WO1993007169A1 - Peptide inhibitors of platelet adhesion - Google Patents

Peptide inhibitors of platelet adhesion Download PDF

Info

Publication number
WO1993007169A1
WO1993007169A1 PCT/US1992/008481 US9208481W WO9307169A1 WO 1993007169 A1 WO1993007169 A1 WO 1993007169A1 US 9208481 W US9208481 W US 9208481W WO 9307169 A1 WO9307169 A1 WO 9307169A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
peptide
phage
peptides
sequence
Prior art date
Application number
PCT/US1992/008481
Other languages
French (fr)
Inventor
James J. Devlin
Michael Doyle
Susan Fong
Original Assignee
Chiron Corporation
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 Chiron Corporation filed Critical Chiron Corporation
Publication of WO1993007169A1 publication Critical patent/WO1993007169A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • This invention relates generally to the field of molecular biology/biochem- istry, and more specifically to compositions that inhibit cell adhesion or aggre ⁇ gation. Such compositions have applications as medicaments for the treatment of diseases, preferably diseases involving undesirable platelet aggregation or adhesion.
  • Platelets may also self-aggregate, commonly in the formation of a platelet plug at an injured vessel wall to arrest bleeding: Colman, R. W. and Walsh, P. N. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman, R.
  • platelets can adhere to other (non-platelet) cells via various cell surface molecules.
  • platelets can bind to neu- trophils. See Hamburger, S.A. and McEver, R.P. (1990) Blood. 75: 550-554.
  • integrin proteins associated with platelets include glycoproteins GPEb-IIIa, GPIa-Ea, GPIc- ⁇ a, and the vitronectin receptor.
  • the GPHbLTIa receptor has been shown to be involved in platelet-plate ⁇ let aggregation, while the others are primarily associated with establishing the adhesive interaction of platelets with extracellular matrix proteins.
  • Other gene families that encode proteins involved in platelet adhesion/ aggregation phenomena include the leucine-rich glycoprotein gene family, or LRG. The most notable members are the GPIb-LX complex and GPV.
  • the for ⁇ mer complex binds to and is a receptor for von Willebrand factor.
  • Other gene families include the selectins such as GMP-140 and the immunoglobulin super- family such as the CAM molecules.
  • GPIV glycoprotein that is not a member of the other gene families and this is GPIV.
  • GPIIb-IIIa Perhaps the most studied of the platelet plasma membrane adhesion/recep ⁇ tor proteins is GPIIb-IIIa. No doubt, at least in part, this is because platelet aggregates are not only involved in maintaining hemostasis, they also are respons ⁇ ible for causing life-threatening thrombotic complications. Colman, R. W. and Walsh, P. N. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman, R. W., et aL, eds. (J.B. Lippincott Company, Philadelphia), pages 594-605. There are about 50,000 GPHb-HIa molecules on the surface of normal platelets.
  • the GPHb-HIa complex consists of one molecule of GPUb, which has a molecular weight of about 140,000, and consists of a large polypep- tide having a molecular weight of 125,000 which is disulfide linked to a small polypeptide, molecular weight 22,000, and one molecule of GPHIa with a molec ⁇ ular weight of 150,000.
  • GPIJIa is a single polypeptide chain. L.K. Jennings and D. Phillips, 1982, J. of Biological Chemistry. 257:10458-10463.
  • GPHb-HIa complex binds fibrinogen, fibronectin, von Willebrand factor, vitronectin, and thrombospondin. Binding of fibrinogen or von Willebrand factor is thought to result in platelet aggregation, while binding to these and the other proteins enhances platelet adhesion. Studies over the past ten years have identi ⁇ fied a region on these proteins that is responsible for binding to GPnb-ILTa. This receptor recognition sequence is associated with adhesive proteins such as those mentioned above, and further including laminin, and collagen, and is Arg-Gly- Asp (RGD in single-letter code). The presence of this sequence in numerous adhesive proteins accounts for the broad binding activity of GPHb-HIa to many adhesive proteins.
  • snake venom toxins contain inhibitors that interfere with the binding of proteins that contain the RGD sequence to GPHb-HIa. Shebuski, R. et aL, 1989, J Biol Chern, 264:21550-21556.
  • the snake venom toxin inhibitors are disulfide-cross- linked peptides of about 48-83 amino acids long that exhibit RGD sequences. It has been established that blocking GPHb-HIa-mediated platelet aggrega ⁇ tion inhibits thrombus formation using certain monoclonal antibodies.
  • a first object of the invention is the description of a family of peptides that interferes with cell aggregation or adhesion, preferably platelet adhesion or aggregation, and includes the following peptides:
  • a second object of the invention is the description of a 17-mer peptide that inhibits the aggregation of platelets and has the amino acid sequence: (SEQ ID NO: 3).
  • a third object of the invention is the description of fusion peptides consist ⁇ ing of the amino acid sequence RGD fused to one or more peptides of the instant invention.
  • a fourth object of the invention is the description of a fusion peptide including the structure RGD-Spacer-Peptide, wherein the spacer consists of flex ⁇ ible chemical residues, preferably amino acids that have one or more extended structure portions (segments in which the peptide bond angles are enlarged) linked by segments that are flexible. Each end of the spacer terminates with a flexible segment.
  • the spacer thus links RGD and peptides of the instant invention and the flexible segments permit three dimensional movement of both.
  • Such fusion pep ⁇ tides may have the RGD sequence at the amino or carboxyl terminal end of the peptide.
  • a fifth object of the invention is the description of a fusion peptide includ ⁇ ing the structure (SEQ ID NO: 4) and (SEQ ID NO: 34).
  • a sixth object of the invention is a description of methods of using thera ⁇ Cookic compositions consisting of the above-mentioned peptides alone, or in com ⁇ bination to treat disease resulting from abnormal or undesirable cell aggregation or adhesion, and preferably such compositions will be used to interfere with or block the aggregation/adhesion of platelets.
  • Figures 1 and 2 show the construction of M13LP67.
  • a degenerate oligonucleotide having the following sequence was synthe- sized, and purified using methods known in the art: (SEQ ID NO: 6) and (SEQ ID NO: 7).
  • NSS 15 a mixture consisting of equal amounts of the deoxynucleotides A, C and T, and about 30% more G was used for N, and an equal mixture of C and G was used for S.
  • X stands for deoxyinosine, and was used because of its capacity to base pair with each of the four bases A,G,C,and T. Reidhaar-Olson, J.F., and Sauer, R. T., 1988, Science. 24:53.
  • other base analogs may be used as described by Habener, J.,_et aL, 1988, Proc Natl Acad Sci USA 85:1735.
  • nucleotide sequence that encodes the random peptide sequence Immediately preceding the nucleotide sequence that encodes the random peptide sequence is a nucleotide sequence that encodes alanine and glutamic acid residues. These amino acids were included because they correspond to the first two amino terminal residues of the wild type mature gene HI protein of M13, and thus may facilitate producing the fusion protein produced as described below.
  • oligonucleotide encodes the following amino acid sequence: (SEQ ED NO: 8).
  • Xaa denotes amino acids encoded by the degenerate DNA sequence.
  • the oligonucleotides were cloned into a der ⁇ ivative of M13 to produce a mature fusion protein having the above amino acid sequence, and additionally, following the proline residues, the entire wild-type mature gene HI protein.
  • the plasmid M13LP67 was used to express the random peptide/gene HI fusion protein construct.
  • M13LP67 was derived from M13 mpl9 as shown in Figures 1 and 2. Briefly, M13mpl9 was altered in two ways. The first altera ⁇ tion consisted of inserting the marker gene, ⁇ -lactamase, into the polylinker region of the virion. This consisted of obtaining the gene by PCR amplification from the plasmid pAc5. The oligonucleotide primers that were annealed to the pAc5 template have the following sequence: (SEQ ID NO: 9) and (SEQ ED NO: 10).
  • Amplified copies of the ⁇ -lactamase gene were digested with the restriction enzymes BglH and EcoRI. and the replicative form of the modified M13mpl9 was digested with BamHI and EcoRI.
  • the desired fragments were purified by gel electrophoresis, ligated, and transformed into E. coli strain DH5 a (BRL).
  • E. c ⁇ fi transformed with phage that carried the insert were selected on ampicillin plates. The phage so produced were termed JD32.
  • the plasmid form of the phage, pJD32 M13mpl9Amp , was mutagenized so that two restriction sites, EagI and KpnI. were introduced into gene HI without altering the amino acids encoded in this region.
  • the restriction sites were intro ⁇ quizd using standard PCR in vitro mutagenesis techniques as described by Innis, M., et al. in PCR Protocols—A Guide to Methods and Applications. 1990, Aca ⁇ demic Press, Inc.
  • the KpnI site was constructed by converting the sequence, TGTTCC, at position 1611 to GGTACC.
  • the two oligonucleotides used to effect the mutagen ⁇ esis have the following sequence: (SEQ ID NO: 11) and (SEQ ID NO: 12).
  • LP162 and LP160 and LP161 were digested with BspHI and KpnI. and KpnI and AlwNI. respectively. These were ligated with T4 ligase to M13mpl9 previously cut with BspHI and AlwNI to yield M13mpLP66.
  • This vector contains the desired EagI and KpnI restriction sites, but lacks the ampicillin resistance gene, ⁇ -lactamase.
  • the vector M13mpLP67 which contains the EagI and KpnI restriction sites and ⁇ lactamase was produced by removing the ⁇ -lactamase sequences from pJD32 by digesting the vector with Xbal and EcoRI.
  • M13mpLP67 The ⁇ -lacta ⁇ mase gene was then inserted into the polylinker region of M13mpLP66 which was previously digested with Xbal and EcoRI. Subsequent ligation with T4 ligase produced M13mpLP67, which was used to generate the random peptide library.
  • Figures 1 and 2 schematically set forth the construction of M13mpLP67. To produce phage having DNA sequences that encode random peptide sequences, M13LP67 was digested with EagI and KpnI. and ligated to the oligo ⁇ nucleotides produced as described above.
  • the ligation mixture consisted of digested M13LP67 DNA at 45 ng/ ⁇ L, a 5-fold molar excess of oligonucleotides, 3.6 U//.L of T4 ligase (New England Biolabs), 25 mM Tris, pH 7.8, 10 mM MgCl 2 , 2 mM DTT, 0.4 mM ATP, and 0.1 mg/mL BSA.
  • T4 ligase New England Biolabs
  • 25 mM Tris pH 7.8, 10 mM MgCl 2 , 2 mM DTT, 0.4 mM ATP, and 0.1 mg/mL BSA.
  • the individual oligonucleotides Prior to being added to the ligation mixture, the individual oligonucleotides were combined and heated to 95 °C for 5 minutes, and subsequently cooled to room temperature in 15 ⁇ L aliquots. Next, the ligation mixture was incubated for 4 hours at room tempera- ture
  • H249 cells were prepared essentially as described by Dower, W. et aL, 1988, Nuc Acids Res .16:6127.
  • H249 cells are a recA, su ⁇ °, F' kan R derivative of MM294. Briefly, 4 x 10 9 H249 cells and 1 ⁇ g of M13LP67 DNA were combined in 85 ⁇ L of a low con ⁇ ductivity solution consisting of 1 mM Hepes. The cell/M13LP67 DNA mixture was positioned in a chilled 0.56 mm gap electrode of a BTX electroporation device (BTX Corp.) and subjected to a 5 millisecond pulse of 560 volts.
  • BTX electroporation device BTX Corp.
  • the cells were removed from the electrode assembly, mixed with fresh H249 lawn cells, and plated at a density of about 2 x 10 5 plaques per 400 cm 2 plate.
  • the next day phage from each plate were eluted with 30 mL of fresh media, PEG precipitated, resuspended in 20% glycerol, and stored frozen at -70°C.
  • About 2.8 x 10 7 plaques were harvested and several hundred analyzed to determine the approximate number that harbor random peptide sequences. Using the polymerase chain reaction to amplify DNA in the region that encodes the random peptide sequence, it was determined that about 50-90% of the phage contained a 69 base pair insert at the 5' end of gene HI.
  • oligonucleotides that encode the random peptide sequences.
  • the PCR reaction was conducted using standard techniques and with the following oligonucleotides: (SEQ ID NO: 15) and (SEQ ID NO: 16). The reaction was run for 40 cycles after which the products were resolved by electrophoresis in a 2% agarose gel. Based on these results, it was calculated that phage from the 2.8 x 10 7 plaques encode about 2 x 10 7 different random amino acid sequences.
  • the random peptide library is advantageous for identifying random pep ⁇ tides that bind to biological receptors, preferably plasma membrane-associated receptors, that are involved in cell adhesion/aggregation. For either cell adhesion or aggregation, it is thought that the initial event is the recognition of a defined amino acid sequence by a plasma membrane associated receptor.
  • assays that would be useful to identify phage that encode random peptides that affect cell aggregation/adhesion would consist of identifying phage by their capacity to spec ⁇ ifically bind to the plasma membrane of a selected target cell, and/or the capacity of the phage to directly or indirectly block the aggregation/adhesion properties of the target cell.
  • phage encoded random peptides may be identified that interact with platelet receptors that interfere with the aggregation/adhesion activities of platelets.
  • the first consists of identifying phage that bind to selected tar- get cells.
  • this method consists of contacting the phage library to an appropriate target cell under suitable conditions that enhance phage binding, and subsequently removing unbound phage by washing, centrifugation, filtration, or other methods.
  • Bound phage may be dissociated from the target cells using reagents known not to adversely affect the biological activities of the phage, including their infectivity. Because of background non-specific binding of phage to the target, the isolated phage consists of those that express the desired random peptide sequence and those that do not. Consequently it may be desirable to enrich for phage that bind to the target by repeating the initial selection step, followed by amplification of the phage.
  • a preferred method for screening the random peptide phage library is to screen the library directly against cells, either in suspension or cell monolayer, to detect phage that encode random peptides that bind to plasma membrane recep ⁇ tors.
  • cell monolayers a variety of biopanning techniques can be employed, and these are generally described by Devlin J. et aL, 1990, Science. 249:404-406 and in Colowick and Kaplan, 1983, Methods in Enzvmologv. 92, Academic Press, N.Y.
  • cell lysates can be prepared and used to biopan. After phage are identified that have the desired properties, phage DNA that encodes the peptides can be amplified using PCR, as described above, and the DNA sequenced using established tech ⁇ niques.
  • the cells may be cultured on an appropriate substratum and in a suitable cell culture medium.
  • the culture substratum may be pre-coated with an appropriate sub ⁇ stance that enhances the adherence of the cells.
  • An appropriate culture sub ⁇ stratum is a 96 well microtiter plate, and a suitable medium is Dulbecco's Modi ⁇ fied Eagles medium containing fetal calf serum and other supplements known to be beneficial for the growth and maintenance of cells in culture, and these are well known to those skilled in the art.
  • each of the random peptide encoding phage isolates is mixed with M13mpl9, and the mix ⁇ tures are panned on plates containing the target cells, and adherent virus eluted, using an appropriate solution.
  • the ratio of random peptide encoding phage to M13mpl9 in the initial mixture before biopanning, and the ratio in the eluate is compared by plating the two phage populations on Xgal plates.
  • the two popula- tions can be distinguished because M13mpl9 phage form blue plaques, while
  • M13LP67 random peptide sequence expressing phage form white plaques.
  • Var ⁇ ious controls are run to insure the reliability of the results, including a control in which both populations of phage are biopanned, as described above, with the exception that the dishes do not contain adherent target cells.
  • the phage are adsorbed onto the cell monolayer followed by washing the cells a suitable number of times with an appropriate solution to remove phage that do not bind to the cell monolayer.
  • the solutions used to adsorb the phage, or wash the cell monolayer after adsorbtion may vary and are a function of the type of target cells being screened. For example, TBS which consists of 50 mM Tris- HCl, pH 7.5, 150 M NaCl, is favored for screening endothelial cells, while Tyrode's buffer is favored for screening platelets.
  • the adherent phage remaining after the cell monolayer is washed are eluted from the plates, preferably with a sterile solution consisting of 6 M urea in 0.1 N HCl (pH adjusted to 2.2 with glycine), or any other reagent having similar properties.
  • the phage are eluted in this solution, and the pH of the solution neu ⁇ tralized.
  • a stock of the phage is prepared by reinfection of E. coH and prepara ⁇ tion of a plate stock, using standard procedures, and the biopanning selection pro- cedure repeated until white plaques have been enriched.
  • the resulting phage are plated at low density, and separate phage stocks prepared from randomly selected, individual plaques.
  • assays may be employed to identify phage that encode random peptides that interfere with the aggregation/adhesion of cells. This may be achieved in its most general form by determining the capacity of the peptide encoding phage to interfere with, or block the adhesion of one cell population to another that is adherent to a solid substratum. This assay can be carried out essentially as described by Springer, T. A., 1990, Annu. Rev. Cell BioL. 6:359-402, or as is known by those skilled in cell-cell adhesion assays.
  • Another assay that may be useful to identify phage that encode random peptides in the instance where a peptide having a desired activity is sought, and the corresponding plasma membrane receptor is known and available, is to affix the purified receptor to a solid substratum and measure the capacity of phage to inhibit binding of cells to the receptor.
  • the polymerase chain reaction may be used to amplify phage DNA in the region that encodes the random peptide sequence. From these data the percentage of the phage that contain a nucleotide insert in gene HI can be determined, and thus confirm the presence of the oligonucleotides that encode the random peptides sequences.
  • the PCR reaction is conducted using standard tech ⁇ niques and with the following oligonucleotides: (SEQ ID NO: 15) and (SEQ ID NO: 16).
  • Phage DNA is sequenced by the dideoxy method of Sanger et al., 1977, Proc Natl Acad Sci USA 74:5463 as further described by Messing et aj. , 1981 , Nuc Acids Res 9:309, or by the method of Maxam et aL, 1980, Meth Enzymol 65:499.
  • sequencing of amplified phage DNA can be performed by direct sequencing of single stranded DNA produced by PCR. The use of PCR to generate single stranded DNA is described in a co-pending U.S. Serial No. 248,896, entitled "Method for Generating Single Stranded DNA by the Polymer ⁇ ase Chain Reaction", filed September 23, 1988.
  • peptides may be synthesized by methods well known in the art and tested in cell aggrega ⁇ tion/adhesion assays to determine if they affect cell-cell interactions.
  • the pre ⁇ ferred method of peptide synthesis is the solid-phase method, described in detail in Merrifield R.B., 1985, Science. 232:341-347.
  • Peptides can be synthesized on a Biosearch 9500 automated peptide machine, cleaved with hydrogen fluoride, and purified by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-20 ⁇ m Vydac C4 PrepPAK column.
  • An alternative method is to use an ABI Automatic Synthesizer.
  • a preferred fusion peptide would consist of the sequence RGD fused to a peptide of the instant invention.
  • RGD-containing peptides or proteins are fusible to peptides of the instant invention to produce a molecule having synergistic anti-platelet aggregation activ ⁇ ity.
  • SEQ ID NO: 3 is fused to snake venom known to contain the RGD sequence.
  • the venoms trigramin, echistatin, applaggin, kistrin or bitan can be fused to (SEQ ID NO: 3) to produce a molecule having high anti- aggregation activity.
  • SEQ ID NO: 3 the venoms trigramin, echistatin, applaggin, kistrin or bitan.
  • These venoms are described by Huang et aL., 1987, J. Biol. Chem.. 261:16157-16163; Garsky et aL, 1989, Proc Natl Acad Sci USA 8£: 4022-4026; Gan et aL, 1988, J. Biol. Chem..
  • a fusion peptide having the RGD sequence and a pep ⁇ tide of the instant invention linked by a spacer moiety comprises a sequence of amino acids that can be described in one of three non-mutually exclu ⁇ sive ways.
  • the spacer is described as having one or more extended structure portions (segments in which the peptide bond angles are enlarged) linked by segments that are flexible. Each end of the spacer terminates with a flexible segment. The spacer thus links the two peptides and the flexible segments permit three dimensional movement of both peptides.
  • the spacer can be described as being formed from a series of extended structure portions in tandem with inter ⁇ mediate flexible regions (the flexible regions lie on either side of the extended structure regions), e.., has the general formula flex.
  • the extended (rigid) portion(s) of the spacer is preferably formed of a series of 4-8 prolines while the flexible portions are preferably composed of 4-8 amino acid residues each selected individually from the group consisting of serine, glycine, or threo- nine.
  • the series of prolines form a left-handed proline H helix.
  • spacers may be represented by the formula ⁇ -(Pro ⁇ F, wherein F represents a flexible sequence composed of amino acids each selected independently from the group consisting of serine, glycine, or threo- nine, n, is an integer from 4 to 8 inclusive, and m is an integer from 1 to 4 inclu ⁇ sive.
  • the flexible sequences may be the same or different.
  • a particularly pre ⁇ ferred spacer domain is defined by the sequence: (SEQ ID NO: 17) where n is an integer from 4 to 8, inclusive, most preferably 6.
  • the spacer is substantially nonhydrophobic so that it has a neutral or positive effect on the water solubility of the conjugate.
  • the spacer's hydrophobicity may be determined by summing the hydrophobicities of the individual amino acids (measured by partition coefficient tests) of which it is composed.
  • a substantially nonhydrophobic sequence will measure neutral or hydrophilic.
  • the spacer can also be described in functional terms as substantially stable in human serum, having a length selected such that it provides an extended structure link at least about 15 A long, preferably about 30 to 100 A long, between the two peptides.
  • Peptides of the instant invention may be used as therapeutic compositions to treat a variety of diseases resulting from undesirable or abnormal cell adhesion or aggregation, and will be used to treat diseases, preferably resulting from plate ⁇ let aggregation adhesion.
  • these compositions may be prepared as injectables in either suspension or liquid form, however, solid forms may also be utilized that may first be solubilized or suspended prior to injection.
  • the peptides can be emulsified prior to administration.
  • Peptides will often be admixed with various excipients which are pharmaceutically acceptable and com ⁇ patible with such peptides. Examples of such excipients include water, saline, dextrose, glycerol, ethanol, or the like in combination thereof.
  • a peptide therapeutic composition useful in the practice of the instant invention can contain such peptides in a neutral pharmaceu- tically acceptable salt form.
  • acceptable salts include those formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids including acetic, oxalic, tartaric, and the like. Such acids form salts with the free amino groups of the peptides. Salts formed with the free carboxyl groups of the peptides can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases including histidine, trimethylamine, and the like.
  • the peptide compositions of the instant invention are administered in a medicinally effective amount. At least partially determinative of the quantity to be administered is the nature of the disease that the subject has, the size of the subject, and other factors well known to those skilled in the art. In those instances where the disease being treated involves abnormal or undesirable plate ⁇ let aggregation, and it is sought to reverse or control this condition, also deter ⁇ minative of the amount being admmistered is the capacity of the subject's blood hemostatic system to utilize the active peptides. Although the precise amount of peptide composition that will be administered will vary depending on the peculiar needs of each individual, it is anticipated that dosages in the range of 1 to 100 nanomoles of a particular peptide will be administered per kilogram of body weight. Moreover, this amount may vary depending on the route of administra ⁇ tion, and the particular formulation used to administer the peptides.
  • Example 1 Identification of Phage that Encode Peptides that bind to Platelets
  • the initial step in identifying peptides that would be useful for treating diseases arising from abnormal or undesirable platelet aggregation/adhesion is to identify peptides in a random peptide library that bind to platelets. Subsequently, the capacity of such peptides to interfere with aggregation/adhesion can be deter ⁇ mined in a suitable assay, and this is described in more detail in the following examples.
  • the procedures as applied to the instant invention consist of drawing human blood into tubes containing 0.1 volume of 3.8% citrate buffer, and isolat- ing the platelet rich plasma (PP ?) by centrifuging twice at 120 xg to pellet the RBC and leukocytes and then pelleting the platelets by centrifugation at 1300 xg after diluting the PRP with citrate buffer.
  • the platelets were resuspended and washed twice with citrate buffer.
  • Platelet aggregation is enhanced by the presence of extracellular ADP, a process termed platelet activation.
  • ADP is thought to enhance the expression of binding sites for adhesive proteins by receptor complexes on the cell surface.
  • 5 x 10 10 plaque forming units (pfu) of phage from the phage ran ⁇ dom peptide library (7.1) were mixed with 5 X 10 10 pfu of M13mpl9 phage in Tyrode's buffer containing 2 mM Ca ++ and 0.35% BSA and the mixture incu ⁇ bated with the adhered platelets. After 30 minutes at 4°C, the unattached phage were removed by multiple washings. The attached phage were eluted from the platelets using 6 M urea in 0.1 N HCl, pH 2.2, and neutralized to pH 7.8 with Tris-base.
  • the eluted and input phage were plaqued to determine the number of library versus M13mpl9 phage present, as described above. Approximately 2.7 x 10 6 eluted phage were expanded and concentrated by plaquing on 530 cm 2 NUNC trays, eluting the phage and PEG precipitation. 1 X 10" of these phage were then used to do a second selection cycle.
  • Table 1 shows the number of library phage, 7.1 versus M13mpl9 phage, that were used, the corresponding ratio of the two phage populations, the ratio in the urea eluate, and the percentage yield of each population.
  • selection cycle 5 phage are enriched for bind ⁇ ing to platelets compared to M13 phage.
  • the DNA from 20 clones was extracted and sequenced.
  • Nineteen out of the 20 clones had the identical rjeptide-en ⁇ hng sequence, termed Wilma.
  • the peptide sequence of Wilma is: (SEQ ID NO: 3).
  • the other clone had a peptide- encoding sequence, termed Pebbles.
  • the peptide sequence of Pebbles is: (SEQ ID NO: 5).
  • Phage encoding (SEQ ID NO: 3) and (SEQ ID NO: 5) were tested for specificity of cell-binding activity using freshly isolated, human platelets, periph ⁇ eral blood lymphocytes (PBLs) and neutrophils (PMNs) as target cells on a bind ⁇ ing and elution protocol similar to the phage selection protocol described above for platelets.
  • PBLs periph ⁇ eral blood lymphocytes
  • PMNs neutrophils
  • Example 2 rSEO ID NO: 3 Inhibits Platelet Aggregation
  • (SEQ ID NO: 3) or (SEQ ID NO: 5) would affect platelet aggregation, and, if so, to compare their effects to the peptide LRGDSP, which is known to inhibit platelet aggregation because of the presence of the RGD sequence.
  • the procedure for measuring platelet aggre ⁇ gation is generally described by Joist, J.H., (1987), "Tests of Platelet Function", pp. 1002-1009, in Gradmple's, Clinical Laboratory Methods/Diagnosis. 8th edi- tion Sonnwirth and Jarett, eds. Similar methods are described by Mazoyer, E.
  • the procedure consisted of isolating platelets from human blood collected in citrate coated tubes by centrifuging the blood at 120 Xg for 10 min ⁇ utes. Plasma containing the platelets was transferred to a clean centrifuge tube and the centrifugation step repeated. About 4 x 10 8 platelets/mL were diluted 1:2 with phosphate buffered saline, or 0.15 M saline, with or without the peptide sought to be tested. Next, the platelets were activated by the addition of ADP to a final concentration of 2 ⁇ M. ADP was made up as a stock solution 1000 X in distilled water, and diluted accordingly. The assay was conducted in 12 x 75 mm glass tubes.
  • Platelets incubated without ADP and without peptide do not aggregate.
  • the platelets were activated by the addition of ADP to a final concentration of 4 ⁇ M or by the addition of ristocetin to a final concentration of 1 mg/mL.
  • ADP was made up as a stock solution of 500 x in distilled water.
  • Ristocetin was stored as a stock solution at 15 mg/mL.
  • Aggregation reactions were recorded as changes in percent light transmission using a Chronolog Corporation Model 440 Dual Aggregometer set at 37°C and a 10,000 rpm stirring speed. The results were quantitated and compared by measuring the slope of the initial reaction and the value of the percent transmission levels at one and three minutes after addition of the agonist. The data show similar trends regardless of the method of calculation. The results shown in Table 3 were based on the values for the initial reaction slopes.
  • the (SEQ ID NO: 5) peptide had no effect on aggregation induced by either ADP or ristocetin.
  • the LRGDSP peptide demonstrated a dose dependent inhibition of ADP-induced aggregation consistent with published results from similar experiments. (Adelman, B. et aL, 1990, Circulation Research. 67:941- 947.)
  • LRGDSP had no effect on aggregation induced by ristocetin.
  • Ristocetin is an antibiotic that depends on the presence of von Willebrand's factor (vWf) to cause platelet aggregation by enhancing the binding of vWf to the LRG membrane receptor GPIb, Kao, K.J.
  • the dose of (SEQ ID NO: 3) required to inhibit platelet aggregation by 50% (I o) was approximately 30 ⁇ M for ADP-induced aggregation and 4-8 ⁇ M for ristocetin-induced aggregation.
  • LRGDSP and fSEO ID NO: 3 both inhibit platelet aggregation at about the same concentrations.
  • Ristocetin is an antibiotic that depends on the presence of von Willebrand factor to cause platelet aggregation by binding of von Willebrand factor to the LRG member, GPlb. Kao, K.J. et aL, 1979. Proc Natl Acad Sci USA 76:5317: and Coller, B.S. et aL, 1977, J. Clin. Invest.. 60:302.
  • Example 2 The procedures described in Example 2 were used to isolate platelets and measure aggregation. (SEQ ID NO: 5) was used as a control peptide.
  • the experiment consisted of determining if (SEQ ID NO: 3) or fibrinogen would compete with phage that encode (SEQ ID NO: 3) for binding to platelet lysates.
  • the procedure consisted of coating plates with platelet lysates prepared as fol ⁇ lows. Platelets were purified from whole, citrated human blood and suspended at 5 x 10 8 - 1 X 10 9 cell mL in phosphate-buffered saline containing calcium and magnesium with or without 10 ⁇ M ADP to activate the platelets. The activated platelets were incubated for 3-5 minutes in a 37° C water bath. At the end of this period the protease inhibitor, leupeptin, was added to a concentration of 50 ⁇ M and the platelets lysed with 0.5 % NP40. The lysates were quick frozen and stored at -20°C until used.
  • the platelet lysates were thawed at room temperature, and diluted 1:20 with phosphate buffered saline lacking calcium and magnesium.
  • 96 well tissue culture plates were coated with 100 ⁇ l of lysate solution followed by incu ⁇ bating the plates overnight at 4°C. The following day the lysate solution was removed and the wells washed three times with Tyrode's buffer and blocked with 200 ⁇ L Tyrode's buffer containing 0.35% BSA for one hour at room tempera- ture. The blocking solution was then removed, and the wells washed twice with Tyrode's buffer.
  • SF refers to phage encoding (SEQ ID NO:3)
  • Example 6 Structural Alternative Which Retain Anti-Aggregation Activity
  • SEQ ID NO: 3 Structural Alternative Which Retain Anti-Aggregation Activity
  • (SEQ ID NO: 3) peptide to inhibit arterial restenosis is dem ⁇ onstrated in a baboon model of in vivo arterial graft insertion and endarterect- omy.
  • a small vascular prothesis in the form of 3 cm long Goretex graft (4 mm i.d.,), is operatively inserted into the carotid artery of a male baboon.
  • (SEQ ID NO: 3) peptide is intravenously administered to the baboon at a rate of about 100 nmol/kg/minute.
  • a control animal is run that does not receive (SEQ ID NO: 3) peptide.
  • U1 ln-platelet deposition is monitored upon reconstituting arterial blood flow as described in U.S. Patent No. 4,929,602.
  • baboon carotid arteries are endarterectomized accord ⁇ ing to standard surgical procedures.
  • (SEQ ID NO: 3) is administered intravenously to the baboon at a rate of about 100 nmol/kg/minute.
  • a control animal is run that does not receive (SEQ ID NO: 3) peptide.
  • Example 5 Based on the results shown in Example 5, which establishes that (SEQ ID NO: 3) peptide and peptides containing the RGD sequence do not compete for binding to a platelet associated receptor, and thus bind to separate receptors, the (SEQ ID NO: 3) peptide would have enhanced therapeutic or prophylactic effi ⁇ cacy for the treatment of disease when used in combination with, or fused to a peptide containing RDG. This is demonstrated by the effect of (SEQ ID NO: 3) in combination with, or fused to RGD containing peptides, preferably LRGDSP, on collagen induced thrombocytopenia in vivo using a rat model system.
  • RGD containing peptides preferably LRGDSP
  • rats are anesthetized with Na pentobarbital (65 mg/kg, Vet Labs, Limited, Inc., Lenexa, KA), and two incisions are made to expose both jugular veins.
  • an infusion pump Harvard Apparatus, South Natick, Mass.
  • a 5 cc syringe with a 19 g butterfly
  • the test mixture containing either (SEQ ID NO: 1
  • a Bifimctional Fusion Peptide A bifimctional fusion peptide was synthesized with the following sequence:
  • Amino acids 1-17 are identical to (SEQ ID NO: 3) or WILMA. Amino acids 18-
  • the KRISTRIN sequence amino acids 22-29, is an "RGD" containing sequence which was previously selected from the random library and was active as a peptide against aggretation.
  • the spacer consists of flexible chemical residues, preferably amino acids that have one or more extended structure portions.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:15:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Peptides and methods for identifying the same using a random peptide library are described wherein the peptides have the property of affecting cell adhesion or aggregation, including interfering with the aggregation of platelets.

Description

PEPTIDE INHIBITORS OF PLATELET ADHESION
Field of the Invention This invention relates generally to the field of molecular biology/biochem- istry, and more specifically to compositions that inhibit cell adhesion or aggre¬ gation. Such compositions have applications as medicaments for the treatment of diseases, preferably diseases involving undesirable platelet aggregation or adhesion.
Background of the Invention
Cell aggregation and adhesion are key phenomena that are responsible for a myriad number of both normal and abnormal biological events. A subset of such events is the interaction of platelets with the extracellular matrix which involves the adhesion of platelets to such extracellular matrix proteins as von Willebrand factor, collagen, fibronectin, and vitronectin. Phillips, D. R., et aL, 1988, Blood. 71:831-834. Platelets may also self-aggregate, commonly in the formation of a platelet plug at an injured vessel wall to arrest bleeding: Colman, R. W. and Walsh, P. N. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman, R. W., et aL , eds. (J.B. Lippincott Company, Philadelphia), pages 594-605. Finally, platelets can adhere to other (non-platelet) cells via various cell surface molecules. For example, platelets can bind to neu- trophils. See Hamburger, S.A. and McEver, R.P. (1990) Blood. 75: 550-554.
The adhesion/aggregation properties of platelets that are essential for hemostatic plug formation are also thought to be responsible for life-threatening pathological conditions including acute myocardial infarction and thrombotic stroke. Stein, B., et aL, 1989, J. Am. Coll. CardioL. 14:813-836. Since both normal and abnormal platelet activities are attributable to their adhesion proper- ties, considerable research has been and continues to be focused on platelet mem¬ brane proteins that act as receptors that affect adhesion/aggregation with extra¬ cellular matrix proteins that are known to bind to certain of these receptors either when associated with the extracellular matrix, or with cellular plasma membranes. Over the past years several platelet membrane receptors have been cloned and sequenced and shown to be involved in cell adhesion/aggregation. Most of these receptors are members of established gene families. One example of a gene family having many members on the platelet plasma membrane is the integrin family. Ginsberg, M. H., et aL, 1988, Thrombosis and Haemostasis. 52:1-6; and Hynes, R. O., 1987, Cell 48:549-554. Integrin proteins associated with platelets include glycoproteins GPEb-IIIa, GPIa-Ea, GPIc-ϋa, and the vitronectin receptor. The GPHbLTIa receptor has been shown to be involved in platelet-plate¬ let aggregation, while the others are primarily associated with establishing the adhesive interaction of platelets with extracellular matrix proteins. Other gene families that encode proteins involved in platelet adhesion/ aggregation phenomena include the leucine-rich glycoprotein gene family, or LRG. The most notable members are the GPIb-LX complex and GPV. The for¬ mer complex binds to and is a receptor for von Willebrand factor. Other gene families include the selectins such as GMP-140 and the immunoglobulin super- family such as the CAM molecules. Finally, there is present on the surface of platelets a glycoprotein that is not a member of the other gene families and this is GPIV.
Perhaps the most studied of the platelet plasma membrane adhesion/recep¬ tor proteins is GPIIb-IIIa. No doubt, at least in part, this is because platelet aggregates are not only involved in maintaining hemostasis, they also are respons¬ ible for causing life-threatening thrombotic complications. Colman, R. W. and Walsh, P. N. (1987) in Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman, R. W., et aL, eds. (J.B. Lippincott Company, Philadelphia), pages 594-605. There are about 50,000 GPHb-HIa molecules on the surface of normal platelets. The GPHb-HIa complex consists of one molecule of GPUb, which has a molecular weight of about 140,000, and consists of a large polypep- tide having a molecular weight of 125,000 which is disulfide linked to a small polypeptide, molecular weight 22,000, and one molecule of GPHIa with a molec¬ ular weight of 150,000. GPIJIa is a single polypeptide chain. L.K. Jennings and D. Phillips, 1982, J. of Biological Chemistry. 257:10458-10463.
GPHb-HIa complex binds fibrinogen, fibronectin, von Willebrand factor, vitronectin, and thrombospondin. Binding of fibrinogen or von Willebrand factor is thought to result in platelet aggregation, while binding to these and the other proteins enhances platelet adhesion. Studies over the past ten years have identi¬ fied a region on these proteins that is responsible for binding to GPnb-ILTa. This receptor recognition sequence is associated with adhesive proteins such as those mentioned above, and further including laminin, and collagen, and is Arg-Gly- Asp (RGD in single-letter code). The presence of this sequence in numerous adhesive proteins accounts for the broad binding activity of GPHb-HIa to many adhesive proteins. Because platelets play a key role in life-threatening conditions such as thrombotic stroke, significant research is directed to identifying molecules that inhibit the adhesion aggregation properties of platelets that occur by binding of RGD-containing proteins to GPHb-HIa. Ruoslahti, E. and Pierschbacher, M. D., 1987, Science. 238:491-497. For instance, peptides containing the RGD sequence have been shown to inhibit the binding of fibrinogen, von Willebrand factor, and fibronectin to the GPHb-HIa complex and simultaneously block plate- let aggregation. E. Plow et aL, 1985, Proc Natl Acad Sci USA £2:8057-8061; T. Gartner and J. Bennett, 1985, J. of Biological Chemistry. 260:11891-11894; and M. Kloczewiak e| a , 1989, Biochemistry. 28:2915-2919. Curiously, snake venom toxins contain inhibitors that interfere with the binding of proteins that contain the RGD sequence to GPHb-HIa. Shebuski, R. et aL, 1989, J Biol Chern, 264:21550-21556. The snake venom toxin inhibitors are disulfide-cross- linked peptides of about 48-83 amino acids long that exhibit RGD sequences. It has been established that blocking GPHb-HIa-mediated platelet aggrega¬ tion inhibits thrombus formation using certain monoclonal antibodies. Shattil, S. J. e aL, 1985, Biol. Chem.. 260:11107-11114; and Cavagnaro, J. gt aL, 1987, Blood. 70:337A. Based partly on the success of these studies, the search has intensified to identify additional inhibitors of platelet aggregation/adhesion, partic- ularly inhibitors of platelet aggregation that exert their effects on receptors other than GPHb-HIa.
Summary of the Invention
A first object of the invention is the description of a family of peptides that interferes with cell aggregation or adhesion, preferably platelet adhesion or aggregation, and includes the following peptides:
(SEQH>NO: 1), (SEQIDNO: 2), (SEQIDNO: 19), (SEQIDNO: 20), (SEQ IDNO: 22), (SEQIDNO: 23), (SEQIDNO: 24), (SEQIDNO: 25), (SEQID NO: 26), (SEQ IDNO: 27), (SEQIDNO: 28), (SEQ IDNO: 29), (SEQIDNO: 30), (SEQH)NO: 31), (SEQID NO: 32), (SEQIDNO: 33), and(SEQIDNO: 34).
Unless stated otherwise, the amino and carboxy terminal ends of the pep¬ tides described herein are at the left and right ends of the sequence, respectively. A second object of the invention is the description of a 17-mer peptide that inhibits the aggregation of platelets and has the amino acid sequence: (SEQ ID NO: 3). A third object of the invention is the description of fusion peptides consist¬ ing of the amino acid sequence RGD fused to one or more peptides of the instant invention.
A fourth object of the invention is the description of a fusion peptide including the structure RGD-Spacer-Peptide, wherein the spacer consists of flex¬ ible chemical residues, preferably amino acids that have one or more extended structure portions (segments in which the peptide bond angles are enlarged) linked by segments that are flexible. Each end of the spacer terminates with a flexible segment. The spacer thus links RGD and peptides of the instant invention and the flexible segments permit three dimensional movement of both. Such fusion pep¬ tides may have the RGD sequence at the amino or carboxyl terminal end of the peptide.
A fifth object of the invention is the description of a fusion peptide includ¬ ing the structure (SEQ ID NO: 4) and (SEQ ID NO: 34). A sixth object of the invention is a description of methods of using thera¬ peutic compositions consisting of the above-mentioned peptides alone, or in com¬ bination to treat disease resulting from abnormal or undesirable cell aggregation or adhesion, and preferably such compositions will be used to interfere with or block the aggregation/adhesion of platelets. These and other objects of the invention will become apparent upon a con¬ sideration of the full invention described below.
Brief Description of the Drawings
Figures 1 and 2 show the construction of M13LP67. General Methods
The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference. The family of peptides described herein that affect cell aggregation or adhesion, including the aggregation/adhesion activities of platelets, was identified using a random peptide M13 phage library. Methods for producing the library and a description of its properties are presented in co-owned PCT application no. WO91/18980 and by J. Devlin et aL, 1990, Science. 241:404-406. A brief des¬ cription of how the Kbrary is made and screened for phage that encode the instant peptides follows.
Production of Random Peptide Library General cloning and molecular biology techniques are described by
Maniatis et el, T., et aL, 1989, Molecular Cloning. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., volumes 1 and 2. The methods used to perform PCR are described in U.S. Patents Nos. 4,683,202, and 4,683,195.
A degenerate oligonucleotide having the following sequence was synthe- sized, and purified using methods known in the art: (SEQ ID NO: 6) and (SEQ ID NO: 7).
During the synthesis of (NNS)15, a mixture consisting of equal amounts of the deoxynucleotides A, C and T, and about 30% more G was used for N, and an equal mixture of C and G was used for S. X stands for deoxyinosine, and was used because of its capacity to base pair with each of the four bases A,G,C,and T. Reidhaar-Olson, J.F., and Sauer, R. T., 1988, Science. 24:53. Alternatively, other base analogs may be used as described by Habener, J.,_et aL, 1988, Proc Natl Acad Sci USA 85:1735.
Immediately preceding the nucleotide sequence that encodes the random peptide sequence is a nucleotide sequence that encodes alanine and glutamic acid residues. These amino acids were included because they correspond to the first two amino terminal residues of the wild type mature gene HI protein of M13, and thus may facilitate producing the fusion protein produced as described below.
Immediately following the random peptide sequence, is a nucleotide sequence that encodes six proline residues. Thus, the oligonucleotide encodes the following amino acid sequence: (SEQ ED NO: 8). Xaa denotes amino acids encoded by the degenerate DNA sequence.
As will be described below, the oligonucleotides were cloned into a der¬ ivative of M13 to produce a mature fusion protein having the above amino acid sequence, and additionally, following the proline residues, the entire wild-type mature gene HI protein.
The plasmid M13LP67 was used to express the random peptide/gene HI fusion protein construct. M13LP67 was derived from M13 mpl9 as shown in Figures 1 and 2. Briefly, M13mpl9 was altered in two ways. The first altera¬ tion consisted of inserting the marker gene, β-lactamase, into the polylinker region of the virion. This consisted of obtaining the gene by PCR amplification from the plasmid pAc5. The oligonucleotide primers that were annealed to the pAc5 template have the following sequence: (SEQ ID NO: 9) and (SEQ ED NO: 10).
Amplified copies of the β-lactamase gene were digested with the restriction enzymes BglH and EcoRI. and the replicative form of the modified M13mpl9 was digested with BamHI and EcoRI. The desired fragments were purified by gel electrophoresis, ligated, and transformed into E. coli strain DH5 a (BRL). E. cρfi transformed with phage that carried the insert were selected on ampicillin plates. The phage so produced were termed JD32.
The plasmid form of the phage, pJD32 M13mpl9Amp,), was mutagenized so that two restriction sites, EagI and KpnI. were introduced into gene HI without altering the amino acids encoded in this region. The restriction sites were intro¬ duced using standard PCR in vitro mutagenesis techniques as described by Innis, M., et al. in PCR Protocols—A Guide to Methods and Applications. 1990, Aca¬ demic Press, Inc.
The KpnI site was constructed by converting the sequence, TGTTCC, at position 1611 to GGTACC. The two oligonucleotides used to effect the mutagen¬ esis have the following sequence: (SEQ ID NO: 11) and (SEQ ID NO: 12).
To construct the EagI restriction site, the sequence at position 1631 of pJD32, CCGCTG, was changed to CGGCCG using the following two oligonuc¬ leotides: (SEQ H5 NO: 13) and (SEQ ID NO: 14). More specifically, the PCR products obtained using the primers LP159,
LP162 and LP160 and LP161 were digested with BspHI and KpnI. and KpnI and AlwNI. respectively. These were ligated with T4 ligase to M13mpl9 previously cut with BspHI and AlwNI to yield M13mpLP66. This vector contains the desired EagI and KpnI restriction sites, but lacks the ampicillin resistance gene, β-lactamase. Thus, the vector M13mpLP67, which contains the EagI and KpnI restriction sites and βlactamase was produced by removing the β-lactamase sequences from pJD32 by digesting the vector with Xbal and EcoRI. The β-lacta¬ mase gene was then inserted into the polylinker region of M13mpLP66 which was previously digested with Xbal and EcoRI. Subsequent ligation with T4 ligase produced M13mpLP67, which was used to generate the random peptide library. Figures 1 and 2 schematically set forth the construction of M13mpLP67. To produce phage having DNA sequences that encode random peptide sequences, M13LP67 was digested with EagI and KpnI. and ligated to the oligo¬ nucleotides produced as described above. The ligation mixture consisted of digested M13LP67 DNA at 45 ng/μL, a 5-fold molar excess of oligonucleotides, 3.6 U//.L of T4 ligase (New England Biolabs), 25 mM Tris, pH 7.8, 10 mM MgCl2, 2 mM DTT, 0.4 mM ATP, and 0.1 mg/mL BSA. Prior to being added to the ligation mixture, the individual oligonucleotides were combined and heated to 95 °C for 5 minutes, and subsequently cooled to room temperature in 15 μL aliquots. Next, the ligation mixture was incubated for 4 hours at room tempera- ture and subsequently overnight at 15 °C. This mixture was then electroporated into E. coli as described below.
M13LP67 DNA was electroporated into H249 cells that were prepared essentially as described by Dower, W. et aL, 1988, Nuc Acids Res .16:6127. H249 cells are a recA, suρ°, F' kanR derivative of MM294. Briefly, 4 x 109 H249 cells and 1 μg of M13LP67 DNA were combined in 85 μL of a low con¬ ductivity solution consisting of 1 mM Hepes. The cell/M13LP67 DNA mixture was positioned in a chilled 0.56 mm gap electrode of a BTX electroporation device (BTX Corp.) and subjected to a 5 millisecond pulse of 560 volts.
Immediately following electroporation, the cells were removed from the electrode assembly, mixed with fresh H249 lawn cells, and plated at a density of about 2 x 105 plaques per 400 cm2 plate. The next day phage from each plate were eluted with 30 mL of fresh media, PEG precipitated, resuspended in 20% glycerol, and stored frozen at -70°C. About 2.8 x 107 plaques were harvested and several hundred analyzed to determine the approximate number that harbor random peptide sequences. Using the polymerase chain reaction to amplify DNA in the region that encodes the random peptide sequence, it was determined that about 50-90% of the phage contained a 69 base pair insert at the 5' end of gene HI. This confirmed the presence of the oligonucleotides that encode the random peptide sequences. The PCR reaction was conducted using standard techniques and with the following oligonucleotides: (SEQ ID NO: 15) and (SEQ ID NO: 16). The reaction was run for 40 cycles after which the products were resolved by electrophoresis in a 2% agarose gel. Based on these results, it was calculated that phage from the 2.8 x 107 plaques encode about 2 x 107 different random amino acid sequences.
Properties of Phage Encoded Random Peptides
The random peptide library is advantageous for identifying random pep¬ tides that bind to biological receptors, preferably plasma membrane-associated receptors, that are involved in cell adhesion/aggregation. For either cell adhesion or aggregation, it is thought that the initial event is the recognition of a defined amino acid sequence by a plasma membrane associated receptor. Thus, assays that would be useful to identify phage that encode random peptides that affect cell aggregation/adhesion would consist of identifying phage by their capacity to spec¬ ifically bind to the plasma membrane of a selected target cell, and/or the capacity of the phage to directly or indirectly block the aggregation/adhesion properties of the target cell. For example, phage encoded random peptides may be identified that interact with platelet receptors that interfere with the aggregation/adhesion activities of platelets.
Several methods may be used to identify phage that encode random pep¬ tides of interest. The first consists of identifying phage that bind to selected tar- get cells. In its most general foπn this method consists of contacting the phage library to an appropriate target cell under suitable conditions that enhance phage binding, and subsequently removing unbound phage by washing, centrifugation, filtration, or other methods. Bound phage may be dissociated from the target cells using reagents known not to adversely affect the biological activities of the phage, including their infectivity. Because of background non-specific binding of phage to the target, the isolated phage consists of those that express the desired random peptide sequence and those that do not. Consequently it may be desirable to enrich for phage that bind to the target by repeating the initial selection step, followed by amplification of the phage.
A preferred method for screening the random peptide phage library is to screen the library directly against cells, either in suspension or cell monolayer, to detect phage that encode random peptides that bind to plasma membrane recep¬ tors. If cell monolayers are used, a variety of biopanning techniques can be employed, and these are generally described by Devlin J. et aL, 1990, Science. 249:404-406 and in Colowick and Kaplan, 1983, Methods in Enzvmologv. 92, Academic Press, N.Y. Alternatively, in lieu of using cell monolayers, cell lysates can be prepared and used to biopan. After phage are identified that have the desired properties, phage DNA that encodes the peptides can be amplified using PCR, as described above, and the DNA sequenced using established tech¬ niques.
More specifically, if the phage library is screened on cell monolayers, the cells may be cultured on an appropriate substratum and in a suitable cell culture medium. The culture substratum may be pre-coated with an appropriate sub¬ stance that enhances the adherence of the cells. An appropriate culture sub¬ stratum is a 96 well microtiter plate, and a suitable medium is Dulbecco's Modi¬ fied Eagles medium containing fetal calf serum and other supplements known to be beneficial for the growth and maintenance of cells in culture, and these are well known to those skilled in the art. After the cells have had sufficient time to adhere to the substratum, they are washed with a physiologically balanced salt solution and incubated with a mixture of phage consisting of phage that encode random peptides, and M13pml9 phage. By having M13mpl9 phage present in the mixture it is possible to determine if the random peptide phage isolates are enriched from phage that do not carry random peptide inserts. Thus, each of the random peptide encoding phage isolates is mixed with M13mpl9, and the mix¬ tures are panned on plates containing the target cells, and adherent virus eluted, using an appropriate solution. The ratio of random peptide encoding phage to M13mpl9 in the initial mixture before biopanning, and the ratio in the eluate is compared by plating the two phage populations on Xgal plates. The two popula- tions can be distinguished because M13mpl9 phage form blue plaques, while
M13LP67 random peptide sequence expressing phage form white plaques. Var¬ ious controls are run to insure the reliability of the results, including a control in which both populations of phage are biopanned, as described above, with the exception that the dishes do not contain adherent target cells. The phage are adsorbed onto the cell monolayer followed by washing the cells a suitable number of times with an appropriate solution to remove phage that do not bind to the cell monolayer. The solutions used to adsorb the phage, or wash the cell monolayer after adsorbtion may vary and are a function of the type of target cells being screened. For example, TBS which consists of 50 mM Tris- HCl, pH 7.5, 150 M NaCl, is favored for screening endothelial cells, while Tyrode's buffer is favored for screening platelets.
The adherent phage remaining after the cell monolayer is washed are eluted from the plates, preferably with a sterile solution consisting of 6 M urea in 0.1 N HCl (pH adjusted to 2.2 with glycine), or any other reagent having similar properties. The phage are eluted in this solution, and the pH of the solution neu¬ tralized. A stock of the phage is prepared by reinfection of E. coH and prepara¬ tion of a plate stock, using standard procedures, and the biopanning selection pro- cedure repeated until white plaques have been enriched. The resulting phage are plated at low density, and separate phage stocks prepared from randomly selected, individual plaques.
Several assays may be employed to identify phage that encode random peptides that interfere with the aggregation/adhesion of cells. This may be achieved in its most general form by determining the capacity of the peptide encoding phage to interfere with, or block the adhesion of one cell population to another that is adherent to a solid substratum. This assay can be carried out essentially as described by Springer, T. A., 1990, Annu. Rev. Cell BioL. 6:359-402, or as is known by those skilled in cell-cell adhesion assays.
Another assay that may be useful to identify phage that encode random peptides in the instance where a peptide having a desired activity is sought, and the corresponding plasma membrane receptor is known and available, is to affix the purified receptor to a solid substratum and measure the capacity of phage to inhibit binding of cells to the receptor.
Regardless of which method is used to identify phage that encode random peptides, the polymerase chain reaction (PCR) may be used to amplify phage DNA in the region that encodes the random peptide sequence. From these data the percentage of the phage that contain a nucleotide insert in gene HI can be determined, and thus confirm the presence of the oligonucleotides that encode the random peptides sequences. The PCR reaction is conducted using standard tech¬ niques and with the following oligonucleotides: (SEQ ID NO: 15) and (SEQ ID NO: 16).
Phage DNA is sequenced by the dideoxy method of Sanger et al., 1977, Proc Natl Acad Sci USA 74:5463 as further described by Messing et aj. , 1981 , Nuc Acids Res 9:309, or by the method of Maxam et aL, 1980, Meth Enzymol 65:499. Alternatively, sequencing of amplified phage DNA can be performed by direct sequencing of single stranded DNA produced by PCR. The use of PCR to generate single stranded DNA is described in a co-pending U.S. Serial No. 248,896, entitled "Method for Generating Single Stranded DNA by the Polymer¬ ase Chain Reaction", filed September 23, 1988. Based on the phage DNA sequences that encode random peptides, peptides may be synthesized by methods well known in the art and tested in cell aggrega¬ tion/adhesion assays to determine if they affect cell-cell interactions. The pre¬ ferred method of peptide synthesis is the solid-phase method, described in detail in Merrifield R.B., 1985, Science. 232:341-347. Peptides can be synthesized on a Biosearch 9500 automated peptide machine, cleaved with hydrogen fluoride, and purified by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-20 μm Vydac C4 PrepPAK column. An alternative method is to use an ABI Automatic Synthesizer.
It will be appreciated that once a peptide is identified that regulates cell adhesion or aggregation interactions, variations of the peptide can be constructed that have more or less biological activity. Also important is the construction of fusion peptides that exhibit synergistic activity. A preferred fusion peptide would consist of the sequence RGD fused to a peptide of the instant invention. In addi¬ tion, RGD-containing peptides or proteins are fusible to peptides of the instant invention to produce a molecule having synergistic anti-platelet aggregation activ¬ ity. Preferably, (SEQ ID NO: 3) is fused to snake venom known to contain the RGD sequence. For example, the venoms trigramin, echistatin, applaggin, kistrin or bitan can be fused to (SEQ ID NO: 3) to produce a molecule having high anti- aggregation activity. These venoms are described by Huang et aL., 1987, J. Biol. Chem.. 261:16157-16163; Garsky et aL, 1989, Proc Natl Acad Sci USA 8£: 4022-4026; Gan et aL, 1988, J. Biol. Chem.. 263:19827-19832; Chao et aL, 1989, Proc Natl Acad Sci USA , 86:8050-8054; and Gartner et aL, 1985, J. Biol. Chem.. 260:11891-11894.
Most preferred is a fusion peptide having the RGD sequence and a pep¬ tide of the instant invention linked by a spacer moiety. The spacer comprises a sequence of amino acids that can be described in one of three non-mutually exclu¬ sive ways. In one aspect, the spacer is described as having one or more extended structure portions (segments in which the peptide bond angles are enlarged) linked by segments that are flexible. Each end of the spacer terminates with a flexible segment. The spacer thus links the two peptides and the flexible segments permit three dimensional movement of both peptides. Thus the spacer can be described as being formed from a series of extended structure portions in tandem with inter¬ mediate flexible regions (the flexible regions lie on either side of the extended structure regions), e.., has the general formula
Figure imgf000017_0001
flex. The extended (rigid) portion(s) of the spacer is preferably formed of a series of 4-8 prolines while the flexible portions are preferably composed of 4-8 amino acid residues each selected individually from the group consisting of serine, glycine, or threo- nine. The series of prolines form a left-handed proline H helix. These preferred spacers (less terminal reactive residue) may be represented by the formula ^-(Pro ^F, wherein F represents a flexible sequence composed of amino acids each selected independently from the group consisting of serine, glycine, or threo- nine, n, is an integer from 4 to 8 inclusive, and m is an integer from 1 to 4 inclu¬ sive. The flexible sequences may be the same or different. A particularly pre¬ ferred spacer domain is defined by the sequence: (SEQ ID NO: 17) where n is an integer from 4 to 8, inclusive, most preferably 6. In a second aspect, the spacer is substantially nonhydrophobic so that it has a neutral or positive effect on the water solubility of the conjugate. The spacer's hydrophobicity may be determined by summing the hydrophobicities of the individual amino acids (measured by partition coefficient tests) of which it is composed. A substantially nonhydrophobic sequence will measure neutral or hydrophilic.
In a third aspect, the spacer can also be described in functional terms as substantially stable in human serum, having a length selected such that it provides an extended structure link at least about 15 A long, preferably about 30 to 100 A long, between the two peptides.
Peptides of the instant invention may be used as therapeutic compositions to treat a variety of diseases resulting from undesirable or abnormal cell adhesion or aggregation, and will be used to treat diseases, preferably resulting from plate¬ let aggregation adhesion. Generally, these compositions may be prepared as injectables in either suspension or liquid form, however, solid forms may also be utilized that may first be solubilized or suspended prior to injection. Also, the peptides can be emulsified prior to administration. Peptides will often be admixed with various excipients which are pharmaceutically acceptable and com¬ patible with such peptides. Examples of such excipients include water, saline, dextrose, glycerol, ethanol, or the like in combination thereof.
It will be appreciated that a peptide therapeutic composition useful in the practice of the instant invention can contain such peptides in a neutral pharmaceu- tically acceptable salt form. Examples of acceptable salts include those formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids including acetic, oxalic, tartaric, and the like. Such acids form salts with the free amino groups of the peptides. Salts formed with the free carboxyl groups of the peptides can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases including histidine, trimethylamine, and the like. The peptide compositions of the instant invention are administered in a medicinally effective amount. At least partially determinative of the quantity to be administered is the nature of the disease that the subject has, the size of the subject, and other factors well known to those skilled in the art. In those instances where the disease being treated involves abnormal or undesirable plate¬ let aggregation, and it is sought to reverse or control this condition, also deter¬ minative of the amount being admmistered is the capacity of the subject's blood hemostatic system to utilize the active peptides. Although the precise amount of peptide composition that will be administered will vary depending on the peculiar needs of each individual, it is anticipated that dosages in the range of 1 to 100 nanomoles of a particular peptide will be administered per kilogram of body weight. Moreover, this amount may vary depending on the route of administra¬ tion, and the particular formulation used to administer the peptides.
The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be con¬ strued as restricting the invention in any way.
Example 1 Identification of Phage that Encode Peptides that bind to Platelets The initial step in identifying peptides that would be useful for treating diseases arising from abnormal or undesirable platelet aggregation/adhesion is to identify peptides in a random peptide library that bind to platelets. Subsequently, the capacity of such peptides to interfere with aggregation/adhesion can be deter¬ mined in a suitable assay, and this is described in more detail in the following examples.
The procedures for isolating and activating platelets preparative to using the platelets to screen the phage random peptide library are generally described by Dennis, M.S. et aL, 1990, Proc Natl Acad Sci USA , 87:2471-2475; and Seymour, J.L. et aL, 1990, J. Biol. Chem.. 265:10143-10147.
The procedures as applied to the instant invention consist of drawing human blood into tubes containing 0.1 volume of 3.8% citrate buffer, and isolat- ing the platelet rich plasma (PP ?) by centrifuging twice at 120 xg to pellet the RBC and leukocytes and then pelleting the platelets by centrifugation at 1300 xg after diluting the PRP with citrate buffer. The platelets were resuspended and washed twice with citrate buffer.
Platelet aggregation is enhanced by the presence of extracellular ADP, a process termed platelet activation. ADP is thought to enhance the expression of binding sites for adhesive proteins by receptor complexes on the cell surface. Thus, to identify phage that encode random peptides that inhibit platelet aggrega¬ tion that occurs by way of protein binding to such receptors, the platelets were first activated prior to screening the library. This was achieved by resuspending the platelets at 5-7 x 107/mL in Tyrode's buffer consisting of 137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose, 5 mM Hepes pH 7.3, 0.35% BSA, and 10 μM ADP. The platelets were dispensed into a 24 well polystyrene tray and incubated for 1 hour at 37°C. At the end of this period, unadhered platelets were removed by gentle washing. Next, 5 x 1010 plaque forming units (pfu) of phage from the phage ran¬ dom peptide library (7.1) were mixed with 5 X 1010 pfu of M13mpl9 phage in Tyrode's buffer containing 2 mM Ca++ and 0.35% BSA and the mixture incu¬ bated with the adhered platelets. After 30 minutes at 4°C, the unattached phage were removed by multiple washings. The attached phage were eluted from the platelets using 6 M urea in 0.1 N HCl, pH 2.2, and neutralized to pH 7.8 with Tris-base. The eluted and input phage were plaqued to determine the number of library versus M13mpl9 phage present, as described above. Approximately 2.7 x 106 eluted phage were expanded and concentrated by plaquing on 530 cm2 NUNC trays, eluting the phage and PEG precipitation. 1 X 10" of these phage were then used to do a second selection cycle.
For each selection cycle 1-5, Table 1 shows the number of library phage, 7.1 versus M13mpl9 phage, that were used, the corresponding ratio of the two phage populations, the ratio in the urea eluate, and the percentage yield of each population. As shown in the table, selection cycle 5 phage are enriched for bind¬ ing to platelets compared to M13 phage.
Figure imgf000021_0001
Next, 50 individual phage clones were selected from the cycle 5 urea elu- tion and tested for the presence of the 15-mer peptide encoding DNA sequence using PCR and the following primers: (SEQ ID NO: 15) and (SEQ ID NO: 16). PCR analysis revealed 49 out of 50 clones contained inserts. The DNA from these 20 clones was extracted and sequenced. The 19 clones which bound both targets had identical peptide-encoding sequences, termed Wilma (from the N-teπninal sequence, WTLMA, in single-letter code). The pep¬ tide sequence of Wilma is: (SEQ ID NO: 3).
The DNA from 20 clones was extracted and sequenced. Nineteen out of the 20 clones had the identical rjeptide-enακhng sequence, termed Wilma. The peptide sequence of Wilma is: (SEQ ID NO: 3). The other clone had a peptide- encoding sequence, termed Pebbles. The peptide sequence of Pebbles is: (SEQ ID NO: 5).
Phage encoding (SEQ ID NO: 3) and (SEQ ID NO: 5) were tested for specificity of cell-binding activity using freshly isolated, human platelets, periph¬ eral blood lymphocytes (PBLs) and neutrophils (PMNs) as target cells on a bind¬ ing and elution protocol similar to the phage selection protocol described above for platelets. In the case of the PBLs and PMNs, the cells were incubated with the phage, washed, and then eluted with 6 M urea, 0.1 N HCl, pH 2.2 as cell suspensions. The platelets were adhered to plastic wells throughout the protocol. The results are shown in Table 2. (SEQ ID NO: 5) showed a 200-fold enrichment on platelets compared to M13. There was no specific enrichment on PMNs or PBLs. (SEQ ID NO: 3) demonstrated a 500-fold enrichment on plate¬ lets and, again, no enrichment on the PBLs or PMNs. Thus (SEQ ID NO: 3) and (SEQ H) NO: 5) bind specifically to platelets both as compared to the parental phage, M13, and as compared to other relevant types of peripheral blood cells. Table 2
Total PFU % Yield
Figure imgf000023_0001
Example 2 rSEO ID NO: 3) Inhibits Platelet Aggregation Experiments were conducted to determine if (SEQ ID NO: 3) or (SEQ ID NO: 5) would affect platelet aggregation, and, if so, to compare their effects to the peptide LRGDSP, which is known to inhibit platelet aggregation because of the presence of the RGD sequence. The procedure for measuring platelet aggre¬ gation is generally described by Joist, J.H., (1987), "Tests of Platelet Function", pp. 1002-1009, in Gradwohl's, Clinical Laboratory Methods/Diagnosis. 8th edi- tion Sonnwirth and Jarett, eds. Similar methods are described by Mazoyer, E. et aL, 1990, Eur. J. Biochem.. 194:43-49, Dennis, M.S. et aL, 1990, Proc Natl Acad Sci USA 87:2471-2475: and Seymour, J.L. et aL, 1990, J. Biol. Chem.. 26^:10143-10147.
Briefly, the procedure consisted of isolating platelets from human blood collected in citrate coated tubes by centrifuging the blood at 120 Xg for 10 min¬ utes. Plasma containing the platelets was transferred to a clean centrifuge tube and the centrifugation step repeated. About 4 x 108 platelets/mL were diluted 1:2 with phosphate buffered saline, or 0.15 M saline, with or without the peptide sought to be tested. Next, the platelets were activated by the addition of ADP to a final concentration of 2 μM. ADP was made up as a stock solution 1000 X in distilled water, and diluted accordingly. The assay was conducted in 12 x 75 mm glass tubes.
The tubes were agitated by hand in a 37°C water bath until platelets in the control tube lacking peptide aggregated. At this time, the presence or absence, and the size of aggregates was scored visually, with or without the aid of a microscope at 150 x magnification. The results are shown below in Table 2. It is apparent that (SEQ ID NO: 3) and LRGDSP inhibit aggregation as determined by both methods of scoring over the concentration range 125-500 μM. Peptide with (SEQ ID NO: 5) did not inhibit aggregation.
Platelets incubated without ADP and without peptide do not aggregate.
Table 2 Inhibition of Aggregation by (SEQ ID NO: 3) and LRGDSP
Gross Microscopic
Peptide μM Aggregation* Aggregation
Figure imgf000025_0001
* - and + denotes little or no aggregation, and substantial or complete aggregation, respectively. Example 3 Differential Inhibition of Platelet Aggregation by LRGDSP. (SEQ ID NO: 3) and (SEQ ID NO: 5)
Experiments were conducted to determine if (SEQ ID NO: 3) or (SEQ ID
NO: 5) would affect platelet aggregation, and if so, to compare their effects to the peptide LRGDSP, which is known to inhibit platelet aggregation because of the presence of the RGD sequence. The procedure for measuring platelet aggre¬ gation is generally described by Joist, J.H., (1987), "Tests of Platelet Function", pages 1002-1009, in Gradwohl's, Clinical Laboratory Methods/Diagnosis. 8th edition Sonnenwirth and Jarett, eds. Similar methods are described by Mazoyer, E. et aL, 1990, Eur. J. Biochem.. 194: 43-49, Dennis, M.S. et. aL, 1990, Proc Natl Acad Sci USA 87, 2471-2475, and Seymour, J.L. et aL, 1990, J. Biol. Chem.. 265:10134-10147. Briefly, the procedure consisted of isolating platelet rich plasma (PRP) from human blood collected in citrate buffer as described above. About 4 X 10s platelets/mL in PRP were diluted 1:2 with phosphate buffered saline in 10 mM HEPPSO (N-[2-hydroxyethyl] piperazine-N'-[2-hydroxypropane sulfonic acid]) buffered saline, pH 8, with or without the peptide to be tested. The platelets were activated by the addition of ADP to a final concentration of 4 μM or by the addition of ristocetin to a final concentration of 1 mg/mL. ADP was made up as a stock solution of 500 x in distilled water. Ristocetin was stored as a stock solution at 15 mg/mL. Aggregation reactions were recorded as changes in percent light transmission using a Chronolog Corporation Model 440 Dual Aggregometer set at 37°C and a 10,000 rpm stirring speed. The results were quantitated and compared by measuring the slope of the initial reaction and the value of the percent transmission levels at one and three minutes after addition of the agonist. The data show similar trends regardless of the method of calculation. The results shown in Table 3 were based on the values for the initial reaction slopes. The (SEQ ID NO: 5) peptide had no effect on aggregation induced by either ADP or ristocetin. The LRGDSP peptide demonstrated a dose dependent inhibition of ADP-induced aggregation consistent with published results from similar experiments. (Adelman, B. et aL, 1990, Circulation Research. 67:941- 947.) LRGDSP had no effect on aggregation induced by ristocetin. Ristocetin is an antibiotic that depends on the presence of von Willebrand's factor (vWf) to cause platelet aggregation by enhancing the binding of vWf to the LRG membrane receptor GPIb, Kao, K.J. et aL, 1979, Proc Natl Acad Sci USA 76:5317 and Coller, B.S. et aL, 1977, J. Clin. Invest. 60:302. The (SEQ ID NO: 3) peptide inhibited aggregation induced by both ADP and ristocetin. The dose of (SEQ ID NO: 3) required to inhibit platelet aggregation by 50% (I o) was approximately 30 μM for ADP-induced aggregation and 4-8 μM for ristocetin-induced aggregation. These data indicate that LRGDSP and (SEQ ID NO: 3) exert their effects by different mechanisms.
Table 3
Figure imgf000027_0001
Figure imgf000028_0001
Example 4
Differential Inhibition of Platelet Aggregation bv LRGDSP and fSEO ID NO: 3) As shown in the preceding examples, LRGDSP and (SEQ ID NO: 3) both inhibit platelet aggregation at about the same concentrations. Thus, experiments were undertaken to distinguish the two peptides using ristocetin to activate plate- lets. Ristocetin is an antibiotic that depends on the presence of von Willebrand factor to cause platelet aggregation by binding of von Willebrand factor to the LRG member, GPlb. Kao, K.J. et aL, 1979. Proc Natl Acad Sci USA 76:5317: and Coller, B.S. et aL, 1977, J. Clin. Invest.. 60:302.
The procedures described in Example 2 were used to isolate platelets and measure aggregation. (SEQ ID NO: 5) was used as a control peptide.
It is apparent that of the three peptides tested, only (SEQ ID NO: 3) and LRGDSP inhibited platelet aggregation when the platelets were activated with ADP, and, importantly, the concentrations of (SEQ ID NO: 3) and LRGDSP that inhibited platelet aggregation differed regardless if the platelets were activated with ADP or ristocetin. However, it is important to point out that the concentra¬ tions of (SEQ ID NO: 3) and LRGDSP that inhibited ADP activated platelet aggregation differed in this experiment compared to the preceding experiment. It is well known that the aggregation properties of platelets are donor dependent, and the platelets used in this and the preceding experiment were from different donors. Thus, the variability in the two experiments is a result of using different donors to conduct the experiments. Indeed, additional experiments have shown that for numerous donors the +/- concentrations of (SEQ ID NO: 3) and LRGDSP varied from 7.5-60 μM and 125-250 μM, respectively.
Finally, it must be noted that (SEQ ID NO: 3) prevented the aggregation of ristocetin activated platelets, but that LRGDSP did not. This indicates that (SEQ ID NO: 3) and LRGDSP exert their effects by two distinct mechanisms.
Table 4
Figure imgf000029_0001
Example 5 - do -
Failure of Fibrinogen or LRGDSP to Inhibit Platelet Binding by Phage Encoding (SEO ID NO: 3) Peptides or proteins that have the RGD sequence have been shown to inhibit the binding of fibrinogen, von Willebrand factor, and fibronectin to the GPHb-HIa complex and simultaneously block platelet aggregation. Ellis, S. G. et aL, 1991, J. Am. Coll. CardioL. 17 (6):89B-95B. Since the (SEQ ID NO: 3) peptide similarly blocks platelet aggregation, an experiment was conducted to deteimine if it also inhibits aggregation by binding to the GPHb-HIa complex. The experiment consisted of determining if (SEQ ID NO: 3) or fibrinogen would compete with phage that encode (SEQ ID NO: 3) for binding to platelet lysates. The procedure consisted of coating plates with platelet lysates prepared as fol¬ lows. Platelets were purified from whole, citrated human blood and suspended at 5 x 108 - 1 X 109 cell mL in phosphate-buffered saline containing calcium and magnesium with or without 10 μM ADP to activate the platelets. The activated platelets were incubated for 3-5 minutes in a 37° C water bath. At the end of this period the protease inhibitor, leupeptin, was added to a concentration of 50 μM and the platelets lysed with 0.5 % NP40. The lysates were quick frozen and stored at -20°C until used.
The platelet lysates were thawed at room temperature, and diluted 1:20 with phosphate buffered saline lacking calcium and magnesium. Next, 96 well tissue culture plates were coated with 100 μl of lysate solution followed by incu¬ bating the plates overnight at 4°C. The following day the lysate solution was removed and the wells washed three times with Tyrode's buffer and blocked with 200 μL Tyrode's buffer containing 0.35% BSA for one hour at room tempera- ture. The blocking solution was then removed, and the wells washed twice with Tyrode's buffer. Next, 100 μL of a mixture containing M13mpl9 phage, (SEQ ID NO: 3) encoding phage and (SEQ ID NO: 3) peptide, LRGDSP or fibrinogen was added to the 96 well plates after removing the second Tyrode's buffer wash. The final titers of virus present in the mixture and the concentrations of (SEQ ID NO: 3) peptide and fibrinogen are shown in Table 4, below. The mixture was incubated for 30 minutes at room temperature, followed by washing the wells ten times with Tyrode's buffer and eluting phage bound to the platelet lysate with 100 μL of 6 M urea in 0.1 N HCl, pH 2.2. The urea was neutralized with Tris-base after 15 minutes and the samples plaqued for phage titers, and the (SEQ ID NO: 3) versus M13 phage ratios determined.
It is apparent from the results presented in Table 5, that (SEQ ID NO: 3) peptide competes with (SEQ ID NO: 3) encoding phage for binding to the platelet lysate but that fibrinogen and LRGDSP do not. These results further establish that (SEQ ID NO: 3) is binding to a site on the platelets that is distinct form the site that is bound by fibrinogen or LRGDSP.
Table 5
Figure imgf000032_0001
Plastic 15(10-*) 7(H)-2) 1.5 x lO4 7.0 x 103 .467
4.1 l phage mix 211(1(T6) 24(10"6) 2.1 x 109 2.4 x 10s .114 —
SF refers to phage encoding (SEQ ID NO:3)
Example 6 (SEO ID NO: 3 Structural Alternative Which Retain Anti-Aggregation Activity To investigate the structure/function relationship of (SEQ ID NO: 3) and its effects on ristocetin-induced platelet aggregation, a phage library encod¬ ing a number of mutants of the original peptide was synthesized. The mutants contained substitutions or deletions in the original sequence. These peptides were tested for their ability to compete with (SEQ ID NO: 3) to inhibit ristocetin- induced platelet aggregation. Table 6 lists the peptide sequences of the mutants which retained significant activity in that assay.
Table 6
10
Figure imgf000034_0001
Figure imgf000035_0001
Example 7 Use of fSEO ID NO: 3) for In Vivn Tnhihirion of Platelet Deposition and Arterial Restenosis
The ability of (SEQ ID NO: 3) peptide to inhibit arterial restenosis is dem¬ onstrated in a baboon model of in vivo arterial graft insertion and endarterect- omy. For graft insertion, a small vascular prothesis in the form of 3 cm long Goretex graft (4 mm i.d.,), is operatively inserted into the carotid artery of a male baboon. Immediately prior to circulating arterial blood through the graft (past the prosthetic surface), and for a time period of about 1 hour thereafter, (SEQ ID NO: 3) peptide is intravenously administered to the baboon at a rate of about 100 nmol/kg/minute. A control animal is run that does not receive (SEQ ID NO: 3) peptide. U1ln-platelet deposition is monitored upon reconstituting arterial blood flow as described in U.S. Patent No. 4,929,602.
For endarterectomy, baboon carotid arteries are endarterectomized accord¬ ing to standard surgical procedures. Immediately prior to circulating blood though the endarterectomized (treated) artery, and for a time period of about 1 hour thereafter, (SEQ ID NO: 3) is administered intravenously to the baboon at a rate of about 100 nmol/kg/minute. A control animal is run that does not receive (SEQ ID NO: 3) peptide.
The results from both studies would indicate that platelet deposition (restenosis) is inhibited 90% in the animal receiving (SEQ ID NO: 3) peptide administration as compared to the control animal. Example 8 Use of (SEO ID NO: 3) in Combination with, or fused to RGD Peptides for the Treatment of Disease
Based on the results shown in Example 5, which establishes that (SEQ ID NO: 3) peptide and peptides containing the RGD sequence do not compete for binding to a platelet associated receptor, and thus bind to separate receptors, the (SEQ ID NO: 3) peptide would have enhanced therapeutic or prophylactic effi¬ cacy for the treatment of disease when used in combination with, or fused to a peptide containing RDG. This is demonstrated by the effect of (SEQ ID NO: 3) in combination with, or fused to RGD containing peptides, preferably LRGDSP, on collagen induced thrombocytopenia in vivo using a rat model system.
Briefly, rats are anesthetized with Na pentobarbital (65 mg/kg, Vet Labs, Limited, Inc., Lenexa, KA), and two incisions are made to expose both jugular veins. Using an infusion pump (Harvard Apparatus, South Natick, Mass.) and a 5 cc syringe with a 19 g butterfly, the test mixture containing either (SEQ ID
NO: 3) or LRGDSP, or a mixture of the two, or (SEQ ID NO: 18) is infused into the left jugular vein at 100 nmol kg/minute for 3 minutes. After 2 minutes of compound/vehicle infusion, collagen (60 μg/kg) (Helena Laboratories, Beaumont, TX) is injected with a 1 mL syringe into the right jugular vein. The body cavity is opened and the vena cava is exposed for blood sampling. One minute after the collagen injection, compound infusion is stopped and blood is sampled from the vena cava (within 30 seconds) with a 3 cc syringe containing 0.3 mg of 4.5 % EDTA/Tris (0.1 M) (pH 7.35) plus 150 mM indomethacin. Platelet rich plasma is prepared by centrifuging the blood at 126 xg for 10 minutes, and 5 mL counted in 20 ml of Isoton® HI in a Coulter Counter. Percent inhibition of collagen induced aggregation is calculated by comparison of the number of plate- lets counted in treated animals with numbers from animals receiving no collagen and with counts from animals receiving vehicle and collagen.
A calculation of the percent inhibition of platelet aggregation jn vivo would reveal that (SEQ ID NO: 3), plus LRGDSP, or (SEQ ID NO: 18) is 3-4 times more effective at preventing aggregation than either (SEQ ID NO: 3) or LRGDSP alone.
Example 9
A Bifimctional Fusion Peptide A bifimctional fusion peptide was synthesized with the following sequence:
AELFSTHYLAF EDYSQ PPGG IPRGDMPT < WILMA- > <KISTRIN>
Amino acids 1-17 are identical to (SEQ ID NO: 3) or WILMA. Amino acids 18-
21 constitute a linker between the WHMA and the KRISTRIN sequences. The KRISTRIN sequence, amino acids 22-29, is an "RGD" containing sequence which was previously selected from the random library and was active as a peptide against aggretation.
This fusion peptide was tested in the platelet aggregation assays described in Example 3. The results are included in Table 7. The WH.MA-KISTRIN fusion was less active per micromole than the WILMA peptide when inhibiting aggregation induced by two different agonists, ristocetin and ADP. The fusion construct was also less active against ADp-aggregation than the original, full- length KISTRIN peptide. TABLE7
Figure imgf000039_0001
Figure imgf000040_0001
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: DEVLIN, JAMES J DOYLE, MICHAEL FONG, SUSAN (ii) TITLE OF INVENTION: PEPTIDE INHIBITORS FOR THE TREATMENT OF DISEASE
(iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CHIRON CORPORATION
(B) STREET: 4560 HORTON STREET
(C) CITY: EMERYVILLE
(D) STATE: CA (E) COUNTRY: USA
(F) ZIP: 94608
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GREEN, GRANT D.
(B) REGISTRATION NUMBER: 31,259
(C) REFERENCE/DOCKET NUMBER: 2617 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (510) 601-2706
(B) TELEFAX: (510) 655-3542
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr 15
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Phe Ser Thr His Tyr Leu Ala Phe Lys Glu Asp Tyr Ser 1 5 10
Gin 14 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin
15
(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 3..4
(D) OTHER INFORMATION: /label= spacer
/note= "The spacer consists of flexible chemical residues, preferably amino acids that have one or more extended structure portions. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Arg Gly Asp Ala Glu Leu Phe Ser Thr His Tyr Leu Ala 1 5 10
Phe Lys Glu Asp Tyr Ser Gin 15 20
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Ala Glu Asn Ala lie Leu Met Arg Thr Trp Ser Pro Tyr 1 5 10
Ser Arg Asp Met 15
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: unsure
(B) LOCATION: replace(24..68, "nns") (D) OTHER INFORMATION: /note= nS can be C or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CTTTCTATTC TCACTCCGCT GAANNSNNSN NSNNSNNSNN SNNSNNSNNS 10 20 30 40 50
NNSNNSNNSN NSNNSNNSCC GCCTCCACCT CCACC 60 70 80
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 22..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGCCGGTGGA GGTGGAGGCG GNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10 20 30 40 50
NNNNNNNNNN NNNNNNTTCA GCGGAGTGAG AATAGAAAGG TAC 60 70 80 90
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ala Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
Xaa Xaa Xaa Xaa Pro Pro Pro Pro Pro Pro 15 20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTGCCCGAG AGATCTGTAT ATATGAGTAA ACTTGG 10 20 30
(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GCAGGCTCGG GAATTCGGGA AATGTGCGCG GAACCC 10 20 30
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AAACTTCCTC ATGAAAAAGT C 10 20
(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AGAATAGAAA GGTACCACTA AAGGA 10 20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13
TTTAGTGGTA CCTTTCTATT CTCACTCGGC CGAAACTGT 10 20 30
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AAAGCGCAGT CTCTGAATTT ACCG 10 20
(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCGAAAGCAA GCTGATAAAC CG 10 20
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16
ACAGACAGCC CTCATAGTTA GCG 10 20
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 6..7
(D) OTHER INFORMATION: /label= N
/note= πnN" is an integer from 4 to 8, inclusive. most preferably 6. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Gly Thr Gly Ser Gly Pro Ser Gly Ser Gly Thr -5 1 5 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin Gly Thr Gly Ser Gly Pro Pro Pro Pro 15 20 25
Ser Gly Ser Gly Thr Arg Gly Asp 30
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ala Glu Leu Phe Ser Gly Tyr Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:20; -48-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Ala Glu Leu Phe Gly Gly Tyr Tyr Leu Ala Phe Lys Glu 1 5 10
Val Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ala Glu Val Pro Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10 Asp Tyr Ser
15
(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Ser Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10 Asp Tyr Ser Gin
15
(2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Ala Glu Leu Tyr Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Ala Glu Leu Phe Ser Ser His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin
15
(2) INFORMATION FOR SEQ ID^NO:26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ala Glu Leu Phe Ser Thr His Tyr Met Ala Phe Lys Glu 1 5 10 Asp Tyr Ser Gin
15 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Ala Glu Leu Phe Ser Thr His Tyr Leu Ser Phe Lys Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Tyr Lys Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:. (A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Arg Glu 1 5 10
Asp Tyr Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Phe Ser Gin 15
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Leu Gin 15
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Thr Gin 15
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10 Asp Tyr Ser His 15
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Ala Glu Leu Phe Ser Thr His Tyr Leu Ala Phe Lys Glu 1 5 10
Asp Tyr Ser Gin Pro Pro Gly Gly lie Pro Arg Gly Asp 15 20 25
Met Pro Thr
(2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:35; lie Pro Arg Gly Asp Met Pro Thr 1 5 The present invention has been described with reference to specific embodiments. However, this application is intended to cover those changes and substitutions which may be made by those skilled in the art without departing from the spirit and the scope of the appended claims.

Claims

WE CLAIM:
1. A peptide comprising an amino acid sequence selected from the group consisting of (SEQ ID NO: 3), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 24), (SEQ ID NO: 25),
(SEQ H) NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), and (SEQ H> NO: 35).
2. A peptide of claim 1 comprising the amino acid sequence: (SEQ ID
NO: 3).
3. A fusion peptide comprising an amino acid sequence: (SEQ ID NO: 4), wherein said spacer is defined by the formula (F-fPτoli e )^, and F represents a flexible sequence composed of amino acids each selected independently from the group consisting of serine, glycine, or threonine, n is an integer from 4-8 inclusive, and m is an integer from 1-4 inclusive.
4. A fusion peptide as described in claim 3, wherein said spacer comprises the following sequence: (SEQ ID NO: 17), and n is an integer from
4-8, inclusive.
5. A fusion peptide comprising an amino acid sequence: (SEQ ID O: 34).
6. A method of treating or preventing disease in a patient comprising administering to said patient an effective amount of peptide represented by the sequence: (SEQ ID NO: 3).
7. A method of treating or preventing disease as described in claim 6, wherein said patient is diagnosed with a disease is selected from the group consisting of platelet dependent thrombosis and restenosis.
8. A method of treating or preventing disease in a patient comprising administering to said patient an effective amount of peptide represented by the structure: (SEQ ID NO: 4), wherein said spacer is defined by the formula (F-ζProlineJJmF, and F represents a flexible sequence composed of amino acids each selected independently from the group consisting of serine, glycine, or threonine, and n is an integer from 4-8 inclusive, and m is an integer from 1-4 inclusive.
9. A method of treating or preventing disease in a patient as described in claim 8, wherein said spacer comprises the following sequence: (SEQ ID NO: 17), and n is an integer from 4-8, inclusive.
10. A method of treating or preventing disease as described in claim 9, wherein said patient is diagnosed with a disease selected from the group consisting of platelet dependent thrombosis and restenosis.
11. A composition comprising an RGD containing peptide and a peptide comprising (SEQ ID NO: 3).
12. A method of treating or preventing disease in a patient comprising administering an effective amount of a composition comprising a RGD peptide and a peptide comprising (SEQ ID NO: 3).
13. A method of treating or preventing disease as described in claim
12, wherein said RGD peptide and said peptide (SEQ ID NO: 3) are administered alone or in combination.
14. A method for identifying peptides that bind to a target cell and affect cell adhesion or aggregation, comprising the steps of: a) forming a target cell peptide complex by contacting a random peptide library comprising a first and a second population of peptides, said first population capable of binding to said target cell to form said complex and said second population substantially incapable of binding to said target cell; b) separating said target cell peptide complex from said second population of peptides; and c) deteπnining the amino acid sequence of said first population of peptides bound to said target cell.
15. A method as described in claim 14, wherein said random peptide library is a phage random peptide library comprising a first and a second population of phage that encode and express on their surface said first and said second population of peptides, respectively.
16. A method as described in claim 15, wherein the expression of said first and said second population of peptides comprises expressing said peptides as part of a phage filamentous fusion protein.
17. A method as described in claim 14, wherein said target cell comprises platelets.
18. A method as described in claim 17, wherein said target cell complex comprises platelets and phage that express said first population of peptides bound to said platelets.
PCT/US1992/008481 1991-10-04 1992-10-05 Peptide inhibitors of platelet adhesion WO1993007169A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US77176891A 1991-10-04 1991-10-04
US771,768 1991-10-04

Publications (1)

Publication Number Publication Date
WO1993007169A1 true WO1993007169A1 (en) 1993-04-15

Family

ID=25092919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/008481 WO1993007169A1 (en) 1991-10-04 1992-10-05 Peptide inhibitors of platelet adhesion

Country Status (1)

Country Link
WO (1) WO1993007169A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532167A (en) * 1994-01-07 1996-07-02 Beth Israel Hospital Substrate specificity of protein kinases
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5705606A (en) * 1988-06-30 1998-01-06 Cor Therapeutics, Inc. Peptides derived from GPIIIa
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US12037421B2 (en) 2022-06-24 2024-07-16 Chevron Phillips Chemical Company Lp Processes for preparing metallocene-based catalyst systems with an alcohol compound

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011458A1 (en) * 1990-02-02 1991-08-08 Genentech, Inc. CYCLIC PEPTIDES CONTAINING Arg-Gly-Asp FLANKED BY PROLINE
WO1991018980A1 (en) * 1990-06-01 1991-12-12 Cetus Corporation Compositions and methods for identifying biologically active molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011458A1 (en) * 1990-02-02 1991-08-08 Genentech, Inc. CYCLIC PEPTIDES CONTAINING Arg-Gly-Asp FLANKED BY PROLINE
WO1991018980A1 (en) * 1990-06-01 1991-12-12 Cetus Corporation Compositions and methods for identifying biologically active molecules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87, no. 7, April 1990, WASHINGTON US pages 2471 - 2475 MARK S. DENNIS ET AL. 'Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: Evidence for a family of platelet-aggregation inhibitors' *
SCIENCE vol. 249, 27 July 1990, LANCASTER, PA US pages 404 - 406 JAMES J. DEVLIN ET AL. 'Random peptide libraries: A source of specific protein binding molecules' cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705606A (en) * 1988-06-30 1998-01-06 Cor Therapeutics, Inc. Peptides derived from GPIIIa
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
US5532167A (en) * 1994-01-07 1996-07-02 Beth Israel Hospital Substrate specificity of protein kinases
US6004757A (en) * 1994-01-07 1999-12-21 Beth Israel Hospital Substrate specificity of a protein kinases
US12037421B2 (en) 2022-06-24 2024-07-16 Chevron Phillips Chemical Company Lp Processes for preparing metallocene-based catalyst systems with an alcohol compound

Similar Documents

Publication Publication Date Title
AU684510B2 (en) Method for selection of biologically active peptide sequences
JP2659781B2 (en) DNA sequence, recombinant DNA molecule and method for producing lymphocyte function-related antigen-3 (LFA-3)
US4683291A (en) Platelet binding inhibitors
US5340727A (en) GPIbα fragments and recombinant DNA expression vectors
US5866363A (en) Method and means for sorting and identifying biological information
CN1329512C (en) Modulator of TNF/NGF superfamily receptors and soluble oligomeric TNF/NGF superfamily receptors
KR100865602B1 (en) Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains
CN109678965B (en) Chimeric antigen receptor, gene and recombinant expression vector thereof, CD22-CD19 dual-targeting T cell and application thereof
AU643306B2 (en) Anticoagulant polypeptides
US5648458A (en) Peptides and compounds that bind to ELAM-1
CA2117747A1 (en) A novel hyaluronan receptor protein and novel hyaluronan binding peptides
WO1993007169A1 (en) Peptide inhibitors of platelet adhesion
KR0150000B1 (en) Genes coding for a protein having human macif activity, expression vectors containing said genes trans formant geues, trans formant cells
CA2544764A1 (en) Protein binding miniature proteins
US5661005A (en) β3 integrin subunit specific polypeptides, cDNAS which encode these polypeptides and methods of producing these polypeptides
Liu et al. Comparison of protein structure and genomic structure of human, rabbit, and Limulus C-reactive proteins: possible implications for function and evolution
EP0333517A2 (en) Method and agent for inhibiting the binding of human polymorphonuclear leukocytes to endothelium and compositions therefor
US20030167129A1 (en) Binding compounds and methods for identifying binding compounds
KR100692226B1 (en) NOVEL POLYPEPTIDES, cDNAS ENCODING THE SAME AND UTILIZATION THEREOF
JPH09500371A (en) Thrombin Receptor Radioligand and Thrombin Receptor Antagonist Identification Assay
WO1993011778A1 (en) Bifunctional antithrombotic molecules and antithrombotic polypeptides
JPH06502541A (en) Von Willebrand factor therapeutic fragment
AU2001247675A1 (en) Binding compounds and methods for identifying binding compounds
US20030018438A1 (en) Binding compounds and methods for identifying binding compounds
JPH08224087A (en) Inhibitor for suppressing activity of protein exhibiting ciita activity,its method for identification and its pharmacological composition

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA DK FI JP NL NO SE US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)

Free format text: DK,NL,SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA