CA2136531A1 - Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity - Google Patents
Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinityInfo
- Publication number
- CA2136531A1 CA2136531A1 CA 2136531 CA2136531A CA2136531A1 CA 2136531 A1 CA2136531 A1 CA 2136531A1 CA 2136531 CA2136531 CA 2136531 CA 2136531 A CA2136531 A CA 2136531A CA 2136531 A1 CA2136531 A1 CA 2136531A1
- Authority
- CA
- Canada
- Prior art keywords
- oligosaccharide
- hgf
- chains
- idoa
- oligosaccharide preparation
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Oligosaccharides having a high specific binding affinity for HGF
growth factors and made up of relatively low molecular weight sulphated oligosaccharide chains are disclosed. The chains are composed of at least three disaccharide units including one or more internal sequences of an N-sulphated D-glucosamine 6-sulphate residue and an L-iduronic acid residue. A method is also disclosed for preparing these oligosaccharides in a purified and relatively homogeneous state from heparan sulphate. For the best HGF-binding affinity there are preferably at least five disaccharide units.
The most favoured structures contain twelve or fourteen monosaccharide residues in all and include a relatively high proportion of 6-0-sulphated hexosamines, e.g. more than 30 % or even 50 %, as compared to oligosaccharide chains of unmodified native heparan sulphate. These oligosaccharides can modulate HGF activity, and uses thereof as drugs for therapeutic purposes in medicine are also disclosed.
growth factors and made up of relatively low molecular weight sulphated oligosaccharide chains are disclosed. The chains are composed of at least three disaccharide units including one or more internal sequences of an N-sulphated D-glucosamine 6-sulphate residue and an L-iduronic acid residue. A method is also disclosed for preparing these oligosaccharides in a purified and relatively homogeneous state from heparan sulphate. For the best HGF-binding affinity there are preferably at least five disaccharide units.
The most favoured structures contain twelve or fourteen monosaccharide residues in all and include a relatively high proportion of 6-0-sulphated hexosamines, e.g. more than 30 % or even 50 %, as compared to oligosaccharide chains of unmodified native heparan sulphate. These oligosaccharides can modulate HGF activity, and uses thereof as drugs for therapeutic purposes in medicine are also disclosed.
Description
VO91/21689 ~ 13 G 5 3 1 PCT/GB9-1/00615 HEPARAN SULPHATE OLIGOSACCHARIDES HAVING
_ HEPATOCYTE GROWTH FACTOR BINDING AFFINITY
The present invention relates to certain novel oligosaccharide products and preparations thereof, useful in the field of biochemistry and medicine, which have particular binding affinity for cer~ain growth factors or cytokines, in particular hepatccyte growth factor (XGF).
BACKGROUND
Various growth factors, often structurally unrelated, are characterised by a strong affinity for heparin. One such growth factor is hepatocyte growth factor (HGF), al;so known as "scatter factor". HGF is an unusually large (82kDa) and structurally complex growth factor that is synthesised as a biologically inactive single chain precursor. This is then proteolytically cleaved at a single site between linked cysteine residues giving rise to a disulphide-bonded heterodimer comprising a large a-chain (54kDa) containing a hairpin loop close to the N-terminus and a sequence of four Kringle domains, together with a smaller ~-chain (26kDa). HGF is produced by various cells including fibroblasts, smooth muscle cells, kidney mesangial cells and liver non-parenchymal cells.
Its target cells are primarily epithelial cells, although it also acts on endothelial cells, hepatocytes and melanocy~es. It is believed to play an important role as a paracrine mediator of epithelial-mesenchymal inter-actions. Cellular responses, however, to HGF are complexand, as well as being mitogenic, it can also stimulate cell migration and morphogenesis depending on the cellular target and its milieu Interestingly, it can have an anti-proliferative effect on some tumour cells, including hepatoma cells, in vitro. It is likely that HGF is an important factor in embryonic organ development. In the adult it has been demonstrated to have a major role in the regenerat~on of damaged organs, such as liver and kidney.
~!0 91121689 ~ 3 6 ~ 3 1 PCT/GB94/0061 The cellular signal response to HGF appears to be mediated by binding (probably through the N-terminal part of the a-chain) to a single high affinity (Kd about 25pM) tyrosine kinase receptor, the product of the c-Met proto-oncogene. However, it has been demonstrated, at leastwith cultured cells, that a much larger number of lower affinity HGF binding sites (Kd about 350-400pM) also exist on the cell surface. It is thought possible that these lower affinity binding sites involve cell surface heparan sulphate proteoglycans, and that HGF interacts with the heparan sulphate (~S) component of such proteoglycans to cause a conformational change leading to a modulation of HGF's interaction with the c-Met receptor protein, in a way that might be analogous to the known dependence of the bFGF-receptor interaction on prior activation of the bFGF
growth factor by heparan sulphate.
The~present invention is based on studies in which we have shown that HGF does in fact interact in vitro with ; 20 heparan ;sulphate. This has led to the isolation and at least partial chàracterisation of novel heparan sulphate oligosaccharides which exhibit significant binding ;affinity ~ for ~HGF and which have certain structural ~feat~res that contra~st with those of other known growth ~fa~ctor binding oligosaccharides.
AB3REVIA~10NS
Throughout the~present specification the following abbreviations are used:
heparan sulphate;
HSPG - - heparan sulphate proteoglycan;
HGF - hepatocyte growth factor;
35 ~dp - degree of polymerisation (e.g. for a disaccharide, dp-2, etc);
G~cA - ~-D-glucuronic acid (or glucuronate);
IdoA - a-L-iduronic acid (or iduronate);
~136~1 Og~ 689 PCT/GB9~/0061;
IdoA(2S) - a-~-iduronic acid 2-sulphate (or iduronate);
GlcNAc N-acetyl a-D-glucosamine;
GlcNAc(6S) - N-acetyl a-D-glucosamine 6-sulphate;
GlcNS~3 - N-sulphated a-D-glucosamine;
GlcNSO3(6S)- N-sulphated ~-D-glucosamine 6-sulphate;
GlcNR - a-D-glucosamine with unspeciied N-substituent;
nUA - unsaturated uronic acid residue (e.g. nGlc for unsaturated D-glucuronic acid and nHex A
for unsaturated unspecified hexuronic acid residue);
SAX - strong-anion exchange;
HPLC - high performance liquid chromatography The symbols (+2S) and (+6S) are used to denote, respectively, that a residue may or may not be sulphated at the C2 or C6 position.
SUMMARY OF THE INVENTION.
.. . __. _ . . I
The invention provides novel oligosaccharides or preparations thereof which have a specific binding affinity for HGF.- Such oligosaccharides will generally be in the form of ~substantially homogenous preparations consisting of oligosaccharide chains composed of a sequence of at leas-t three-- disaccharide units (dp>6), preferably at least five disaccharide units (dp>lO), and including a plurality of disaccharide units which each contai~n an IdoA(~2S) and a GlcNSO3(~6S) residue and which preferably are arranged in between the terminal sugar residues of the oligosaccharide chains but not necessarily contiguously. In -~referred embodiments, the oligo-saccharide chains w~ generally also be resistant to further depolym~risation by heparinase III (heparitinase -EC 4.2.2.8), and ~ill be obtained from heparan sulphate orfrom other natural heparan type material.
More specifically, in one aspect the invention 3 ~
~09~/1689 ~ PCT/GB9~/0061 - consists in an oligosaccharide preparation obtainable from partially depolymerised heparan sulphate (HS) or other natural heparin ~ype material as a fraction thereof, characterised in that the oligosacchari.de preparation consists essentially of oligosaccharide chains which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of a sequence of at least three disaccharide units (dp>6~ that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(+2S) and an N-sulphated D-glucosamine residue GlcNS03(+6S).
In another aspect the invention can alternatively be defined as an oli~osaccharide preparation comprising heparan sulphate ~HS) fragments which have a specific binding affinity for hepatocyte growth factor (HGF~ and which are composed of oligosaccharide chains containing a : sequence of at least three disaccharide units (dp>6) that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(+2S) and an N-sulphated D-glucosamine residue GlcNS03(+6S).
~ ~ Preferably, one or more of the above-mentioned at least two disaccharide units in the oligosaccharide chains is IdoA-al,4-GlcNS03(6S), and the preparations may be such that t.h~--HGF-binding affinity is not completely destroyed - by trea~men~ under depolymerising conditions with heparinase I. At least the majority of the oligosacc-. ~ haride chains may have substantially the same length as a resuIt of carrying out a size fractionation separation procedure, and in preferred embodiments at least the .mai~r-ity of the oligosaccharide chains each have a degree -o~ polymerisation tdp) of lO or more, but with the maximum size being no greater than ten disaccharide units in total. More preferably, ~he oligosaccharide chains each ha~e a degree of polymerisation (dp) of 12 or 14.
As already indicated, in preferred embodiments the ~-'O9~/~1689 ~ 3 31 , CT/GB91/0061;
oligosaccharide chains of the preparations of this invention are substantially completely resistant to further depolymerisation ~pon treatment under enzymic depolymerising conditions ~ith heparinase III (heparit-inase I). Also, the IdoA(2S) content, if any, of saidoligosaccharide chains will be less than the unsulphated IdoA content thereof, and in general the oligosaccharide chains will usually contain a relatively high proportion of 6-O-sulphated hexosamines compared to oligosaccharide chains of unmodified native heparan sulphate. Thus, it may be expected that the content of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 will usually be greater than 24%, for example about 35% or greater. More specifically, the GlcNS~3(6S) content of the oligosaccharide chains, i.e. number of residues per lOO disaccharides, is preferably at least 30% and may be ~ ~ 50~ or more.
- In at least most embodiments the structure of the oligosaccharide chains will include internal repeat æequences of IdoA(+2S) and GlcNS03(6S) interrupted by occasional GlcNAc(+6S) components, and in presently prefer-red ~ embodiments substantially all said oligo-saccharide chains consist~of a sequence of six or seven ~ 25 disaccharlde units in all.
" ~
,, ~ .
Oligosaccharide preparations~ of this invention will generally be obtainable by enzymic partial depolymerisa-tion' to the fullest extent of heparan sulphate using heparinase III (heparitinasè-~ I), followed by size fractionation using, for example, gel filtration size exclusion chromatography,_~an~ then, in respect of a selected fraction or fracti-ons recovered from the size fractionating stage, affinity chromatography using an HGF
growth factor as the immobilised ligand in order to separate out HGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient to fractionate said fragments in respect of -,~
-'O9~/21689 ~ 13 6 ~ PCT/GB9~/0061~
~GF binding affinity, followed by recovering the most strongly bound fragments and, optionally, further purifying the recovered product by carrying out at least one additional step of size fractionation and selection of recovered product. The heparan sulphate (HS) may be derived from human fibroblast heparan sulphate proteoglycan (HSPG) or any other suitable biological source.
The invention may also be defined as providing an oligosaccharide preparation made up of oligosaccharide chains having a specific binding affinity for human hepatocyte growth factor (HGF), characterised in that (a) it is composed predominantly of a molecular species:
: r 1 ~, x~Y~Z
in which X is nHexA-GlcNS03 Y is IdoA(+2S)-GlcR(+6S), : Z is IdoA-GlcR
where R is NSO3 or NAc, and n is in the range l to 5 .
with the proviso that when n is ~hree or more -then at least for the majorlty of said ; --mo~ecular species two or more of the GlcR3 residues in Y are N-sulphated glycosamines . I sulphated at C-6, i.e. GlcNSO3(6S); and 30 _~(b) it is obtainable by a process comprising the - - steps of digesting a heparan sulphate with . t heparinase III (heparitinase I) so as to bring - about partial depolymerisation thereof to the fullest extent, followed by size fractionating 35 ~ the oligosaccharide mixture produced using for ~ example gel filtration size exclusion -~ chromatography, collecting a fraction or fractions containing oligosaccharide chains :
09l/21689 ~ 1~ 6 5 31 PCT/GB9~/0061 having a part~cular size selected within the range of lO to 20 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using ~n im~.obilised HGF ligand and recovering the more strongly HGF-binding constituents by eluting under a salt gradient over a range of salt concentrations and collecting a selected fraction or fractions containing the bound material which desorbs only at the highest salt concentrations.
In at least most preferred embodiments the s~nbol Y
in the above structural formula will represent primarily or exclusively IdoA-GlcNSO3(+6S), and n is the range 3 to 5, preferably 4 or 5 so ~hat said molecular species consists of a total of six or seven disaccharide units in all. In addition, the content of glucosamine residues having a 6-O-sulphate group will be greater than 24%.
Usually, the content of IdoA12S), if any, will be small in these embodimentsO
The invention also provides an oligosaccharide preparation having a specific binding affinity for hepato-cyte growth factors (HGF's) that is substantially whollycomposed of oligosaccharide chains--which are twelve or fourteen monosaccharide residues -in ~length and which contain an internal se~uence comprising at least 2 di-saccharide units each consisting of an IdoA residue linked to a GlcNSO3(~6S) residue, -wlth more than 20~ of the glucosamine residues (terminal- or internal) bein~ 6-O-sulphated. In accordance .-~ith this aspect of the invention, substantially all: the oligosaccharide chains may have the following sequence nGlcA-GlcNSO3-[IdoA-GlcNSO3(+6S)]n~IdoA-GlcR
where R is NS03 or NAc, and n is 4 or 5.
In a further aspect, the present invention embraces a ~O9~/'1689 ~ 1 3 6 ~ ~ ~ PCT/GB9~/00615 method or process for obtaining oligosaccharides that have a particular binding affinity for hepatocyte growth factor, characterised in that partial depolymerisation products of heparan sulphate, produced by treatment with a selective scission reagent that cleaves the polysaccharide chains thereof selectively in regions of relatively low sulphation, are sub;ected to affinity chromatography using HGF as the immobilised ligand so as to separate out HGF-binding fragments, the more strongly binding constituents then being recovered by eluting under a salt gradient and collecting a selected fraction or fractions containing the bound material which desorbs at the higher salt concen-trations. More specifically from this aspect the invention provides a method of isolating from heparan lS sulphate derived from heparan sulphate proteoglycan of mammalian cells low molecular weight oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for hepatocyte growth factor, - said method comprising the steps of (a) preparing an affinity chromatographic matrix or substrate incorporating a sample of hepatocyte growth factor (HGF) as the affinity ligand immobilised thereon;
(b)- treating said heparan sulphate with a selective scission reagent so as to cleave the ---polysaccharide chains thereof -~electively in -regions of relatively low sulphation;
(c) subjecting the product of step (b) to size fractionation, for example by gel filtration . .
.~ - size exclusion chromatography, and collecting selectively therefrom fractions that appear to contain oligosaccharides composed of less than ten disaccharide units, (d) contacting the affinity chromatographic matrix 35 ~ or substrate from step (a) with a selected fraction, or set of fractions, from step (c) containing a specific num~er of disaccharide units in the range of five to seven in order to g~/21689 ~ 6 ~ 3 ~ PCT/GB9-1/00615 extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the heparan sulphate glycosaminoglycan that have at least some binding affinity for the immobilised HGF;
(e) eluting the affinity chromatographic matrix or substrate using a progressively increasing salt concentration or gradient in the eluant;
. (f) collecting the fraction or set of fractions containing oligosaccharide fragments eluting in selected highest ranges of eluant salt concentration; and optionally, (g) further purifying the product of the selected fraction, or set of fractions, from step (f)~by selectively repeating step (c) using said selected fraction or set of fractions collected in step (f) instead of the reaction mixture obtained from step (b), and optionally also repeating steps (d), (e) and (f).
In carrying out the above-specified method it will be appreciated that preferably the selective scission reagent is heparinase III (heparitinase I) and the heparan sulphate is partially depolymerised-to the fullest extent by digesting therewith until cleavage of the heparitase III sensitive linkages is completQ~ Also, the fractions collected from the size ~ractionation stage will preferably be those that appear to contain oligo-saccharides composed of six or seven disaccharide units.
The oligosaccharide preparations are applicable for therapeutic use, acting as--an HGF-activity modulating agent for controlling - OE -- reducing cell growth, proliferation or migration in treating mammals in need of such treatment. Thus, the invention also provides pharmaceutical formulations or compositions for medical use comprising a therapeutically effective non-toxic amount of an HGF-activity modulating agent comprising an ~091/21689 PCT/GB9~/0061~
X1'~ 1 10 oligosaccharide preparation as herein specified, or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier or vehicle.
A pharmaceutical composition or formulation in accordance with the invention for use in controlling the activity of hepatocyte growth factors in mammals may also be defined as comprising a therapeutically useful amount of an essentially pure oligosaccharide preparation having a specific binding affinity for hepatocyte growth actors (HGF's), consisting essentially of linear oligosaccharide chains which are substantially homogeneous with respect to HGF binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of disaccharide units each composed of an N-sulphated glucosamine residue (+6S) and an unsulphated iduronic acid residue.
The invention will be further described, with reference to the accompanying drawings, in relation to some of .the background experimental work carried out by the inventors which brings out various further features of the invention and illustrates the way in which HGF-binding oligos~c~harides in accordance with the invention may be isolated and characterised. Accordingly, from this description. th~.. skilled person in the art will more readily be abIe-to-appreciate the nature of the invention and will more- readily be able to put it into practical effect.
.
BRIEF DESCRIPTION OF THE DRAWINGS:
.
FIGURE -l~ This shows the comparative affinities of 3H- I
heparin (panel A), 3H35S-liver HSPG (panel B) and 3H35S-liver- HS chains (panel C) for an HGF affinity column, samples being applied in 0.15M NaCl and eluted with a step gradient of 0.2 - l.0M NaCl as shown by the arrows in panel A.
~0 91/21689 ' I 3 6 5 31 PCT/GB9~10061;
FIGURE 2: This shows the effect of various specific modifications or depolymerlsations of fibroblast HS on its affinity for HGF. Samples of 3H-labelled fibroblast HS, intact (panel A) or after low pH nitrous acid degradation (panel B), or after solvolytic de-N-sulphation/re-N-acetylation (panel C), or after heparinase III digestion (panel D), or after heparinase I digestion (panel E), were applied to the HGF affinity column in 0.15M NaCl. Bound material was then eluted with a step gradient of 0.2 -l.OM NaCl as shown by the arrows in panel A.
FIGURE 3: This shows a size fractionation of heparinaseIII-resistant oligosaccharides. 3H-fibroblast HS was exhaustively digested with heparinase III and the digest was fractionated into its constituent oligosaccharide sizes by gel filtration chromatography on Bio-Gel PlO.
Oligosaccharide fractions corresponding to dp2 - dpl2/14 (where dp is the number of monosaccharide units) were individually recovered.
FIGURE 4: This shows the effect of HS oligosaccharide size on HGF affinity. 3H-Fibroblast HS was digested with heparinase III and size fractionated -on a Bio-Gel PlO
column. Fractions corresponding to origosaccharide sizes of dp6 (panel B), dp8 (panel C), dplO (panel D) and combined dpl2/14 (panel E) were tested for aff~nity to HGF
and compared with the affinity of intact parent HS (panel A). Samples were applied in 0.15M NaCl and bound material was step eluted with increasing 0.2 - l.OM NaCl concen-trations as shown by the arrows in panel-A.
~ORE DETAILED DESCRIPTION ~ -.
Background experimental work was carried out using,35 as source materials, recombinant ~human HGF which was purified from the culture medium of cells transfected with a plasmid containing the human HGF cDNA (see Nakamura et al, ( 1989 ) Nature 342, 440-443), and HSPG which was ~09l/2168g PCT/GB9~/00615 2136~
prepared- from both (a) culture medium from confluent cultures of a human foetal s~in fibroblast cell line biosynthetically radiolabelled with 3H-glucosamine (see Turnbull, J.E. and Gallagher, J.T. (1991), Biochem, J.
272, 553-559) and (b) rat livers biosynthetically radiolabelled in vivo with 3H-glucosamine and Na23~04 (see Lyon, M. and Gallagher, J.T. (1991), Biochem, J. 273, 415-422. From the HSPG, radiolabelled heparan sulphate (HS) chains were prepared by exhaustive proteolytic digestion with Pronase. Thus, in an example of one particular procedure HS chains were obtained from cultured foetal skin fibroblasts grown in MEM containing 10% (v/v) heat-inactivated donor calf serum (Gibco) and lmM glutamine.
Confluent cultures were metabolically radiolabelled with lO~Ci/ml of D-~6-3H~-glucosamine hydrochloride for 72 hours. The culture medium was removed and kept to one : side whilst the cell layers were extracted with 0.15M
NaCl, 20mM sodium phosphate, i~ (v/v) Triton X-100 pH 7.0 ~: for 1 hour at room temperature with agitation. The cell 20~layer extracts were recombined with the culture supernatants and the whole was digested with Pronase (lOO~g/ml) for 3 hours at 37C. The digest was heated to 100C for 5- minutes, clarified by centrifugation and then ~-~ applied to-a small DEAE-SephaceL column. This was washed extensively with 0.3M NaC1, 20mM sodium phosphate, 1 (v/v) Triton -X-lQ0- pH 7.0 after which the 3H-labelled ~`:` sulphated GAGs were--recovered by step elution with l.SM
- ~ NaCl, 20mM sodium phosphate, 1% (v/v) Triton X-100, pH
-:. 7.0,.l The recovered material (comprising mixed H5 and CS/DS) was.dialysed against 50mM NaCl, 50mM Tris HC1, pH
8.0, concentràted to approximately lml by reverse osmosis against po-}~(ethylene glycol) and then digested with O.lunit/mi ~-chondroitinase ABC for 4 hours at 37C. The -` intact HS chains were recovered by re-application of the ~: :35 digest to a small DEAE-Sephacel column, which was eluted -: as described above but omitting the Triton X-lO0. The 3H-~ HS chains were precipitated from the 1.5M NaC1 eluant by Wog~/21689 ~-? 6 ~ 31 PCT/GB91/OOSl~
addition of 3 vols of 95% (v/v) ethanol, air-dried and re-dissolved in distilled water.
The HS chains were selectively depolymerised either with heparinases or low pH nitrous acid, using methods performed essentially as described in Turnbull and Gallagher (Biochem, J. (1991), 273, 553-559). Solvolytic N-desulphation of fibroblast HS, followed by re-N-acetylation with acetic anhydride was also performed using the method of Inoue and Nagasawa (Carbohydr. Res. (1976), _ , 87-95). The content of the above-mentioned papers are incorporated herein by reference.
Heparinase Enzymes The polysaccharide lyase enzyme heparinase (Flavobacterium heparinum; EC 4.2.2.7) referred to herein was supplied by Seikagaku Kogyo Co., Tokyo, Japan, but heparinase II (F. heparinum; no EC number assigned) and heparinase III (F. heparinium; EC 4.2.2.8) were from Grampian Enzymes of Aberdeen, Scotland. Heparinase III is in fact substantially the same as the enzyme supplied under the designation heparitinase I by Seikagaku Kogyo Co. Heparinase III (hepari-tinase:-I) will selectively cleave glycosidic linkages on the non-reducing side of GlcA-containing disaccharides, such as in GlcNAc-al,4-GlcA
present in regions of low sulpXation, but in general it will not cleave bonds of ~sulphated disaccharides containing L-iduronic acid or 2-sulphated L-iduronic acid, i.e. IdoA or IdoA(2S). This is in- contrast to the enzyme heparlnase I (EC 4.2.2.7) which cleaves glycosidic linkages of disaccharides cont=aining 2-sulphated L-iduronic acid. For a review of these enzymes see R J
Linhardt et al ( 1990) Biochemistry 29, 2611-2617.
In connection with the cleavage of polysaccharide or oligosaccharide glycosidic linkages, e.g. 1,4 linkages, by enzymes such as heparinase I and heparinase III, it should -O9~/21689 PCT/GB91/0061;
1~
incidentally be appreciated that in one of the fragments produced the monosaccharide residue at the non-reducing end which is immediately adjacent the cleaved bond will generally become unsaturated with a double-bond formed between C4 and C5. This unsaturation, however, is not likely to affect significantly the growth factor binding affinity of the fragment concerned, although it may perhaps affect stability of the molecule.
HGF - affinity chromatography In the course of this work, HGF binding affinity of the HSPG, HS and oligosaccharide HS depolymerisation products was investigated usin~ affinity chromatography with an HGF-affinity matrix or substrate, from which the HGF binding constituents were eluted and selectively recovered using a salt gradient.
For preparing the HGF affinity matrix, Affi-Gel lO
(RTM) activated affinity gel (from Bio-Rad Laboratories) was washed following the supplier's instructions. A
portion of the recombinant human HGF (lOO~g) was pre-mixed with an exce s of heparin (500,ug) in lOO,ul of coupling buffer (O.lM-HEPE~, 80mM NaCl, pH 7.0) and incubated for 20 minutes at room temperature. The mixture was then added to 300,ul of the washed Affi-Gel lO and the volume adjusted to lml -wit~ the coupling buffer. This was mixed .
end-over-end for lO minutes at room temperature before the addition of 0.5ml of lM ethanolamine to block remaining active groups on the gel. After a further l hour of mixing the geI-was transferred to a small column, washed extensively wi-th 1.5M NaCl, 20mM sodium phosphate pH 7.0, _ . ..
and then re-equilibrated in 0.15M NaCl, 20mM sodium phosphate, 0.2mM sodium azide pH 7Ø When not in use the column was stored in this solution at 4C. A control column was also prepared exactly as described above, but omitting the HGF.
WO9~/2168~ 6 ~ 31 PCT/GB9~/0061 In performing the affinity chromatography, the radiolabelled samples, diluted where necessary to an ionic strength <0.15M NaCl, were each applied to the column and recirculated a number of times e.g. at a flow rate of 0.5ml/min. and at room temperature, so as to maximise opportunity to bind to the HGF. The column was then washed with 5ml of 0.15M NaCl, 20mM sodium phosphate pH
7.0, followed sequentially with 5ml volumes of 0.2, 0.4, O.6, 0.8 and 1.OM NaCl in 20mM sodium phosphate pH 7Ø
When liver HSPG was chromatographed on the HGF column O.l~
(w/v) CHAPS was included in all the solutions. Fractions of lml were collected and monitored for radioactivity.
In carrying out the selective depolymerisation operations, enzymatic digestions of HS with either heparinase I or heparinase III were performed with ~ additions of 20mIU/ml of enzyme in O.lM sodium acetate, O.lmM calcium acetate, lmg bovine serum albumin/ml, pH 7.0 at 37~C. In order to ensure maximum breakdown of the HS
three additions of enzyme were made over an 18 hour periad. For the de-N-sulphation of HS this was carried - out by solvolysis of the pyridinium salt in 95% (v/v) dimethyl sulphoxide/5~ (v/v) methanol,- followed by acetylation of the resulting free amine gr~ups with acetic anhydride in accordance with the method of Inoue and Nagasawa previously referred to. - _ To prepare the HS oligosaccharides, 3H fibroblast HS
; was degraded with heparinase III as described above. The digest was then separated into its- - constituent oligosaccharide size fractions by gel filtration chromatography on a Bio-Gel PlO column ~lxll5cm) eluted with 0.2M NH4HC03 at a flow rate of 5ml/-hr.- The peaks corresponding to oligosaccharides from dp2 to a combined dpl2/14 fraction were individually pooled and repeatedly lyophilised to remove the NH4HC03.
~091l21689 ~ ~ 3 1 l6 PCT/GB91/0061 Disaccharide composition of HS oligosaccharides -Disaccharide compositions of specific HS
oligosaccharide fractions recovered from the affinity chromatography stage were analysed after exhaustive digestion and complete depolymerisation with a combination of the enzymes heparina~e I, II and III. The digestion mixture was generally mada up of 20mIU/ml each of heparinases I, II and III in 0.lM sodium acetate, 0.lmM
calcium acetate, lmg bovine serum albumin/ml pH 7.0 at 37C. Three additions of enzymes were made over an 18 hour digestion period. The digest was then chromatographed on a Bio-Gel P2 column (l x lllcm) eluted with 0.2M NH4~CO3 at a flow rate of 4ml/hr. Fractions corresponding to disaccharides were pooled, repeatedly lyophilised and finally re-dissolved in distilled water adjusted to pH 3.5 by the addition of HCl. Samples were then injected onto a Spherisorb (RTM) 5~m SAX (strong anion-exchange) column (Technicol, Stockport, UK) linked to a Dionex HPLC system. The column was washed with 5ml of acidified water pH 3.5 followed by elution of the constituent disaccharides with a 40ml gradient of 0-0.75M
- NaCl, pH 3.5 at a flow rate of lml/min. The eluant was monitored with~~n on--line Radiomatic Flo-One/Beta Series A-200 radioactivity detector (Canberra Packard) using a 0.5ml flow-through liquid cell and a scintillant:sample - ratio of 3:l. ~The - identities of the constituent disaccharides were determined by comparison with the elution positions of eight known disaccharide standards monitored by W detéction at 232nm.
. . ~ , . "
Interaction of intact and partially depolymerised HS with . .
HGF affinity column--It was f~und that liver HSPG bound strongly to the HGF affinity column with the majority of the bound material requiring 0.6 and 0.8M NaCl for eluting as shown in FIGURE 18. The abundant unbound fraction would also ~'09~/21689 ~1 3 6 5 31 PCT/GB94/006t;
bind if re-applied and was due to. overloading of the column. Pronase-released HS chains also bound strongly, although the proportion that eluted at the higher step (0.8M NaCl) was reduced (see FIGURE lC). It is believed that the higher affinity of the intact HSPG may reflect the polyvalency of the HSPG and the greater possibility of bridging more than one immobilised HGF molecule.
Nevertheless, the similar affinities of the HSPG and HS
chains demonstrated that the affinity resides in the HS
moiety with little, if any, contribution from the protein core. In comparison, commercial 3H-heparin was found to bind with an apparen~ affinity similar to that of the intact HSPG (see FIGURE lA).
Identification of_the ma~or structural determ~inants for HS
binding to HGF
Elucidation of the principal structural determinants for interaction with HGF was undertaken by comparison of the effects of various specific chemical and enzymic modifications or depolymerisations on the ability of fibroblast HS (which binds to HGF with characteristics similar to liver HS) to bind to the HGF-affinity column.
~ Deaminitive scission with low pH nitrous--~acid, which :- -25 specifically cleaves N-sulphated disaccharides with concomitant loss of the N-sulphate group, ...completely abolished binding to the column (see FIGURE-23--and-compare with FIGURE 2A). This demonstrated the inability of the nitrous acid resistant, mainly non-sulphated, blocks of GlcA- GlcNAc to support interaction and- suggested a .requirement for N-sulphate groups. However, alternative chemical de-N-sulphation of HS by -solvolysis twith replacement of the N-sulphates by N-acetyl groups), without the concomitant depolymerisation of the HS chain that occurs with nitrous acid, had relatively little effect on the HGF binding ( see FIGURE 2C), elution occuring at just one step lower, i.e. 0.6M, compared to O.8M NaCl for the native unmodified HS. This indicated ~O9~/21689 ~. 3 ~ 5 3 1 PCT/GBg~/00615 that N-sulphates per se make no more than a minor contribution to the binding activity and that the major binding determinants are other structural features spatially associated with the N-sulphation or GlcNSO3 residues in such a way as to be similarly disrupted by nitrous acid depolymerisation treatment. Since both iduronate residues IdoA(+2S) and 6-0-sulphated hexosamines are biosynthetically linked to the presence of N-sulphate, it was deduced that these are likely to provide the major binding detarminants.
Further elucidation was gained from the analysis of individual enzymic depolymerisations with heparinases I
and III. Heparinase I, which specifically cleaves N-sulphated disaccharides containing IdoA(2S) residues,especially GlcNSO3(+6S)-IdoA(2S), generated relatively large resistant fragments from fibroblast HS which would have internal sequences containing non-sulphated GlcA/IdoA. However, the treatment had relatively little effect on HGF binding with most material eluting at 0.4M
and 0.6M NaCl (see FIGURE 2E). This indicated that interaction with HGF does not require clusters or contiguous sequences of two or more disaccharides containing IdoA(2S)- residues. In comparison, digestion with heparinase III, which cleaves HS in regions of low sulphation so as -to excise and depolymerise nearly all GlcA-containing disaccharides (mainly GlcNAc-GlcA) to give rise to enzyme resistant oligosaccharide sequences which are of a generally smaller size than with heparinase I
(being made up- -primarily of contiguous sequences of GlcNSO3-IdoA with- variable O-sulphation), generated a more complex pattern-~see FIGURE 2D) with fragments of mixed HGF affinities.----Here, the majority of the material did not bind, but that which did eluted predominantly at 0.4M
and 0.6M with only a small amount at 0.2M. Since Heparinase III-resistant oligosaccharides will be enriched in IdoA(+2S), in the light of previous observations including the fact that HGF binding is not particularly ~09~/21689 1 3 fi ~ 3 1 PCTlGBg~/00615 sensitive to heparinase I, this result further indicates that non-sulphated IdoA and/or 6-0-sulphates are the most important determinants for HGF binding.
Analysis of HGF-binding oligosaccharides The various HGF-binding fractions from a heparinase III digest of 3H-fibroblast HS (FIGURE 2D) were analysed for their relative size distribution by gel filtration exclusion chromatography on Bio-Gel P10 (not shown). It was found that the non-binding ( O.l5M NaCl) and weakly bound (0.2M NaCl) frac~ions comprised predominantly dp2-4 oligosaccharides. In contrast, the medium (0.4M NaCl) and high (0.6M NaCl) affinity fractions contained oligosacch-arides of dp6-10 and dp>10 (mostly dpl2) respectively.
Size dependence was analysed in more depth by collecting individual oligosaccharide fractions from a preparative gel filtration chromatography fractionation of a large-scale heparinase III digest of 3H-fibroblast HS.
The digest was fractionated on Bio-Gel P10 into oligo-saccharides ranging in size from dp2 to a mixed dpl2/14 fraction (see Fig. 3). These oligosaccharide fractions were individually assayed for HGF-bindi~g activity. The general trend was for HGF affinity to increase with oligosaccharide size (see FIGURE 4). Dp2 and dp4 oligo-saccharides did not bind to HGF in Q.15M NaCl--(data not shown). The smallest oligosaccharides which exhibited some binding to HGF at NaCl concentrations above 0.15M
NaCl were dp6 in which a small proportion -of oligosacc-harides eluted with 0.4M NaCl (see Fig. 4B). The ma~ority of octasaccharide-~ (dp-8) eluted with 0.4M--NaCl (see Fig.
4C). Only with dplO, and more substa~t~ially with the dpl2/14 fraction, was higher affinity demonstrated such that 0.6M NaCl was required for elution (Figs. 4D and E
I respectively). Thus, oligosaccharides within the dplO-12 ¦ size range probably comprise the smallest high affinity ~ HGF-binding oligosaccharides.
~:, t~
~'O9ll~1689 PCT/GB94/0061~
~13(~31 The disaccharide compositions of dplO and dpl2/14 oligosaccharides with different binding affinities were further analysed to more positively identify structural features correlating with HGF affinity. Oligosaccharides fractions recovered from the HGF column (Figs. 4D and 4E) were depolymerised by digesting using a combination of heparinases I, II and III and the resulting disaccharides were recovered by Bio-Gel P2 gel filtraticn chromato-graphy. The fractions were then analysed and identified using a SAX-HPLC column calibrated with known disaccharide standards.
These analyses, of which the results are set out in Table 1, showed that although the total content of both GlcNS03 and IdoA(2S) increased slightly with increasing HGF affinity, a most dramatic correlation appeared in the content of 6-O-sulphates, in particular the GlcNSO3(6S) - residues. In the dplO oligosaccharides the percentage of 6-O-sulphation was 18.4%, 29.8~ and 50.6~ in the low (0.2M), medium (0.4M) and high (0.6M) affinity fractions respectively. Similarly, in the dpl2/14 oligosaccharides the corresponding medium and high affinity fractions contained 24.2%~and 36.8% 6-O-sulphates respectively. As might be expected, these increases were only associated with N-sulphated disaccharides (primarily nUA-GlcNSO3(6S) and nUA(2S)-GlcNSO3(-6~)-disaccharides), and not with N-acetylated (6S) disaccharides whose abundance remained relatively constant. These two N-sulphated(6S) di-saccharides would be expected to contain IdoA or IdoA(2S) respectively in the original oligosaccharides and to be located internally. The N-acetylated disaccharides would be expected to contain GlcA and could be derived from the reducing or n~n-reducing end (i.e. the sites of heparinase III cleavage), or might possibly be in an internal position where its environment may impart resistance to the enzyme.
It has thus been found not only that HS (and HSPG) Og~/~1689 r3~ 1 3 ~ PCT/GB9~/0061 does bind to HGF under physiologic~l conditions of pH and ionic strength, but Heparinase III digestion of HS will excise oligosaccharides which still retain most of the affinity or HGF. The smallest such oligosaccharides found to have the high HGF affinity are decasaccharides (dplO), although the minimum binding sequence (minimum core sequence which retains useful high affinity) could perhaps be shorter than this. Heparinase III-resistant sequences comprise mainly IdoA-containing disaccharides (with or without 2-sulphation) except for the non-reducing terminal, but non-sulphated IdoA residues appear to be important structural determinants of the HGF interaction.
Clusters of IdoA(2S) residues are clearly not essential though there is some possibility that single residues may give some enhancement of binding. Heparinase III-resistant oligosaccharides will contain GlcNSO3 residues internally, but the results of the desulphation experiment indicate that these are not absolutely essential for the interaction and make only a modest contribution to the 20 ~binding process. In these oligosaccharides, GlcNSO3 residues~ will, by necessity, be present in combination wlth IdoA(~2S) residues as the latter can only be introduced into the polysaccharide adjacent to existing G1cNSO3 residues. In addition, it- is clear from the 25~ dlsaccharide compositions of HS oligosaccharides with differing~ affinities for HGF that there i~ a strong ~~~r ~ correlation~ between the presence of 6-`O-su~phation (of GlcNSO3~, although presumably GlcNAc would sùffice) and high affinity. It is therefore deduced that HGF~binding to HS requires repeat sequences of IdoA(-+2S) - GlcNSO3(6S) disaccharides occuring in oligosaccharides of dp~lO, as prepared from HS by partial depo~ymerisation with heparinase III. Moreover, although--IdoA- residues are ;; considered to be essential IdoAt2S) residues may be regarded as optional for binding a ffinity.
It wilI accordingly be appreciated that oligo-~; - saccharide preparations with a specific HGF-binding O9~/21689 PCT/GB91/0061~
~1~6~31 22 affinity have been obtained which are composed predom-inantly of oligosaccharide chains possessing one or more of the following features:
(a) a degree of polymerisation (dp) of at least '0 (preferably lO, 12 or 14, but not greater than 20);
(b) heparinase III resistance;
(c) HGF-binding affinity not destroyed by heparinase I, (d) a relatively high proportion of 6-O-sulphated hexosamines;
(e) a structure that includes (preferably internally) repeat sequences (not necessarily all arranged contiguously) of IdoA(+2S)-GlcNSO3(6S), possibly interrupted by occasional GlcNAc(+6S) components;
(f) an IdoA(2S) content, if any, which is less than the unsulphated IdoA content;
(g) a GlcNSO3(6S) content which is greater than 24~, for example about 30~ or preferably greater, up to say about 50% or more.
In practice, to produce the HGF-binding oligo-saccharide products or preparations of the present invention, the same basic techniques can be used as described abo~e in connection with the background experi-mental work.~- Thus-,- using a purified heparan sulphate as a starting material, this can be partially depolymerised by treatment with hep~rinase III (or other equivalent selective scission -reagent) and subjected to affinity chromatography- using an HGF-affinity matrix or substrate and eluting under a salt gradient, then selectively collecting fracttons eluting at the higher salt concent-rations to recover the material having the highest ~HGF-binding affinit-y, thereby providing a preparation of relatively shor-t- oligosaccharides which is substantially homogeneous with respect to HGF-binding affinity. By combining -the affini~y chromatography with a preceding and/or subsequent stage of gel filtration size exclusion chromatography and selecting fractions corresponding to a particular size or sizes, preferably dp>lO up to, say, ~091/~1689 ~1 ~ 6 ~ ~ 1 PCT/GB9~/00615 dp=20, and if desired carrying out further purifications by repeating these stages and/or using other purification methods such as SAX HPLC chromatography or gradient PAGE
for example, well defined and pu~ified preparations of the oligosaccharide products can be obtained which are substantially homogeneous both with respect to HGF-binding affinity and oligosaccharide chain size.
It is, however, also envisaged that sources other than heparan sulphate (or HSPG's), even for example heparin using an appropriate selective scission reagent for depolymerisation, may be used. Moreover, it may be possible to prepare equivalent HGF-binding oligo-saccharides synthetically.
Since the oligosaccharides or preparations thereof in accordance with the invention can have a well defined composition and are readily capable of further purificat-ion if necessary, and considering also their relatively small sizes and specific HGF growth factor binding affinity, they can be very well suited for pharmaceutical use to exploit a potential in the field of medicine, e.g.
as hepatocyte growth factor inhibitors or activators and --~ mobilising agents. Accordingly,-they are expected to have valuable applications as therapeutic drugs, particularly for controlling or regulating the activity of HGF. This may arise for example where there is à~ need to control or modulate HGF-activity dependent cell growth and prolifer-ation or migration in clinical treatment of various conditions. For these purposes,~ ~he oligosaccharide products (or pharmaceutically-acceptable salts thereof) may be made up into pharmaceutical formulations as re~uired, and such uses are also-with-in- the scope of the invention.
As will be seen, the invention provides a number of different aspects and, in general, it embraces all novel and inventive features and aspects, including novel ~-'O9~/21689 PCT/GB9~/0061S
_ HEPATOCYTE GROWTH FACTOR BINDING AFFINITY
The present invention relates to certain novel oligosaccharide products and preparations thereof, useful in the field of biochemistry and medicine, which have particular binding affinity for cer~ain growth factors or cytokines, in particular hepatccyte growth factor (XGF).
BACKGROUND
Various growth factors, often structurally unrelated, are characterised by a strong affinity for heparin. One such growth factor is hepatocyte growth factor (HGF), al;so known as "scatter factor". HGF is an unusually large (82kDa) and structurally complex growth factor that is synthesised as a biologically inactive single chain precursor. This is then proteolytically cleaved at a single site between linked cysteine residues giving rise to a disulphide-bonded heterodimer comprising a large a-chain (54kDa) containing a hairpin loop close to the N-terminus and a sequence of four Kringle domains, together with a smaller ~-chain (26kDa). HGF is produced by various cells including fibroblasts, smooth muscle cells, kidney mesangial cells and liver non-parenchymal cells.
Its target cells are primarily epithelial cells, although it also acts on endothelial cells, hepatocytes and melanocy~es. It is believed to play an important role as a paracrine mediator of epithelial-mesenchymal inter-actions. Cellular responses, however, to HGF are complexand, as well as being mitogenic, it can also stimulate cell migration and morphogenesis depending on the cellular target and its milieu Interestingly, it can have an anti-proliferative effect on some tumour cells, including hepatoma cells, in vitro. It is likely that HGF is an important factor in embryonic organ development. In the adult it has been demonstrated to have a major role in the regenerat~on of damaged organs, such as liver and kidney.
~!0 91121689 ~ 3 6 ~ 3 1 PCT/GB94/0061 The cellular signal response to HGF appears to be mediated by binding (probably through the N-terminal part of the a-chain) to a single high affinity (Kd about 25pM) tyrosine kinase receptor, the product of the c-Met proto-oncogene. However, it has been demonstrated, at leastwith cultured cells, that a much larger number of lower affinity HGF binding sites (Kd about 350-400pM) also exist on the cell surface. It is thought possible that these lower affinity binding sites involve cell surface heparan sulphate proteoglycans, and that HGF interacts with the heparan sulphate (~S) component of such proteoglycans to cause a conformational change leading to a modulation of HGF's interaction with the c-Met receptor protein, in a way that might be analogous to the known dependence of the bFGF-receptor interaction on prior activation of the bFGF
growth factor by heparan sulphate.
The~present invention is based on studies in which we have shown that HGF does in fact interact in vitro with ; 20 heparan ;sulphate. This has led to the isolation and at least partial chàracterisation of novel heparan sulphate oligosaccharides which exhibit significant binding ;affinity ~ for ~HGF and which have certain structural ~feat~res that contra~st with those of other known growth ~fa~ctor binding oligosaccharides.
AB3REVIA~10NS
Throughout the~present specification the following abbreviations are used:
heparan sulphate;
HSPG - - heparan sulphate proteoglycan;
HGF - hepatocyte growth factor;
35 ~dp - degree of polymerisation (e.g. for a disaccharide, dp-2, etc);
G~cA - ~-D-glucuronic acid (or glucuronate);
IdoA - a-L-iduronic acid (or iduronate);
~136~1 Og~ 689 PCT/GB9~/0061;
IdoA(2S) - a-~-iduronic acid 2-sulphate (or iduronate);
GlcNAc N-acetyl a-D-glucosamine;
GlcNAc(6S) - N-acetyl a-D-glucosamine 6-sulphate;
GlcNS~3 - N-sulphated a-D-glucosamine;
GlcNSO3(6S)- N-sulphated ~-D-glucosamine 6-sulphate;
GlcNR - a-D-glucosamine with unspeciied N-substituent;
nUA - unsaturated uronic acid residue (e.g. nGlc for unsaturated D-glucuronic acid and nHex A
for unsaturated unspecified hexuronic acid residue);
SAX - strong-anion exchange;
HPLC - high performance liquid chromatography The symbols (+2S) and (+6S) are used to denote, respectively, that a residue may or may not be sulphated at the C2 or C6 position.
SUMMARY OF THE INVENTION.
.. . __. _ . . I
The invention provides novel oligosaccharides or preparations thereof which have a specific binding affinity for HGF.- Such oligosaccharides will generally be in the form of ~substantially homogenous preparations consisting of oligosaccharide chains composed of a sequence of at leas-t three-- disaccharide units (dp>6), preferably at least five disaccharide units (dp>lO), and including a plurality of disaccharide units which each contai~n an IdoA(~2S) and a GlcNSO3(~6S) residue and which preferably are arranged in between the terminal sugar residues of the oligosaccharide chains but not necessarily contiguously. In -~referred embodiments, the oligo-saccharide chains w~ generally also be resistant to further depolym~risation by heparinase III (heparitinase -EC 4.2.2.8), and ~ill be obtained from heparan sulphate orfrom other natural heparan type material.
More specifically, in one aspect the invention 3 ~
~09~/1689 ~ PCT/GB9~/0061 - consists in an oligosaccharide preparation obtainable from partially depolymerised heparan sulphate (HS) or other natural heparin ~ype material as a fraction thereof, characterised in that the oligosacchari.de preparation consists essentially of oligosaccharide chains which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of a sequence of at least three disaccharide units (dp>6~ that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(+2S) and an N-sulphated D-glucosamine residue GlcNS03(+6S).
In another aspect the invention can alternatively be defined as an oli~osaccharide preparation comprising heparan sulphate ~HS) fragments which have a specific binding affinity for hepatocyte growth factor (HGF~ and which are composed of oligosaccharide chains containing a : sequence of at least three disaccharide units (dp>6) that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(+2S) and an N-sulphated D-glucosamine residue GlcNS03(+6S).
~ ~ Preferably, one or more of the above-mentioned at least two disaccharide units in the oligosaccharide chains is IdoA-al,4-GlcNS03(6S), and the preparations may be such that t.h~--HGF-binding affinity is not completely destroyed - by trea~men~ under depolymerising conditions with heparinase I. At least the majority of the oligosacc-. ~ haride chains may have substantially the same length as a resuIt of carrying out a size fractionation separation procedure, and in preferred embodiments at least the .mai~r-ity of the oligosaccharide chains each have a degree -o~ polymerisation tdp) of lO or more, but with the maximum size being no greater than ten disaccharide units in total. More preferably, ~he oligosaccharide chains each ha~e a degree of polymerisation (dp) of 12 or 14.
As already indicated, in preferred embodiments the ~-'O9~/~1689 ~ 3 31 , CT/GB91/0061;
oligosaccharide chains of the preparations of this invention are substantially completely resistant to further depolymerisation ~pon treatment under enzymic depolymerising conditions ~ith heparinase III (heparit-inase I). Also, the IdoA(2S) content, if any, of saidoligosaccharide chains will be less than the unsulphated IdoA content thereof, and in general the oligosaccharide chains will usually contain a relatively high proportion of 6-O-sulphated hexosamines compared to oligosaccharide chains of unmodified native heparan sulphate. Thus, it may be expected that the content of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 will usually be greater than 24%, for example about 35% or greater. More specifically, the GlcNS~3(6S) content of the oligosaccharide chains, i.e. number of residues per lOO disaccharides, is preferably at least 30% and may be ~ ~ 50~ or more.
- In at least most embodiments the structure of the oligosaccharide chains will include internal repeat æequences of IdoA(+2S) and GlcNS03(6S) interrupted by occasional GlcNAc(+6S) components, and in presently prefer-red ~ embodiments substantially all said oligo-saccharide chains consist~of a sequence of six or seven ~ 25 disaccharlde units in all.
" ~
,, ~ .
Oligosaccharide preparations~ of this invention will generally be obtainable by enzymic partial depolymerisa-tion' to the fullest extent of heparan sulphate using heparinase III (heparitinasè-~ I), followed by size fractionation using, for example, gel filtration size exclusion chromatography,_~an~ then, in respect of a selected fraction or fracti-ons recovered from the size fractionating stage, affinity chromatography using an HGF
growth factor as the immobilised ligand in order to separate out HGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient to fractionate said fragments in respect of -,~
-'O9~/21689 ~ 13 6 ~ PCT/GB9~/0061~
~GF binding affinity, followed by recovering the most strongly bound fragments and, optionally, further purifying the recovered product by carrying out at least one additional step of size fractionation and selection of recovered product. The heparan sulphate (HS) may be derived from human fibroblast heparan sulphate proteoglycan (HSPG) or any other suitable biological source.
The invention may also be defined as providing an oligosaccharide preparation made up of oligosaccharide chains having a specific binding affinity for human hepatocyte growth factor (HGF), characterised in that (a) it is composed predominantly of a molecular species:
: r 1 ~, x~Y~Z
in which X is nHexA-GlcNS03 Y is IdoA(+2S)-GlcR(+6S), : Z is IdoA-GlcR
where R is NSO3 or NAc, and n is in the range l to 5 .
with the proviso that when n is ~hree or more -then at least for the majorlty of said ; --mo~ecular species two or more of the GlcR3 residues in Y are N-sulphated glycosamines . I sulphated at C-6, i.e. GlcNSO3(6S); and 30 _~(b) it is obtainable by a process comprising the - - steps of digesting a heparan sulphate with . t heparinase III (heparitinase I) so as to bring - about partial depolymerisation thereof to the fullest extent, followed by size fractionating 35 ~ the oligosaccharide mixture produced using for ~ example gel filtration size exclusion -~ chromatography, collecting a fraction or fractions containing oligosaccharide chains :
09l/21689 ~ 1~ 6 5 31 PCT/GB9~/0061 having a part~cular size selected within the range of lO to 20 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using ~n im~.obilised HGF ligand and recovering the more strongly HGF-binding constituents by eluting under a salt gradient over a range of salt concentrations and collecting a selected fraction or fractions containing the bound material which desorbs only at the highest salt concentrations.
In at least most preferred embodiments the s~nbol Y
in the above structural formula will represent primarily or exclusively IdoA-GlcNSO3(+6S), and n is the range 3 to 5, preferably 4 or 5 so ~hat said molecular species consists of a total of six or seven disaccharide units in all. In addition, the content of glucosamine residues having a 6-O-sulphate group will be greater than 24%.
Usually, the content of IdoA12S), if any, will be small in these embodimentsO
The invention also provides an oligosaccharide preparation having a specific binding affinity for hepato-cyte growth factors (HGF's) that is substantially whollycomposed of oligosaccharide chains--which are twelve or fourteen monosaccharide residues -in ~length and which contain an internal se~uence comprising at least 2 di-saccharide units each consisting of an IdoA residue linked to a GlcNSO3(~6S) residue, -wlth more than 20~ of the glucosamine residues (terminal- or internal) bein~ 6-O-sulphated. In accordance .-~ith this aspect of the invention, substantially all: the oligosaccharide chains may have the following sequence nGlcA-GlcNSO3-[IdoA-GlcNSO3(+6S)]n~IdoA-GlcR
where R is NS03 or NAc, and n is 4 or 5.
In a further aspect, the present invention embraces a ~O9~/'1689 ~ 1 3 6 ~ ~ ~ PCT/GB9~/00615 method or process for obtaining oligosaccharides that have a particular binding affinity for hepatocyte growth factor, characterised in that partial depolymerisation products of heparan sulphate, produced by treatment with a selective scission reagent that cleaves the polysaccharide chains thereof selectively in regions of relatively low sulphation, are sub;ected to affinity chromatography using HGF as the immobilised ligand so as to separate out HGF-binding fragments, the more strongly binding constituents then being recovered by eluting under a salt gradient and collecting a selected fraction or fractions containing the bound material which desorbs at the higher salt concen-trations. More specifically from this aspect the invention provides a method of isolating from heparan lS sulphate derived from heparan sulphate proteoglycan of mammalian cells low molecular weight oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for hepatocyte growth factor, - said method comprising the steps of (a) preparing an affinity chromatographic matrix or substrate incorporating a sample of hepatocyte growth factor (HGF) as the affinity ligand immobilised thereon;
(b)- treating said heparan sulphate with a selective scission reagent so as to cleave the ---polysaccharide chains thereof -~electively in -regions of relatively low sulphation;
(c) subjecting the product of step (b) to size fractionation, for example by gel filtration . .
.~ - size exclusion chromatography, and collecting selectively therefrom fractions that appear to contain oligosaccharides composed of less than ten disaccharide units, (d) contacting the affinity chromatographic matrix 35 ~ or substrate from step (a) with a selected fraction, or set of fractions, from step (c) containing a specific num~er of disaccharide units in the range of five to seven in order to g~/21689 ~ 6 ~ 3 ~ PCT/GB9-1/00615 extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the heparan sulphate glycosaminoglycan that have at least some binding affinity for the immobilised HGF;
(e) eluting the affinity chromatographic matrix or substrate using a progressively increasing salt concentration or gradient in the eluant;
. (f) collecting the fraction or set of fractions containing oligosaccharide fragments eluting in selected highest ranges of eluant salt concentration; and optionally, (g) further purifying the product of the selected fraction, or set of fractions, from step (f)~by selectively repeating step (c) using said selected fraction or set of fractions collected in step (f) instead of the reaction mixture obtained from step (b), and optionally also repeating steps (d), (e) and (f).
In carrying out the above-specified method it will be appreciated that preferably the selective scission reagent is heparinase III (heparitinase I) and the heparan sulphate is partially depolymerised-to the fullest extent by digesting therewith until cleavage of the heparitase III sensitive linkages is completQ~ Also, the fractions collected from the size ~ractionation stage will preferably be those that appear to contain oligo-saccharides composed of six or seven disaccharide units.
The oligosaccharide preparations are applicable for therapeutic use, acting as--an HGF-activity modulating agent for controlling - OE -- reducing cell growth, proliferation or migration in treating mammals in need of such treatment. Thus, the invention also provides pharmaceutical formulations or compositions for medical use comprising a therapeutically effective non-toxic amount of an HGF-activity modulating agent comprising an ~091/21689 PCT/GB9~/0061~
X1'~ 1 10 oligosaccharide preparation as herein specified, or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier or vehicle.
A pharmaceutical composition or formulation in accordance with the invention for use in controlling the activity of hepatocyte growth factors in mammals may also be defined as comprising a therapeutically useful amount of an essentially pure oligosaccharide preparation having a specific binding affinity for hepatocyte growth actors (HGF's), consisting essentially of linear oligosaccharide chains which are substantially homogeneous with respect to HGF binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of disaccharide units each composed of an N-sulphated glucosamine residue (+6S) and an unsulphated iduronic acid residue.
The invention will be further described, with reference to the accompanying drawings, in relation to some of .the background experimental work carried out by the inventors which brings out various further features of the invention and illustrates the way in which HGF-binding oligos~c~harides in accordance with the invention may be isolated and characterised. Accordingly, from this description. th~.. skilled person in the art will more readily be abIe-to-appreciate the nature of the invention and will more- readily be able to put it into practical effect.
.
BRIEF DESCRIPTION OF THE DRAWINGS:
.
FIGURE -l~ This shows the comparative affinities of 3H- I
heparin (panel A), 3H35S-liver HSPG (panel B) and 3H35S-liver- HS chains (panel C) for an HGF affinity column, samples being applied in 0.15M NaCl and eluted with a step gradient of 0.2 - l.0M NaCl as shown by the arrows in panel A.
~0 91/21689 ' I 3 6 5 31 PCT/GB9~10061;
FIGURE 2: This shows the effect of various specific modifications or depolymerlsations of fibroblast HS on its affinity for HGF. Samples of 3H-labelled fibroblast HS, intact (panel A) or after low pH nitrous acid degradation (panel B), or after solvolytic de-N-sulphation/re-N-acetylation (panel C), or after heparinase III digestion (panel D), or after heparinase I digestion (panel E), were applied to the HGF affinity column in 0.15M NaCl. Bound material was then eluted with a step gradient of 0.2 -l.OM NaCl as shown by the arrows in panel A.
FIGURE 3: This shows a size fractionation of heparinaseIII-resistant oligosaccharides. 3H-fibroblast HS was exhaustively digested with heparinase III and the digest was fractionated into its constituent oligosaccharide sizes by gel filtration chromatography on Bio-Gel PlO.
Oligosaccharide fractions corresponding to dp2 - dpl2/14 (where dp is the number of monosaccharide units) were individually recovered.
FIGURE 4: This shows the effect of HS oligosaccharide size on HGF affinity. 3H-Fibroblast HS was digested with heparinase III and size fractionated -on a Bio-Gel PlO
column. Fractions corresponding to origosaccharide sizes of dp6 (panel B), dp8 (panel C), dplO (panel D) and combined dpl2/14 (panel E) were tested for aff~nity to HGF
and compared with the affinity of intact parent HS (panel A). Samples were applied in 0.15M NaCl and bound material was step eluted with increasing 0.2 - l.OM NaCl concen-trations as shown by the arrows in panel-A.
~ORE DETAILED DESCRIPTION ~ -.
Background experimental work was carried out using,35 as source materials, recombinant ~human HGF which was purified from the culture medium of cells transfected with a plasmid containing the human HGF cDNA (see Nakamura et al, ( 1989 ) Nature 342, 440-443), and HSPG which was ~09l/2168g PCT/GB9~/00615 2136~
prepared- from both (a) culture medium from confluent cultures of a human foetal s~in fibroblast cell line biosynthetically radiolabelled with 3H-glucosamine (see Turnbull, J.E. and Gallagher, J.T. (1991), Biochem, J.
272, 553-559) and (b) rat livers biosynthetically radiolabelled in vivo with 3H-glucosamine and Na23~04 (see Lyon, M. and Gallagher, J.T. (1991), Biochem, J. 273, 415-422. From the HSPG, radiolabelled heparan sulphate (HS) chains were prepared by exhaustive proteolytic digestion with Pronase. Thus, in an example of one particular procedure HS chains were obtained from cultured foetal skin fibroblasts grown in MEM containing 10% (v/v) heat-inactivated donor calf serum (Gibco) and lmM glutamine.
Confluent cultures were metabolically radiolabelled with lO~Ci/ml of D-~6-3H~-glucosamine hydrochloride for 72 hours. The culture medium was removed and kept to one : side whilst the cell layers were extracted with 0.15M
NaCl, 20mM sodium phosphate, i~ (v/v) Triton X-100 pH 7.0 ~: for 1 hour at room temperature with agitation. The cell 20~layer extracts were recombined with the culture supernatants and the whole was digested with Pronase (lOO~g/ml) for 3 hours at 37C. The digest was heated to 100C for 5- minutes, clarified by centrifugation and then ~-~ applied to-a small DEAE-SephaceL column. This was washed extensively with 0.3M NaC1, 20mM sodium phosphate, 1 (v/v) Triton -X-lQ0- pH 7.0 after which the 3H-labelled ~`:` sulphated GAGs were--recovered by step elution with l.SM
- ~ NaCl, 20mM sodium phosphate, 1% (v/v) Triton X-100, pH
-:. 7.0,.l The recovered material (comprising mixed H5 and CS/DS) was.dialysed against 50mM NaCl, 50mM Tris HC1, pH
8.0, concentràted to approximately lml by reverse osmosis against po-}~(ethylene glycol) and then digested with O.lunit/mi ~-chondroitinase ABC for 4 hours at 37C. The -` intact HS chains were recovered by re-application of the ~: :35 digest to a small DEAE-Sephacel column, which was eluted -: as described above but omitting the Triton X-lO0. The 3H-~ HS chains were precipitated from the 1.5M NaC1 eluant by Wog~/21689 ~-? 6 ~ 31 PCT/GB91/OOSl~
addition of 3 vols of 95% (v/v) ethanol, air-dried and re-dissolved in distilled water.
The HS chains were selectively depolymerised either with heparinases or low pH nitrous acid, using methods performed essentially as described in Turnbull and Gallagher (Biochem, J. (1991), 273, 553-559). Solvolytic N-desulphation of fibroblast HS, followed by re-N-acetylation with acetic anhydride was also performed using the method of Inoue and Nagasawa (Carbohydr. Res. (1976), _ , 87-95). The content of the above-mentioned papers are incorporated herein by reference.
Heparinase Enzymes The polysaccharide lyase enzyme heparinase (Flavobacterium heparinum; EC 4.2.2.7) referred to herein was supplied by Seikagaku Kogyo Co., Tokyo, Japan, but heparinase II (F. heparinum; no EC number assigned) and heparinase III (F. heparinium; EC 4.2.2.8) were from Grampian Enzymes of Aberdeen, Scotland. Heparinase III is in fact substantially the same as the enzyme supplied under the designation heparitinase I by Seikagaku Kogyo Co. Heparinase III (hepari-tinase:-I) will selectively cleave glycosidic linkages on the non-reducing side of GlcA-containing disaccharides, such as in GlcNAc-al,4-GlcA
present in regions of low sulpXation, but in general it will not cleave bonds of ~sulphated disaccharides containing L-iduronic acid or 2-sulphated L-iduronic acid, i.e. IdoA or IdoA(2S). This is in- contrast to the enzyme heparlnase I (EC 4.2.2.7) which cleaves glycosidic linkages of disaccharides cont=aining 2-sulphated L-iduronic acid. For a review of these enzymes see R J
Linhardt et al ( 1990) Biochemistry 29, 2611-2617.
In connection with the cleavage of polysaccharide or oligosaccharide glycosidic linkages, e.g. 1,4 linkages, by enzymes such as heparinase I and heparinase III, it should -O9~/21689 PCT/GB91/0061;
1~
incidentally be appreciated that in one of the fragments produced the monosaccharide residue at the non-reducing end which is immediately adjacent the cleaved bond will generally become unsaturated with a double-bond formed between C4 and C5. This unsaturation, however, is not likely to affect significantly the growth factor binding affinity of the fragment concerned, although it may perhaps affect stability of the molecule.
HGF - affinity chromatography In the course of this work, HGF binding affinity of the HSPG, HS and oligosaccharide HS depolymerisation products was investigated usin~ affinity chromatography with an HGF-affinity matrix or substrate, from which the HGF binding constituents were eluted and selectively recovered using a salt gradient.
For preparing the HGF affinity matrix, Affi-Gel lO
(RTM) activated affinity gel (from Bio-Rad Laboratories) was washed following the supplier's instructions. A
portion of the recombinant human HGF (lOO~g) was pre-mixed with an exce s of heparin (500,ug) in lOO,ul of coupling buffer (O.lM-HEPE~, 80mM NaCl, pH 7.0) and incubated for 20 minutes at room temperature. The mixture was then added to 300,ul of the washed Affi-Gel lO and the volume adjusted to lml -wit~ the coupling buffer. This was mixed .
end-over-end for lO minutes at room temperature before the addition of 0.5ml of lM ethanolamine to block remaining active groups on the gel. After a further l hour of mixing the geI-was transferred to a small column, washed extensively wi-th 1.5M NaCl, 20mM sodium phosphate pH 7.0, _ . ..
and then re-equilibrated in 0.15M NaCl, 20mM sodium phosphate, 0.2mM sodium azide pH 7Ø When not in use the column was stored in this solution at 4C. A control column was also prepared exactly as described above, but omitting the HGF.
WO9~/2168~ 6 ~ 31 PCT/GB9~/0061 In performing the affinity chromatography, the radiolabelled samples, diluted where necessary to an ionic strength <0.15M NaCl, were each applied to the column and recirculated a number of times e.g. at a flow rate of 0.5ml/min. and at room temperature, so as to maximise opportunity to bind to the HGF. The column was then washed with 5ml of 0.15M NaCl, 20mM sodium phosphate pH
7.0, followed sequentially with 5ml volumes of 0.2, 0.4, O.6, 0.8 and 1.OM NaCl in 20mM sodium phosphate pH 7Ø
When liver HSPG was chromatographed on the HGF column O.l~
(w/v) CHAPS was included in all the solutions. Fractions of lml were collected and monitored for radioactivity.
In carrying out the selective depolymerisation operations, enzymatic digestions of HS with either heparinase I or heparinase III were performed with ~ additions of 20mIU/ml of enzyme in O.lM sodium acetate, O.lmM calcium acetate, lmg bovine serum albumin/ml, pH 7.0 at 37~C. In order to ensure maximum breakdown of the HS
three additions of enzyme were made over an 18 hour periad. For the de-N-sulphation of HS this was carried - out by solvolysis of the pyridinium salt in 95% (v/v) dimethyl sulphoxide/5~ (v/v) methanol,- followed by acetylation of the resulting free amine gr~ups with acetic anhydride in accordance with the method of Inoue and Nagasawa previously referred to. - _ To prepare the HS oligosaccharides, 3H fibroblast HS
; was degraded with heparinase III as described above. The digest was then separated into its- - constituent oligosaccharide size fractions by gel filtration chromatography on a Bio-Gel PlO column ~lxll5cm) eluted with 0.2M NH4HC03 at a flow rate of 5ml/-hr.- The peaks corresponding to oligosaccharides from dp2 to a combined dpl2/14 fraction were individually pooled and repeatedly lyophilised to remove the NH4HC03.
~091l21689 ~ ~ 3 1 l6 PCT/GB91/0061 Disaccharide composition of HS oligosaccharides -Disaccharide compositions of specific HS
oligosaccharide fractions recovered from the affinity chromatography stage were analysed after exhaustive digestion and complete depolymerisation with a combination of the enzymes heparina~e I, II and III. The digestion mixture was generally mada up of 20mIU/ml each of heparinases I, II and III in 0.lM sodium acetate, 0.lmM
calcium acetate, lmg bovine serum albumin/ml pH 7.0 at 37C. Three additions of enzymes were made over an 18 hour digestion period. The digest was then chromatographed on a Bio-Gel P2 column (l x lllcm) eluted with 0.2M NH4~CO3 at a flow rate of 4ml/hr. Fractions corresponding to disaccharides were pooled, repeatedly lyophilised and finally re-dissolved in distilled water adjusted to pH 3.5 by the addition of HCl. Samples were then injected onto a Spherisorb (RTM) 5~m SAX (strong anion-exchange) column (Technicol, Stockport, UK) linked to a Dionex HPLC system. The column was washed with 5ml of acidified water pH 3.5 followed by elution of the constituent disaccharides with a 40ml gradient of 0-0.75M
- NaCl, pH 3.5 at a flow rate of lml/min. The eluant was monitored with~~n on--line Radiomatic Flo-One/Beta Series A-200 radioactivity detector (Canberra Packard) using a 0.5ml flow-through liquid cell and a scintillant:sample - ratio of 3:l. ~The - identities of the constituent disaccharides were determined by comparison with the elution positions of eight known disaccharide standards monitored by W detéction at 232nm.
. . ~ , . "
Interaction of intact and partially depolymerised HS with . .
HGF affinity column--It was f~und that liver HSPG bound strongly to the HGF affinity column with the majority of the bound material requiring 0.6 and 0.8M NaCl for eluting as shown in FIGURE 18. The abundant unbound fraction would also ~'09~/21689 ~1 3 6 5 31 PCT/GB94/006t;
bind if re-applied and was due to. overloading of the column. Pronase-released HS chains also bound strongly, although the proportion that eluted at the higher step (0.8M NaCl) was reduced (see FIGURE lC). It is believed that the higher affinity of the intact HSPG may reflect the polyvalency of the HSPG and the greater possibility of bridging more than one immobilised HGF molecule.
Nevertheless, the similar affinities of the HSPG and HS
chains demonstrated that the affinity resides in the HS
moiety with little, if any, contribution from the protein core. In comparison, commercial 3H-heparin was found to bind with an apparen~ affinity similar to that of the intact HSPG (see FIGURE lA).
Identification of_the ma~or structural determ~inants for HS
binding to HGF
Elucidation of the principal structural determinants for interaction with HGF was undertaken by comparison of the effects of various specific chemical and enzymic modifications or depolymerisations on the ability of fibroblast HS (which binds to HGF with characteristics similar to liver HS) to bind to the HGF-affinity column.
~ Deaminitive scission with low pH nitrous--~acid, which :- -25 specifically cleaves N-sulphated disaccharides with concomitant loss of the N-sulphate group, ...completely abolished binding to the column (see FIGURE-23--and-compare with FIGURE 2A). This demonstrated the inability of the nitrous acid resistant, mainly non-sulphated, blocks of GlcA- GlcNAc to support interaction and- suggested a .requirement for N-sulphate groups. However, alternative chemical de-N-sulphation of HS by -solvolysis twith replacement of the N-sulphates by N-acetyl groups), without the concomitant depolymerisation of the HS chain that occurs with nitrous acid, had relatively little effect on the HGF binding ( see FIGURE 2C), elution occuring at just one step lower, i.e. 0.6M, compared to O.8M NaCl for the native unmodified HS. This indicated ~O9~/21689 ~. 3 ~ 5 3 1 PCT/GBg~/00615 that N-sulphates per se make no more than a minor contribution to the binding activity and that the major binding determinants are other structural features spatially associated with the N-sulphation or GlcNSO3 residues in such a way as to be similarly disrupted by nitrous acid depolymerisation treatment. Since both iduronate residues IdoA(+2S) and 6-0-sulphated hexosamines are biosynthetically linked to the presence of N-sulphate, it was deduced that these are likely to provide the major binding detarminants.
Further elucidation was gained from the analysis of individual enzymic depolymerisations with heparinases I
and III. Heparinase I, which specifically cleaves N-sulphated disaccharides containing IdoA(2S) residues,especially GlcNSO3(+6S)-IdoA(2S), generated relatively large resistant fragments from fibroblast HS which would have internal sequences containing non-sulphated GlcA/IdoA. However, the treatment had relatively little effect on HGF binding with most material eluting at 0.4M
and 0.6M NaCl (see FIGURE 2E). This indicated that interaction with HGF does not require clusters or contiguous sequences of two or more disaccharides containing IdoA(2S)- residues. In comparison, digestion with heparinase III, which cleaves HS in regions of low sulphation so as -to excise and depolymerise nearly all GlcA-containing disaccharides (mainly GlcNAc-GlcA) to give rise to enzyme resistant oligosaccharide sequences which are of a generally smaller size than with heparinase I
(being made up- -primarily of contiguous sequences of GlcNSO3-IdoA with- variable O-sulphation), generated a more complex pattern-~see FIGURE 2D) with fragments of mixed HGF affinities.----Here, the majority of the material did not bind, but that which did eluted predominantly at 0.4M
and 0.6M with only a small amount at 0.2M. Since Heparinase III-resistant oligosaccharides will be enriched in IdoA(+2S), in the light of previous observations including the fact that HGF binding is not particularly ~09~/21689 1 3 fi ~ 3 1 PCTlGBg~/00615 sensitive to heparinase I, this result further indicates that non-sulphated IdoA and/or 6-0-sulphates are the most important determinants for HGF binding.
Analysis of HGF-binding oligosaccharides The various HGF-binding fractions from a heparinase III digest of 3H-fibroblast HS (FIGURE 2D) were analysed for their relative size distribution by gel filtration exclusion chromatography on Bio-Gel P10 (not shown). It was found that the non-binding ( O.l5M NaCl) and weakly bound (0.2M NaCl) frac~ions comprised predominantly dp2-4 oligosaccharides. In contrast, the medium (0.4M NaCl) and high (0.6M NaCl) affinity fractions contained oligosacch-arides of dp6-10 and dp>10 (mostly dpl2) respectively.
Size dependence was analysed in more depth by collecting individual oligosaccharide fractions from a preparative gel filtration chromatography fractionation of a large-scale heparinase III digest of 3H-fibroblast HS.
The digest was fractionated on Bio-Gel P10 into oligo-saccharides ranging in size from dp2 to a mixed dpl2/14 fraction (see Fig. 3). These oligosaccharide fractions were individually assayed for HGF-bindi~g activity. The general trend was for HGF affinity to increase with oligosaccharide size (see FIGURE 4). Dp2 and dp4 oligo-saccharides did not bind to HGF in Q.15M NaCl--(data not shown). The smallest oligosaccharides which exhibited some binding to HGF at NaCl concentrations above 0.15M
NaCl were dp6 in which a small proportion -of oligosacc-harides eluted with 0.4M NaCl (see Fig. 4B). The ma~ority of octasaccharide-~ (dp-8) eluted with 0.4M--NaCl (see Fig.
4C). Only with dplO, and more substa~t~ially with the dpl2/14 fraction, was higher affinity demonstrated such that 0.6M NaCl was required for elution (Figs. 4D and E
I respectively). Thus, oligosaccharides within the dplO-12 ¦ size range probably comprise the smallest high affinity ~ HGF-binding oligosaccharides.
~:, t~
~'O9ll~1689 PCT/GB94/0061~
~13(~31 The disaccharide compositions of dplO and dpl2/14 oligosaccharides with different binding affinities were further analysed to more positively identify structural features correlating with HGF affinity. Oligosaccharides fractions recovered from the HGF column (Figs. 4D and 4E) were depolymerised by digesting using a combination of heparinases I, II and III and the resulting disaccharides were recovered by Bio-Gel P2 gel filtraticn chromato-graphy. The fractions were then analysed and identified using a SAX-HPLC column calibrated with known disaccharide standards.
These analyses, of which the results are set out in Table 1, showed that although the total content of both GlcNS03 and IdoA(2S) increased slightly with increasing HGF affinity, a most dramatic correlation appeared in the content of 6-O-sulphates, in particular the GlcNSO3(6S) - residues. In the dplO oligosaccharides the percentage of 6-O-sulphation was 18.4%, 29.8~ and 50.6~ in the low (0.2M), medium (0.4M) and high (0.6M) affinity fractions respectively. Similarly, in the dpl2/14 oligosaccharides the corresponding medium and high affinity fractions contained 24.2%~and 36.8% 6-O-sulphates respectively. As might be expected, these increases were only associated with N-sulphated disaccharides (primarily nUA-GlcNSO3(6S) and nUA(2S)-GlcNSO3(-6~)-disaccharides), and not with N-acetylated (6S) disaccharides whose abundance remained relatively constant. These two N-sulphated(6S) di-saccharides would be expected to contain IdoA or IdoA(2S) respectively in the original oligosaccharides and to be located internally. The N-acetylated disaccharides would be expected to contain GlcA and could be derived from the reducing or n~n-reducing end (i.e. the sites of heparinase III cleavage), or might possibly be in an internal position where its environment may impart resistance to the enzyme.
It has thus been found not only that HS (and HSPG) Og~/~1689 r3~ 1 3 ~ PCT/GB9~/0061 does bind to HGF under physiologic~l conditions of pH and ionic strength, but Heparinase III digestion of HS will excise oligosaccharides which still retain most of the affinity or HGF. The smallest such oligosaccharides found to have the high HGF affinity are decasaccharides (dplO), although the minimum binding sequence (minimum core sequence which retains useful high affinity) could perhaps be shorter than this. Heparinase III-resistant sequences comprise mainly IdoA-containing disaccharides (with or without 2-sulphation) except for the non-reducing terminal, but non-sulphated IdoA residues appear to be important structural determinants of the HGF interaction.
Clusters of IdoA(2S) residues are clearly not essential though there is some possibility that single residues may give some enhancement of binding. Heparinase III-resistant oligosaccharides will contain GlcNSO3 residues internally, but the results of the desulphation experiment indicate that these are not absolutely essential for the interaction and make only a modest contribution to the 20 ~binding process. In these oligosaccharides, GlcNSO3 residues~ will, by necessity, be present in combination wlth IdoA(~2S) residues as the latter can only be introduced into the polysaccharide adjacent to existing G1cNSO3 residues. In addition, it- is clear from the 25~ dlsaccharide compositions of HS oligosaccharides with differing~ affinities for HGF that there i~ a strong ~~~r ~ correlation~ between the presence of 6-`O-su~phation (of GlcNSO3~, although presumably GlcNAc would sùffice) and high affinity. It is therefore deduced that HGF~binding to HS requires repeat sequences of IdoA(-+2S) - GlcNSO3(6S) disaccharides occuring in oligosaccharides of dp~lO, as prepared from HS by partial depo~ymerisation with heparinase III. Moreover, although--IdoA- residues are ;; considered to be essential IdoAt2S) residues may be regarded as optional for binding a ffinity.
It wilI accordingly be appreciated that oligo-~; - saccharide preparations with a specific HGF-binding O9~/21689 PCT/GB91/0061~
~1~6~31 22 affinity have been obtained which are composed predom-inantly of oligosaccharide chains possessing one or more of the following features:
(a) a degree of polymerisation (dp) of at least '0 (preferably lO, 12 or 14, but not greater than 20);
(b) heparinase III resistance;
(c) HGF-binding affinity not destroyed by heparinase I, (d) a relatively high proportion of 6-O-sulphated hexosamines;
(e) a structure that includes (preferably internally) repeat sequences (not necessarily all arranged contiguously) of IdoA(+2S)-GlcNSO3(6S), possibly interrupted by occasional GlcNAc(+6S) components;
(f) an IdoA(2S) content, if any, which is less than the unsulphated IdoA content;
(g) a GlcNSO3(6S) content which is greater than 24~, for example about 30~ or preferably greater, up to say about 50% or more.
In practice, to produce the HGF-binding oligo-saccharide products or preparations of the present invention, the same basic techniques can be used as described abo~e in connection with the background experi-mental work.~- Thus-,- using a purified heparan sulphate as a starting material, this can be partially depolymerised by treatment with hep~rinase III (or other equivalent selective scission -reagent) and subjected to affinity chromatography- using an HGF-affinity matrix or substrate and eluting under a salt gradient, then selectively collecting fracttons eluting at the higher salt concent-rations to recover the material having the highest ~HGF-binding affinit-y, thereby providing a preparation of relatively shor-t- oligosaccharides which is substantially homogeneous with respect to HGF-binding affinity. By combining -the affini~y chromatography with a preceding and/or subsequent stage of gel filtration size exclusion chromatography and selecting fractions corresponding to a particular size or sizes, preferably dp>lO up to, say, ~091/~1689 ~1 ~ 6 ~ ~ 1 PCT/GB9~/00615 dp=20, and if desired carrying out further purifications by repeating these stages and/or using other purification methods such as SAX HPLC chromatography or gradient PAGE
for example, well defined and pu~ified preparations of the oligosaccharide products can be obtained which are substantially homogeneous both with respect to HGF-binding affinity and oligosaccharide chain size.
It is, however, also envisaged that sources other than heparan sulphate (or HSPG's), even for example heparin using an appropriate selective scission reagent for depolymerisation, may be used. Moreover, it may be possible to prepare equivalent HGF-binding oligo-saccharides synthetically.
Since the oligosaccharides or preparations thereof in accordance with the invention can have a well defined composition and are readily capable of further purificat-ion if necessary, and considering also their relatively small sizes and specific HGF growth factor binding affinity, they can be very well suited for pharmaceutical use to exploit a potential in the field of medicine, e.g.
as hepatocyte growth factor inhibitors or activators and --~ mobilising agents. Accordingly,-they are expected to have valuable applications as therapeutic drugs, particularly for controlling or regulating the activity of HGF. This may arise for example where there is à~ need to control or modulate HGF-activity dependent cell growth and prolifer-ation or migration in clinical treatment of various conditions. For these purposes,~ ~he oligosaccharide products (or pharmaceutically-acceptable salts thereof) may be made up into pharmaceutical formulations as re~uired, and such uses are also-with-in- the scope of the invention.
As will be seen, the invention provides a number of different aspects and, in general, it embraces all novel and inventive features and aspects, including novel ~-'O9~/21689 PCT/GB9~/0061S
2 1 3 ~a 31 24 compounds, herein disclosed either expllcitly or implicit-ly and either singly or in combination with one another.
Moreover, the scope of the invention is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense.
: 20 .
: 25 .
-' . ' ' .
_ . . . ..
' ~O 9~/21689 ~ ~ 3 6 ~ ~1 PCT/GB9~/00615 _ TABLE I Disaccharide composition of HS oligosaccharides ~Ivith differing arfinilies for ~IGF
Hepannase III-resis~ant oligosacchandes of size dplO and dpl2/14 were f~ctionated byaf~lnity on HGF-Affigel. Oligosaccharide fraclions elutcd wi~h 0.2M, 0.4M and 0.6M ~laCI s~eps were recovered. Thesc were depolymerised using a combination of hcpannases and the resulling disaccharides were resolved by SAX-HPLC
~nd quantillcd.
Disaccharide % Total Disaccharides Stlueture Dpl0 oligosaccharides DplVl4 oligosac~har~desa elut~d w~lh: eluted wi~h:
0.2M 0.4M 0.6M 0.4M 0.6M
~HexA ~ GlcNAc 38.4 26.3 17.0 25.4 16.4 ~HcxA - G1c~Ac(6-OS03) 9.1 1 1.611.1 10.9 11.7 ~HexA - Glcl~SO3 28.3 21.6 14.9 23.2 19.8 ~HexA - Glrl~lSO3(6-OSO3) 6.3 11.9 22.9 8.9 15.3 ~IcxA(2-OSO3) - GlcNS03 14.9 22.3 12.4 27.1 24.8 ~FlcxA~2-OSO3)- GlcNSO3(6-OSO3) 3.0 6.3 16.6 4.4 9.8 unknownb . - 5.1 - 2.3 ~IS03/100 disaccharid~s S2.S 62.1 -66.8 ~ - ~63.6 69.7 2-OSO3J100 disaccharides 17.9 28.6 29.0 ~1.5 34.6 6-OSO3/100 disaccharides 18.4 29.8 $0.6 24.2 36.8 a There was insufrlcient of the 0.2M rracSlon ror analysis b Frorn ils elution posi~ion this is p~obably a disulpha~ed disaccharide species
Moreover, the scope of the invention is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense.
: 20 .
: 25 .
-' . ' ' .
_ . . . ..
' ~O 9~/21689 ~ ~ 3 6 ~ ~1 PCT/GB9~/00615 _ TABLE I Disaccharide composition of HS oligosaccharides ~Ivith differing arfinilies for ~IGF
Hepannase III-resis~ant oligosacchandes of size dplO and dpl2/14 were f~ctionated byaf~lnity on HGF-Affigel. Oligosaccharide fraclions elutcd wi~h 0.2M, 0.4M and 0.6M ~laCI s~eps were recovered. Thesc were depolymerised using a combination of hcpannases and the resulling disaccharides were resolved by SAX-HPLC
~nd quantillcd.
Disaccharide % Total Disaccharides Stlueture Dpl0 oligosaccharides DplVl4 oligosac~har~desa elut~d w~lh: eluted wi~h:
0.2M 0.4M 0.6M 0.4M 0.6M
~HexA ~ GlcNAc 38.4 26.3 17.0 25.4 16.4 ~HcxA - G1c~Ac(6-OS03) 9.1 1 1.611.1 10.9 11.7 ~HexA - Glcl~SO3 28.3 21.6 14.9 23.2 19.8 ~HexA - Glrl~lSO3(6-OSO3) 6.3 11.9 22.9 8.9 15.3 ~IcxA(2-OSO3) - GlcNS03 14.9 22.3 12.4 27.1 24.8 ~FlcxA~2-OSO3)- GlcNSO3(6-OSO3) 3.0 6.3 16.6 4.4 9.8 unknownb . - 5.1 - 2.3 ~IS03/100 disaccharid~s S2.S 62.1 -66.8 ~ - ~63.6 69.7 2-OSO3J100 disaccharides 17.9 28.6 29.0 ~1.5 34.6 6-OSO3/100 disaccharides 18.4 29.8 $0.6 24.2 36.8 a There was insufrlcient of the 0.2M rracSlon ror analysis b Frorn ils elution posi~ion this is p~obably a disulpha~ed disaccharide species
Claims (33)
1. An oligosaccharide preparation obtainable from partially depolymerised heparan sulphate (HS) or other natural heparin type material as a fraction thereof, characterised in that it consists essentially of oligosaccharide chains which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of a sequence of at least three disaccharide units (dp?6) that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(?2S) and an N-sulphated D-glucosamine residue G1cNSO3(?6S).
2. An oligosaccharide preparation comprising heparan sulphate (HS) fragments which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of oligosaccharide chains containing a sequence of at least three disaccharide units (dp?6) that includes at least two disaccharide units containing an L-iduronic acid residue IdoA(?2S) and an N-sulphated D-glucosamine residue GlcN5O3(?6S).
3. An oligosaccharide preparation as claimed in Claim 1 or 2 in which said disaccharide units containing the IdoA(?2S) and GlcNSO3(?6S) residues are disposed in between the terminal sugar residues of the oligosaccharide chains.
4. An oligosaccharide preparation as claimed in any of the preceding claims in which one or more of said at least two disaccharide units of the oligosaccharide chains is IdoA-.alpha.1,4-GlcNSO3(6S).
5. An oligosaccharide preparation as claimed in any of the preceding claims further characterised in that the HGF-binding affinity is not completely destroyed by treatment under depolymerising conditions with heparinase I.
6. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that at least the majority of the oligosaccharide chains each have substantially the same length as a result of carrying out a size fractionation separation procedure.
7. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that at least the majority of the oligosaccharide chains each have a degree of polymerisation (dp) of 10 or more.
8. An oligosaccharide preparation as claimed in any of the preceding claims in which said oligosaccharide chains consist of a sequence of not more than ten disaccharide units in total.
9. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that at least the majority of the oligosaccharide chains each have a degree of polymerisation (dp) of 12 or 14.
10. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that said oligosaccharide chains are substantially completely resistant to further depolymerisation upon treatment under enzymic depolymerising conditions with heparinase III
(heparitinase I).
(heparitinase I).
11. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that the IdoA(2S) content, if any, of said oligosaccharide chains is less than the unsulphated IdoA content thereof.
12. An oligosaccharide preparation as claimed in any of the preceding claims further characterised in that the oligosaccharide chains contain a relatively high proportion of 6-O-sulphated hexosamines compared to oligosaccharide chains of unmodified native heparan sulphate,
13. An oligosaccharide preparation as claimed in Claim 12, further characterised in that the GlcNSO3(6S) content of the oligosaccharide chains, i.e. number of residues per 100 disaccharides, is at least 30%.
14. An oligosaccharide preparation as claimed in Claim 12, further characterised in that the 6-O-sulphated hexosamine content of the oligosaccharide chains, i.e.
number of residues per 100 disaccharides, is 50% or more.
number of residues per 100 disaccharides, is 50% or more.
15. An oligosaccharide preparation as claimed in any of the preceding claims in which the content of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 is greater than 24%.
16. An oligosaccharide preparation as claimed in Claim 15 in which the content of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 is about 35% or greater.
17. An oligosaccharide preparation as claimed in any of the preceding claims, further characterised in that the structure of the oligosaccharide chains includes internal repeat sequences of IdoA(?2S) and GlcNSO3(6S) interrupted by occasional GlcNAc(?6S) components.
18. An oligosaccharide preparation as claimed in any of the preceding claims in which substantially all said oligosaccharide chains consist of a sequence of six or seven disaccharide units in all.
19. An oligosaccharide preparation as claimed in any of the preceding claims further characterised in that it is obtainable from heparan sulphate (HS) of human fibroblast heparan sulphate proteoglycan (HSPG) by enzymic partial depolymerisation to the fullest extent with heparinase III
(heparitinase I) followed by size fractionation, using for example gel filtration size exclusion chromatography, followed by, in respect of a selected fraction or fractions recovered from the size fractionating stage, affinity chromatography using an HGF growth factor as the immobilised ligand in order to separate out HGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient to fractionate said fragments in respect of HGF binding affinity, followed by recovering the most strongly bound fragments and, optionally, further purifying the recovered product by carrying out at least one additional step of size fractionation and selection of recovered product.
(heparitinase I) followed by size fractionation, using for example gel filtration size exclusion chromatography, followed by, in respect of a selected fraction or fractions recovered from the size fractionating stage, affinity chromatography using an HGF growth factor as the immobilised ligand in order to separate out HGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient to fractionate said fragments in respect of HGF binding affinity, followed by recovering the most strongly bound fragments and, optionally, further purifying the recovered product by carrying out at least one additional step of size fractionation and selection of recovered product.
20. An oligosaccharide preparation made up of oligo-saccharide chains having a specific binding affinity for human hepatocyte growth factor (HGF), characterised in that (a) it is composed predominantly of a molecular species:
in which X is ?HexA-GlcNSO3 Y is IdoA(?2S)-GlcR(?6S), Z is IdoA-GlcR
where R is NSO3 or NAc, and n is in the range 1 to 5 with the proviso that when n is three or more then at least for the majority of said molecular species two or more of the GlcR3 residues in Y are N-sulphated -glycosamines sulphated at C-6, i.e. GlcNSO3(6S);
(b) it is obtainable by a process comprising the steps of digesting a heparan sulphate with heparinase III (heparitinase I) so as to bring about partial depolymerisation thereof to the fullest extent, followed by size fractionating the oligosaccharide mixture produced using for example gel filtration size exclusion chroma-tography, collecting a fraction or fractions containing oligosaccharide chains having a particular size selected within the range of 10 to 20 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using an immobilised HGF ligand and recovering the more strongly HGF-binding constituents by eluting under a salt gradient over a range of salt concentrations and collecting a selected fraction or fractions containing the bound material which desorbs only at the highest salt concentrations.
in which X is ?HexA-GlcNSO3 Y is IdoA(?2S)-GlcR(?6S), Z is IdoA-GlcR
where R is NSO3 or NAc, and n is in the range 1 to 5 with the proviso that when n is three or more then at least for the majority of said molecular species two or more of the GlcR3 residues in Y are N-sulphated -glycosamines sulphated at C-6, i.e. GlcNSO3(6S);
(b) it is obtainable by a process comprising the steps of digesting a heparan sulphate with heparinase III (heparitinase I) so as to bring about partial depolymerisation thereof to the fullest extent, followed by size fractionating the oligosaccharide mixture produced using for example gel filtration size exclusion chroma-tography, collecting a fraction or fractions containing oligosaccharide chains having a particular size selected within the range of 10 to 20 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using an immobilised HGF ligand and recovering the more strongly HGF-binding constituents by eluting under a salt gradient over a range of salt concentrations and collecting a selected fraction or fractions containing the bound material which desorbs only at the highest salt concentrations.
21. An oligosaccharide preparation as claimed in Claim 20, wherein Y is primarily IdoA-GlcNSO3(?6S).
22. An oligosaccharide preparation as claimed in Claim 20 or 21, wherein n is the range 3 - 5.
23. An oligosaccharide preparation as claimed in Claim 22 wherein said molecular species consists of a total of six or seven disaccharide units in all.
24. An oligosaccharide preparation as claimed in any of Claims 20 to 23 in which the content of glucosamine residues having a 6-O-sulphate group is greater than 24%.
25. An oligosaccharide preparation having a specific binding affinity for hepatocyte growth factors (HGF's) and substantially all composed of oligosaccharide chains which are twelve or fourteen monosaccharide residues in length and which contain an internal sequence comprising at least 2 disaccharide units each consisting of an IdoA residue linked to a GlcNSO3(?6S) residue, with more than 20% of the glucosamine residues (terminal or internal) being 6-O-sulphated.
26. An oligosaccharide preparation as claimed in Claim 25 wherein substantially all the oligosaccharide chains have the following sequence ?GlcA-GlcNSO3-[IdoA-GlcNSO3(?6S)]n-IdoA-GlcR
where R is NSO3 or NAc, and n is 4 or 5.
where R is NSO3 or NAc, and n is 4 or 5.
27. A method of isolating from heparan sulphate derived from heparan sulphate proteoglycan of mammalian cells low molecular weight oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for hepatocyte growth factor, said method comprising the steps of (a) preparing an affinity chromatographic matrix or substrate incorporating a sample of hepatocyte growth factor (HGF) as the affinity ligand immobilised thereon;
(b) treating said heparan sulphate with a selective scission reagent so as to cleave the polysaccharide chains thereof selectively in regions of relatively low sulphation;
(c) subjecting the product of step (b) to size fractionation, for example by gel filtration size exclusion chromatography, and collecting selectively therefrom fractions that appear to contain oligosaccharides composed of less than ten disaccharide units, (d) contacting the affinity chromatographic matrix or substrate from step (a) with a selected fraction, or set of fractions, from step (c) containing a specific number of disaccharide units in the range of five to seven in order to extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the heparan sulphate glycosaminoglycan that have at least some binding affinity for the immobilised HGF;
(e) eluting the affinity chromatographic matrix or substrate using a progressively increasing salt concentration or gradient in the eluant;
(f) collecting the fraction or set of fractions containing oligosaccharide fragments eluting in selected highest ranges of eluant salt concentration; and optionally (g) further purifying the product of the selected fraction, or set of fractions, from step (f) by selectively repeating step (c) using said selected fraction or set of fractions collected in step (f) instead of the reaction mixture obtained from step (b), and optionally also repeating steps (d), (e) and (f).
(b) treating said heparan sulphate with a selective scission reagent so as to cleave the polysaccharide chains thereof selectively in regions of relatively low sulphation;
(c) subjecting the product of step (b) to size fractionation, for example by gel filtration size exclusion chromatography, and collecting selectively therefrom fractions that appear to contain oligosaccharides composed of less than ten disaccharide units, (d) contacting the affinity chromatographic matrix or substrate from step (a) with a selected fraction, or set of fractions, from step (c) containing a specific number of disaccharide units in the range of five to seven in order to extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the heparan sulphate glycosaminoglycan that have at least some binding affinity for the immobilised HGF;
(e) eluting the affinity chromatographic matrix or substrate using a progressively increasing salt concentration or gradient in the eluant;
(f) collecting the fraction or set of fractions containing oligosaccharide fragments eluting in selected highest ranges of eluant salt concentration; and optionally (g) further purifying the product of the selected fraction, or set of fractions, from step (f) by selectively repeating step (c) using said selected fraction or set of fractions collected in step (f) instead of the reaction mixture obtained from step (b), and optionally also repeating steps (d), (e) and (f).
28. A method as claimed in Claim 27 in which the selective scission reagent is heparinase III (heparitinase I) and the heparan sulphate is partially depolymerised to the fullest extent by digesting therewith until cleavage of the heparitase III sensitive linkages is complete.
29. A method as claimed in Claim 27 to 28, wherein the fractions collected from the size fractionation stage are those that appear to contain oligosaccharides composed of six or seven disaccharide units.
30. An oligosaccharide preparation as claimed in any one of Claims 1 to 26 for therapeutic use as an active HGF-activity modulating agent for controlling or reducing cell growth, proliferation or migration in treating mammals in need of such treatment.
31. A pharmaceutical formulation or composition for medical use comprising a therapeutically effective non-toxic amount of an HGF-activity modulating agent comprising an oligosaccharide preparation as claimed in any of Claims 1 to 26 or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier or vehicle.
32. An oligosaccharide preparation having a specific binding affinity for hepatocyte growth factors (HGF's), consisting essentially of linear oligosaccharide chains which are substantially homogeneous with respect to HGF
binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of disaccharide units each composed of an N-sulphated glucosamine residue (?6S) and an unsulphated iduronic acid residue.
binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of disaccharide units each composed of an N-sulphated glucosamine residue (?6S) and an unsulphated iduronic acid residue.
33. A pharmaceutical composition or formulation for use in controlling the activity of hepatocyte growth factors in mammals, said composition or formulation comprising a therapeutically useful amount of an essentially pure oligosaccharide preparation as claimed in Claim 32.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB939306255A GB9306255D0 (en) | 1993-03-25 | 1993-03-25 | Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity |
| GB9306255.2 | 1993-03-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2136531A1 true CA2136531A1 (en) | 1994-09-29 |
Family
ID=10732759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2136531 Abandoned CA2136531A1 (en) | 1993-03-25 | 1994-03-24 | Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0642533A1 (en) |
| JP (1) | JPH07507596A (en) |
| AU (1) | AU6287594A (en) |
| CA (1) | CA2136531A1 (en) |
| GB (1) | GB9306255D0 (en) |
| WO (1) | WO1994021689A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976534A (en) * | 1993-02-25 | 1999-11-02 | Zymogenetics, Inc. | Inhibition of intimal hyperplasia using antibodies to PDGF receptors and heparin |
| US5620687A (en) * | 1993-02-25 | 1997-04-15 | Zymogenetics, Inc. | Inhibition of intimal hyperplasia using antibodies to PDGF beta receptors |
| US6217863B1 (en) | 1995-10-30 | 2001-04-17 | Massachusetts Institute Of Technology | Rationally designed polysaccharide lyases derived from heparinase I |
| EP0861903A1 (en) * | 1997-02-22 | 1998-09-02 | Lansing, Manfred | Method for reversible immobilizing oligo and/or polysaccharides |
| US6861254B1 (en) | 1997-10-24 | 2005-03-01 | Massachusetts Institute Of Technology | Heparan sulfate D-glucosaminyl 3-O-sulfotransferases, and uses therefor |
| US7056504B1 (en) | 1998-08-27 | 2006-06-06 | Massachusetts Institute Of Technology | Rationally designed heparinases derived from heparinase I and II |
| AU3837300A (en) * | 1999-04-15 | 2000-11-02 | Takara Shuzo Co., Ltd. | Remedies |
| CN1192780C (en) * | 1999-04-15 | 2005-03-16 | 宝生物工程株式会社 | Therapeutical agent |
| KR100776956B1 (en) | 1999-04-15 | 2007-11-28 | 다카라 바이오 가부시키가이샤 | Remedies |
| CA2643162C (en) | 1999-04-23 | 2018-01-02 | Massachusetts Institute Of Technology | Polymer identification, compositional analysis and sequencing, based on property comparison |
| WO2001066772A2 (en) | 2000-03-08 | 2001-09-13 | Massachusetts Institute Of Technology | Heparinase iii and uses thereof |
| WO2002023190A2 (en) | 2000-09-12 | 2002-03-21 | Massachusetts Institute Of Technology | Methods and products related to low molecular weight heparin |
| JP2004523479A (en) | 2000-10-18 | 2004-08-05 | マサチューセッツ インスティテュート オブ テクノロジー | Methods and products for pulmonary delivery of polysaccharides |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1136620A (en) * | 1979-01-08 | 1982-11-30 | Ulf P.F. Lindahl | Heparin fragments having selective anticoagulation activity |
| FR2597484B1 (en) * | 1986-04-17 | 1988-12-23 | Sanofi Sa | GLYCOSAMINOGLYCANS OF THE HEPARIN OR HEPARANE-SULPHATE TYPE WITH ACTIVITY ON CELL DIVISION AND DIFFERENTIATION, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATIONS. |
| NO921495L (en) * | 1991-04-16 | 1992-10-19 | Seikagaku Kogyo Co Ltd | OLIGOSACCARID AND PROCEDURE FOR ITS PREPARATION |
| TW318142B (en) * | 1991-06-03 | 1997-10-21 | Mitsubishi Chemicals Co Ltd | |
| GB9206291D0 (en) * | 1992-03-23 | 1992-05-06 | Cancer Res Campaign Tech | Oligosaccharides having growth factor binding affinity |
-
1993
- 1993-03-25 GB GB939306255A patent/GB9306255D0/en active Pending
-
1994
- 1994-03-24 WO PCT/GB1994/000615 patent/WO1994021689A1/en not_active Application Discontinuation
- 1994-03-24 JP JP6520835A patent/JPH07507596A/en active Pending
- 1994-03-24 CA CA 2136531 patent/CA2136531A1/en not_active Abandoned
- 1994-03-24 AU AU62875/94A patent/AU6287594A/en not_active Abandoned
- 1994-03-24 EP EP94910470A patent/EP0642533A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07507596A (en) | 1995-08-24 |
| EP0642533A1 (en) | 1995-03-15 |
| GB9306255D0 (en) | 1993-05-19 |
| WO1994021689A1 (en) | 1994-09-29 |
| AU6287594A (en) | 1994-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Toida et al. | Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species | |
| Oldberg et al. | Characterization of platelet endoglycosidase degrading heparin-like polysaccharides | |
| AU634199B2 (en) | Heparin fragments as inhibitors of smooth muscle cell proliferation | |
| Kusche et al. | Biosynthesis of heparin. O-sulfation of the antithrombin-binding region. | |
| Jacobsson et al. | Identification of N-sulphated disaccharide units in heparin-like polysaccharides | |
| Goupille et al. | Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides | |
| CA2136531A1 (en) | Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity | |
| Ishihara et al. | Structure and function of heparin and heparan sulfate; heparinoid library and modification of FGF-activities | |
| WO2009014715A2 (en) | Enzymatic synthesis of sulfated polysaccharides without iduronic acid residues | |
| EP0509517B1 (en) | Oligosaccharide having affinity for fibroblast growth factor and process for producing same | |
| Pejler et al. | Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells | |
| Sugahara et al. | Structure determination of the octa-and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestinal heparin | |
| Lindblom et al. | Domain structure of endothelial heparan sulphate | |
| Suzuki et al. | Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides | |
| Zhang et al. | Design and synthesis of neutralizable fondaparinux | |
| US20080207895A1 (en) | Methods for synthesizing polysaccharides | |
| Chavante et al. | A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana | |
| Zhang et al. | Enzymatic production of chondroitin oligosaccharides and its sulfate derivatives | |
| Garg et al. | Effect of fully sulfated glycosaminoglycans on pulmonary artery smooth muscle cell proliferation | |
| BAKER et al. | Specificity of the hyaluronate lyase of group-B streptococcus toward unsulphated regions of chondroitin sulphate | |
| Fugedi | The potential of the molecular diversity of heparin and heparan sulfate for drug development | |
| Kraemer | Production of heparin related glycosaminoglycans by an established mammalian cell line | |
| Joseph et al. | Influence of molecular weight, protein core and charge of native heparin fractions on pulmonary artery smooth muscle cell proliferation | |
| HAUSSER et al. | Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis | |
| JP5344785B2 (en) | Pleiotrophin-binding and non-binding chondroitin sulfate / dermatan sulfate oligosaccharides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |