 W.R. Grace & Co.-Conn. Purification et caractérisation d'un récepteur d'adhésion de cellules tumorales reconnaissant la séquence CSVTCG
EP0578342A3 (en) * 1992-05-14 1994-06-29 Grace W R & Co Purification and characterization of a csvtcg-specific tumor cell adhesion receptor
WO1996033218A1 (fr) * 1995-04-21 1996-10-24 Allelix Biopharmaceuticals Inc. Peptides anti-hemorragiques
WO1996036349A1 (fr) * 1995-05-17 1996-11-21 Manitoba Cancer Treatment And Research Foundation ACTIVATION POST-TRANSLATIVE DU FACTEUR DE CROISSANCE TRANSFORMANT BETA1 (TGFβ1) FAISANT INTERVENIR LE RECEPTEUR CD36 DU TSP-1
US6090367A (en) * 1995-05-17 2000-07-18 Manitoba Cancer Treatment And Research Foundation Post-translational activation of TGF-β1 involving the TSP-1 receptor CD36
EP0778014A1 (fr) 1995-07-07 1997-06-11 Jerome Douglas Muchin Dilatateur nasale
EP0941723A1 (fr) 1998-03-13 1999-09-15 Charles E. Lundy, Jr. Dilatateur nasal non-lineaire
CN106279362A (zh) * 2015-06-23 2017-01-04 首都医科大学 Arg-Leu-Val-Cys-Val,其合成,药理活性和应用
CN106317191A (zh) * 2015-06-23 2017-01-11 首都医科大学 Thr-Val-Gly-Cys-Ser,其合成,活性和应用
CN106279362B (zh) * 2015-06-23 2019-07-12 首都医科大学 Arg-Leu-Val-Cys-Val，其合成，药理活性和应用
EP3117804A1 (fr) 2015-07-13 2017-01-18 Avataneo, Elisa Dispositif de dilatation nasale
EP4226900A1 (fr) 2015-07-13 2023-08-16 Euprana S.R.L. Dispositif dilatateur nasal

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