EP0364481A1 - Wachstumsfaktoren - Google Patents

Wachstumsfaktoren

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Publication number
EP0364481A1
EP0364481A1 EP88905578A EP88905578A EP0364481A1 EP 0364481 A1 EP0364481 A1 EP 0364481A1 EP 88905578 A EP88905578 A EP 88905578A EP 88905578 A EP88905578 A EP 88905578A EP 0364481 A1 EP0364481 A1 EP 0364481A1
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EP
European Patent Office
Prior art keywords
hbgf
pro
gly
peptide analogue
peptide
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Withdrawn
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EP88905578A
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English (en)
French (fr)
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EP0364481A4 (en
Inventor
Milton Thomas William Hearn
Joseph Bertolini
Mark Andrew Guthridge
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Monash University
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Monash University
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Publication of EP0364481A1 publication Critical patent/EP0364481A1/de
Publication of EP0364481A4 publication Critical patent/EP0364481A4/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GROWTH FACTORS This invention relates to mammalian growth factors and in particular to heparin-binding growth factors.
  • the heparin-binding growth factors are polypeptide mitogens, found in brain, pituitary and other mesenchymal, neuroectodermal and epithelial derived tissues, which induce proliferation of cells in vitro and in vivo. These factors include basic fibroblast growth factor (FGF-1), acidic fibroblast growth factor, and transforming growth factors o and / . A number of these molecules have angiogenic activity, and this has been summarized in a review by Polkman and lagsbrun (Science 235 442-447, 1987).
  • Heparin binding growth f ctor-/. (HBGH- 2 ). a mesenchy ally directed growth factor with angiogenic properties, has been purified to homogeneity from tissue extracts or serum from several species (for example bovine, porcine, human, rat) .
  • a form of heparin binding growth factor (HBGF- ⁇ 1) isolated from bovine brain has been shown to have an identical amino acid seguence to bovine fibroblast growth factor - A (FGF- ⁇ 1-146) so these two are now considered synonymous.
  • the total amino acid seguence established for bHBGF-/5 1, beginning with th ⁇ e amino-terminal residue Pro is
  • This amino acid seguence corresponds to the complete seguence for the mature 146 residue form of basic FGF- ⁇ derived from bovine pituitary glands as described by Esch, F., et al Proc. Natl. Acad. Sci (USA) (1985) S2_ 6507-6511.
  • the characterisation and biological properties of this HBGF- ⁇ form has been recently reviewed by Gospodarowicz, D. et al. Mol. Cell Endocr. (1986) 46 187-204 and Fiddes J.C. et al. Int. Patent Publ. WO 87/01728.
  • HBGF-A growth factors with related biological activity but different primary structures to HBGF-A , including endothelial cell growth factor, tumor angiogenesis factor, and acidic fibroblast growth factors, 5 have been characterised (see for example, Lobb R.R. et al. Biochemistry (1984) 2 _, 6295-6299; Maciag T et al. Science (1984) 225, 932-935; Klagsbrun et al., Proc. Natl. Acad. Sci. USA (1985) 8_2 805-809; Bohlen P et al. EMBO J (1985) _4, 1951-1956; Gimenez-Gallego G. et al. Science (1985) 230, 10 1385-1388; Esch. P.
  • HBGF- in impure form has been alleged to be effective in wound healing of tissue subjected to trauma (Davidson et. al. Journal of Cell Biology (1985) 100, 1219-1227; Thomas K.A. et al. op. cit.), including the 0 treatment of myocardial infarction (Svet-Moldavsky G et. al. Lancet 1977, 1913, US Patents 4296100, 4378347), in neuronal survival in fetal rat hippocampal neurons (Walicke, P., Proc. Natl. Acad Sci (USA) 1986) _83_, 3012-3016 Schubert D et al. , J.C.B.
  • polypeptide mitogens which we have isolated and characterized are biologically active, having the activity of HBGF- h in a variety of systems in vitro and in vivo, including their ability to promote angiogenesis. It is thus likely that these variant polypeptides may be general mesodermal cell/tissue growth factors arising from tissue-specific post translational processing, although other modes of expression and synthesis cannot be excluded. They are maximally active, depending on the in vitro cell culture assay system, between K 10 —12 and 10—16 mole.
  • HBGF- ⁇ variants appear to exhibit a tissue specific processing capability which might underlie enhanced biological potency as an autocrine factor compared to exogenous components derived from other morphologically and physically distant sources, it is an object of the present invention to overcome, or at least alleviate, one or more difficulties related to the prior art and provide the opportunity to utilize these HBFG- ⁇ variants in specific and general biological applications.
  • tissue specific form of these HBGF-/J variants, or derivatives made by chemical or genetic manipulation in adeguate guantities in a high degree of purity, and presented in a form compatible with use as a slow release therapeutic substance suitable for topical or internal modes of delivery.
  • HBGF- ⁇ mammalian heparin-binding growth factor
  • polypeptide analogue is a human, bovine, porcine, sheep, horse or rat HBGF-6 analogue.
  • polypeptide analogue has an amino terminal structure selected from: Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys
  • a method of isolation of truncated forms of HBGF- ⁇ from a mammalian source comprising the steps of: subjecting a sample containing HBGF-5 truncated forms to ammonium sulphate fractionation, isolating and pooling fractions having HBGF-.0 activity, and subjecting the pooled fractions to seguential steps of cation exchange chromatography, triazine dye affinity chromatography or heparin affinity chromatography, and size exclusion chromatography, and optionally reversed phase chromatography.
  • the triazine dye affinity chromatography step is immediately after the cation exchange chromatography step.
  • the cation exchange chromatographic step involves a rigid, macroporous, monodisperse polymeric chromatographic support derivatised with carboxylmethyl or sulphonic acid groups, such as the Pharmacia Fast Flow S or Accell ⁇ .
  • the triazine dye affinity chromatography involves either a single or a tandem affinity chromatographic system selected from the following: Procion triazine dyes Yellow MX4R, Yellow MX3R, Yellow MXGR, Yellow MX8G, Blue MXG, Blue MX4GD, Blue MX3G, Blue MX7RX, or Turquoise MXG, Brown
  • MX5BR Red MX5B
  • Rubine MXB or equivalent or similar triazine dyes obtained from ICI, Ciba Geigy and other manufacturers of such triazine dyes.
  • the heparin affinity chromatographic step involves heparin immobilised to carbonyldiimidazole (CDI) activated hydroxylic matrices such as Fractogel HW65, Trisacryl GW3000, Superose 12 or glycidylpropoxylsilicas available in various porosities and particle diameters.
  • CDI carbonyldiimidazole
  • the reversed phase chromatography involves butyl -, octyl -, or octadecyl - groups chemically bonded to monodisperse non-porous or porous silica particles with particle sizes ranging from 0.7 - 60 ⁇ m with particle size distribution from +_ 1% to + 30%, depending on the sizing procedure used to sieve the silica particles.
  • the source of truncated forms of HBGF-/. is selected from tissue ho ogenates, plasma, tissue or cell cultures, cell or tissue culture conditioned medium, and tumour cells.
  • a method for the preparation of polypeptide analogues of mammalian HBGF- ⁇ comprising transformation of a susceptible bacterial, yeast, baculovirus, insect or mammalian cell host with a DNA sequence capable of directing the expression of the truncated analogues of HBGF- A .
  • the DNA sequences may be recombinant, synthetic, chromosomal fragments, cDNA or combinations thereof, and may be incorporated into a suitable cloning vehicle such as a plasmid.
  • the inserted coding sequences may incorporate deletions or omissions to account for differences between the sequences of the truncated analogues (e.g. destetrapeptido-HBGF- A and related polypeptides) and the complete compound.
  • polypeptide analogues may have an amino- or carboxy- terminal extension which includes a biological binding site which is recognised by heparin, heparan sulphate, chondroitin sulphate, or a binding or transport protein or substance involved in the chemical or biological recognition of HBGF-5 analogues in vivo or in vitro
  • the complete polypeptide chain of HBGF- A may be cleaved by appropriate aminopeptidases as part of the downstream processing or modification of natural or recombinant material to generate truncated variants in accordance with the present invention.
  • a method for the preparation of a peptide analogue of mammalian HBGF-5 wherein from 1 to 12 amino acid residues are absent from the N-terminal comprises the steps of
  • the peptide analogues can be produced by appropriate modifications to conventional de novo or semi-synthetic methods based on solid-phase or solution peptide synthesis methods existing for the production of the intact HBGF- 5 peptide. These modifications would be readily apparant to those skilled in the art.
  • despeptido HBGF-/* may be synthesised chemically using procedures suitable for HBGF- but with the final 1 to 12 cycles of amino acid ligation omitted.
  • Synthetic despeptido and related peptides specific for other mammalian species may be produced by techniques similar to those used for bovine HBGF- > using amino acid sequence information for these peptides.
  • an antibody specific for the polypeptide analogues according to the invention is provided.
  • a method of immunoassay of heparin-binding growth factors comprising the step of using either the polypeptide analogues according to the invention or antibody specific thereto, coupled to a suitable marker.
  • a sixth aspect of the invention there are provided methods of diagnosis and treatment of disease in a mammal comprising the step of utilizing either the peptide analogues according to the invention or antibody specific thereto.
  • HBGF-/-S variants and related polypeptide analogues may be administered alone or together with a pharmaceutically acceptable diluent, carrier, or excipient suitable for the intended method and site of administration.
  • the dose rates and times for the administration of despeptido HBGF- may be set at levels proportionally less than for intact HBGF-. in accordance with the increased potency of the present invention. Dose rates of approximately 1 to 1000 micrograms/kilogram/day may be used. Dose rates of approximately 10 to 100 micrograms/kilogram/day are preferred.
  • a composition for enhancing the healing of an internal or external wound in a mammal in need of such treatment comprising a peptide analogue of HBGF-/S as defined above adsorbed on or entrapped in a physiologically compatible carrier material, whereby a therapeutically effective amount of the peptide is provided at the wound site.
  • the carrier material is selected from a synthetic or natural organic polymer and chemically modified inorganic molecules. More preferably the carrier is cellulose or another polysaccharide.
  • the amount of peptide is 1 to 1000 ⁇ cg/Kg, more preferably 1 to 100 ⁇ g/Kg body weight of the mammal.
  • a cell or tissue culture medium comprising a polypeptide analogue according to the invention.
  • the medium is a chemically-defined medium.
  • the number of deletions resulting in maximal activity is species-specific, and also to a certain extent tissue-specific. In rat the despentapeptide form is most active, whereas in bovine the destri- or destetrapeptides have greatest activity.
  • the compound bovine destripeptide HBGF- ⁇ having the formula of bovine fibroblast-growth factor (FGF-1) as described by Esch et al. (1985), but lacking the amino acid residues Pro-Ala-Leu from the N-terminal region, is effective in promoting cell proliferation, and in stimulating both protein synthesis and DNA synthesis in cellular systems at concentrations between 10 and 50-fold lower than required for the entire bovine HBGF- ⁇ .
  • FGF-1 bovine fibroblast-growth factor
  • the sites of truncation are not predictable from the amino acid sequence of entire bovine HBGF- ⁇ .
  • Figure 1 shows elution profiles of tissue homogenates chromatographed on CM-Sephadex G50;
  • Figure 2 shows elution profiles of partially-purified HBGF- despeptides from different sources on.heparin-Sepharose
  • Figure 4 shows results of titration of rabbit antiserum to bovine HBGF-. destripeptide using the radioimmunoassay according to the invention.
  • Figure 5 shows the displacement of
  • I-destetrapeptido HBGF-/S from antibody by purified HBGF-/. from bovine brain (a), pig brain (b) and bovine lung, rat brain, and rat chondrosarcoma (c);
  • Figure 6 shows the levels of destetrapeptido HBGF-/*> detected by radioimmunoassay (a) in extracts of bovine lung and ovine pituitary and (b) in rat serum and extracts of rat brain and pituitary;
  • Figure 7 shows the effect of various hormones and growth factors on the destetrapeptido HBGF-5 content of cultured bovine ovarian theca cells;
  • Figure 8 shows the effect of destetrapeptido HBGF-A on progesterone production by cultured bovine ovarian granulosa cells.
  • HBGF- f> heparin-binding growth factor isolated from various mammalian tissues exhibits considerable N-terminal amino acid sequence heterogeneity.
  • tissue-specific post-tran ⁇ lational processing in each tissue represents an important mechanism for the specific production of a unique form of HBGF-/. which manifests optimal biological activity with the adjacent cell bodies, i.e. functions dominantly as an autocrine factor such that truncation represents a unique biological mechanism to permit and discriminate specific proliferative response activity from a generalised cell mitogenic response.
  • the evidence for this conclusion is based on the following observations: (1) the abundance of a specific truncated form is characteristic of the tissue type from which it is isolated;
  • truncated forms can be isolated from tissue extracts when protease inhibitors are present in high concentration throughout the purification;
  • the site(s) of cleavage for truncation are not those normally associated with currently-recognised proteolytic enzymes found in mammalian cell extracts, whether or not the extracts are contaminated with microorganisms;
  • Example 1 Purification of despeptido analogues from tissue Isolation of destripeptide HBGF-_ destetrapeptide HBGF- ? , despentapeptide HBGF-. and other despeptido forms of HBGF-5 has been achieved from bovine, pig and rat brain, lung, pituitary, ovary, corpus luteum and plasma, and from human plasma and a rat chondrosarcoma tumour cell line.
  • polypeptides were purified to homogeneity from tissue homogenates or plasma in the presence of protease inhibitors (ImM disodium ethylenediamine-tetraacetate (EDTA) , 2mM phenylmethylsulphonylfluoride, 2.5 mg/1 leupeptin, 2.5 mg/1 pepstatin, and 2.5-5.0 mg/1 benzamidine) by solubilisation at pH 3.5-4.5 with phosphate buffer (O.lM NaH_PO. containing O.lM NaCl) and partially fractionated using ammonium sulphate fractionation (1-4.IM), followed by sequential use of cation exchange chromatography, heparin affinity chromatography and size exclusion chromatography.
  • protease inhibitors ImM disodium ethylenediamine-tetraacetate (EDTA) , 2mM phenylmethylsulphonylfluoride, 2.5 mg/1 leupeptin, 2.5 mg/1 pepstatin, and 2.5-5.0 mg/1 benzam
  • Triazine dye affinity chromatography and heparin affinity chromatography may be used as alternatives. For large scale purification triazine dye columns are cheaper, and are preferred for this reason. A reversed phase chromatography step may optionally be used for final purification.
  • Chromato g raphy media found to be suitable for the purification procedure are summarized in Table 1. Other suitable media will be apparent to the person skilled in the art .
  • Typical elution profiles of tissue homogenates on CM-Sephadex G-50 and heparin-Sepharose are shown in Figures 1 and 2.
  • the bar indicates fractions with HBGF activity.
  • Elution from the cation exchange column may be performed stepwise or using a gradient, depending upon the sample.
  • HBGF- fractions were pooled and desalted using Centricon-10 microconcentrators.
  • amino and composition the samples were evaporated to dryness or precipitated with 4 volumes of AR grade methanol followed by centrifugation, resuspended in methanol and transferred to an hydrolysis tube.
  • the samples were hydrolysed at 110°C with 5M hydrochloric acid containing 1.25 nmol or norleucine standard and 0.1% phenol for 24 hours.
  • Amino acid analysis was performed on a Durrum D-500 or Waters Pico-Tag amino acid analyser system.
  • DMEM Dulbecco's minimum essential medium
  • FCS fetal calf serum
  • RPMI 1640 tissue culture medium containing 5% fetal calf serum at a concentration of 1.5 x 10 cells/ml. After culture for
  • the bovine lung endothelial cell culture protocol was based on modifications of the Ryan and White procedure (Tissue Culture Methods 3-0(1), 9-13, 1986) using the Jacobson and Ryan microcarriers (Tissue and Cell, 14, 69-83, 1982).
  • Cell proliferation activity and endogenous activity with the myoblast L6 cell line, and collagenase dispersed rat hepatocyte, chrondrocyte and chrondrosarcoma cells were measured using the following procedures: Culture of the myoblast L6 cell line was carried out in enriched Dulbecco's minimal essential medium (DMEM) supplemented with 3.6g glucose, 4.Og Hepes, 3.7g sodium bicarbonate, 200 mg glutamine, 10000U penicillin, lOOOOug streptomycin and 250 ug amphotericin B/ml per litre of medium.
  • DMEM Dulbecco's minimal essential medium
  • Confluent cells were subcultured after 5-7 days, washed twice with phosphate buffered saline (PBS) and trypsinised for 3 min., then immediately diluted with DMEM and aliquoted to calO cells/ml prior to assay. Similar culture procedures were utilised for the hepatocyte and other cell lines. Culture of the rat chondrocyte culture involved the following procedures. The sternum and costal ribs were removed from rats under sterile conditions. After removing adhering connective tissue, the sternum and ribs were minced and digested in 10 ml 0.25% (w/v) trypsin in Ham's F12 medium for 30 min. at 37°C.
  • PBS phosphate buffered saline
  • Example 4 Production of Polyclonal Antisera against Despeptido-HBGF-A -
  • Adult female and male New Zealand white rabbits were immunised with the destripeptide HBGF-A .... desdodecapeptide HBGF- emulsified in Marcol 52-Montanide 888 (9:1 v/v) by intramuscular or subcutaneous administration.
  • the despeptide was given alone, or coupled to ovalbumin or keyhole limpet haemocyanin by conventional methods.
  • Polypeptide coupling to carrier proteins for immunisation used the bifunctional reagent 6-maleimidocapronic - acyl - N-hydroxysuccinimide ester essentially as described by Stevens, V.C. et al. Immunol Letts.
  • Antibodies against the carrier proteins were removed from the recovered antisera by adsorption with the specific carrier protein.
  • a specific radioimmunoassay utilizing heparin-Sepharose purified bovine pituitary destripeptide HBGF-, . or destetrapeptide HBGF-S as standard and purified iodinated bovine pituitary FGF as tracer was developed and characterized.
  • the assay used polyclonal antibodies prepared as in Example 4. It will be appreciated that the use of monoclonal antibodies prepared by methods well-known in the art, and other well-known types of immunoassay such as enzyme-linked immunoassay (ELISA) and fluorescent immunoassay, are within the scope of the invention.
  • Tracer was labelled with 125I by the lactoperoxidase method (Thorell, J.I. and B.F. Johansson [1971]: Biochim. Biophys. Acta 251 ;63). The tracer was used at approximately
  • Results of a typical assay are shown in Figure 4.
  • the tracer was 125I-bHBGF-4 destripeptide, and the antiserum was directed against bHBGF-/i destripeptide.
  • a final dilution of antiserum of 1:5000 resulted in 36% binding.
  • Scatchard and logit plots derived from antiserum binding curves were linear, indicating that the antiserum was monospecific.
  • the radioimmunoassay has been used to determine the abundance of related truncated forms of HBGF-/3 in a variety of samples, including a) tissue homogenates - bovine ovarian theca rat brain, pituitary ovine pituitary
  • HBGF-A from antibody by bovine, pig and rat brain, brain lung and rat chondrosarcoma are shown in Figure 5.
  • the assay could detect HBGF-/_ in extracts of rat chondrosarcoma, a tumour cell line.
  • HBGF-/S form was documented through the down-regulation of response when exogenous stimulation of the granulosa cells in culture was carried out with the factor at higher concentrations (Figure 8). Progesterone production by granulosa cells was significantly decreased by doses of destetrapeptido HBGF- ) greater than 100 pg per ml culture. This effect was observed at 2, 4 and 6 days of culture.
  • Example 7 Effect of Despeptido HBGF-j on Angiogenesis The functional sequelae of the angiogenic role of these HBGF-/5 polypeptides are being assessed by determination of their dose dependent effect on
  • Example 8 Adsorption to carriers for local administration
  • heparin and other sulphated support carriers such as heparansulphate proteoglycans and SP-cellulose, SP-agarose, or SP-derivatised polymethacrylate copolymers etc.
  • the procedure is based on coupling heparin or heparansulphate proteoglycans to preactivated cellulose and adsorptively binding the factor(s) at a subsequent step.
  • the preferred method of preactivation involves the use of carbonyldiimidazole (Hearn, Meth. Enzym, 1987, 135, 102-131).
  • the rat implant system based on the Schilling Hunt polyvinylsponge insert impregnated with exogenous factor(s) or control substances was used to establish assessment with external wounds. Additional endogenous factor activity was found in wound fluid as well as follicular fluid, with a 2.5 fold increase in growth rate of stomatised cells mediated by these factors being observed.
  • HBGF-/S variants are batch adsorbed to heparin derivatised polymers prepared for example from -.-aminocaproyl derivatised cellulose, agarose, jj-glycidoxyl propylsilica, hydromethylated polymethacrylate, derivatised polyvinylalcohol (Fractogels) of tris-acrylamide copolymers (Trisacryl) essentially according to the methods of Finlay et al Anal Biochem., 109 (1980) 354; Schutyser et al Affinity Chromatography and Related Techniques (T.C. Gribnan, J. Visser and R.J.F. Nivard eds) Elsevier Sci Publ.
  • Trisacryl tris-acrylamide copolymers
  • HBGF- A in tissues and extracts were used to monitor and quantify the presence of the HBGF- variants in various tissues from different mammalian species.
  • the oligonucleotide probe with the sequence such as CGGTTTGCACACACTCC, GACACAACCCCTCTCTCTTCTGCTTG and CTAGTAATCTTCCATCTTCTTTCATAG, when 3'- labelled with phosphonucleotidyltransferase and S-ATP to a high specific activity, permits the detection and quantitation of mRNA levels corresponding to the HBGF-/S in both normal and transformed cells such as chondrocytes, chondrosarcomas, hepatoma and colorectal carcinoma specimens, using in situ hybridisation technigues, dot blot methods or agarose gel probe procedures.
  • HBGF- f> despeptides have been found in hepatoma and in colorectal carcinoma specimens by these methods.
  • Oligonucleotide probes corresponding to HBGF- ⁇ were synthesized on the Model 380A DNA synthesizer (Applied Biosystems Inc.) using phosphoramidite chemistry and purified on a Dupont Zorbax Bio Series Oligo column (8 cm x 6.2 mm) followed by desalting on a G-25 column (2.8 cm x 9 mm).
  • Oligonucleotides were either 5' end labelled using T. polynucleotide kinase and 32P-ATP or 3' end labelled using phosphonucleotidyl transferase and 35S-ATP. All probes had final specific activities of 5 x 10 8 - 8 x 109 dpm/ug.
  • Microscope slides were baked, coated with poly-L-lysine, treated with 0.1% diethyl pyrocarbonate and allowed to air dry. Mono dispersed cells in culture medium were spun onto slides using a Cytospin (10 - 3x10 cells/slide) . Transverse 5-10um sections were cut from OCT embedded tissue on a cryostat at -20°C and mounted on microscope slides. Both cells and tissue slices were left on dry-ice blocks for 20 min. and then fixed for 10 min. in 4% glut raldehyde, lOOmM Na N- P0. , 20% ethylene glycol pH 7.3.
  • HBGF-/4 was detected in rat chondrocytes, rat chondrosarcoma, bovine aortic and lung endothelial cells, and rat ganglion neurons, and in human hepatoma and colorectal carcinoma.
  • Synthetic peptides corresponding to the internal sequence of the destetrapeptido-HBGF-/i have been synthesised by solid phase methods on the benzhydrylamine/divinylbenzene resin.
  • the synthesis of the peptide Leu Arg He His Pro Asp Gly Arg Val Asp Gly is representative of the methods.
  • the initial coupling to the benzhydramine resin employed a 3-fold excess of the designated side chain protected amino acid dissolved in N, N'- dimethyl-formamide (DMF) .
  • Subsequent couplings employed a two-fold excess of side chain protected amino acid in DMF.
  • the amino acids were sequentially activated using tert-butanol and dicyclohexylcarbodiimide in DMF and allowing the reactants to stir at room temperature for 5-10 min.
  • the acylurea was removed by filtration and the asymmetric anhydride added to the resin.
  • the suspended resin was stirred for the reaction time of 120 min. at room temperature. Each coupling was monitored for unreacted amino groups with 2, 4, 6-trinitrobenzenesulphonic acid.
  • the crude product was chromatographed by anion exchange on Whatman DE52 and then desalted on Biogel P2 gels. Further purification was achieved using reversed phase HPLC with octadecylsilica as stationary phase (or other n-alkylsilica) and aguo-organic solvent gradients such as 0-75% water-acetonitrile -0.1% trifluoracetic acid or 0-75% water-acetonitrile-lOOmM ammonium bicarbonate as the mobile phase.
  • Synthetic peptides were characterised by amino acid compositional analysis, analytical reversed phase HPLC with several elution systems, fast atom bombardment - mass spectroscopy (FAB-MS), FTIR-infrared spectroscopy, and paper electrophoresis. Peptides were conjugated to carrier proteins for use as antigens, as described above, for production of polyclonal and monoclonal antibodies.
  • the actions of these despeptido HBGFs are distinct from those of other growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) , transforming growth factors K or ⁇ (TGF- ⁇ *. or - ) or fibroblast growth factor - « (FGF-.-X) , which show different time-course profiles for cell proliferation and different molar responsiveness for maximal stimulation.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF- ⁇ *. or - transforming growth factors K or ⁇
  • FGF-.-X fibroblast growth factor - «
  • the heparin-binding growth factors probably represent a key class of growth factors in wound healing and in maintenance of normal and pathological vascularisation of tissue, including corneal cells, smooth muscles and glial cells.
  • mitogenic activity exhibited by the heparin-binding growth factors it is likely that they stimulate and co-ordinate mitogenesis of multiple cell types via an autocrine/paracrine mechanism during normal cellular growth and tissue repair or during growth of transformed cells such as tumour cells.
  • peptidic antagonists of HBGF-/S or alternatively immunosuppression of HBGF- thus has potential to either arrest or reverse the growth of solid tumors, many of which are not responsive to surgery, chemotherapy or radiotherapy.
  • DNA polymerase activity and de novo DNA and protein biosynthesis are all indices of proliferation and outgrowth of mesodermal cells such as endothelial cells in lung or small capillaries, or granulosa cells in ovarian follicles, and insofar as such functions are indices of neovascularisation or angiogenesis and follicular growth, the presence of the despeptido-HBGF-/3s promotes folliculogenesis and neovascularisation of placental tissue, and may be involved in blastocyst implantation.
  • Despeptide HBGF-/S may be involved in the control of neuronal growth in central and peripheral nervous tissue, and in regulation of differentiation of specialised tissues such as muscle.
  • use of synthetic peptides related to these despeptido-HBGF-5s is likely to enhance cell proliferation and growth and so provide methods for selective tissue repair, regeneration or proliferation.
  • HBGF- Comparison of structure of HBGF-,. isolated from different species shows that the amino acid seguence is strongly conserved, with differences in only one or two amino acids. These differences are probably the result of single nucleotide changes. Because of the sites of these changes, the bovine and human despeptido HBGF-/6 show differences only at residues 112 and 128. Similarly in sheep, at residue 10 gly is replaced by ser. This suggests that bovine HBGF-/5 analogues may not be immunogenic in humans, and that sheep HBGF- ⁇ analogues will either be slightly immunogenic or will have different potency from the human peptide. A high degree of cross-species activity would in general be predicted. This is confirmed through the ability of oligonucleotide probes constructed from the amino acid sequence of one species form of the HBGF- ⁇ variants to hybridise with the corresponding mRNA or DNA isolated from the tissues of other species.
  • tissue specificity As regards endogenous truncated forms of HBGF-i , it appears that this does not affect the activity of exogenously administered despeptide HBGF- J other than in relation to potency at a given site.
  • the significance of the endogenous specificity probably lies in the resulting ability to mount a localized response to a stimulus such as trauma. There may also be a functional amplication, for example by a further processing step in situ.

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EP19880905578 1987-06-18 1988-06-15 Growth factors Withdrawn EP0364481A4 (en)

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EP0409858B1 (de) * 1988-03-17 1993-12-08 Novo Nordisk A/S Heparin bindende proteine, dafür kodierende dna, verfahren zu ihrer herstellung sowie sie enthaltende therapeutische präparate
US5814602A (en) * 1988-03-17 1998-09-29 Novo Nordisk A/S Heparin-binding proteins
AU6605690A (en) * 1989-10-27 1991-05-31 Du Pont Merck Pharmaceutical Company, The Monoclonal antibodies to basic fibroblast growth factor that inhibit its biological activity
AU659723B2 (en) * 1992-02-14 1995-05-25 Kaken Pharmaceutical Co., Ltd. Remedy for airway diseases
PL214284B1 (pl) * 2002-07-09 2013-07-31 Baxter Healthcare Sa Sposób wytwarzania wirusa oraz sposób wytwarzania immunogennej kompozycji zawierajacej wirusa albo antygen wirusa
US7598356B2 (en) 2004-07-08 2009-10-06 Board of Regents of the University of Nebraska by and on behalf of the University of Nebraska Medical Center Method for purifying a protein of the cystine-knot superfamily

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EP0288687A2 (de) * 1987-03-03 1988-11-02 Takeda Chemical Industries, Ltd. Monoklonaler Antikörper, Hybridomen, ihre Herstellung und ihre Verwendung

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US4444760A (en) * 1983-06-17 1984-04-24 Merck & Co., Inc. Purification and characterization of a protein fibroblast growth factor
ATE105868T1 (de) * 1985-09-12 1994-06-15 Scios Nova Inc Rekombinanter fibroblast-wachstumsfaktor.
JP2526965B2 (ja) * 1987-02-24 1996-08-21 武田薬品工業株式会社 ムテイン，ｄｎａおよびその用途

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Title
MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 51, no. 3, 1987, pages 187-200, Elsevier Scientific Publishers Ireland, Ltd; J. BERTOLINI et al.: "Isolation, characterisation and tissue localisation of an N-terminal-truncated variant of fibroblast growth factor" *
PROC. NATL. ACAD. SCI. USA, vol. 84, April 1987, pages 1839-1843; M. KLAGSBRUN et al.: "Multiple forms of basic fibroblast growth factor: Amino-terminal cleavages by tumor cell- and brain cell- derived acid proteinases" *
REGULATORY PEPTIDES, vol. 16, no. 2, 1986, pages 135-145, Elsevier Science Publishers B.V. (Biomedical Devision); N. UENO et al.: "Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor" *
See also references of WO8810269A1 *

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