WO1997010839A1 - Compositions and methods for inhibiting the binding of pecam - Google Patents

Compositions and methods for inhibiting the binding of pecam Download PDF

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Publication number
WO1997010839A1
WO1997010839A1 PCT/US1996/014940 US9614940W WO9710839A1 WO 1997010839 A1 WO1997010839 A1 WO 1997010839A1 US 9614940 W US9614940 W US 9614940W WO 9710839 A1 WO9710839 A1 WO 9710839A1
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WIPO (PCT)
Prior art keywords
xaa
seq
ala
amino acid
peptide
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PCT/US1996/014940
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French (fr)
Inventor
Pamela Beck
Phi Nga Kint
B. Mitch Revelle
Sid Sherwood
Robert J. Bjercke
Kaijun Ren
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Texas Biotechnology Corporation
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Priority to AU71610/96A priority Critical patent/AU7161096A/en
Publication of WO1997010839A1 publication Critical patent/WO1997010839A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates to compounds that inhibit the binding of PECAM-l (platelet endothelial cellular adhesion molecule- 1 ) to itself. More particularly, this invention pertains to linear and cyclic peptides that inhibit that binding.
  • PECAM-l platelet endothelial cellular adhesion molecule- 1
  • PECAM-l which has also been called platelet/endothelial cell adhesion molecule- 1 and CD31, is a glycoprotein that is constitutively expressed on the surface of endothelial cells, platelets, and most leukocytes. PECAM-l recognizes and binds to other PECAM-l molecules that are present on the surface of adjacent cells.
  • white blood cells also called leukocytes
  • a tissue has been invaded by a microorganism or has been damaged
  • white blood cells also called leukocytes
  • One important aspect of the inflammatory response involves leukocyte adhesion and subsequent emigration or extravasation across the endothelial cell wall that lines the blood vessel.
  • leukocytes are found circulating through the bloodstream.
  • the white blood cells must be able to recognize the invaded or damaged tissue and be able to bind to the wall of the capillary near the affected tissue and subsequently migrate through the capillary wall into the affected tissue.
  • PECAM-l has been shown to bind to specific types of white blood cells and to enable these cells to then migrate from the blood vessel into the affected tissue.
  • granulocytes There are three main types of white blood cells: granulocytes, monocytes and lymphocytes.
  • PECAM-l is thought to recognize PECAM-l and heparin-related oligosaccharides presented as glycoproteins on the surface of each of these types of cells, including neutrophils, monocytes, a specific T lymphocytes, eosinophils, and basophils.
  • Neutrophils are a subclass of granulocytes that phagocytose and destroy small organisms, especially bacteria.
  • Monocytes after leaving the bloodstream through the wall of a capillary, mature into macrophages that phagocytose and digest invading microorganisms, foreign bodies and senescent cells. Lymphocytes produce antibodies and kill infected cells. Eosinophils and basophils secrete mediators of various inflammatory reactions.
  • Monocytes and neutrophils are able to localize the site where tissue has been damaged by binding to selectins, which are expressed on the surface of the endothelial cells lining capillaries when the tissue surrounding a capillary has been infected or damaged. This binding slows the flow of white blood cells through the bloodstream, prior to integrin mediated firm attachment and
  • PECAM-l mediated transmigration, all of which help to localize white blood cells to areas of injury or infection.
  • some of the diseases that might be treated by the inhibition of PECAM-l binding to PECAM-l include, but are not limited to, allergy, ARDS, Crohn's disease, septic shock, traumatic shock, multi-organ failure, auto-immune diseases, asthma, inflammatory bowel disease, psoriasis, rheumatoid arthritis and reperfusion injury following heart attacks, strokes, cancer and organ transplants.
  • the present invention provides an isolated and purified peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight linked through an amide bond to the N-terminal residue; and (b) a C-terminal carboxylic acid group or amide group.
  • the peptide includes a sequence that is identical to a contiguous stretch of at least four amino acid residues of the extracellular domain of the expression product of the human PECAM-l gene, or a single amino acid residue substituent thereof.
  • the contiguous stretch of at least four amino acid residues has the sequence of SEQ ID NO:2, or a single amino acid residue substituent thereof.
  • the single amino acid residue substituent has the sequence of any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
  • a preferred peptide according to this embodiment has the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1 or SEQ
  • the contiguous stretch of at least four amino acid residues has the sequence of SEQ ID NO: 13.
  • the single amino acid residue substituent has the sequence of any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 14
  • the contiguous stretch of at least four amino acid residues has the amino acid residue sequence SEQ ID NO:22, or a single amino acid residue substituent thereof.
  • the single amino acid residue substituent has the sequence of any of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ
  • the peptide has the sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.
  • a peptide of the present invention can be linear or cyclic.
  • a cyclic peptide of the present invention can be cyclized via formation of a lactam or can be cyclized with the use of one or more cysteine or modified cysteine residues.
  • a peptide of the present invention comprises a cysteine or modified cysteine residue and a -CH 2 CO- group at the N-terminal position, wherein the sulfur atom of the cysteine or modified cysteine residue is attached to the CH 2 group of -CH 2 CO-.
  • a peptide comprises at least two cysteine or modified cysteine residues, wherein at least one of the cysteine or modified cysteine residues is located at the N- or C-te ⁇ ninal position.
  • a preferred peptide of the present invention has the amino acid residue sequence of any of SEQ ID NOs:33-105.
  • the present invention further provides a pharmaceutical composition containing a physiologically acceptable diluent and any peptide of the invention.
  • the present invention still further provides a process of selectively inhibiting the binding of PECAM-l to itself.
  • a first cell that expresses PECAM-l is exposed to a second cell that expresses PECAM-l in the presence of an effective inhibiting amount of a peptides of this invention.
  • the first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell.
  • the present invention provides a process of selectively inhibiting the adhesion of a first cell that expresses PECAM-l to a second cell that PECAM- 1.
  • the process includes the step of exposing at least one of the first and second cells to an effective inhibiting amount of a peptide of the present invention.
  • the first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell.
  • at least one of the first and second cells is a vascular endothelial cell.
  • the first and second cells are located in a living organism.
  • the present invention provides a process of inhibiting the binding of PECAM-l to itself.
  • the invention also provides peptides that inhibit that binding.
  • PECAM-l Platelet endothelial cellular adhesion molecule-1
  • cytokines such as tumor necrosis factor- ⁇ ,, phorbol ester, thrombin and calcium ionophores.
  • Antibodies that block PECAM-l binding have been shown to inhibit leukocyte extravasation both in vitro and in vivo.
  • PECAM-l is hypothesized to contribute to leukocyte extravasion in inflammatory conditions such as ARDS, Crohn's disease, septic shock, traumatic shock, multi-organ failure, auto-immune diseases, rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis, and reperfusion injury that follows heart attacks, strokes and organ transplants.
  • inflammatory conditions such as ARDS, Crohn's disease, septic shock, traumatic shock, multi-organ failure, auto-immune diseases, rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis, and reperfusion injury that follows heart attacks, strokes and organ transplants.
  • the expression product of the human PECAM-l gene is a polypeptide of
  • PECAM-l comprises a number of functional domains.
  • PECAM-l comprises an extracellular domain (residues 28-601 of SEQ ID NO:l), a transmembrane domain (residues 602-620 of SEQ ID NO:l) and a cytoplamic domain (residues 621 -738 of SEQ ID NO: l ).
  • the extracellular domain further comprises six, Ig-like, C2-type domains.
  • the present invention provides peptides that inhibit binding of PECAM-l to itself.
  • a peptide of the present invention is modeled after a portion of one of the C2-type, IG domains of PECAM-l , which domain is presented in such a way by the peptide to produce a potent PECAM-l binding inhibitor.
  • amino acid residue sequences are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino (N) to the carboxyl (C) terminus. Amino acid residue sequences are denominated by either a single letter or a three letter code. The meanings of those codes as well as various other abbreviations used herein are in accordance with the recommendation of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, and are shown below.
  • Modifications and changes can be made in the structure of a peptide of the present invention and still obtain a molecule that inhibits the binding of PECAM-l to itself.
  • certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity; likewise, D- or L- amino acid residues can be used.
  • D-amino acids are indicated herein as d- Xaa, where Xaa is the three-letter amino acid code (e.g., dTrp).
  • certain amino acids can be substituted or added which greatly enhance binding inhibition.
  • a peptide of the present invention contains from 4 to about 13 amino acid residues.
  • the N-terminal amino acid residue has a free terminal amine group (NH 2 ), acetyl group (Ac) or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight (MPEG X000 ) linked through an amide bond to the N-terminal residue.
  • the C-terminal amino acid residue has a terminal carboxylic acid group (OH) or amide group.
  • a peptide of this invention is modeled after a short segment of one of the C2-type IG domains of PECAM-l. More particularly, a peptide is modeled after a segment of Domain 1 or Domain 2.
  • a peptide is modeled after a short segment of Domain 1.
  • Preferred segments of Domain 1 include amino acid residues 58-63 or 1 15- 123 of SEQ ID NO:l.
  • a peptide of the present invention includes an amino acid residue sequence that is identical to a contiguous stretch of at least four amino acid residues of residues 58-63 or 1 15-
  • the contiguous stretch of at least four amino acid residues preferably has the sequence Phe-AIa-Asp-Val (SEQ ID NO:2).
  • the single amino acid residue substituent of SEQ ID NO:2 has the sequence of any of Xaa 4 -Ala-Asp- Val (SEQ ID NO:3), Phe-Xaa'-Asp-Val (SEQ ID NO:4), Phe-Ala-Xaa 2 -Val (SEQ ID NO:5) or Phe-Ala-Asp-Xaa 3 (SEQ ID NO:6), wherein Xaa 1 , Xaa 2 , Xaa 3 and Xaa 4 are any L-amino acid residue.
  • a preferred peptide according to this embodiment has the sequence Phe-Ala-Asp-Val-Ser (SEQ ID NO:7), Xaa 4 -Ala-Asp- Val-S
  • the contiguous stretch of at least four amino acid residues preferably has the sequence Asn-Lys-Glu-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 13).
  • the single amino acid residue substituent has lhe sequence of any of Xaa'-Lys-Glu-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 14), Asn-Xaa 2 -Glu-
  • Lys-Thr-Thr-Ala-Glu SEQ ID NO: 15
  • Asn-Lys-Xaa 3 -Lys-Thr-Thr-Ala-Glu SEQ ID NO: 16
  • Asn-Lys-Glu-Xaa 4 -Thr-Thr-Ala-Glu SEQ ID NO: 17
  • Asn- Lys-Glu-Lys-Xaa 5 -Thr-Ala-Glu SEQ ID NO: 18
  • Asn-Lys-Glu-Lys-Thr-Xaa 6 - Ala-Glu SEQ ID NO: 19
  • Asn-Lys-Glu-Lys-Thr-Thr-Xaa 7 -Glu SEQ ID NO:20
  • Asn-Lys-Glu-Lys-Thr-Thr-Thr-Ala-Xaa 8 SEQ ID NO:21
  • Xaa 1'8 are each independently any L-amino acid residue.
  • Xaa' is Ala
  • Xaa 2 is Ala
  • Xaa 3 is Ala, Gin or Lys
  • Xaa 4 is Ala
  • Xaa 5 is Ala
  • Xaa 6 is Ala
  • Xaa 7 and Xaa 8 are Ala.
  • a peptide of this invention is modeled after a short segment of Domain 2.
  • Such a peptide includes an amino acid residue sequence that is identical to a contiguous stretch of at least four amino acid residues of residues 212-227 of SEQ ID NO:l, or a single amino acid residue substituent thereof
  • the contiguous stretch of at least four amino acid residues preferably that short segment includes residues 212-227 of SEQ ID NO:l and, more preferably residues 212-217 of SEQ ID NO:l .
  • An exemplary and preferred peptide comprises the amino acid residue sequence of Ser-Gly-lle-His (SEQ ID NO:22), or a single amino acid residue substituent thereof.
  • the single amino acid residue substituent has the sequence of any of Xaa'-Gly-Ile-His (SEQ ID NO:23), Ser- Xaa 2 -Ile-His (SEQ ID NO:24), Ser-Gly-Xaa 3 -His (SEQ ID NO:25) or Ser-Gly- Ile-Xaa 4 (SEQ ID NO:26), wherein Xaa 1 , Xaa 2 , Xaa 3 and Xaa 4 are any L- ⁇ - amino acid residue.
  • a peptide of the present invention comprises the amino acid residue sequence of Ser-Gly-Ile-His-Met (SEQ ID NO:27), or a single amino acid residue substituent thereof.
  • a single amino acid residue substituent has the sequence of any of Xaa'-Gly-Ile-His-Met (SEQ ID NO:28), Ser-Xaa 2 -Ile-His-Met (SEQ ID NO:29), Ser-Gly-Xaa 3 -His-Met (SEQ ID NO:30), Ser-Gly-Ile-Xaa 4 -Met (SEQ ID NO:31) or Ser-Gly-Ile-His-Xaa 5 (SEQ ID NO:32), wherein Xaa', Xaa 2 , Xaa 3 , Xaa 4 and Xaa 5 are any L- ⁇ -amino acid residue.
  • each of Xaa'-Xaa 5 in the above sequences is Ala.
  • a peptide in accordance with the sequences set forth above can be extended in the N-terminal direction by the addition of from 1 to 5 L- or D- ⁇ -amino acids and, in the C-terminal direction, by the addition of from 1 to 5 L- or D- ⁇ -amino acids.
  • the C-terminal amide peptides were prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the same general method as the other couplings.
  • the C-terminal carboxylic acid peptide were prepared by purchasing Wang resin to which the C-terminal amino acid was bound as a carboxylic ester.
  • the ⁇ -amino protecting group was removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by simultaneous treatment of the resin with the Fmoc-amino acid, a coupling reagent such as DIC or HBTU, and if necessary HOBT.
  • a coupling reagent such as DIC or HBTU
  • Fmoc protecting group was removed by treatment with a 20% solution of piperidine in DMF.
  • piperidine can be replaced by other bases, furthermore the coupling reagents and protocols used can be substituted with any of those known in the field of peptide synthesis (including the use of Boc chemistry based solid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural amino acids, D-amino acids and other compounds were coupled by manual addition of li e reagent, following the same procedure as for automated operation.
  • TFA cocktail After cleavage the resin was removed by filtration and cold ether added to the solution to give a precipitate. The precipitate was collected and washed a few times with ether to remove residual TFA and scavengers. The precipitate was redissolved in aqueous solution for lyophilization to give the crude product.
  • a gradient of increasing percentage of solution B was used to elute the peptide from the solid support, however the gradient used was sequence dependent, and can be selected by someone skilled in the art of peptide purification. Other methods of purification are equally acceptable.
  • the purity of the peptides was checked by C analytical HPLC (300A, 4.6 mm x 25 cm, 5 ⁇ m spherical packing) at a flow rate of 1 ml/min.
  • Especially preferred linear peptides of the present invention modeled after residues 58-63 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO:33 (Phe-Ala-Asp-Val-Ser-Thr-amide), SEQ ID NO:34 (Ala- Ala- Asp- Val-Ser-Thr-amide), SEQ ID NO: 35 (Phe- Ala-Ala- Val-Ser-Thr- amide), SEQ ID NO:36 (Phe-Ala-Asp-Ala-Ser-Thr-amide), SEQ ID NO:37 (Phe-Ala-Asp-Val-Ala-Thr-amide), SEQ ID NO:38 (Phe-Ala-Asp-Val-Ser- Ala-amide), SEQ ID NO:39 (Phe-Ala-Asp-Val-Ser-amide), SEQ ID NO:40
  • Al-Asp-Val-Ser-amide SEQ ID NO:41 (Phe- Ala-Asp- Val-amide), SEQ ID NO:42 (Phe-Ala-Asp-Val-Ser-Thr-OH), SEQ ID NO:43 (Phe-Ala-Asp-Val- Ser-Ala-OH), SEQ ID NO:44 (Phe-Ala-Asp-Val-Ser-OH), or SEQ ID NO:45 (Ala-Asp-Val-Ser-OH).
  • Especially preferred linear peptides of the present invention modeled after residues 115-123 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO: 46 (Asn-Lys-Glu-Lys-Thr-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:47 (Asn-Lys-Glu-Lys-Thr-amide), SEQ ID NO:48 (Lys-Thr- Thr- Ala-Glu- Ala-amide), SEQ ID NO:49 (Ala-Lys-Glu-Lys-Thr- Thr- Ala-
  • Glu-Tyr-amide SEQ ID NO: 50 (Asn-Ala-Glu-Lys-Thr-Thr-Ala-Glu-Tyr- amide), SEQ ID NO: 51 (Asn-Lys-Ala-Lys-Thr-Thr-Ala-Glu-Tyr -amide), SEQ ID NO: 52 (Asn-Lys-Glu-Ala-Thr-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:53 (Asn-Lys-Glu-Lys-Ala-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:54 (Asn-Lys-Glu-Lys-Thr-Ala-Ala-Glu-Tyr-amide), SEQ ID NO:55 (Asn-Lys-Lys-Thr-Ala-Ala-Glu-Tyr-amide), SEQ ID NO:55 (Asn-Lys-Lys-Thr-Ala-Ala-Glu
  • Glu-Lys-Thr-Thr-Ala-Ala-Tyr-amide SEQ ID NO:56 (Glu-Lys-Thr-Ala- Ala-Glu-amide), SEQ ID NO:57 (Glu-Lys-Thr-Thr-Ala-Glu-amide), SEQ ID NO:58 (Lys-Lys-Thr-Ala-Ala-Glu-amide), SEQ ID NO:59 (Gln-Lys-Thr- Ala-Ala-Glu-amide), SEQ ID NO:60 (Ala-Lys-Thr- Ala-Ala-Glu-amide), SEQ ID NO:61 (Thr- Ala-Ala-Glu-amide), SEQ ID NO:62 (Lys-Thr-Ala-Ala-Glu- amide), SEQ ID NO:63 (Ala-Lys-Thr- Ala-amide), SEQ ID NO:64 (Glu-Ala- Thr-Thr-Ala-Glu-amide), SEQ ID NO:65 (Glu-Lys-Ala
  • Thr-Ala-Ala-Glu-OH SEQ ID NO:71 (Glu-Lys-Thr-Ala-Ala-Glu-OH), SEQ ID NO:72 (Thr-Ala-Ala-Glu-OH), SEQ ID NO:73 (Lys-Thr-Ala-Ala-Glu- OH), SEQ ID NO:74 (Ala-Lys-Thr-Ala-OH), or SEQ ID NO:75 (Lys-Thr- Thr-Ala-Glu-Ala-OH).
  • Especially preferred linear peptides of the present invention modeled after residues 212-227 of SEQ ID NO:l have the amino acid residue sequence of SEQ ID NO:76 (Ser-Gly-Ile-His-Met-Gln-amide), SEQ ID NO:77 (Ala-Gly-Ile-His-Met-Gln-amide), SEQ ID NO:78 (Ser-Ala-Ile-His- Met-Gln-amide), SEQ ID NO:79 (Ser-Gly-Ala-His-Met-Gln-amide), SEQ ID NO:80 (Ser-Gly-Ile-Ala-Met-Gln-amide), SEQ ID NO:81 (Ser-Gly-Ile-His- Ala-Gln-amide), SEQ ID NO:82 (Ser-Gly-Ile-His-Met-Ala-amide), SEQ ID NO:76 (Ser-Gly-Ile-His-Met-Gln-amide), SEQ ID NO
  • a cyclic peptide can be cyclized without or with a sulfur containing bridge. Where a cyclic peptide does not comprise a sulfur containing bridge, the N- and C-terminal amino acid residues are joined together with an amide bond (formally a lactam in the case of cyclization) . Where a cyclic peptide comprises a sulfur containing bridge, either one or two amino acid residues of the corresponding linear peptide is a Cys or modified Cys residue (dCys or dPen).
  • Such a cyclic peptide can comprise a cyclic sulfide, sulfoxide or sulfone (one Cys residue in the corresponding linear peptide) or a cyclic disulfide (two Cys residues in the corresponding linear peptide).
  • a peptide of the present invention contains a sulfide, sulfoxide or sulfone bridge
  • that peptide comprises a cysteine or modified cysteine residue at one position and a -CH 2 CO- group at the N-terminal position.
  • modified cysteine refers to D-cysteine (dCys) or D- penicillamine (dPen).
  • the sulfur atom of the cysteine or modified cysteine residue is attached to the CH 2 group forming the cyclic peptide.
  • a peptide of the present invention contains a disulfide bridge
  • that peptide contains two cysteine or modified cysteine residues.
  • one of the cysteine or modified cysteine residues is located at the N- or C- terminal position and at least one of those amino acid residues is Cys.
  • a cyclic peptide of the present invention can be made using standard peptide synthetic procedures well known in the art. Typically, peptides were made with Fmoc-amino acids. However, peptides can also be made using Boc protecting groups by methods well known to those skilled in the art. Side chain protecting groups of trifunction amino acids used in the synthetic procedure include Arginine (Pmc), Aspartic acid (tBu), Cysteine (Trt), Glutamic acid (tBu), Histidine (Boc), Lysine (Boc), Serine (tBu), Threonine
  • the C-terminal amide peptides are prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the same general method as the other couplings.
  • the C-terminal carboxylic acid peptides are prepared by purchasing Wang resin to which the C-terminal amino acid was bound to the resin as a carboxylic ester.
  • the ⁇ -amino protecting group is removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by simultaneous treatment of the resin with the Fmoc-amino acid, a coupling reagent such as DIC or HBTU, and if necessary HOBT.
  • a coupling reagent such as DIC or HBTU
  • Fmoc protecting group is removed by treatment with a 20% solution of piperidine in DMF.
  • piperidine in DMF.
  • the exact percentage of piperidine is not critical and should not be considered limiting in this invention.
  • piperidine can be replaced by other bases, furthermore the coupling reagents and protocols used can be substimted with any of those known in the field of peptide synthesis (including the use of Boc chemistry based solid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural amino acids, D-amino acids and other compounds are coupled by manual addition of the reagent, following the same procedure as for automated operation.
  • the peptides can be synthesized on an insoluble carrier such as p-benzyloxybenzyl alcohol resin (Wang resin), whereas the equivalent C-terminal amides were prepared on 4-(2',4'-dimethoxyphenyl- Fmoc-aminomethyl)-phenoxy resin (Rink resin).
  • Wang resin p-benzyloxybenzyl alcohol resin
  • Rink resin 4-(2',4'-dimethoxyphenyl- Fmoc-aminomethyl)-phenoxy resin
  • two cysteines are present, mild acid removal of the trityl protecting groups and oxidative cyclization on the resin using DMSO or NIS forms the disulfide bond, and this compound was cleaved from the resin in the normal way.
  • disulfides can be prepared by solution phase cyclization of the linear sequence in guanidine hydrochloride.
  • N-terminus can be acylated with bromoacetic acid, the cysteine trityl group removed and cyclization achieved by NMM in DMF treatment.
  • the head-tail lactams were synthesized on a chlorotrityl resin which forms a carboxylic ester linkage between the C- terminal amino acid and the resin.
  • the linear peptide was cleaved from the resin with acetic acid in DCM and cyclized in solution to form the lactam.
  • Peptides are cleaved from the resin with a TFA cocktail after the removal of the N-terminal Fmoc protecting group.
  • the exact composition of the TFA cocktail is varied depending on the side chain protecting groups present, and is well known to those skilled in the art.
  • the range of TFA is typically from 85 to 95 % , and the remainder comprised of a mixture of scavengers selected from a combination of anisole, thioanisole, cresol, thiocresol, phenol, thiophenol, EDT, trimethylsilane and water.
  • the time of the cleavage reaction required is sequence dependant, normally being from 1 to 3 hours.
  • the resin is removed by filtration and cold ether added to the solution to give a precipitate.
  • the precipitate is collected and washed a few times with ether to remove residual TFA and scavengers.
  • the precipitate is redissolved in aqueous solution for lyophilization to give the crude product.
  • Purification is typically carried out by reverse-phase HPLC on a C lg - preparative column (30 ⁇ A, 21.4 mm x 25 cm, 5 ⁇ m spherical packing) at a flow rate of 10 ml/min. The selection of any other suitable packing known to one skilled in the art is equally acceptable. Products are detected by UV absorption at 214 nm. Two mobile phases are used in the HPLC system, solution A and B using a gradient elution. Solution A is comprised of 5% acetonitrile in deionized water containing 0.15% TFA, while solution B is comprised of 5% deionized water in acetonitrile containing 0.1 % of TFA.
  • a gradient of increasing percentage of solution B is used to elute the peptide from the solid support, however the gradient used is sequence dependant. Other methods of purification known to one skilled in the art are equally acceptable.
  • the purity of the peptide is checked by C lg analytical HPLC (300 A, 4.6 mm x 25 cm, 5 ⁇ m spherical packing) at a flow rate of 1 ml/min.
  • Especially preferred cyclic peptides of the present invention modeled after residues 58-63 of SEQ ID NO: l have the amino acid resiue sequence of
  • SEQ ID NO:92 cyclic Phe-Ala-Asp-Val-Ser
  • SEQ ID NO:93 cyclic Phe- Ala-Asp-Val-Ser-Thr
  • SEQ ID NO: 94 Cys-Phe- Ala-Asp- Val-Cys- Amide, cyclic disulfide.
  • Especially preferred cyclic peptides of the present invention modeled after residues 115-123 of SEQ ID NO: l have the amino acid resiue sequence of SEQ ID NO:95 (cyclic Thr-Ala-Ala-Glu), SEQ ID NO:96 (cyclic Ala- Lys-Thr- Ala- Ala-Glu), SEQ ID NO:97 (cyclic Glu-Lys-Thr-Thr- Ala-Glu), SEQ ID NO: 98 (cyclic Lys-Thr- Ala- Ala-Glu), SEQ ID NO: 99 (cyclic Ala- Lys-Thr- Ala), SEQ ID NO: 100 (cyclic Glu-Lys-Thr- Ala- Ala-Glu), or SEQ
  • Especially preferred cyclic peptides of the present invention modeled after residues 212-227 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO: 102 (cyclic Ser-Gly-Ile-His-Met-Gln), SEQ ID NO: 102 (cyclic Ser-Gly-Ile-His-Met-Gln), SEQ ID NO: 102 (cyclic Ser-Gly-Ile-His-Met-Gln), SEQ ID NO: 102 (cyclic Ser-Gly-Ile-His-Met-Gln), SEQ ID
  • the present invention provides a pharmaceutical composition comprising a peptide of the present invention and a physiologically tolerable diluent.
  • the present invention includes one or more peptides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral administration in solid or liquid form, for rectal or topical admimstration, or the like.
  • compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol.
  • the compositions can also be delivered through a catheter for local delivery at a target site, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer.
  • the compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions.
  • the compositions are preferably administered by catheter, i.v. or subcutaneous injection, or intranasally via a spray or aerosol.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged abso ⁇ tion of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents.
  • adjuvants such as preserving, wetting, emulsifying and suspending agents.
  • Suspensions in addition to the active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • Dosage forms for topical admimstration of a compound of this invention include ointments, powders, sprays and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required.
  • Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • the present invention contemplates a process of selectively inhibiting the binding of PECAM-l to itself (i.e. , PECAM-l to PECAM-l).
  • a process of the present invention can be used in vitro or in vivo in a living organism.
  • a first cell expressing PECAM-l is exposed to a second cell expressing PECAM-l in the presence of an effective inhibiting amount of a peptide of the present invention.
  • Means for determining an effective inhibiting amount are well known in the art.
  • a preferred linear peptide of the present invention for use in the process has the sequence of any of SEQ ID NOs: 33, 42-46, 49-55, 57, 64-76, 87- 91. More preferably, a useful linear peptide has the sequence of any of SEQ ID NOs: 42, 46, 49, 50, 52-55, 67-76, 87,88. Most preferably, a useful linear peptide has the sequence of any of SEQ ID NOs: 67-72.
  • a preferred cyclic peptide of the present invention for use in the process has the sequence of any of SEQ ID NOs: 92-98, 100-103.
  • a useful cyclic peptide has the sequence of any of SEQ ID NOs: 92, 93, 95- 98, 100, 102, 103. Most preferably, a useful cyclic peptide has the sequence of any of SEQ ID NOs: 93, 95-97, 102,103.
  • a cell expressing PECAM-l can be a naturally occurring white blood cell, platelet, endothelial cell, mast cell or other cell type that naturally expresses PECAM-l on the cell surface, or a cell transfected with an expression vector that contains a polynucleotide (e.g. , genomic DNA or cDNA) that encodes PECAM-l .
  • PECAM-l is present on the surface of a vascular endothelial cell or white blood cell such as a monocyte, a lymphocyte or a granulocyte (e.g. , an eosinophil or a basophil).
  • a peptide is administered in an effective amount to the living organism.
  • the peptide is in a pharmaceutical composition of this invention.
  • Administering is preferably accomplished via intravascular injection or intranasal administration. The ability of peptides of the present invention to inhibit binding are described in detail hereinafter in the Examples.
  • a process of the present invention is especially useful in treating diseases associated with uncontrolled migration of white blood cells to damaged tissue.
  • diseases include, but are not limited to, asthma, atherosclerosis, rheumatoid arthritis, allergy, multiple sclerosis, leukemia, and cancer.
  • a process of inhibiting PECAM-l binding uses a peptide of the present invention as set forth hereinbefore. Preferred such peptides are the same as set forth above.
  • the present invention also provides a process of selectively inhibiting the adhesion of a cell that expresses PECAM-l to another cell that expresses PECAM-l .
  • at least one the cells expressing PECAM-l is exposed to the other cell in the presence of an effective inhibiting amount of a peptide of the present invention.
  • at least one of the cells expressing PECAM-l is a vascular endothelial cell.
  • Preferred peptides for use in such a process are the same as set forth above.
  • EXAMPLE 1 Synthesis of Phe-Ala-Asp-Val-Ser-Thr-NH, (SEO ID NO:33)
  • the Fmoc-amino acids and an equimolar amount of HBTU were dissolved in DMF. DIC in DCM was used as the coupling reagent.
  • the Fmoc-Wang resin was swollen by treatment with DMF for 15-20 min, then deprotected by treatment with 20% piperidine in DMF and the resin was washed with DMF. The first amide bond was formed using a 1-1.2 hour coupling time. The resin was washed with DMF, and this procedure was repeated for each amino acid until the peptide was complete.
  • the N- terminal Fmoc group was deprotected with piperidine and washed with
  • the Fmoc-amino acids and an equimolar amount of HBTU were dissolved in DMF. DIC in DCM was used as the coupling reagent.
  • the Fmoc-Wang resin was swollen by treatment with DMF for 15-20 min, then deprotected by treatment with 20% piperidine in DMF, and the resin was washed with DMF. The first amide bond was formed using a 1-1.2 hour coupling time. The resin was washed with DMF, and this procedure was repeated for each amino acid until the peptide was complete.
  • the N- terminal Fmoc group was deprotected with piperidine and washed with
  • the Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF.
  • DIC in DCM was used as the coupling reagent.
  • Fmoc-Rink resin 25 nM was swollen by treatment with DMF (1.25 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3 x
  • EXAMPLE 5 Synthesis of Cyclic Phe-Ala-Asp-Val-Ser (SEO ID NO: 92)
  • the Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF.
  • DIC in DCM was used as the coupling reagent with 1- 1.2 hour reaction times.
  • the Fmoc-Ser Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3x, 8 min each), and the resin was washed with DMF (6x).
  • the first amide bond was formed using Fmoc- Val (150 nM) and DIC (150 nM), and this procedure was repeated until all amino acids were coupled.
  • the peptide was cleaved from the resin with a TFA cocktail (containing 5 % anisole and 5% EDT) for 1 hour at room temperature.
  • the TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether. After washing with ether (3x), the peptide was lyophilized from aqueous solution to give the crude linear peptide.
  • EXAMPLE 7 Synthesis of Cyclic Ala-Lvs-Thr-Ala-Ala-Glu (SEO ID NO:96)
  • the Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF.
  • DIC in DCM was used as the coupling reagent with 1- 1.2 hour reaction times.
  • the Fmoc-Glu Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3x, 8 min each), and the resin was washed with DMF (6x).
  • the first amide bond was formed using Fmoc- Ala (150 nM) and DIC (150 nM), and this procedure was repeated until all amino acids were coupled.
  • the peptide was cleaved from the resin with a TFA cocktail (containing 5 % anisole and 5% EDT) for 1 hour at room temperamre.
  • the TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether.
  • EXAMPLE 8 Synthesis of Cyclic Thr-Ala-Ala-Glu (SEO ID NO:95). Cyclic Glu-Lvs-Thr-Thr- Ala-Glu (SEO ID NO: 97). Cyclic
  • the TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether. After washing with ether (3x), the peptide was lyophilized from aqueous solution to give the crude linear peptide. Cyclization was achieved by dissolving in water (20 ml) containing guanidine # HCl (2.0 g) and ammonium acetate (1.6 g) at pH 7.8, and the solution stirred at 4°C for 48 hours and then lyophilized. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization.
  • This peptide was prepared using the same general procedure as in
  • Peptides were assayed for their ability to inhibit the binding of the PECAM-l to itself.
  • the assay is described below.
  • PECAM-l was expressed in recombinant form as a fusion protein possessing all six immunoglobulin domains fused to the hinge and constant heavy chain regions 1 and 2 of the mouse IgG 2A cDNA.
  • the fusion cassette was generated by polymerase chain reaction (PCR) from the PECAM-l cDNA that was PCR cloned from total RNA extracted from human placenta.
  • the mouse IgG cDNA was cloned from PCR amplified cDNA generated from RNA extracted from the hybridoma cell line 402C10. All fusion cassettes were expressed frombaculovirus vectors using the BakPAK method and SF21 cells purchased from Clonetech.
  • Recombinant fusion protein was purified from baculovirus infected culture supernatants by immunoprecipitation using Dynal TM goat anti-mouse IgG coated magnetic beads. Mock beads were generated from uninfected SF21 culture supernatants. Following immunoprecipitation, beads incubated with mock culture supernatants did not bind HL60 cells that express PECAM-l served as controls. Beads incubated from PECAM-l culture supernatants did bind HL60 cells.
  • HL-60 cells were fluorescently labeled with Calcein AM C-3099
  • the HL60 cells remaining bound to the beads were inspected by microscopy and then lysed by adding about 50 ⁇ l of a 1.0% solution of NP-40 in PBS. Binding was quantified by fluorimetry using a Millipore Cytofluor 2350 fluorimeter. Dose response curves were calculated and IC 50 values determined.
  • Peptides having the amino acid residue sequence of SEQ ID NOs: 33- 105 were found to significantly inhibit the binding of PECAM-l to itself with an IC 50 values ranging from 0.1 ⁇ M to 150 ⁇ M.
  • mice Six to eight week old female BALB/c mice weighing approximately 25 gm were injected intraperitoneally at time zero with 1ml of a 2% solution of oyster glycogen (Type II, Sigma Chemical Co.) in Dulbecco's phosphate buffered saline (PBS) (Gibco/BRL). Control animals received 1ml of PBS alone. Each dose of 0.1 ml of solution was administered in NS at neutral pH of the compound of interest (SEQ ID NO: 97) was injected subcutaneously at: -15 minutes, time 0, + 15 minutes, +45 minutes, and +75 minutes. Mice were sacrificed at 180 minutes and a 10ml peritoneal lavage composed of PBS, EDTA, BSA, and gelatin was performed. The total number of cells in the exudate was determined using a hemocytometer and the number of polymorphonuclear leukocytes (PMN's) determined using cytospin preparations treated with Wright-Giemsa stain.
  • ADDRESSEE DRESSLER, GOLDSMITH, MILNAMOW
  • NAME NORTHRUP, THOMAS E.

Abstract

The present invention is directed to an isolated and purified linear peptide of from 4 to about 13 residues modeled after a C2-type, IG domain of the PECAM-1 peptide. A peptide of this invention can be linear or cyclic and has the amino acid residue sequence of any SEQ ID NOs: 2-105. The present invention is further directed to a process of selectively inhibiting the binding of PECAM-1 to itself comprising exposing a first cell that expresses PECAM-1 to a second cell that expresses PECAM-1 in the presence of an effective inhibiting amount of such a peptide. The present invention is still further directed to a pharmaceutical composition comprising a physiologically acceptable carrier and a peptide of the invention.

Description

COMPOSITIONS AND METHODS FOR INHIBITING THE BINDING OF PECAM
Field of the Invention
This invention relates to compounds that inhibit the binding of PECAM-l (platelet endothelial cellular adhesion molecule- 1 ) to itself. More particularly, this invention pertains to linear and cyclic peptides that inhibit that binding.
Background of the Invention
PECAM-l , which has also been called platelet/endothelial cell adhesion molecule- 1 and CD31, is a glycoprotein that is constitutively expressed on the surface of endothelial cells, platelets, and most leukocytes. PECAM-l recognizes and binds to other PECAM-l molecules that are present on the surface of adjacent cells.
When a tissue has been invaded by a microorganism or has been damaged, white blood cells, also called leukocytes, play a major role in the inflammatory response. One important aspect of the inflammatory response involves leukocyte adhesion and subsequent emigration or extravasation across the endothelial cell wall that lines the blood vessel. Generally, leukocytes are found circulating through the bloodstream. However, when a tissue is infected or becomes damaged, the white blood cells must be able to recognize the invaded or damaged tissue and be able to bind to the wall of the capillary near the affected tissue and subsequently migrate through the capillary wall into the affected tissue. PECAM-l has been shown to bind to specific types of white blood cells and to enable these cells to then migrate from the blood vessel into the affected tissue.
There are three main types of white blood cells: granulocytes, monocytes and lymphocytes. PECAM-l is thought to recognize PECAM-l and heparin-related oligosaccharides presented as glycoproteins on the surface of each of these types of cells, including neutrophils, monocytes, a specific T lymphocytes, eosinophils, and basophils. Neutrophils are a subclass of granulocytes that phagocytose and destroy small organisms, especially bacteria. Monocytes, after leaving the bloodstream through the wall of a capillary, mature into macrophages that phagocytose and digest invading microorganisms, foreign bodies and senescent cells. Lymphocytes produce antibodies and kill infected cells. Eosinophils and basophils secrete mediators of various inflammatory reactions.
Monocytes and neutrophils are able to localize the site where tissue has been damaged by binding to selectins, which are expressed on the surface of the endothelial cells lining capillaries when the tissue surrounding a capillary has been infected or damaged. This binding slows the flow of white blood cells through the bloodstream, prior to integrin mediated firm attachment and
PECAM-l mediated transmigration, all of which help to localize white blood cells to areas of injury or infection.
While white blood cell migration to the site of injury helps fight infection and destroy foreign material, in many instances this migration can get out of control, with white blood cells flooding to the scene and causing widespread tissue damage. Therefore, compounds capable of blocking this process, may be beneficial as therapeutic agents. Thus, it would be useful to develop inhibitors that would prevent the migration of white blood cells into tissues by blocking binding to PECAM-l . For example, some of the diseases that might be treated by the inhibition of PECAM-l binding to PECAM-l include, but are not limited to, allergy, ARDS, Crohn's disease, septic shock, traumatic shock, multi-organ failure, auto-immune diseases, asthma, inflammatory bowel disease, psoriasis, rheumatoid arthritis and reperfusion injury following heart attacks, strokes, cancer and organ transplants.
Brief Summary of the Invention
In one aspect, the present invention provides an isolated and purified peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight linked through an amide bond to the N-terminal residue; and (b) a C-terminal carboxylic acid group or amide group. The peptide includes a sequence that is identical to a contiguous stretch of at least four amino acid residues of the extracellular domain of the expression product of the human PECAM-l gene, or a single amino acid residue substituent thereof.
In one embodiment, the contiguous stretch of at least four amino acid residues has the sequence of SEQ ID NO:2, or a single amino acid residue substituent thereof. The single amino acid residue substituent has the sequence of any of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. A preferred peptide according to this embodiment has the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1 or SEQ
ID NO:12.
In another embodiment, the contiguous stretch of at least four amino acid residues has the sequence of SEQ ID NO: 13. In accordance with this embodiment, the single amino acid residue substituent has the sequence of any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
In another embodiment, the contiguous stretch of at least four amino acid residues has the amino acid residue sequence SEQ ID NO:22, or a single amino acid residue substituent thereof. The single amino acid residue substituent has the sequence of any of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ
ID NO:26. Preferably, the peptide has the sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.
A peptide of the present invention can be linear or cyclic. A cyclic peptide of the present invention can be cyclized via formation of a lactam or can be cyclized with the use of one or more cysteine or modified cysteine residues.
In one embodiment, a peptide of the present invention comprises a cysteine or modified cysteine residue and a -CH2CO- group at the N-terminal position, wherein the sulfur atom of the cysteine or modified cysteine residue is attached to the CH2 group of -CH2CO-. In another embodiment, a peptide comprises at least two cysteine or modified cysteine residues, wherein at least one of the cysteine or modified cysteine residues is located at the N- or C-teπninal position.
A preferred peptide of the present invention has the amino acid residue sequence of any of SEQ ID NOs:33-105. The present invention further provides a pharmaceutical composition containing a physiologically acceptable diluent and any peptide of the invention.
The present invention still further provides a process of selectively inhibiting the binding of PECAM-l to itself. In accordance with that process, a first cell that expresses PECAM-l is exposed to a second cell that expresses PECAM-l in the presence of an effective inhibiting amount of a peptides of this invention. Preferably, the first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell.
In a related aspect, the present invention provides a process of selectively inhibiting the adhesion of a first cell that expresses PECAM-l to a second cell that PECAM- 1. The process includes the step of exposing at least one of the first and second cells to an effective inhibiting amount of a peptide of the present invention. The first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell. In an especially preferred embodiment, at least one of the first and second cells is a vascular endothelial cell. Preferably, the first and second cells are located in a living organism.
Detailed Description of the Invention I. The Invention
The present invention provides a process of inhibiting the binding of PECAM-l to itself. The invention also provides peptides that inhibit that binding.
The adhesion of leukocytes to the vascular endothelium and their subsequent extravasation into tissues are critical steps in the inflammatory response. Platelet endothelial cellular adhesion molecule-1 (PECAM-l) is a member of the immunoglobulin superfamily and is constitutively expressed on the surface of endothelial cells and a restricted number of other cell types. Its surface distribution and molecular interactions can be altered by cytokines such as tumor necrosis factor-α,, phorbol ester, thrombin and calcium ionophores. Antibodies that block PECAM-l binding have been shown to inhibit leukocyte extravasation both in vitro and in vivo. Therefore, PECAM-l is hypothesized to contribute to leukocyte extravasion in inflammatory conditions such as ARDS, Crohn's disease, septic shock, traumatic shock, multi-organ failure, auto-immune diseases, rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis, and reperfusion injury that follows heart attacks, strokes and organ transplants.
The expression product of the human PECAM-l gene is a polypeptide of
738 amino acid residues (See FIG. 1 , SEQ ID NO: l). Residues 1 -27 of that polypeptide represent a signal peptide; residues 28-738 represent the mature peptide. PECAM-l comprises a number of functional domains. By way of example, PECAM-l comprises an extracellular domain (residues 28-601 of SEQ ID NO:l), a transmembrane domain (residues 602-620 of SEQ ID NO:l) and a cytoplamic domain (residues 621 -738 of SEQ ID NO: l ). The extracellular domain further comprises six, Ig-like, C2-type domains. The locations of those six, C2-type domains are residues 28-144 of SEQ ID NO:l (Domain 1 ). residues 145-248 of SEQ ID NO: l (Domain 2), residues 249-339 of SEQ ID NO:l (Domain 3), residues 340-423 of SEQ ID NO:l (Domain 4), residues 424-515 of
SEQ ID NO: l (Domain 5), and residues 516-601 of SEQ ID NO: l (Domain 6).
II. Peptides
In one aspect, the present invention provides peptides that inhibit binding of PECAM-l to itself. A peptide of the present invention is modeled after a portion of one of the C2-type, IG domains of PECAM-l , which domain is presented in such a way by the peptide to produce a potent PECAM-l binding inhibitor.
Peptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino (N) to the carboxyl (C) terminus. Amino acid residue sequences are denominated by either a single letter or a three letter code. The meanings of those codes as well as various other abbreviations used herein are in accordance with the recommendation of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, and are shown below.
A Ala L-alanine
Ac acetyl
Aie 2-aminoindan-2- carboxylic acid
Acm acetamidomethyl
C Cys L-cysteine dC dCys D-cysteine
C(SO3H) L-cysteic acid tBu tert-butyl
D Asp L-aspartic acid dD dAsp D-aspartic acid
E Glu L-glutamic acid dE dGlu D-glutamic acid
<E L-pyroglutamic acid
F Phe L-phenylalanine
G Gly glycine
H His L-histidine
I lie L-isoleucine
L Leu L-leucine
K Lys L-lysine
M Met L-methionine
N Asn L-asparagine
P Pro L-proline dP dPro D-proline dPen D-penicillamine
Pmc 2,2,5,7,8- pentamethylchroman-6 sulphonyl
Q Gin L-glutamine
R Arg L-arginine
S Ser L-serine
T Thr L-threonine
Trt trityl
V Val L-valine
W Trp L-tryptophan dW dTrp D-tryptophan
Y Tyr L-tyrosine
Boc tert-butoxycarbonyl
DCM methylene chloride
Die N,N'-diisopropyl carbodiimide
DIPEA diisopropylethylamine EDT 1,2-ethanedithiol
Fmoc 9-fluoreny methoxy carbonyl HOBT 1 -hydroxy- 1H- benzotriazole HBTU O-benzotriazole-
N,N,N',N'-tetra- methyluronium- hexafluorophosphate DMF N,N-dimethyl formamide MCPBA /w-chloroperoxy- benzoic acid NMM N-methylmorpholine
TFA trifluoroacetic acid
Modifications and changes can be made in the structure of a peptide of the present invention and still obtain a molecule that inhibits the binding of PECAM-l to itself. For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity; likewise, D- or L- amino acid residues can be used. D-amino acids are indicated herein as d- Xaa, where Xaa is the three-letter amino acid code (e.g., dTrp). In fact, certain amino acids can be substituted or added which greatly enhance binding inhibition. Because it is the interactive capacity and nature of a peptide that defines that peptide's biological functional activity, certain amino acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties, particularly inhibition of the binding of PECAM-l to tiself. Exemplary such peptides are set forth below.
A peptide of the present invention contains from 4 to about 13 amino acid residues. The N-terminal amino acid residue has a free terminal amine group (NH2), acetyl group (Ac) or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight (MPEGX000) linked through an amide bond to the N-terminal residue. The C-terminal amino acid residue has a terminal carboxylic acid group (OH) or amide group. A peptide of this invention is modeled after a short segment of one of the C2-type IG domains of PECAM-l. More particularly, a peptide is modeled after a segment of Domain 1 or Domain 2. In one embodiment, a peptide is modeled after a short segment of Domain 1. Preferred segments of Domain 1 include amino acid residues 58-63 or 1 15- 123 of SEQ ID NO:l. In accordance with this embodiment, a peptide of the present invention includes an amino acid residue sequence that is identical to a contiguous stretch of at least four amino acid residues of residues 58-63 or 1 15-
123 of SEQ ID NO:l , or a single amino acid residue substituent thereof
Where the peptide is modeled after residues 58-63 of SEQ ID NO:l , the contiguous stretch of at least four amino acid residues preferably has the sequence Phe-AIa-Asp-Val (SEQ ID NO:2). The single amino acid residue substituent of SEQ ID NO:2 has the sequence of any of Xaa4 -Ala-Asp- Val (SEQ ID NO:3), Phe-Xaa'-Asp-Val (SEQ ID NO:4), Phe-Ala-Xaa2-Val (SEQ ID NO:5) or Phe-Ala-Asp-Xaa3 (SEQ ID NO:6), wherein Xaa1, Xaa2, Xaa3 and Xaa4 are any L-amino acid residue. A preferred peptide according to this embodiment has the sequence Phe-Ala-Asp-Val-Ser (SEQ ID NO:7), Xaa4 -Ala-Asp- Val-Ser
(SEQ ID NO:8), Phe-Xaa'-Asp-Val-Ser (SEQ ID NO:9), Phe-Ala-Xaa2-Val-Ser (SEQ ID NO:10), Phe-Ala-Asp-Xaa3-Ser (SEQ ID NO:l l) or Phe-A la-Asp- Val- Xaa5 (SEQ ID NO: 12), wherein Xaa1, Xaa2, Xaa3, Xaa4 and Xaa5 are each independently any L-amino acid residue.
Where the peptide is modeled after residues 1 15-123 of SEQ ID NO:l , the contiguous stretch of at least four amino acid residues preferably has the sequence Asn-Lys-Glu-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 13). In accordance with this embodiment, the single amino acid residue substituent has lhe sequence of any of Xaa'-Lys-Glu-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 14), Asn-Xaa2-Glu-
Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 15), Asn-Lys-Xaa3-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 16), Asn-Lys-Glu-Xaa4-Thr-Thr-Ala-Glu (SEQ ID NO: 17), Asn- Lys-Glu-Lys-Xaa5-Thr-Ala-Glu (SEQ ID NO: 18), Asn-Lys-Glu-Lys-Thr-Xaa6- Ala-Glu (SEQ ID NO: 19), Asn-Lys-Glu-Lys-Thr-Thr-Xaa7-Glu (SEQ ID NO:20) or Asn-Lys-Glu-Lys-Thr-Thr-Ala-Xaa8 (SEQ ID NO:21), wherein Xaa1'8 are each independently any L-amino acid residue. Preferably, Xaa' is Ala; Xaa2 is Ala; Xaa3 is Ala, Gin or Lys; Xaa4 is Ala; Xaa5 is Ala; Xaa6 is Ala; Xaa7 and Xaa8 are Ala. In another embodiment, a peptide of this invention is modeled after a short segment of Domain 2. Such a peptide includes an amino acid residue sequence that is identical to a contiguous stretch of at least four amino acid residues of residues 212-227 of SEQ ID NO:l, or a single amino acid residue substituent thereof
Where the peptide is modeled after residues 212-227 of SEQ ID NO:l , the contiguous stretch of at least four amino acid residues preferably that short segment includes residues 212-227 of SEQ ID NO:l and, more preferably residues 212-217 of SEQ ID NO:l . An exemplary and preferred peptide comprises the amino acid residue sequence of Ser-Gly-lle-His (SEQ ID NO:22), or a single amino acid residue substituent thereof. The single amino acid residue substituent has the sequence of any of Xaa'-Gly-Ile-His (SEQ ID NO:23), Ser- Xaa2-Ile-His (SEQ ID NO:24), Ser-Gly-Xaa3-His (SEQ ID NO:25) or Ser-Gly- Ile-Xaa4 (SEQ ID NO:26), wherein Xaa1, Xaa2, Xaa3 and Xaa4 are any L-α- amino acid residue.
In another preferred embodiment, a peptide of the present invention comprises the amino acid residue sequence of Ser-Gly-Ile-His-Met (SEQ ID NO:27), or a single amino acid residue substituent thereof. Such a single amino acid residue substituent has the sequence of any of Xaa'-Gly-Ile-His-Met (SEQ ID NO:28), Ser-Xaa2-Ile-His-Met (SEQ ID NO:29), Ser-Gly-Xaa3-His-Met (SEQ ID NO:30), Ser-Gly-Ile-Xaa4-Met (SEQ ID NO:31) or Ser-Gly-Ile-His-Xaa5 (SEQ ID NO:32), wherein Xaa', Xaa2, Xaa3, Xaa4 and Xaa5 are any L-α-amino acid residue. Preferably, each of Xaa'-Xaa5 in the above sequences is Ala. A peptide of the present invention can be linear or cyclic.
A peptide in accordance with the sequences set forth above can be extended in the N-terminal direction by the addition of from 1 to 5 L- or D-α-amino acids and, in the C-terminal direction, by the addition of from 1 to 5 L- or D-α-amino acids. A. Linear Peptides
The preparation of the linear peptides in this invention by solid phase methodology is well known to those skilled in the art, and can be described as follows. Peptides were synthesized on an insoluble carrier such as p- benzyloxybenzyl alcohol resin for the synthesis of C-terminal carboxylic acid peptides (Wang resin, where normally the resin can be purchased with the first amino acid bound), and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin for C-terminal amide peptides (Rink resin). The peptides were prepared by solid phase synthesis using either HBTU or DIC chemistry procedures on a Protein Technologies Inc. Symphony peptide synthesizer.
The C-terminal amide peptides were prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the same general method as the other couplings. The C-terminal carboxylic acid peptide were prepared by purchasing Wang resin to which the C-terminal amino acid was bound as a carboxylic ester. The α-amino protecting group was removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by simultaneous treatment of the resin with the Fmoc-amino acid, a coupling reagent such as DIC or HBTU, and if necessary HOBT. Such deprotection and couplings were repeated to afford each desired peptide. In all cases the
Fmoc protecting group was removed by treatment with a 20% solution of piperidine in DMF. However, it is understood by those skilled in the art that the exact percentage of piperidine is not critical and should not be considered limiting in this invention. It is also understood by those skilled in the art that piperidine can be replaced by other bases, furthermore the coupling reagents and protocols used can be substituted with any of those known in the field of peptide synthesis (including the use of Boc chemistry based solid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural amino acids, D-amino acids and other compounds were coupled by manual addition of li e reagent, following the same procedure as for automated operation. Peptides were cleaved from the resin with a TFA cocktail after the removal of the N-terminal Fmoc protecting group. The exact composition of the TFA cocktail was varied depending on the side chain protecting groups present, and is well known to those skilled in the art. The range of TFA was from 85 to 95%, and the remainder comprised of a mixture of scavengers selected from a combination of anisole, thioanisole, cresol, thiocresol, phenol, thiophenol, EDT, trimethylsilane and water. The time of the cleavage reaction required was sequence dependant, normally being from 1 to 3 hours. After cleavage the resin was removed by filtration and cold ether added to the solution to give a precipitate. The precipitate was collected and washed a few times with ether to remove residual TFA and scavengers. The precipitate was redissolved in aqueous solution for lyophilization to give the crude product.
Purification of the crude peptide was carried out by reverse-phase HPLC on a C18-column preparative column (300A, 21.4 mm x 25 cm, 5 μm spherical packing) at a flow rate of 10 ml/min. The selection of any other suitable reverse-phase packing known to one skilled in the art is equally acceptable. Products were detected by UV absorption at 214 nm. Two mobile phases were used in the HPLC system, solution A and B using a gradient elution. Solution A was comprised of 5 % acetonitrile in deionized water containing 0.15% TFA, while solution B comprised 5 % of deionized water in acetonitrile containing 0.1% TFA. A gradient of increasing percentage of solution B was used to elute the peptide from the solid support, however the gradient used was sequence dependent, and can be selected by someone skilled in the art of peptide purification. Other methods of purification are equally acceptable. The purity of the peptides was checked by C analytical HPLC (300A, 4.6 mm x 25 cm, 5 μm spherical packing) at a flow rate of 1 ml/min.
Especially preferred linear peptides of the present invention modeled after residues 58-63 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO:33 (Phe-Ala-Asp-Val-Ser-Thr-amide), SEQ ID NO:34 (Ala- Ala- Asp- Val-Ser-Thr-amide), SEQ ID NO: 35 (Phe- Ala-Ala- Val-Ser-Thr- amide), SEQ ID NO:36 (Phe-Ala-Asp-Ala-Ser-Thr-amide), SEQ ID NO:37 (Phe-Ala-Asp-Val-Ala-Thr-amide), SEQ ID NO:38 (Phe-Ala-Asp-Val-Ser- Ala-amide), SEQ ID NO:39 (Phe-Ala-Asp-Val-Ser-amide), SEQ ID NO:40
(Ala-Asp-Val-Ser-amide), SEQ ID NO:41 (Phe- Ala-Asp- Val-amide), SEQ ID NO:42 (Phe-Ala-Asp-Val-Ser-Thr-OH), SEQ ID NO:43 (Phe-Ala-Asp-Val- Ser-Ala-OH), SEQ ID NO:44 (Phe-Ala-Asp-Val-Ser-OH), or SEQ ID NO:45 (Ala-Asp-Val-Ser-OH).
Especially preferred linear peptides of the present invention modeled after residues 115-123 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO: 46 (Asn-Lys-Glu-Lys-Thr-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:47 (Asn-Lys-Glu-Lys-Thr-amide), SEQ ID NO:48 (Lys-Thr- Thr- Ala-Glu- Ala-amide), SEQ ID NO:49 (Ala-Lys-Glu-Lys-Thr- Thr- Ala-
Glu-Tyr-amide), SEQ ID NO: 50 (Asn-Ala-Glu-Lys-Thr-Thr-Ala-Glu-Tyr- amide), SEQ ID NO: 51 (Asn-Lys-Ala-Lys-Thr-Thr-Ala-Glu-Tyr -amide), SEQ ID NO: 52 (Asn-Lys-Glu-Ala-Thr-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:53 (Asn-Lys-Glu-Lys-Ala-Thr-Ala-Glu-Tyr-amide), SEQ ID NO:54 (Asn-Lys-Glu-Lys-Thr-Ala-Ala-Glu-Tyr-amide), SEQ ID NO:55 (Asn-Lys-
Glu-Lys-Thr-Thr-Ala-Ala-Tyr-amide), SEQ ID NO:56 (Glu-Lys-Thr-Ala- Ala-Glu-amide), SEQ ID NO:57 (Glu-Lys-Thr-Thr-Ala-Glu-amide), SEQ ID NO:58 (Lys-Lys-Thr-Ala-Ala-Glu-amide), SEQ ID NO:59 (Gln-Lys-Thr- Ala-Ala-Glu-amide), SEQ ID NO:60 (Ala-Lys-Thr- Ala-Ala-Glu-amide), SEQ ID NO:61 (Thr- Ala-Ala-Glu-amide), SEQ ID NO:62 (Lys-Thr-Ala-Ala-Glu- amide), SEQ ID NO:63 (Ala-Lys-Thr- Ala-amide), SEQ ID NO:64 (Glu-Ala- Thr-Thr-Ala-Glu-amide), SEQ ID NO:65 (Glu-Lys-Ala-Thr-Ala-Glu-amide), SEQ ID NO:66 (Glu-Lys-Thr-Thr-Ala-Ala-amide), SEQ ID NO:67 (Glu- Lys-Thr-Thr-Ala-Glu-OH), SEQ ID NO: 68 (Lys-Lys-Thr-Ala-Ala-Glu-OH), SEQ ID NO:69 (Ala-Lys-Thr- Ala-Ala-Glu-OH), SEQ ID NO: 70 (Gln-Lys-
Thr-Ala-Ala-Glu-OH), SEQ ID NO:71 (Glu-Lys-Thr-Ala-Ala-Glu-OH), SEQ ID NO:72 (Thr-Ala-Ala-Glu-OH), SEQ ID NO:73 (Lys-Thr-Ala-Ala-Glu- OH), SEQ ID NO:74 (Ala-Lys-Thr-Ala-OH), or SEQ ID NO:75 (Lys-Thr- Thr-Ala-Glu-Ala-OH).
Especially preferred linear peptides of the present invention modeled after residues 212-227 of SEQ ID NO:l have the amino acid residue sequence of SEQ ID NO:76 (Ser-Gly-Ile-His-Met-Gln-amide), SEQ ID NO:77 (Ala-Gly-Ile-His-Met-Gln-amide), SEQ ID NO:78 (Ser-Ala-Ile-His- Met-Gln-amide), SEQ ID NO:79 (Ser-Gly-Ala-His-Met-Gln-amide), SEQ ID NO:80 (Ser-Gly-Ile-Ala-Met-Gln-amide), SEQ ID NO:81 (Ser-Gly-Ile-His- Ala-Gln-amide), SEQ ID NO:82 (Ser-Gly-Ile-His-Met-Ala-amide), SEQ ID
NO:83 (Ser-Gly-Ile-His-amide), SEQ ID NO:84 (Ser-Gly-Ile-His-Met- amide), SEQ ID NO:85 (Gly-Ile-His-amide), SEQ ID NO:86 (Ser-Gly-Ile- His-Met-Gln-Thr-Ser-Glu-Ser-amide), SEQ ID NO: 87 (Ser-Gly-Ile-His-Met- OH), SEQ ID NO:88 (Ala-Gly-Ile-His-Met-Gln-OH), SEQ ID NO:89 (Ser- Gly-Ile-Ala-Met-Gln-OH), SEQ ID NO:90 (Ser-Gly-Ile-His-Ala-Gln-OH), or
SEQ ID NO:91 (Ser-Gly-Ile-His-OH).
B. Cyclic Peptides
A cyclic peptide can be cyclized without or with a sulfur containing bridge. Where a cyclic peptide does not comprise a sulfur containing bridge, the N- and C-terminal amino acid residues are joined together with an amide bond (formally a lactam in the case of cyclization) . Where a cyclic peptide comprises a sulfur containing bridge, either one or two amino acid residues of the corresponding linear peptide is a Cys or modified Cys residue (dCys or dPen). Such a cyclic peptide can comprise a cyclic sulfide, sulfoxide or sulfone (one Cys residue in the corresponding linear peptide) or a cyclic disulfide (two Cys residues in the corresponding linear peptide).
Where a peptide of the present invention contains a sulfide, sulfoxide or sulfone bridge, that peptide comprises a cysteine or modified cysteine residue at one position and a -CH2CO- group at the N-terminal position. As used herein, the term "modified cysteine" refers to D-cysteine (dCys) or D- penicillamine (dPen). The sulfur atom of the cysteine or modified cysteine residue is attached to the CH2 group forming the cyclic peptide.
Where a peptide of the present invention contains a disulfide bridge, that peptide contains two cysteine or modified cysteine residues. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C- terminal position and at least one of those amino acid residues is Cys.
A cyclic peptide of the present invention can be made using standard peptide synthetic procedures well known in the art. Typically, peptides were made with Fmoc-amino acids. However, peptides can also be made using Boc protecting groups by methods well known to those skilled in the art. Side chain protecting groups of trifunction amino acids used in the synthetic procedure include Arginine (Pmc), Aspartic acid (tBu), Cysteine (Trt), Glutamic acid (tBu), Histidine (Boc), Lysine (Boc), Serine (tBu), Threonine
(tBu), and Tyrosine (tBu). Other protecting groups are specifically described.
The preparation of the peptides in this invention by solid phase methodology is well known to those skilled in the art, and can be described as follows. Peptides were synthesized on an insoluble carrier such as p- benzyloxybenzyl alcohol resin for the synthesis of C-terminal carboxylic acid peptides (Wang resin, where normally the resin can be purchased with the first amino acid bound), and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy resin for C-terminal amide peptides (Rink resin). The peptides were prepared by solid phase synthesis using either HBTU or DIC chemistry procedures on a Protein Technologies Inc. Symphony peptide synthesizer.
The C-terminal amide peptides are prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the same general method as the other couplings. The C-terminal carboxylic acid peptides are prepared by purchasing Wang resin to which the C-terminal amino acid was bound to the resin as a carboxylic ester. The α-amino protecting group is removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by simultaneous treatment of the resin with the Fmoc-amino acid, a coupling reagent such as DIC or HBTU, and if necessary HOBT. Such deprotection and couplings are repeated to afford each desired peptide. In all cases the
Fmoc protecting group is removed by treatment with a 20% solution of piperidine in DMF. However, it is understood by those skilled in the art that the exact percentage of piperidine is not critical and should not be considered limiting in this invention.
It is also understood by those skilled in the art that piperidine can be replaced by other bases, furthermore the coupling reagents and protocols used can be substimted with any of those known in the field of peptide synthesis (including the use of Boc chemistry based solid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural amino acids, D-amino acids and other compounds are coupled by manual addition of the reagent, following the same procedure as for automated operation.
Where cyclic sulfides, sulfoxides, sulfones or disulfides with C-terminal carboxylic acids are desired, the peptides can be synthesized on an insoluble carrier such as p-benzyloxybenzyl alcohol resin (Wang resin), whereas the equivalent C-terminal amides were prepared on 4-(2',4'-dimethoxyphenyl- Fmoc-aminomethyl)-phenoxy resin (Rink resin). Where two cysteines are present, mild acid removal of the trityl protecting groups and oxidative cyclization on the resin using DMSO or NIS forms the disulfide bond, and this compound was cleaved from the resin in the normal way. Alternatively disulfides can be prepared by solution phase cyclization of the linear sequence in guanidine hydrochloride. Where cyclic sulfides (and their oxidation products) are desired the N-terminus can be acylated with bromoacetic acid, the cysteine trityl group removed and cyclization achieved by NMM in DMF treatment. The head-tail lactams were synthesized on a chlorotrityl resin which forms a carboxylic ester linkage between the C- terminal amino acid and the resin. The linear peptide was cleaved from the resin with acetic acid in DCM and cyclized in solution to form the lactam.
Peptides are cleaved from the resin with a TFA cocktail after the removal of the N-terminal Fmoc protecting group. The exact composition of the TFA cocktail is varied depending on the side chain protecting groups present, and is well known to those skilled in the art. The range of TFA is typically from 85 to 95 % , and the remainder comprised of a mixture of scavengers selected from a combination of anisole, thioanisole, cresol, thiocresol, phenol, thiophenol, EDT, trimethylsilane and water. The time of the cleavage reaction required is sequence dependant, normally being from 1 to 3 hours. After cleavage, the resin is removed by filtration and cold ether added to the solution to give a precipitate. The precipitate is collected and washed a few times with ether to remove residual TFA and scavengers. The precipitate is redissolved in aqueous solution for lyophilization to give the crude product.
Purification is typically carried out by reverse-phase HPLC on a Clg- preparative column (30θA, 21.4 mm x 25 cm, 5 μm spherical packing) at a flow rate of 10 ml/min. The selection of any other suitable packing known to one skilled in the art is equally acceptable. Products are detected by UV absorption at 214 nm. Two mobile phases are used in the HPLC system, solution A and B using a gradient elution. Solution A is comprised of 5% acetonitrile in deionized water containing 0.15% TFA, while solution B is comprised of 5% deionized water in acetonitrile containing 0.1 % of TFA. A gradient of increasing percentage of solution B is used to elute the peptide from the solid support, however the gradient used is sequence dependant. Other methods of purification known to one skilled in the art are equally acceptable. The purity of the peptide is checked by Clg analytical HPLC (300 A, 4.6 mm x 25 cm, 5 μm spherical packing) at a flow rate of 1 ml/min.
Especially preferred cyclic peptides of the present invention modeled after residues 58-63 of SEQ ID NO: l have the amino acid resiue sequence of
SEQ ID NO:92 (cyclic Phe-Ala-Asp-Val-Ser), SEQ ID NO:93 (cyclic Phe- Ala-Asp-Val-Ser-Thr), or SEQ ID NO: 94 (Cys-Phe- Ala-Asp- Val-Cys- Amide, cyclic disulfide).
Especially preferred cyclic peptides of the present invention modeled after residues 115-123 of SEQ ID NO: l have the amino acid resiue sequence of SEQ ID NO:95 (cyclic Thr-Ala-Ala-Glu), SEQ ID NO:96 (cyclic Ala- Lys-Thr- Ala- Ala-Glu), SEQ ID NO:97 (cyclic Glu-Lys-Thr-Thr- Ala-Glu), SEQ ID NO: 98 (cyclic Lys-Thr- Ala- Ala-Glu), SEQ ID NO: 99 (cyclic Ala- Lys-Thr- Ala), SEQ ID NO: 100 (cyclic Glu-Lys-Thr- Ala- Ala-Glu), or SEQ
ID NO: 101 (Cys-Lys-Thr-Thr-Ala-Glu-Cys-amide, cyclic disulfide).
Especially preferred cyclic peptides of the present invention modeled after residues 212-227 of SEQ ID NO: l have the amino acid residue sequence of SEQ ID NO: 102 (cyclic Ser-Gly-Ile-His-Met-Gln), SEQ ID
NO: 103 (cyclic Ser-Gly-Ile-His-Met), SEQ ID NO: 104 (cyclic Ser-Gly-Ile- His) or SEQ ID NO: 105 (cyclic Gly-Ile-His).
II. Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition comprising a peptide of the present invention and a physiologically tolerable diluent. The present invention includes one or more peptides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral administration in solid or liquid form, for rectal or topical admimstration, or the like.
The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol. The compositions can also be delivered through a catheter for local delivery at a target site, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer. The compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions. The compositions are preferably administered by catheter, i.v. or subcutaneous injection, or intranasally via a spray or aerosol.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absoφtion of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents.
Suspensions, in addition to the active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Dosage forms for topical admimstration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
III. Process of Inhibiting the Binding of PECAM-l
In another aspect, the present invention contemplates a process of selectively inhibiting the binding of PECAM-l to itself (i.e. , PECAM-l to PECAM-l). A process of the present invention can be used in vitro or in vivo in a living organism. In accordance with a process of the present invention, a first cell expressing PECAM-l is exposed to a second cell expressing PECAM-l in the presence of an effective inhibiting amount of a peptide of the present invention. Means for determining an effective inhibiting amount are well known in the art.
A preferred linear peptide of the present invention for use in the process has the sequence of any of SEQ ID NOs: 33, 42-46, 49-55, 57, 64-76, 87- 91. More preferably, a useful linear peptide has the sequence of any of SEQ ID NOs: 42, 46, 49, 50, 52-55, 67-76, 87,88. Most preferably, a useful linear peptide has the sequence of any of SEQ ID NOs: 67-72. A preferred cyclic peptide of the present invention for use in the process has the sequence of any of SEQ ID NOs: 92-98, 100-103. More preferably, a useful cyclic peptide has the sequence of any of SEQ ID NOs: 92, 93, 95- 98, 100, 102, 103. Most preferably, a useful cyclic peptide has the sequence of any of SEQ ID NOs: 93, 95-97, 102,103.
A cell expressing PECAM-l can be a naturally occurring white blood cell, platelet, endothelial cell, mast cell or other cell type that naturally expresses PECAM-l on the cell surface, or a cell transfected with an expression vector that contains a polynucleotide (e.g. , genomic DNA or cDNA) that encodes PECAM-l . In an especially preferred embodiment, PECAM-l is present on the surface of a vascular endothelial cell or white blood cell such as a monocyte, a lymphocyte or a granulocyte (e.g. , an eosinophil or a basophil). Where the cells expressing PECAM-l are in a living organism, a peptide is administered in an effective amount to the living organism. Preferably, the peptide is in a pharmaceutical composition of this invention. Administering is preferably accomplished via intravascular injection or intranasal administration. The ability of peptides of the present invention to inhibit binding are described in detail hereinafter in the Examples.
A process of the present invention is especially useful in treating diseases associated with uncontrolled migration of white blood cells to damaged tissue. Such diseases include, but are not limited to, asthma, atherosclerosis, rheumatoid arthritis, allergy, multiple sclerosis, leukemia, and cancer. A process of inhibiting PECAM-l binding uses a peptide of the present invention as set forth hereinbefore. Preferred such peptides are the same as set forth above.
The present invention also provides a process of selectively inhibiting the adhesion of a cell that expresses PECAM-l to another cell that expresses PECAM-l . In accordance with that process, at least one the cells expressing PECAM-l is exposed to the other cell in the presence of an effective inhibiting amount of a peptide of the present invention. In a preferred embodiment, at least one of the cells expressing PECAM-l is a vascular endothelial cell. Preferred peptides for use in such a process are the same as set forth above.
The following Examples illustrate particular embodiments of the present invention and are not limiting of the specification and claims in any way .
EXAMPLES
EXAMPLE 1: Synthesis of Phe-Ala-Asp-Val-Ser-Thr-NH, (SEO ID NO:33) The Fmoc-amino acids and an equimolar amount of HBTU were dissolved in DMF. DIC in DCM was used as the coupling reagent. The Fmoc-Wang resin was swollen by treatment with DMF for 15-20 min, then deprotected by treatment with 20% piperidine in DMF and the resin was washed with DMF. The first amide bond was formed using a 1-1.2 hour coupling time. The resin was washed with DMF, and this procedure was repeated for each amino acid until the peptide was complete. The N- terminal Fmoc group was deprotected with piperidine and washed with
DMF, and then DCM. After deprotection of the N-terminal Fmoc group, DMF containing 20% acetic anhydride was added to the resin and stirred for 0.5 hours at room temperature and the peptide subsequently cleaved with TFA cocktail (1.5 ml containing 4% thioansole, 4% thiophenol and 4% EDT) for 1 hour. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization (9.1 mg., > 99% pure by HPLC). EXAMPLE 2: Synthesis of Asn-Lvs-Glu-Lvs-Thr-Thr-Ala-Glu-Tyr-NH2 (SEO ID NO.46)
The Fmoc-amino acids and an equimolar amount of HBTU were dissolved in DMF. DIC in DCM was used as the coupling reagent. The Fmoc-Wang resin was swollen by treatment with DMF for 15-20 min, then deprotected by treatment with 20% piperidine in DMF, and the resin was washed with DMF. The first amide bond was formed using a 1-1.2 hour coupling time. The resin was washed with DMF, and this procedure was repeated for each amino acid until the peptide was complete. The N- terminal Fmoc group was deprotected with piperidine and washed with
DMF, and then DCM. After deprotection of the N-terminal Fmoc group, DMF containing 20% acetic anhydride was added to the resin and stirred for 0.5 hours at room temperature and the peptide subsequently cleaved with TFA cocktail (1.5 ml containing 4% thioansole, 4% thiophenol and 4% EDT) for 1 hour. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization (9.1 mg., > 99% pure by HPLC).
EXAMPLE 3: Synthesis of Ser-Glv-Ile-His-Met-Gln-NH2 (SEO ID
NO:76)
The Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF. DIC in DCM was used as the coupling reagent. The
Fmoc-Rink resin (25 nM) was swollen by treatment with DMF (1.25 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3 x
8 min each), and the resin was washed with DMF (6x). The first amide bond was formed using Fmoc-Thr and a 1-1.2 hour coupling time. The resin was washed with DMF (3x), and this procedure was repeated until for each amino acid. The N-terminal Fmoc group was deprotected with piperidine (3x, 8 minutes each) and washed with DMF (6x), and then DCM
(6x). After deprotection of the N-terminal Fmoc group, DMF containing 20% acetic anhydride (3 ml) was added to the resin and stirred for 0.5 hours at room temperature and the peptide subsequently cleaved with TFA cocktail (1.5 ml containing 4% thioansole, 4% thiophenol and 4% EDT) for 1 hour. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization (9.1 mg. , > 99% pure by HPLC).
EXAMPLE 4: Synthesis of SEO ID NOs: 34-45. 47-75 and 77-91
These peptides were prepared using the same general procedure as in EXAMPLES 1-3 for the couplings except using different Fmoc-residue Wang resins and forming the first amide bond with different Fmoc-residues.
EXAMPLE 5: Synthesis of Cyclic Phe-Ala-Asp-Val-Ser (SEO ID NO: 92) The Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF. DIC in DCM was used as the coupling reagent with 1- 1.2 hour reaction times. The Fmoc-Ser Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3x, 8 min each), and the resin was washed with DMF (6x). The first amide bond was formed using Fmoc- Val (150 nM) and DIC (150 nM), and this procedure was repeated until all amino acids were coupled. After deprotection of the N-terminal protecting group, the peptide was cleaved from the resin with a TFA cocktail (containing 5 % anisole and 5% EDT) for 1 hour at room temperature. The TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether. After washing with ether (3x), the peptide was lyophilized from aqueous solution to give the crude linear peptide. Cyclization was achieved by dissolving in water (20 ml) containing guanidine»HCl (2.0 g) and ammonium acetate (1.6 g) at pH 7.8, and the solution stirred at 4°C for 48 hours and then lyophilized. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization. EXAMPLE 6: Synthesis of Cyclic Phe- Ala-Asp- Val-Ser-Thr (SEO ID NO:93)
This peptide was prepared using the same general proceduie as in
EXAMPLE 1 for the couplings except using Fmoc-Thr Wang resin and forming the first amide bond with Fmoc-Ser. Cyclization, isolation and purification were carried out as in Example 1.
EXAMPLE 7: Synthesis of Cyclic Ala-Lvs-Thr-Ala-Ala-Glu (SEO ID NO:96) The Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF. DIC in DCM was used as the coupling reagent with 1- 1.2 hour reaction times. The Fmoc-Glu Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3x, 8 min each), and the resin was washed with DMF (6x). The first amide bond was formed using Fmoc- Ala (150 nM) and DIC (150 nM), and this procedure was repeated until all amino acids were coupled. After deprotection of the N-terminal protecting group, the peptide was cleaved from the resin with a TFA cocktail (containing 5 % anisole and 5% EDT) for 1 hour at room temperamre. The TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether.
After washing with ether (3x), the peptide was lyophilized from aqueous solution to give the crude linear peptide. Cyclization was achieved by dissolving in water (20 ml) containing guanidine*HCl (2.0 g) and ammonium acetate (1.6 g) at pH 7.8, and the solution stirred at 4°C for 48 hours and then lyophilized. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization.
EXAMPLE 8: Synthesis of Cyclic Thr-Ala-Ala-Glu (SEO ID NO:95). Cyclic Glu-Lvs-Thr-Thr- Ala-Glu (SEO ID NO: 97). Cyclic
Lvs-Thr-Ala-Ala-Glu (SEO ID NO:98) and Cyclic Ala- Lvs-Thr-Ala (SEO ID NO:99):
These peptides were prepared using the same general procedure as in
EXAMPLE 1 for the couplings except using different Fmoc-(residue) Wang resins and forming the first amide bond with differing Fmoc-residues. Cyclization, isolation and purification were carried out as in Example 1.
EXAMPLE 9: Synthesis of Cyclic Ser-Glv-Ile-His-Met-Gln (SEO ID NO: 102)
The Fmoc-amino acids and an equimolar amount of HOBT were dissolved in DMF. DIC in DCM was used as the coupling reagent with 1-
1.2 hour reaction times. The Fmoc-Gln Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then deprotected by treatment with 20% piperidine in DMF (3x, 8 min each), and the resin was washed with DMF (6x). The first amide bond was formed using Fmoc-Met (150 nM) and DIC (150 nM), and this procedure was repeated until all amino acids were coupled. After deprotection of the N-terminal protecting group, the peptide was cleaved from the resin with a TFA cocktail (containing 5 % anisole and 5% EDT) for 1 hour at room temperature. The TFA solution was reduced to about 0.5 ml and the product precipitated with cold ether. After washing with ether (3x), the peptide was lyophilized from aqueous solution to give the crude linear peptide. Cyclization was achieved by dissolving in water (20 ml) containing guanidine#HCl (2.0 g) and ammonium acetate (1.6 g) at pH 7.8, and the solution stirred at 4°C for 48 hours and then lyophilized. Purification was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyophilization.
EXAMPLE 10: Synthesis of Cyclic Ser-Glv-He-His-Met (SEO ID
NO: 103). Cyclic Ser-Glv-Ile-His (SEO ID NO: 104). Cyclic Glv-Ile-His (SEO ID NO: 105)
This peptide was prepared using the same general procedure as in
EXAMPLE 1 for the couplings except using different Fmoc-residue Wang resins and forming the first amide bond with different Fmoc-residues.
Cyclization, isolation and purification were carried out as in Example 1. EXAMPLE 11: Binding Assays
Peptides were assayed for their ability to inhibit the binding of the PECAM-l to itself. The assay is described below.
Cell-Protein Assay
PECAM-l was expressed in recombinant form as a fusion protein possessing all six immunoglobulin domains fused to the hinge and constant heavy chain regions 1 and 2 of the mouse IgG2A cDNA. The fusion cassette was generated by polymerase chain reaction (PCR) from the PECAM-l cDNA that was PCR cloned from total RNA extracted from human placenta. The mouse IgG cDNA was cloned from PCR amplified cDNA generated from RNA extracted from the hybridoma cell line 402C10. All fusion cassettes were expressed frombaculovirus vectors using the BakPAK method and SF21 cells purchased from Clonetech.
Recombinant fusion protein was purified from baculovirus infected culture supernatants by immunoprecipitation using Dynal ™ goat anti-mouse IgG coated magnetic beads. Mock beads were generated from uninfected SF21 culture supernatants. Following immunoprecipitation, beads incubated with mock culture supernatants did not bind HL60 cells that express PECAM-l served as controls. Beads incubated from PECAM-l culture supernatants did bind HL60 cells.
HL-60 cells were fluorescently labeled with Calcein AM C-3099
(Molecular Probes) in RPMI 1640 with 10% fetal calf serum (FCS). The magnetic beads (7 μl, 8xl06 beads/ml) were placed in wells of a 96-well microtiter dish with 7 μl of peptide at various concentrations and 7 μl of calcein-labeled HL60 cells. The plates were incubated for about 25 minutes at about 37 °C. The plate was then placed on a magnetic separatoe and incubated for an additional 2 minutes. While the assay plate remained on the separator, excess, unbound HL60 cells were removed and the wells washed twice with phosphate buffered saline (PBS) to remove any remaining unbound cells. The HL60 cells remaining bound to the beads were inspected by microscopy and then lysed by adding about 50 μl of a 1.0% solution of NP-40 in PBS. Binding was quantified by fluorimetry using a Millipore Cytofluor 2350 fluorimeter. Dose response curves were calculated and IC50 values determined.
Peptides having the amino acid residue sequence of SEQ ID NOs: 33- 105 were found to significantly inhibit the binding of PECAM-l to itself with an IC50 values ranging from 0.1 μM to 150 μM.
EXAMPLE: 12 IN VIVO EFFICACY: ANIMAL MODEL
1. ACUTE INFLAMMATION : Mouse Peritoneal Exudate Model
Six to eight week old female BALB/c mice weighing approximately 25 gm were injected intraperitoneally at time zero with 1ml of a 2% solution of oyster glycogen (Type II, Sigma Chemical Co.) in Dulbecco's phosphate buffered saline (PBS) (Gibco/BRL). Control animals received 1ml of PBS alone. Each dose of 0.1 ml of solution was administered in NS at neutral pH of the compound of interest (SEQ ID NO: 97) was injected subcutaneously at: -15 minutes, time 0, + 15 minutes, +45 minutes, and +75 minutes. Mice were sacrificed at 180 minutes and a 10ml peritoneal lavage composed of PBS, EDTA, BSA, and gelatin was performed. The total number of cells in the exudate was determined using a hemocytometer and the number of polymorphonuclear leukocytes (PMN's) determined using cytospin preparations treated with Wright-Giemsa stain.
Treatment of animals with therapeutic levels of Seq ID No. 97 reduced total cell and neutrophil migration to the inflammatory stimulus and this reduction was statistically significant (p,0.02).
The foregoing Examples illustrate particular embodiments of the present invention. One of ordinary skill in the art will readily appreciate that changes, modification and alterations can be made in those embodiments without departing from true scope and spirit of the invention.
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPUCANT: Beck, Pam Kint, Phi Nga Revelle, B. Mitch Ren, Kai Bjercke, Robert Sherwood, Sid
(ii) TITLE OF INVENTION: PEPTIDES AND METHODS FOR
INHIBITING THE BINDING OF PECAM
(iii) NUMBER OF SEQUENCES: 105
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DRESSLER, GOLDSMITH, MILNAMOW
& KATZ, LTD.
(B) STREET: 180 N. STETSON, SUITE 4700
(C) CITY: CHICAGO
(D) STATE: ILLINOIS
(E) COUNTRY: U.S.A.
(F) ZIP: 60601
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: NORTHRUP, THOMAS E.
(B) REGISTRATION NUMBER: 33,268
(C) REFERENCE/DOCKET NUMBER: TEX4542P0280US
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (312) 546-5400
(B) TELEFAX: (312) 546-5460
(2) INFORMATION FOR SEQ ID NO: l: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 738 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 1..27
(D) OTHER INFORMAΗON: /label = Signal
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 28..738
(D) OTHER INFORMATION: /label = Mature peptide
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 28..601
(D) OTHER INFORMATION: /label = Extracellular
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 602..620
(D) OTHER INFORMATION: /label = Potential
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 621..738
(D) OTHER INFORMATION: /label = Cytoplasmic-pot
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 28..144
(D) OTHER INFORMATION: /label = Ig-like
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 145..248
(D) OTHER INFORMATION: /label = Ig-like
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 249..339
(D) OTHER INFORMATION: /label = Ig-like (ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 340..423
(D) OTHER INFORMATION: /label = Ig-like
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 424..515
(D) OTHER INFORMATION: /label = Ig-like
(ix) FEATURE:
(A) NAME/KEY: Domain
(B) LOCATION: 516..601
(D) OTHER INFORMATION: /label = Ig-like
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l :
Met Gin Pro Arg Tφ Ala Gin Gly Ala Thr Met Tφ Leu Gly Val Leu 1 5 10 15
Leu Thi Leu Leu Leu Cys Ser Ser Leu Glu Gly Gin Glu Asn Ser Phe 20 25 30
Thr He Asn Ser Val Asp Met Lys Ser Leu Pro Asp Tφ Thr Val Gin
35 40 45
Asn Gly Lys Asn Leu Thr Leu Gin Cys Phe Ala Asp Val Ser Thr Thr 50 55 60
Ser His Val Lys Pro Gin His Gin Met Leu Phe Tyr Lys Asp Asp Val 65 70 75
Leu Phe Tyr Asn He Ser Ser Met Lys Ser Thr Glu Ser Tyr Phe He 80 85 90 95
Pro Glu Val Arg He Tyr Asp Ser Gly Thr Tyr Lys Cys Thr Val He 100 105 110
Val Asn Asn Lys Glu Lys Thr Thr Ala Glu Tyr Gin Leu Leu Val Glu 115 120 125
Gly Val Pro Ser Pro Arg Val Thr Leu Asp Lys Lys Glu Ala He Gin 130 135 140
Gly Gly He Val Arg Val Asn Cys Ser Val Pro Glu Glu Lys Ala Pro 145 150 155 160 He His Phe Thr He Glu Lys Leu Glu Leu Asn Glu Lys Met Val Lys 165 170 175
Leu Lys Arg Glu Lys Asn Ser Arg Asp Gin Asn Phe Val He Leu Glu 180 185 190
Phe Pro Val Glu Glu Gin Asp Arg Val Leu Ser Phe Arg Cys Gin Ala 195 200 205
Arg He He Ser Gly He His Met Gin Thr Ser Glu Ser Thr Lys Ser 210 215 220
Glu Leu Val Thr Val Thr Glu Ser Phe Ser Thr Pro Lys Phe His He
225 230 235 240
Ser Pro Thr Gly Met He Met Glu Gly Ala Gin Leu His He Lys Cys
245 250 255
Thr He Gin Val Thr His Leu Ala Gin Glu Phe Pro Glu He He He
260 265 270
Gin Lys Asp Lys Ala He Val Ala His Asn Arg His Gly Asn Lys Ala
275 280 285
Val Tyr Ser Val Met Ala Met Val Glu His Ser Gly Asn Tyr Thr Cys 290 295 300
Lys Val Glu Ser Ser Arg He Ser Lys Val Ser Ser He Val Val Asn 305 310 315 320
He Thr Glu Leu Phe Ser Lys Pro Glu Leu Glu Ser Ser Phe Thr His
325 330 335
Leu Asp Gin Gly Glu Arg Leu Asn Leu Ser Cys Ser He Pro Gly Ala
340 345 350
Pro Pro Ala Asn Phe Thr He Gin Lys Glu Asp Thr He Val Ser Gin 355 360 365
Thr Gin Asp Phe Thr Lys He Ala Ser Lys Ser Asp Ser Gly Thr Tyr
370 375 380
He Cys Thr Ala Gly He Asp Lys Val Val Lys Lys Ser Asn Thr Val 385 390 395 400
Gin He Val Val Cys Glu Met Leu Ser Gin Pro Arg He Ser Tyr Asp 405 410 415 Ala Gin Phe Glu Val He Lys Gly Gin Thr He Glu Val Arg Cys Glu
420 425 430
Ser He Ser Gly Thr Leu Pro He Ser Tyr Gin Leu Leu Lys Thr Ser
435 440 445
Lys Val Leu Glu Asn Ser Thr Lys Asn Ser Asn Asp Pro Ala Val Phe 450 455 460
Lys Asp Asn Pro Thr Glu Asp Val Glu Tyr Gin Cys Val Ala Asp Asn 465 470 475 480
Cys His Ser His Ala Lys Met Leu Ser Glu Val Leu Arg Val Lys Val 485 490 495
He Ala Pro Val Asp Glu Val Gin He Ser He Leu Ser Ser Lys Val 500 505 510
Val Glu Ser Gly Glu Asp He Val Leu Gin Cys Ala Val Asn Glu Gly
515 520 525
Ser Gly Pro He Thr Tyr Lys Phe Tyr Arg Glu Lys Glu Gly Lys Pro 530 535 540
Phe Tyr Gin Met Thr Ser Asn Ala Thr Gin Ala Phe Tφ Thr Lys Gin 545 550 555 560
Lys Ala Ser Lys Glu Gin Glu Gly Glu Tyr Tyr Cys Thr Ala Phe Asn 565 570 575
Arg Ala Asn His Ala Ser Ser Val Pro Arg Ser Lys He Leu Thr Val 580 585 590
Arg Val He Leu Ala Pro Tφ Lys Lys Gly Leu He Ala Val Val He 595 600 605
He Gly Val He He Ala Leu Leu He He Ala Ala Lys Cys Tyr Phe 610 615 620
Leu Arg Lys Ala Lys Ala Lys Gin Met Pro Val Glu Met Ser Arg Pro 625 630 635 640
Ala Val Pro Leu Leu Asn Ser Asn Asn Glu Lys Met Ser Asp Pro Asn 645 650 655
Met Glu Ala Asn Ser His Tyr Gly His Asn Asp Asp Val Arg Asn His 660 665 670 Ala Met Lys Pro He Asn Asp Asn Lys Glu Pro Leu Asn Ser Asp Val 675 680 685
Gin Tyr Thr Glu Val Gin Val Ser Ser Ala Glu Ser His Lys Asp Leu 690 695 700
Gly Lys Lys Asp Thr Glu Thr Val Tyr Ser Glu Val Arg Lys Ala Val
705 710 715 720
Pro Asp Ala Val Glu Ser Arg Tyr Ser Arg Thr Glu Gly Ser Leu Asp
725 730 735
Gly Thr
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Phe Ala Asp Val 1
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Ala Asp Val 1
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 2
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Phe Xaa Asp Val 1
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Phe Ala Xaa Val 1 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Ala Asp Xaa
1
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Phe Ala Asp Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 1
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Xaa Ala Asp Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 2
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Phe Xaa Asp Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 3
(D) OTHER INFORMATION, /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Phe Ala Xaa Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l l :
Phe Ala Asp Xaa Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Phe Ala Asp Val Xaa 1 5
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Asn Lys Glu Lys Thr Thr Ala Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 14:
• (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Xaa Lys Glu Lys Thr Thr Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 2
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Asn Xaa Glu Lys Thr Thr Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Asn Lys Xaa Lys Thr Thr Ala Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Asn Lys Glu Xaa Thr Thr Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asn Lys Glu Lys Xaa Thr Ala Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asn Lys Glu Lys Thr Xaa Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 7
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Asn Lys Glu Lys Thr Thr Xaa Glu 1 5
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 8
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = any L-α-amino acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Asn Lys Glu Lys Thr Thr Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ser Gly He His
1 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modif ied-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Xaa Gly He His 1
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ser Xaa He His 1
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Ser Gly Xaa His
1
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:26:
Ser Gly He Xaa
1
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Ser Gly He His Met
1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCAΗON: 1
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Xaa Gly He His Met 1 5
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMAΗON: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ser Xaa He His Met 1 5 (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Ser Gly Xaa His Met
1 5
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Ser Gly He Xaa Met 1 5
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa /note= "Xaa is any L-α-amino acid. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Ser Gly He His Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Phe Ala Asp Val Ser Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Ala Ala Asp Val Ser Xaa 1 5
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Phe Ala Ala Val Ser Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-NH2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Phe Ala Asp Ala Ser Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Phe Ala Asp Val Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ala-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Phe Ala Asp Val Ser Xaa 1 5
(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ser-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Phe Ala Asp Val Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ser-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Ala Asp Val Xaa 1
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Asp-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Phe Ala Asp Val NH2
1
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Phe Ala Asp Val Ser Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = Ala-OH (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Phe Ala Asp Val Ser Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ser-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Phe Ala Asp Val Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ser-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Ala Asp Val Xaa 1 (2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-amide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Asn-Lys-Glu-Lys-Thr-Thr-Ala-Glu-Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Thr-amide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Asn-Lys-Glu-Lys-Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ala-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Lys Thr Thr Ala Glu Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Ala Lys Glu Lys Thr Thr Ala Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Asn Ala Glu Lys Thr Thr Ala Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:51 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51 :
Asn Lys Ala Lys Thr Thr Ala Glu Xaa 1 5 10
(2) INFORMAΗON FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: Asn Lys Glu Ala Thr Thr Ala Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Asn Lys Glu Lys Ala Thr Ala Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION, /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Asn Lys Glu Lys Thr Ala Ala Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Tyr-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Asn Lys Glu Lys Thr Thr Ala Ala Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Glu Lys Thr Ala Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Glu Lys Thr Thr Ala Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Lys Lys Thr Ala Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Gin Lys Thr Ala Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Ala Lys Thr Ala Ala Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:61 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61 :
Thr Ala Ala Xaa 1 5
(2) INFORMAΗON FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Lys Thr Ala Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMAΗON: /label = Xaa
/note= Xaa = Ala-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Ala Lys Thr Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-amide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Glu-Ala-Thr-Thr-Ala-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-amide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
Glu-Lys-Ala-Thr-Ala-Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = Ala-amide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Glu-Lys-Thr-Thr-Ala-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Glu-Lys-Thr-Thr-Ala-Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Lys-Lys-Thr-Ala-Ala-Xaa
1 5 (2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
Ala-Lys-Thr-Ala-Ala-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Gln-Lys-Thr-Ala-Ala-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71 :
Glu-Lys-Thr-Ala-Ala-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Thr-Ala-Ala-Xaa 1
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Glu-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Lys-Thr-Ala-Ala-Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ala-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Ala-Lys-Thr-Xaa 1
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ala-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: Lys-Thr-Thr-Ala-Glu-Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
Ser Gly He His Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Ala Gly He His Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:78: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
Ser Ala He His Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note = Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Ser Gly Ala His Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
Ser Gly He Ala Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:81 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81 :
Ser Gly He His Ala Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ala-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Ser Gly He His Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = His-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Ser Gly He Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = Met-NH2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
Ser Gly He His Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 3
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = His-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
Gly He Xaa 1
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 10
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Ser-NH2
(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 86: Ser Gly He His Met Gin Thr Ser Glu Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Met-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Ser Gly He His Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCAΗON: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
Ala Gly He His Met Xaa 1 5 (2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Ser Gly He Ala Met Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = Gln-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
Ser Gly He His Ala Xaa 1 5
(2) INFORMATION FOR SEQ ID NO:91 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified site
(B) LOCATION: 4
(D) OTHER INFORMATION: /label = Xaa
/note= Xaa = His-OH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91 :
Ser Gly He Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..5
(D) OTHER INFORMATION:
/note= crosslink between Phe at position 1 and Ser at position 5.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Phe Ala Asp Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note = crosslink between Phe at position 1 and Thr at position 6.
(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 93:
Phe Ala Asp Val Ser Thr
1 5
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note = crosslink between Cys at position 1 and Cys at position 6.
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = Cys-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
Cys Phe Ala Asp Val Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:95: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY, cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..4
(D) OTHER INFORMATION:
/note= crosslink between Thr at position 1 and Glu at position 5.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Thr Ala Ala Glu
1
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note = crosslink between Ala at position 1 and Glu at position 6.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Ala Lys Thr Ala Ala Glu
1 5
(2) INFORMAΗON FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note= crosslink between Glu at position 1 and Glu at position 6.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.97:
Glu Lys Thr Thr Ala Glu
1 5
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..5
(D) OTHER INFORMATION:
/note= crosslink between Lys at position 1 and Glu at position 5.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
Lys Thr Ala Ala Glu 1 5
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1. 4
(D) OTHER INFORMATION:
/note= crosslink between Ala at position 1 and Ala at position 4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
Ala Lys Thr Ala 1
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note = crosslink between Glu at position 1 and
Glu at position 6.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Glu Lys Thr Ala Ala Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: (A) NAME/KEY: Modified-site
(B) LOCATION: 1..7
(D) OTHER INFORMATION:
/note= crosslink between Cys at position 1 and Cys at position 7.
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCAΗON: 7
(D) OTHER INFORMATION: /label = Xaa /note= Xaa = Cys-NH2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
Cys Lys Thr Thr Ala Glu Cys
1 5
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..6
(D) OTHER INFORMATION:
/note = crosslink between Ser at position 1 and Gin at position 5.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Ser Gly He His Met Gin
1 5
(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..5
(D) OTHER INFORMATION:
/note= crosslink between Ser at position 1 and Met at position 5.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Ser Gly He His Met 1 5
(2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..4
(D) OTHER INFORMATION:
/note= crosslink between Ser at position 1 and His at position 4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
Ser Gly He His 1
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: cyclic
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..3
(D) OTHER INFORMATION:
/note = crosslink between Gly at position 1 and His at position 3.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: Gly He His

Claims

WHAT IS CLAIMED IS:
1. An synthetic, isolated, and purified peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight linked through an amide bond to the N- terminal residue; and (b) a C-terminal carboxylic acid group or amide group; the peptide comprising a sequence that is identical to a contiguous stretch of at least four amino acid residues of a C2-type, IG domain of SEQ ID NO:l, or a single amino acid residue substiment thereof.
2. The peptide of claim 1 wherein the contiguous stretch of at least four amino acid residues has the sequence Phe-Ala-Asp-Val (SEQ ID NO:2), or a single amino acid residue substiment thereof.
3. The peptide of claim 2 wherein the single amino acid residue substiment has the sequence of any of Xaa4- Ala- Asp- Val (SEQ ID NO:3), Phe-Xaa'-Asp-Val (SEQ ID NO:4), Phe-Ala-Xaa2-Val (SEQ ID NO:5) or Phe-Ala-Asp-Xaa3 (SEQ ID NO:6), wherein Xaa1, Xaa2, Xaa3 and Xaa4 are any L-amino acid residue.
4. The peptide of claim 3 having the sequence Phe-Ala-Asp- Val-Ser (SEQ ID NO:7), or a single amino acid residue substiment thereof.
5. The peptide of claim 4 wherein the single amino acid residue substiment has the sequence of any of Xaa4- Ala-Asp- Val-Ser (SEQ ID NO:8), Phe-Xa^-Asp- Val-Ser (SEQ ID NO:9), Phe-Ala-Xaa2- Val-Ser (SEQ ID NO: 10), Phe-Ala-Asp-Xaa3-Ser (SEQ ID NO: 11) or Phe-Ala-Asp- Val-Xaa5 (SEQ ID NO: 12), wherein Xaa1, Xaa2, Xaa3, Xaa4 and Xaa5 are each independently any L-amino acid residue.
6. The peptide of claim 1 wherein the contiguous stretch of at least four amino acid residues has the sequence Asn-Lys-Glu-Lys-Thr-Thr- Ala-Glu (SEQ ID NO:13).
7. The peptide of claim 6 wherein the single amino acid residue substituent has the sequence of any of Xaa'-Lys-Glu-Lys-Thr-Thr- Ala-Glu (SEQ ID NO: 14), Asn-Xaa -Glu-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 15), Asn-Lys-Xaa3-Lys-Thr-Thr-Ala-Glu (SEQ ID NO: 16), Asn-Lys- Glu-Xaa4-Thr-Thr-Ala-Glu (SEQ ID NO: 17), Asn-Lys-Glu-Lys-Xaa5-Thr- Ala-Glu (SEQ ID NO: 18), Asn-Lys-Glu-Lys-Thr-Xaa6-Ala-Glu (SEQ ID NO: 19), Asn-Lys-Glu-Lys-Thr-Thr-Xaa7-Glu (SEQ ID NO:20) or Asn-Lys- Glu-Lys-Thr-Thr-Ala-Xaa8 (SEQ ID NO:21), wherein Xaa1"8 are each independently any L-amino acid residue.
8. The peptide of claim 7 wherein Xaa1 is Ala; Xaa2 is Ala; Xaa3 is Ala, Gin or Lys; Xaa4 is Ala; Xaa5 is Ala; Xaa6 is Ala; Xaa7 is Ala; and Xaa8 is Ala.
9. The peptide of claim 1 wherein the contiguous stretch of at least four amino acid residues has the amino acid residue sequence Ser- Gly-Ile-His (SEQ ID NO: 22), or a single amino acid residue substiment thereof.
10. The peptide of claim 9 wherein the single amino acid residue substiment has the sequence of any of Xaa'-Gly-He-His (SEQ ID NO:23), Ser-Xaa2-Ile-His (SEQ ID NO:24), Ser-Gly-Xaa3-His (SEQ ID NO:25) or Ser-Gly-Ile-Xaa4 (SEQ ID NO:26), wherein Xaa1, Xaa2, Xaa3 and Xaa4 are each independently any L-amino acid residue.
11. The peptide of claim 10 having the sequence Ser-Gly-Ile- His-Met (SEQ ID NO: 27), or a single amino acid residue substituent thereof.
12. The peptide of claim 11 wherein the single amino acid residue substiment has the sequence of any of Xaa'-Gly-Ile-His-Met (SEQ ID NO:28), Ser-Xaa2-Ile-His-Met (SEQ ID NO:29), Ser-Gly-Xaa3-His-Met (SEQ ID NO:30), Ser-Gly-Ile-Xaa4-Met (SEQ ID NO:31) or Ser-Gly-Ile-His- Xaa5 (SEQ ID NO:32), wherein Xaa1, Xaa2, Xaa3, and Xaa4 are as defmed in claim 1 and Xaa5 is any L-amino acid residue.
13. The peptide of any of claims 1-12 that is a linear peptide.
14. A linear peptide having the amino acid residue sequence of any of SEQ ID NOs:33-91.
15. The peptide of any of claims 1-12 that is a cyclic peptide.
16. A peptide having the amino acid residue sequence of any of SEQ ID NOs:92-105.
17. A pharmaceutical composition comprising a physiologically acceptable diluent and any of the peptides of claims 1-16.
18. A process of selectively inhibiting the binding of PECAM-l to PECAM-l comprising exposing a first cell that expresses PECAM-l to a second cell that expresses PECAM-l in the presence of an effective inhibiting amount of any of the peptides of claims 1-16.
19. The process of claim 18 wherein the first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell.
20. A process of selectively inhibiting the adhesion of a first cell that expresses PECAM-l to a second cell that expresses PECAM-l comprising exposing at least one of the first and second cells to an effective inhibiting amount of any of the peptides of claims 1-16, wherein the first and second cell are independently a white blood cell, a platelet, a mast cell or an endothelial cell.
21. The process of claim 20 wherein at least one of the first and second cells is a vascular endothelial cell.
PCT/US1996/014940 1995-09-19 1996-09-18 Compositions and methods for inhibiting the binding of pecam WO1997010839A1 (en)

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US399695P 1995-09-19 1995-09-19
US395395P 1995-09-19 1995-09-19
US60/003,953 1995-09-19
US60/003,996 1995-09-19
US60/003,985 1995-09-19
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Publication number Priority date Publication date Assignee Title
US5955443A (en) * 1998-03-19 1999-09-21 Isis Pharmaceuticals Inc. Antisense modulation of PECAM-1
JP2007254417A (en) * 2006-03-24 2007-10-04 Snow Brand Milk Prod Co Ltd Peptide
US7566765B2 (en) 2000-03-06 2009-07-28 Rigel Pharmaceuticals, Inc. Heterocyclic compounds containing a nine-membered carbon-nitrogen ring
US11613565B2 (en) * 2015-03-16 2023-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Isolated peptides derived from the B7 ligand dimer interface and uses thereof

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US5264554A (en) * 1990-01-19 1993-11-23 The Blood Center Of Southeastern Wisconsin, Inc. Platelet cell adhesion molecule and variants thereof
US5470831A (en) * 1990-12-21 1995-11-28 Curative Technologies, Inc. Angiogenic peptides
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US5264554A (en) * 1990-01-19 1993-11-23 The Blood Center Of Southeastern Wisconsin, Inc. Platelet cell adhesion molecule and variants thereof
US5470831A (en) * 1990-12-21 1995-11-28 Curative Technologies, Inc. Angiogenic peptides
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5955443A (en) * 1998-03-19 1999-09-21 Isis Pharmaceuticals Inc. Antisense modulation of PECAM-1
US7566765B2 (en) 2000-03-06 2009-07-28 Rigel Pharmaceuticals, Inc. Heterocyclic compounds containing a nine-membered carbon-nitrogen ring
JP2007254417A (en) * 2006-03-24 2007-10-04 Snow Brand Milk Prod Co Ltd Peptide
US11613565B2 (en) * 2015-03-16 2023-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Isolated peptides derived from the B7 ligand dimer interface and uses thereof

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