[go: up one dir, main page]

WO1992013887A1 - Nouveaux peptides d'adhesion cellulaire - Google Patents

Nouveaux peptides d'adhesion cellulaire Download PDF

Info

Publication number
WO1992013887A1
WO1992013887A1 PCT/GB1992/000226 GB9200226W WO9213887A1 WO 1992013887 A1 WO1992013887 A1 WO 1992013887A1 GB 9200226 W GB9200226 W GB 9200226W WO 9213887 A1 WO9213887 A1 WO 9213887A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
peptide
amino acid
cells
cell
Prior art date
Application number
PCT/GB1992/000226
Other languages
English (en)
Inventor
Martin James Humphries
Original Assignee
The Victoria University Of Manchester
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB919102655A external-priority patent/GB9102655D0/en
Priority claimed from GB919102818A external-priority patent/GB9102818D0/en
Application filed by The Victoria University Of Manchester filed Critical The Victoria University Of Manchester
Publication of WO1992013887A1 publication Critical patent/WO1992013887A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to new peptides, which have the properties of regulating or controlling cell adhesion.
  • cell adhesion occurs ubiquitously within the body and is central to many normal events, such as wound healing and cell migration during development. However, it also causes or contributes to many undesirable conditions or effects, for example the pathogenesis of many of the major diseases. These include thrombosis, inflammation, auto-immune diseases, malignant cancer and arthritis.
  • a cell adhesive peptide which may be a polypeptide, useful as a selective antagonist to the agents and processes of cell adhesion, including a novel adhesive signal, namely an amino acid sequence comprising X-Asp-Y-(A)n-Phe, where X and Y are chosen from a, Leu, lie, and Val, A is any amino acid and n is in the range 3 to 10, in which at least a sub-sequence of the said sequence is adherant for OLT-4 human lymphoblastic leukaemia, or A375-SM human metastatic melanoma or H1080 human fibrosarcoma cells.
  • the preferred sequence is that designated as "L-D-V” (leucine-aspartic acid-valine) and peptides containing this are able to promote MOLT-4, A375-SM or H1080 cell adhesion while inhibiting adhesion to the parent molecules, ie to the natural adhesion protein containing the adhesive sequence. By blocking cell receptors, the peptide inhibits natural adehsion proteins from binding to the cell.
  • Alternative useful sequences are those designated as L-D-L (leucine-aspartic acid-leucine) or I-D-A (isoleucine-aspartic acid-alanine) .
  • sequence of amino acid units in a peptide is normally written as starting from the unit with the "free N” (as the -NH- group) and running to that with the "free C” (as the -COOH group). This convention is followed and used throughout this specification for designating the sequences of amino acid units in the peptide compounds of the invention.
  • the peptide sequence containing a novel adhesive signal of the present invention in terms of the number of amino acid units and part of the sequence of such units within them.
  • the number of amino acid units may vary according to particular requirements, for example of manufacture and activity, but we prefer that the number of amino acid units should be in the range 15 to 40, and still more preferably in the range 20 to 30.
  • the number may be larger or smaller than these ranges, but these tend to be less useful and/or more difficult to make or use; building up a large peptide molecule is more expensive as the number of amino acid units increases, so the economic factor then becomes more important.
  • a convenient number of amino acid units is 19 (the compounds then being conveniently referred as as "19-mers”), but this number is not to be taken as the only number permissible.
  • amino acid sequences of importance are indicated by underlining for ease of recognition.
  • the specified amino acid unit sequence* may thus be seen as present as part of the structure of a larger polypeptide (i.e. "buried" within a polypeptide structure) having more amino acid units, but accessible to a cell receptor and thus able to bind thereto.
  • Other proteins and peptide sequences may occur in nature which contain the amino acid sequence of the present invention, however, this does not necessarily mean they are adhesion proteins/peptides and may not therefore be adherant for MOLT-4, A375-SM or H1080 cells.
  • the peptide sequences of the present invention may be made by conventional peptide-forming reactions and processes, well known in the art and used to build up the desired sequence from the individual component amino acids or appropriate combinations of them. For example, they may be made using automated synthesisers which are commercially available, or the peptide sequence may be synthesised using recombinant DNA technology.
  • Peptides will be cleaved from the resin by treatment with anhydrous hydrofluoric acid (t-moc) or by trifluoroacetic acid (f-boc) and purified by reverse-phase HPLC (high pressure liquid chromatography) .
  • t-moc anhydrous hydrofluoric acid
  • f-boc trifluoroacetic acid
  • HPLC reverse-phase HPLC
  • the compounds of the present invention may be made into pharmaceutical compositions by combining one or more of the compounds defined above with one or more pharmaceutically-acceptable excipients and/or diluents.
  • the compounds of the present invention may be administered intravenously or subcutaneously, or they may be incorporated into dressing, wash solutions and the like. Commonly, they may be solubilised or dispersed in normal saline.
  • peptide sequences of the present invention are useful as selective antagonists to the agents and processes of cell adhesion but may be effective in different ways and in different circumstances, depending for example upon such factors as the particular natural processes involved. - 3 -
  • they can be active in various leukocyte functions, for example by flooding the system with peptides to block adhesion protein receptors on cells, they aid in blocking leukocyte extravasation during inflammation, lymphocyte cell-cell recognition during responses to antigens and in inhibiting platelet aggrega ⁇ tion and fibrosis.
  • they can be used to alleviate conditions in which these mechanisms are involved, and particularly in inflammatory or coagulatory conditions.
  • they may be used for treatment of, for example, inflammatory lesions n vivo, rheumatoid arthritis, asthma, inflammatory bowel disease, sepsis, prevention of graft rejection, reperfusion of cardiac tissue after yocardial infarction and dermatoses, and the like.
  • the peptide sequences of the present invention are primarily useful for their effects on animal cells, but their application is not necessarily restricted to these and can be relevant for other types of biological cells according to the particular amino acid sequences required for optimum effect.
  • the mode of action is not fully understood, but is believed to be either through mediation of the adhesion and migration of various leukocyte subsets from the bloodstream into the inflamed tissue, or possibly by interference with the initial adhesion of leukocytes to the endothelial lining of the blood vessel and impairing the ability of the leukocytes to generate an inflammatory response.
  • Figure 1 shows attachment of MOLT-4 cells to peptides.
  • Figure 2 shows the effect of anti-integrin antibodies on peptide mediated MOLT-4 cell attachment and A375-SM cell spreading.
  • the first assay measures simple attachment of cells to the peptide which is first immobilised on the surface of plastic wells, and the second measures the ability of the cells to attach and then spread out and flatten on to a peptide immobilised in the same way.
  • attachment measures simple attachment of cells to the peptide which is first immobilised on the surface of plastic wells
  • spreading measures the ability of the cells to attach and then spread out and flatten on to a peptide immobilised in the same way.
  • the peptide is not used in free solution but is first immobilised onto a plastic substrate. However, the peptide is not coated onto the plastic directly, but prior to the experiment is covalently coupled to normal rabbit IgG and then the resulting conjugate is coated. This is done because large protein molecules bind much better to plastic and because the direct binding of peptides may mask their active sites.
  • proteins containing the novel adhesion signal may be coated directly onto the plastic.
  • Covalent coupling of peptides to IgG via their N-terminal cysteine residues was accomplished by cross- linking with the hetero-bifunctional agent N-succinimidyl-3(2-pyridylthio) propionate (SPDP) .
  • SPDP N-succinimidyl-3(2-pyridylthio) propionate
  • 5PDP was dissolved in ethanol to a concentration of 1.5 mg/ml and mixed with ⁇ mg/ml of rabbit's IgG in Dulbecco's phosphate-buffered saline to give an SPDP:IgG weight ratio of 1:10 (molar ratio of* 50:1).
  • MOLT-4 human lymphoblastic leukaemia cells were activated by addition of 20 nM phorbol-12,13-dibutyrate to their culture medium for 15 minutes at 37°C, and then re-suspended to 10'/ml in Dulbecco's MEM containing 20 mM HEPES and 1 M MnCi 2 . 100 ul aliquots of the cell suspension were then added to adhesive substrates, ie. peptides, coated onto wells of 96-well tissue culture plates and blocked with heat-denatured BSA to block the non-specific adhesion sites on the plastic surface (coating and blocking are as described for the spreading assay below) .
  • adhesive substrates ie. peptides
  • the cells were incubated in the wells for 15 minutes at 37°C, and loosely adhered cells were removed by gentle agitation and aspiration. Remaining attached cells were fixed by addition of 100 ul of 5% (v/v) glutaraldehyde, washed with water and stained with 10 ul of 0.1% (w/v) crystal violet in 200 mM MES, pH 6, for 20 minutes at room temperature. Cells were then de-stained with three water washes and dye released by addition of 100 ul of 10% (v/v) acetic acid. Colour was quantitated by measuring absorbance at 570 nm in an ELISA reader and the results converted to percent attachment based on an appropriate standard curve. The results of this assay are shown in figure 1 and Table 1.
  • the adhesion of MOLT-4 cells to peptides containing the novel adhesive signal increases with increasing peptide concentration, ie. is dose dependent.
  • the optimum peptide concentration resulting in 80 to 100% adhesion of the cells was found to be between 1:1000 and 1:10.
  • a peptide sequence (CSlscrl) which did not contain the adhesive signal failed to attach any MOLT-4 cells (figure 1) .
  • A375-SM cells from a human metastatic melanoma cell line were cultured in Eagle's minimal essential medium containing 10% fetal calf serum, minimal essential medium vitamins, non-essential amino acids, sodium pyruvate and glutamine.
  • Cell spreading assays were performed in 96-well microtitre plates. Wells were coated for 60 minutes at room temperature with 100 ul aliquots of conjugated peptides diluted with Dulbecco's phosphate-buffered saline, and then sites on the plastic for non-specific cell adhesion were blocked for 30 minutes at room temperature with 100 ul of 10 mg/ml heat-denatured bovine serum albumin (BSA) , after which the BSA was removed.
  • BSA heat-denatured bovine serum albumin
  • the cell receptor involved in this specific adhesion to the peptide sequence of the present invention has been identified as the integrin alpha-4,beta-l.
  • integrin alpha-4,beta-l By blocking the receptor with anti-functional anti-integrin subunit monoclonal antibodies and repeating the cell spreading and cell attachment assays, confirmation that the peptide induced cell attachment and spreading was due to adhesion to this receptor may be acheived if the presence of antibody inhibits these effects.

Landscapes

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

Abstract

L'invention porte sur un peptide d'adhésion cellulaire, pouvant être un polypeptide, constitué d'une séquence d'acides aminés comprenant X-Asp-Y-(A)n-Phe, dans laquelle X et Y sont choisis parmi Ala, Leu, Ile et Val, A étant un acide aminé quelconque et n se situant entre 3 et 10, où au moins une sous-séquence de ladite séquence adhère aux cellules de la leucémie humaine lymphoblastique MOLT-4, du mélanome métastatique humain A375-SM ou de fibrosarcome humain H1080.
PCT/GB1992/000226 1991-02-07 1992-02-06 Nouveaux peptides d'adhesion cellulaire WO1992013887A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9102655.9 1991-02-07
GB919102655A GB9102655D0 (en) 1991-02-07 1991-02-07 New peptides
GB9102818.3 1991-02-08
GB919102818A GB9102818D0 (en) 1991-02-08 1991-02-08 New peptides

Publications (1)

Publication Number Publication Date
WO1992013887A1 true WO1992013887A1 (fr) 1992-08-20

Family

ID=26298409

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/000226 WO1992013887A1 (fr) 1991-02-07 1992-02-06 Nouveaux peptides d'adhesion cellulaire

Country Status (2)

Country Link
AU (1) AU1221292A (fr)
WO (1) WO1992013887A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5440015A (en) * 1992-07-21 1995-08-08 Glycomed Incorporated Selectin peptide medicaments for treating disease
WO1996006114A1 (fr) * 1994-08-22 1996-02-29 Beiersdorf Ag Peptides d'adhesion cellulaire destines a modifier le pouvoir d'adhesion intercellulaire de cellules eucaryotes
US5660992A (en) * 1993-06-16 1997-08-26 Glycomed Incorporated Sialic acid/fucose based assay reagents and assay methods
EP0737204A4 (fr) * 1993-12-06 1999-06-23 Cytel Corp Peptidomimetiques cs-1, compositions et procedes pour les utiliser
US6103870A (en) * 1993-12-06 2000-08-15 Cytel Corporation CS-1 peptidomimetic, compositions and methods of using the same
WO2002044207A1 (fr) * 2000-11-30 2002-06-06 Imperial College Innovations Limited Procedes et molecules d'immunotherapie
EP1061939A4 (fr) * 1998-02-18 2002-06-26 Univ New York State Res Found Matrice extracellulaire a base de fibronectine de recombinaison utilisee dans la cicatrisation des blessures
WO2016094764A1 (fr) * 2014-12-12 2016-06-16 Tufts University Les peptides dérivés de la collagénase activent la régénération tissulaire et la cicatrisation des plaies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
The EMBO Journal, vol. 10, no. 13, December 1991, IRL Press, (Oxford, GB), A.P. MOULD et al.: "Identification of a novel recognition sequence for the integrin alpha4beta1 in the COOH-terminal heparin-binding domain of fibronectin, pages 4089-4095, see the whole document *
The Journal of Biological Chemistry, vol. 262, no. 14, 15 May 1987, (Bethesda, Maryland, US), M.J. HUMPHRIES et al.: "Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion", pages 6886-6892, see the whole document *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5440015A (en) * 1992-07-21 1995-08-08 Glycomed Incorporated Selectin peptide medicaments for treating disease
US5660992A (en) * 1993-06-16 1997-08-26 Glycomed Incorporated Sialic acid/fucose based assay reagents and assay methods
EP0737204A4 (fr) * 1993-12-06 1999-06-23 Cytel Corp Peptidomimetiques cs-1, compositions et procedes pour les utiliser
US6103870A (en) * 1993-12-06 2000-08-15 Cytel Corporation CS-1 peptidomimetic, compositions and methods of using the same
US6117840A (en) * 1993-12-06 2000-09-12 Cytel Corporation CS-1 peptidomimetics, compositions and methods of using the same
WO1996006114A1 (fr) * 1994-08-22 1996-02-29 Beiersdorf Ag Peptides d'adhesion cellulaire destines a modifier le pouvoir d'adhesion intercellulaire de cellules eucaryotes
US5994311A (en) * 1994-08-22 1999-11-30 Beiersdorf Ag Cell adhesion peptides for modifying the adhesion capacity of eukaryotic cells between each other
EP1061939A4 (fr) * 1998-02-18 2002-06-26 Univ New York State Res Found Matrice extracellulaire a base de fibronectine de recombinaison utilisee dans la cicatrisation des blessures
WO2002044207A1 (fr) * 2000-11-30 2002-06-06 Imperial College Innovations Limited Procedes et molecules d'immunotherapie
WO2016094764A1 (fr) * 2014-12-12 2016-06-16 Tufts University Les peptides dérivés de la collagénase activent la régénération tissulaire et la cicatrisation des plaies
US10485846B2 (en) 2014-12-12 2019-11-26 Tufts University Collagenase-derived peptides promote tissue regeneration and wound healing

Also Published As

Publication number Publication date
AU1221292A (en) 1992-09-07

Similar Documents

Publication Publication Date Title
Pierschbacher et al. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule
Wong et al. αv integrins mediate adhesion and migration of breast carcinoma cell lines
Nierodzik et al. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo
EP0573545B1 (fr) Anticorps monoclonaux contre des sites de liaison induits par un recepteur
Berndt et al. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin
Whaley et al. C2 synthesis by human monocytes is modulated by a nicotinic cholinergic receptor
EP0328552B1 (fr) Polypeptides derives de la thrombine, compositions les contenant et procedes d'utilisation
CA1245153A (fr) Systeme de recepteurs cellulaires utilise dans le diagnostic et le traitement du cancer
EP0906919A2 (fr) Nouveaux peptides liant les intégrines
EP0205405A2 (fr) Facteur purifié relatif à une immunoglobuline, anticorps monoclonaux, lignées cellulaires d'hybridomes, procédés et applications
CA2000048A1 (fr) Peptides et anticorps inhibant la liaison integrine-ligand
AU600112B2 (en) Peptides with laminin activity
Tomasini et al. On the identity of vitronectin and S-protein: immunological crossreactivity and functional studies
Raugi et al. Location and partial characterization of the heparin-binding fragment of platelet thrombospondin
Garcia-Pardo et al. Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation
WO1992013887A1 (fr) Nouveaux peptides d'adhesion cellulaire
US6544750B1 (en) Peptide analogs as selective inhibitors of thrombin activation of protease activated receptor 1
US5789382A (en) Retro-peptide fibroblast growth factor which blocks FGF receptor
US6825319B1 (en) Synthetic peptides and pharmaceutical compositions comprising them for diagnosis and treatment of anti-phospholipid syndrome
CA2008334A1 (fr) Anticorps monoclonaux specifiques des inhibiteurs de la thrombine
EP0452314B1 (fr) Recepteur d'adhesion pour laminine et son utilisation
JP2000109500A (ja) ヒト尿由来細胞接着糖蛋白質、その製造方法およびそれを 含有する医薬品
JP2001506994A (ja) 免疫グロブリン受容体相互作用の阻害剤を含んでいる医薬組成物
Tan et al. Complement C1q inhibits cellular spreading and stimulates adenylyl cyclase activity of fibroblasts
US6630572B1 (en) Thrombin derived polypeptides: compositions and methods for use

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA FI HU JP KP KR LK MG MW NO PL RO RU SD US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CI CM DE DK ES FR GA GB GN GR IT LU MC ML MR NL SE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA