CA2132750A1 - Oligosaccharides having growth factor binding affinity - Google Patents

Abstract

Oligosaccharides having a high specific binding affinity for FGF
growth factors and made up of less than ten disaccharide units in all are disclosed which include sulphated disaccharide units composed of an N-sulphated glucosamine residue and a 2-0-sulphated iduronic acid residue. A method is also disclosed for preparing these oligosaccharides in a purified and relatively homogeneous state from glycosaminoglycans such as heparan sulphate. For the best FGF-binding affinity there are preferably at least four of the sulphated disaccharide units arranged as an internal contiguous sequence. The most favoured structures contain fourteen monosaccharide residues in all, but structures having twelve monosaccharide residues can also have quite high FGF-binding affinity, at least for bFGF. These oligosaccharides can either activate and stimulate FGF activity or inhibit FGF activity, and uses thereof as drugs for therapeutic purposes in medicine are also disclosed.

Description

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OLIG~SACC~ARIDES HAVING GROWTH FACTOR BINDING AFFINITY
_ The present invention relates to the field of biochemistry and medicine. More particularly, it concerns certain novel oligosaccharide products and preparations thereof which have particular binding affinity for certain bioactive proteinC or polypeptides present in biological systems, especially certain growth factors or cytokines such as fibroblast growth factors ( FGF ' s ) . It also concerns uses of such oligosaccharide products, especially in medicine.

BACKGROUND

In complex multicellular living organisms, for example humans and other mammals, it is well known that various aspects of cell development, migration, growth and/or proliferation, involving in some cases cell-cell interactions, are often under the control of or are regulated by ~arious extracellular mediators or cytokines, commonly referred to as growth factors, which are generally specialised soluble proteins or polypeptides secreted by cells of the tissues concerned.

These growth factors, of which many have already been isolated and subsequently synthesised using recombinant DNA technology, are believed to act through a variety of mechanisms, but in general their effect appears to result from an initial interaction with specific receptors or ~inding sites on the surface of target cells which are thereby activated to bring about a chain or sequence of intracellular biochemical events.

Certain of these protein growth factors, characterised inter alia by a high binding affinity for heparin, are designated by the general term Fibroblast Growth Factor (FGF) of which two main forms having partial amino acid sequence ldentity but differing isoelectric WO93/19096 ~ PCT/CB93/00597 ~;~ 3 ~,?~
points are recognised, acidic Fibroblast Growth Factor (aFGF) and basic Fibroblast Growth Factor (bFGF). FGF's are present in a wide variety of mammalian tissues; they appear to function in both normal and in diseased physiological states as important signalling molecules involved in regulation of cell growth and differentiation and they act as potent mitogens stimulating proliferation for a range of cell types. A review by D Gospodarowicz of some of the characteristics and properties in FGF's is 10 to be found in Cell ~iology Reviews (1991) 25 (4), 305-314.

In particular, basic fibroblast growth factor (bFGF) appears to have an important role in processes such as embryonic development, wound repair and tumour growth, and it has been specifically implicated as being directly concerned in various disorders or degenerative conditions involving cell proliferation, including for example -~ diabetic retinopathy, capsular opacification following cataract operations, restenosis after angioplasty, tumour angiogenesis, and various forms of chronic inflammation.
It delivers its signal to cells ~y binding with specific cell surface tyrosine kinase receptors (Rd 10-500 pM), such as receptors which are the expression products of the gene flg, that generate intracellular signals. However, the mode of action of bFGF and similar growth fact~rs or cytokines is complex and appears also to involve an inter-action with the heparan sulphate component of heparan sulphate proteoglycans (HSPGs) (Kd 5-50 nM) on the cell surface or in the extracellular matrix of mammalian cells.
Recent work has shown, for example, that in cells which are deficient in heparan sulphate (HS) synthesis the f l g receptor will not respond to bFGF, but that addition of heparin or heparan sulphate (HS) can restore responsive-ness. It seems clear that in at least many cases such growth factors need to be activated before they can exert their biological effect. It has been suggested that poly-; saccharides such as HS and heparin induce a conformational ~ .

~WO93/1~6 ~ 1 3 2 7 ~ ~ PCT/GBg3/00597 change in growth factors such as bFGF with which they interact and that this is a prerequisite for binding to the signal transducing receptor. ~hus, on this basis a model invoking a dual-receptor mechanism, at least for the act~on of bFGF, has been proposed. Hitherto, however, the nature of a supposed bFGF binding site in HS has not been fully elucidated. HS is probably the most complex mammalian glycosaminoglycan (GAG), consisting of a linear polysaccharide chain having an ordered arrangement of domains rich in N- and O- sulphate groups, in which the basic disaccharide repeat unit consists of glucuronic acld or iduronic acid linked to an N-sulphated glucosamine `(i.e. GlcA/IdoA-GlcNSO3), spaced apart by regions of low sulphation in which N-acetylated disaccharides (GlcA-GlcNAc) predominate. Since bFGF is a heparin-binding ~rowth factor the sulphated domains which contain some "heparin-like" regions might be expected to provide the most likely location of the bFGF binding site. On the other hand, the slze of these domains, their sulphation , 20 pattern and their iduronic acid content are h~ghly v~riable, and a possib~llty arises that the strong lnter~ction w~th bFGF may re~uire a strictly defined sequence of sulphated monosaccharide isomers providing a special~sed binding domaln in a manner simi}ar to the specif~c pentasaccharide se~uence in heparin which has been found to have high affinity for antlthrombin III.
Endothelial cell derived HS has already baen demonstrated by affinity chromatography to bind strongly to bF~F, and a weaker interaction with HS from the Engelbreth Holm Swarm (EHS) tumour has also been reported, but the full structural requirements for such interactions have not previously been known.

As compared to bFGF, acidic fibroblast growth factor (aFGF) seems generally to be less potent, but nonetheless it is known as an active mitogen and differentiation factor for a wide variety of cells, especially mesodermal derived cell types, it is present in a variety of tissues, W093/19096 ~ PCT/GB93/00597 it binds to the same cell surface receptors as bFGF with substantially the same affinity, it likewise binds strongly to heparin and to the heparan sulphate of cell surface or extracellular matrix heparan sulphate proteoglycans, and the mechanism of interaction would appear to be the same as with bFGF. A number of other growth factors or cytokines also bind to heparan sulphate or similar sulphated glycosaminoglycans of extracellular matrix or cell surface proteo~lycans, and again this may be a necessary prerequisite for their biological activity under physiological conditions.

Since theqe growth factors or cytokines such as FGF
appear to have such an important and wide-ranging role in controlling or regulating cellular processes that are responsible both for maintaining or restoring a normal physiological state or for promoting certain disease states, the possibility of controlling or modulating their activlty for the purpose of therapeutic treatment is an attract~ve proposit~on. Thus, some consideration h~s already been given for example to the development and use of agents which would block the cell surface signal transducing binding receptors in order to inh~bit growth factor activity, and in other cases, such as wound healing for example, where increased growth factor activity may be benef~cial the use and administration of exogenous growth factors as therapeutic drugs has been considered. Another possibility for blocking or reducing acti~ity would be to employ agents that would act as antagonists or agonists to ~0 interfere with the preliminary b~nding interactlon between such growth factors and the proteoglycan or glycosamino-glycan, such as heparan sulphate, wh~ch appears to be necessary before bindlng to the cell surface signal inducing receptors can take place, and for this purpose the possible use and administration of heparin for acting as a competitive inhibitor could be considered, at least in principle. However, heparin (or heparan sulphate itself) is not particularly suitable for use as a drug in wo 93/lgo96 ~ ~ ~ C~ 7 3(~ PCT/GB93/00597 this context, not least because of its complexity and heterogeneity with a large numher of different disaccharide sequences in its molecular composit~on such that it is likely to have multiple activities giving other undesirable effects and it would lack specificity. What is needed for use as a drug is a high purity or substantially homogeneous preparation of a relatively small molecular compound of known composition which can be conveniently administered and which would have a very high degree of specificity for binding to the particular glycosaminoglycan binding sites of the growth factors in question with a low risk of promoting unpredictable or unwelcome side effects. In other words, it would be most desirable to have a molecule of minimal s~ze consistent w~th high specific binding affinity, or a h~gh value for the ratio of binding affinity or biological activity to size. Such a drug could then provide a valuable regulatory therapeutic agent for blocking or inhibiting subsequent binding to the cell surface signal inducing receptors and thus reducing growth factor activlty, or in other cases it might act to stimulate growth factor activity by promoting-su~sequent growth factor binding to the cell surface signal inducing receptors. Also, if it is desired to admln~ster exogeneous growth factors for therapeutic purposes, as perhaps in wound healing or various other tissue repair applications, it may be advantageous for such growth factors at the time of adm~ nistration to b2 complexsd wi~h a protective or actl~ating agent in the form of a relatively small molecular compound as referred to above which could be co administered with the growth factor and which would bind ! ' with a high degree of specificity to the glycosaminoglycan binding sites of the growth factor.

Although it may be expected that, like the parent molecule, at least certain fragments of heparan sulphate would also have some specific binding affinity for FGF
growth factors, and it is known that heparan sulphate can WO93/1~96 ~ PCT/GB93/00597 l ~ 6 be partially depolymerised by selective scission reagents (e.g. enzymes such as heparinase and heparitinase) to yield preparations of relatively short length oligo-saccharides, such oligosaccharide preparations generally comprise a complex mixture of various molecular species having a wide range of different compositions and sizes.
These preparations would therefore be no more suitable for use as drugs than would be heparan sulphate itself or heparin, and whilst varlous fractionations and partial purifications of such oligosaccharide preparations or mixtures have been carried out in the course of experimental work, the lack of more detailed knowledge about the particular structural characteristics that pro~ide high specific binding affinity for FGF growth factors has been a problem that has hindered development of more well defined oligosaccharide products or preparations having optimum efficiency and better suited for possible medical use as drugs or therapeutic agents.

; ~
The present invention has originated in the course of work which was undertaken to investigate human skin flbroblast heparan sulphate and which has led to the isolation and characterisation of distinct oligosaccharide structures having particular spec.ific binding affinity for - FGF's and similar heparin or heparan sulphate binding growth factors. As a consequence, the invention enables oligosaccharide products to be prepared wh~ch, for medical use, especially as FGF growth factor modulatlng agents in connection with the treatment of various conditions herein referred to, are more suitable than any oligosaccharide preparations hitherto known.

ABBREVIA~IONS

Throughout the present specific~tion, including the claims, the following abbreviations are used:

2 ~. 3 ,~ 7 ~ ~ PCT/GB93/00597 GAG - glycosaminoglycan;
HS - heparan sulphate;
HSPG - heparan sulphate proteoglycan;
bFGF - basic fibroblast growth factor;
aFGF - acidic fibroblast growth factor;
dp - degree of polymerisation (e.g. for a disaccharide, dp-2, etc);
GlcA - D-glucuronic acid;
IdoA - L-iduronic acid:
IdoA(2S) - L-iduronic acid 2-sulphate;
GlcNAc - N-acetyl D-glucosamine;
GlcNSO3 - N-sulphated D-glucosamine;
GlcNSO3(6S) - N-sulphated D-glucosamine 6-sulphate;
GlcAt2S) - D-glucuronic acid 2-sulphate;
nHexA - unsaturated uronic acid residue;
GlcA - unsaturated hexuronate residue designated GlcA on the basis that lt is believed to be ~; derived from the saturated residue GlcA in an -~ original polymer chain, e.g. based on the known specificity of heparitinase scission (see later);
aManR ~ 2,5-anhydro-D-mannitol formed by reduction of terminal 2,5-anhydromannose residues with Na~H4.

~he symbol ( n ) is used to indicate that the monosaccharide resldue concerned may or may not be unsaturated, and the symbol ('6S) denotes that a residue may or may not be sulphated at the C6 position.

3 0 SU~IARY OF THE INVENTION

! ~
As indicated, the invention broadly provides novel ollgosaccharide products having a high specific protein or polypeptide binding affinity, especially in respect of HS-binding proteins or polypeptides exemplified by growth factors such as FGF's.

More particularly, in one aspect the invention W093/19096 ,~ PCT/GB93/00597~

provides an oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's), characterised in that it consists essentially of oligo-saccharide chains which are substantially homogeneous with respect to FGF binding affinity and which contain at least four, preferably at least six, disaccharide units including sulphated disaccharide units, preferably arranged as a contiguous sequence, that are each composed of an N-sulphated glucosamine residue ('6S) and a 2-~-sulphated iduronic acid residue.

Also, it is preferred that each of said sulphateddisaccharide units is IdoA(2S)-al,4-GlcNSO3, and that the oligosaccharide chains consist of a sequence of less than ten disaccharide units in all. In preferred em~odiments, the oligosaccharide chains may consist of a sequence of slx disaccharide units in all of which at least four are included in the aforesaid contiguous sequence of sulphated disaccharide units, although in the most preferred embodlments there are a total of seven disaccharide unlts of wh~ch at least five are included in said contiguous sequence of sulphated disaccharide units.
.d It is also preferred that the predominating majority of the oligosaccharide chains should all be of the same length and that the content (if any) of glucosamine residues 0-sulpha~ed at C6 should be less than 20%, or more preferably less than 5%. Oligosaccharides in accordance with the invention will generally be substantially completely resistant to depolymerisation by heparitinase but not by hepsrinase, and may be obtainable from heparan sulphate (HS) of human fibroblast heparan sulphate proteoglycan (HSPG) by enzymic partial depolymerisation to the fullest extent with heparitinase followed by size fractionation, using for example gel filtration size exclusion chromatography, followed by, in re~pect of a selected fraction or fractions recovered from the size fractionating stage, affinity chromatography :

WO93/19096 9 ~ 7 ~ ;~ PCT/GBg3/00597 using an FGF growth factor as ~he immohilised ligand in order to separate out the FGF-~inding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient, advantageously a serially stepped gradient, to fractionate said fragments in respect of FGF
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 using the methods herein referred to.

Alternatively, an oligosaccharide product having a specific binding affinity for fibro~last growth factors (FGF's) in accordance with the invention may be defined as being characterised in that (a) it is composed predominantly of a molecular species:
~: X~Y~Z
in which X is nHexA-GlcNS03(+6S), Y is IdoA(2S)-GlcNS03(+6S), Z is IdoA-Glc~(+6S) or IdoA(2S)-GlcR(+6S) where R is NS03 or NAc, and n is in the range 4 to 7 (b) the content, if any, of monosaccharide res~dues having a 6-0-sulphate group is less than 20~;
(c) it is obtainable by a process comprising the steps of digesting a heparan sulphate with hepari~inase so as to bring about partial depolymerisation thereof to the fullest extent, followed by size fractionating the oligo-saccharide mixture produced using for example gel filtration size exclusion chromatography, collecting a fraction or fractions containing oligosaccharide chains having a particular size ::
.

WO93/19096 ~q~ PCT/GB93/00597 selected within the range of 12 to 18 mono-saccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using an immobilised FGF ligand and recovering the more strongly FGF-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 desor~s only at the highest salt concentrations, and preferably being further characterised in that:

Y is exclusively IdoA(2S)-GlcNSO3, n is 5 or 6 with there ~eing a total of seven disaccharide units in all, or is 4 with there being a total of six disaccharide units in all, and the content, if any, of residues having a 6-O-sulphate group is less than 5~.

The i~vention may also be defined as providing an oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's) that, at least in preferred embodlments, is substantially all composed of oligosaccharide chains which are either fourteen monosaccharide residues in length and which contain an internal contiguous sequence of 5 or 6 disaccharide units each consistin~ ~of an IdoA(2S) residue linked to a GlcNSO3(~6S) residue, wlth less than 20% of the glucosamine residues (terminal or internal) being 6-O-sulphated, or which are twelve monosaccharide residues in length and which contain an internal contiguous sequence of 4 disaccharide units each consisting of an IdoA(2S) residue linked to a GlcNS03(l6S) residue, again with less than 20~ of the ~lucosamine residues (terminal or 1 1~ .t ~ 7 ~ ~
internal) being 6-O-sulphated, the predominant oligo-saccharide chain sequence, accounting for substantially more than 50~ of the component oligosaccharide chains and preferably more than at least 70~ of the component oligo-saccharide chains, being preferably selected from thefollowing:

n )GlcA-GlcNSO3-~IdoA(2S)-GlcNSO3]5-IdoA-GlcR, ( n )GlcA-[GlcNS03-IdoA(2S)]6-Glc~, and 1 o ( n ) GlcA-GlcNSO3('6S)-[IdoA(2S)-GlcNS03]4-IdoA-GlcR(+6S), where R is NSO3 or NAc.

Oligosaccharides in accordance with the invention include in particular the main constituent of the oligosaccharide product or preparation hereinafter designated oligo-H having a disaccharide sequence:

nGlcA-~l,4-GlcNSO3-al,4-[IdoA(2S)-al,4-GlcNSO3]5-al,4-IdoA-al,4-GlcR, or nGlcA-~l,4-~GlcNSO3-al,4-IdoA(2S)~6-al,4-GlcR, where R is NAc or NSO3, and minor variants thereof having at least the same relatively high specific binding affinity to bFGF.

Oligosaccharides in accordance with ths invention also include, however, related hi~hly sulphated oligo-saccharides such as those comprising the main constituent of oligosaccharide preparatlons hereinafter designated oligo-M and oligo-L which have a weaker, but still significant, binding affinity to bFGF. These include, at least for oligo-M, oligosaccharide chains having the sequence nGlcA-GlcNso3(l6s)-~IdoA(2s)-GlcNso3]4-IdoA-GlcR(l6s) where R is generally NAc but may be NSO3 The main components of oligo-L appear to comprise W093/1~96 ~ PCT/GB93/00597 ~ 12 the sequences nGlcA-GlcNso3-IdoA-GlcNAc(6s)-GlcA-GlcNso3(6s) ~IdoA(2S)-GlcNS03]2-IdoA-GlcR(+6S) and nGlcA-GlcNso3-IdoA(2s)-GlcNso3-IdoA-GlcNAc(6s)-GlcA
GlcNS03(6S)- IdoA(2S)-GlcNS03-IdoA-GlcR(~6S) where R is generally NAc but may be NS03 Oligosaccharide products in accordance with the inventlon may either be isolated from natural sources or may be made synthetically.

In respect of isolation from natural sources, in broad terms the invention further provides a method of isolating from a glycosaminoglycan such as heparan sulphate small oligosaccharides in a purified and relatively homogeneous state which have a specific binding affin~ty for a selected bioacttve proteir.. or polypeptide : th~t itself binds to said glycosaminoglycan or to the correspond~ng proteoglycan in multicellular biological systems, said method comprising the steps of:
(a) preparing an affinity chromatographlc matrix or ;: substrate incorporating a sample of said protein or polypeptide as the affinity ligand immobilised thereon;
(b) treating said glycosaminoglycan with a selective scission reagent so as ~o 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) WO93/19096 13 ~ 3 s~ 7 ~ ~ PCT/GB93/00597 containing a specific number of disaccharide units in the range of four to nine in order to extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the glycosaminoglycan that have at least some binding affinity for the immobilised said protein or polypeptide;
(e) eluting the affinity chromatographic matrix or su~strate using a progressively increasing salt concentration or gradient in the eluant;
(f) collecting the frac~ion or set of fractions containing oligosaccharide fragments elut:ing in ~.
selected highest ranges of eluant salt concentration: and op~ional~y, (g) further purifying the product of the .e.lected 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 method, the partial depolymerisation of the glycosami~oglycan may be carried out by a chemic~l method in which the polysaccharide is flrst N-deacetyl~ted, e.g. by hydrazinolysis and is then - treat~d with nitrous acid at about pH 4, this being used as the selective scission reagen~, to bring about deaminitive cleavage at the free amino ~roups ~f the glucosam~ne residues resulting from the N-deacetylatlon.

However, at least in the case of heparan sulphate the pre~erred selective scission reagent is the poly-saccharide lyase enzyme heparitinase which is commercially available from Seikagaku Corporation of Tokyo, Japan under the designation "Heparitinase I", or from Sigma Chemical Co. under the designation "Heparinase III", and which has the classification EC 4.2.2.8. This enzyme will select-WO93/19~6 ~;?~ 14 PCT/GB93/00597 ively cleave glycosidic linkages on the non-reducihg side of GlcA-containing disaccharides, such as in GlcNAc-al,4-GlcA present in regions of low sulphation, but in general it w$11 not cleave bonds of sulphated disaccharides containing L-iduronic acid or 2-sulphated L-iduronic acid, l.e. IdoA or IdoA(2S). This is in contrast to the enzyme known as heparinase (EC 4.2.2.7) which cleaves glycosidlc linkages between disaccharides containing 2-sulphated L-iduronic acid (for a review of these enzymes see R J
Linhardt et al (1990) Blochemistry 29, 2611-2617). There are several known varieties of the heparitinase enzyme which have substantially the same linkage specificlty but which vary for example in depolymerisation efficiency accordin~ to the size of the substrate molecules.
However, in general through`out the present specification, including the claims, unless otherwise stated the term "heparitinase" is used to denote the enzyme supplied by Selkagaku Corporatlon as "Heparitinase I", or any other equivalent enzyme having the same glycosidlc linkage spec~flcity.

In connection wlth the cleavage of polysaccharlde or oligosaccharlde glycosidlc llnkages, e.g. 1,4 llnkages, by enzymes such as heparitlnase and heparinase, it should lncldentally be apprec~ated that in the one fragment 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. Th$s 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. `

Oligosaccharides or oligosaccharide products in accordance with the invention generally have a well defined composit-ion, readily capable of further purification if necessary, and considering also their size and specific growth factor binding affinity they can be very well suited for W093/1~96 PCT/GB93/~597 1~ ~ 3 X 7 `~ o pharmaceutical use to exploit a considerable potential in -the field of medicine, e.g. as growth factor inhibitors or activators and mobilising agents. Accordingly, they are expected to have valuable applications as therapeutic s drugs, particularly for controlling or regulating the activity of FGF's, especially bFGF. This may arise for example where there is a need to control or reduce F~F-activity dependent cell growth or proliferation $n clinlcal treatment of conditions such as diabetic 10 retinopathy, restenosis after angioplasty, capsular ;
opacification, proliferation vitreoretinopathy, arthritis --and other chronic inflammatory condi~ions, cancer cell growth and tumour angiogensis, mild muscular dystrophy, Alzheimer disease and va,rious viral infections (e.g.
15 Herpes Simplex type 1)~ This may also arise where there `~
is a need to stimulate endogeneous FGF's or to administer and activate exogeneous FGF's for promoting healing or tlssue repair, for example in clinical treatment of condltions such as wound healing, bone healing, nerve reg~neration, duodenal or venous ulcers, various ocular and retinal d~sorders, atherosclerosis, degenerative muscle disorders, ischaemia, or for protecting tissues against serious damage during radiation treatment. For these purposes, the oligosaccharide products (or pharmaceutically-acceptable salts thereof) may be made up lnto pharmaceutical formulations as required, and such uses are also within the scope of the invention.

By way of further explanation so that the skilled person in the art will more readily be able to appreciate the nature of the invention and will more readily be able to put it into practical effect, there now follows a fuller description of the invention, including some of the background experimental work carried out by the inventors and various practical details thereof. In connection with this description, reference should also be had to the accompanying drawings.

WO 93/19096 G~ PCI`/GB93/00597 ~" 16 BRIEF DESCRIPTION OF THE DRP.WINGS
. . ~

FIGURE 1: This shows the fractionation of native and partially depolymerised HS on a bFGF-affinity column in experiments in which 3H-labelled samples of HS chains ~control - panel A), or HS treated with heparinase (panel B), or heparitinase (panel C), were fractionated on a bFGF
affinity column as hereinafter described. Bound material was eluted with a step gradient of sodium chloride as shown in panel A (dotted line). Heparitinase-resistant oligosaccharides retaining hi~h affinity for bFGF (see panel C, material eluting at 1 to 1.5M NaC1, fractions 22-35) were pooled, then dialysed (Spectrapor 7 1000-Mr cut-off, Spectrum UK) and freeze dried. Their size was then established by gel filtratron on a Bio-Gel P6 colu~ (1 x 120cm) at a flow rate of 4ml/hour in O.SM NH4HC03 (panel D).

FIGURE 2: This shows the effect of heparinase on the affinity of HS oligosaccharides for bFGF in experiments in which 3H-labelled HS chains were first treated with heparitinase and size fractionated by Bio-Gel P6 chromatography. Fractions of the heparitinase-resistant oligosaccharides of size dpl2 and dpl4 were pooled and 2~ then fractionated by bFGF affinity chromatography. Three major fractions eluting at 0.75M, l.OM and > 1.25M NaCl were obtained, designated oligo-L (low), oligo-M ~mèdium) and oligo-H (high) affinity oligosaccharides respectively.
Th~ affinity of these oligosaccharides for bF~F was tested by re-application to the affinity column either intact ~solid line), or after heparinase treatment (dashed line), and elution with a step gradient of sodium chloride (panel A, dotted line).

FIGURE 3: This shows the results of Bio-Gel P6 chromatography of HS oligosaccharides having differing affinit es for bFGF in experiments in which HS oligo-saccharides (dpl2-14) with relatively low (oligo-L), `~ ? 17 ~ t ~ 7 ~ f~ cr/GB93/oos9~
medium (oligo-M) and high (oligo-H) affinity for bF~F were prepared as in connec~ion with Fig. 2. Their size ~.
distribution was established by Bio-Gel P6 chromatography either intact (solid line) or after heparinase treatmenk 5 (dashed line).

FIGURE 4: This shows the results of Bio-Gel P6 chromato-graphy of bFGF-binding HS oligosaccharides subjected to 10 deaminitive scission in experiments in which HS oligo-saccharides (dp 12-14) with low (oligo-L), medium (oligo-M) and high (oligo-H) affinity for bFGF, prepared as described in connection with Fig. 2, were treated with nitrous acid and fra~tionated by Bio-Gel P6 chromato-15 graphy. The disaccharides (dp2) were partially resolved into mono-sulphated (main peak) and non-sulphated species.

FIGURE 5: Shows the results of ~io-Gel P6 chromatography .~
20 of bFGF-binding oligosaccharides (Oligo-H and Oligo-M) ~:
after ~eing su~jected to heparitinase IV depolymerisation;

FIGURE 6: Shows the results of bFGF affinity chromato-graphy of heparitinase~resistant oligosaccharides for each dlfferent size from dp-2 to dp~l4 after preparation by 8io-Gel P6 gel filtration;

F~GURE 7: Shows graphs "A" and "B", for bFGF and aFGF
respectively, illustrating the effect of size of HS-bindin~ oligosaccharides and binding affinity in relation to growth factor activation:

FIGURE 8: Shows a typical result of Bio-Gel P6 gel filtration of a heparitinase digest of 3H-labelled fibroblast HS prior to bFGF-affinity chromatography, as referred to in the Example described herein.

W093/19096 ~s~ ~ 18 PCT/GB93/OOS97 DETAILED DESCRIPTION and EXAMPLES
.

In the initial background experimental work that led to the present invention, investigations were conducted using as source materials heparan sulphate derived from human skin fibroblasts and human recombinant bFGF.

The human recombinant bFGF was prepared ln a manner similar to that described previously for acidic FGF by Ke, Y. et al, ( 1990) Biochem Btophys. Res. Comm. 171, 963-971.
Briefly, the recombinant bFGF was purified by heparin-Sepharose chromatography and reverse phase or cation~
exchange HPLC from lysates of bactarial cells, harbouring a PKK 233-2-bFG~ construct (see Amann, E et al, (1985) Gene, 40, 183-190) encoding amino acids 1-155 of human ` ~
bFGF (~ee Abraham, J.A. et al, ( 1986 ) EM~O J. 5, 2523- - :
2528), to yield a single compound of MW 17kDa on SDS-PAGE.
The amino acid sequence was consistent with that of human bFGF and the recombinant protein possessed full biological activity.

HSPG and HS chains biosynthetically radiolabelled with 3H-glucosamine were prepared from confluent cultures of adult human skin fibrobl~sts as described in a paper by Turnbull and Gallaghe~ (see Turnbull r J . E . et al, ( 1991) ~lochem. J. 273, 553-559), the content of which is incorporated herein by reference.

Amongst the experimental techniques used, depolymerisation of HS chains with heparitinases, heparinase or low pH nitrous acid, and gel chromatography of oligosaccharides on Bio-Gel P6 or P2 were carried out as also pre~iously described in the above-mentioned paper of Turnbull, J.E. et al, and in another paper of Turnbull, J.E. et al, ( 1991~ ~iochem. J. 277, 297-303. Chemical N-desulphation/re-N-acetylation was carried out as described ~-by Inoue and Nagasawa (see Inoue, Y. et al ( 1976) Carbohydrate Res. 46, 87-95). `:;:

W093/19096 ~ I ~ 2 7 X ~

The work also involved the use of affinity chromatography and strong-anion exchange HPLC of disaccharides, the affinity chromatography involving a bFGF-Affi-Gel 10 affinity matrix. To prepare the latter, lml of packed Affi-Gel 10~SM activated affinity gel from Bio-Rad Laboratories was washed four times with five volumes of double distilled water using centrifugation at 800g for 1 minute. Heparin (500~g) was added to bFGF
(500~g in 3ml 0.6M NaCl, 25 mM Na2HP04, pH 6.6) and mixed with lml of washed and packed Affi-Gel 10 overnight at

4-C. 2ml of 4M Tris-HC1, pH 8.0 was added to block unreacted groups on the gel. 5mg of heparin was added to stabilise bound bFGF and 4.5~1 of 20% (w/v) NaN~ as preservative. The gel was, washed with lO volumes of 2M
NaC~ in lOmM Tris-HCl, pH 6.5. No bFGF was detected in the wash by reverse-phase HPLC, indicat~ng a high coupling efficiency.

The Affinity chromAtography was generally carried out as follows:
Approximately lml of the bFGF-Affi-Gel 10 aff~nlty matrix was packed into a glass column (bed dimensions 6mm x 35mm)). Samples were loaded onto the column in lOmM
~ris-HCl, pH 6.5, at a flow rate of 0.25ml per minute.
Unbound material was eluted by collecting five lml fr~ctions. Bound material was eluted with a step gradient of sodium chloride (O - 2M NaCl in column buffer in 0.25M
steps) at a flow rate of 0.5ml per minute. Five lml fractions were collected at each concentration. The column was stored at 4C in running buffer containing lO~g/ml heparin (Sigma), 0.01% (w/v) sodium azide and 0.2M
NaC1.

To analyse the disaccharide composition of HS oligo-saccharides, the latter were completely depolymerisedenzymically with a mixture of heparitinase, heparitinase II and heparinase (obtained from Seikagaku Kogyo Co., Tokyo, Japan). Disaccharides were recovered by Bio-Gel ~ ~ 20 PCT/GB93/00597 P2RS~ chromatography and separated by HPLC on a ProPac PA1 ~;
analytical column (4 x 250mm; Dionex, UK). After equilibration in mobile phase ~double distilled water ad~usted to pH 3.5 with HCl) at lml/minute samples were injected and disaccharides eluted with a linear gradient of sodium chloride (O - lM over 45 minutes) in the same mobile phase. The eluant was monitored in-line for W
absorbance (A232 for unlabelled disaccharides) and for radioactivity (Radiomatic Flo-one/Beta A-200 detector).
Disaccharides released by nitrous acid treatment were separated by HPLC as has been described previously (see Pejler, G. et al, ( 1987 ) Biochem. J. 248, 67-69 and Bienkowski, M.J. et al, 3iochem. J. 260, 356-365 ) . ..

In initial experiment~, HSPG, metabolically-labelled ~
with 3H-glucosamine, was purified from the medium of `~.-cultures of human skin fibroblasts. HS chains were prepared by Pronase treatment of the HSPG and applied to an affinity column prepared with human recombinant bFGF as ~-hereinbefore described. Bound material was eluted stepwise with NaC1 concentrations ranging from 0.25M - ~;
2.OM in O.25M steps. The ma~ority of the HS bound ~trongly to bFGF, the ma~or peak eluting at 1.25M NaCl (see Fig lA). A similar elution profile was obtained for the intact HSPG (results not shown), indicating that the heparan sulphate (HS) chains are the principal determinan~
of proteoglycan binding to bFGF. Hyaluronic acid did not bind to the bFGF column and fibroblast-derived chondroitin - :`
and dermatan sulphate eluted in the range O - O.SM NaCl: -however commercial heparin eluted mainly at 1.25M and 1.5M
NaC1 (data not shown). These results indicated a specific interaction of bFGF with N-sulphated polysaccharides. The importance of N-sulphate groups was confirmed by the findings that either deaminitive scission with nitrous acid, or N-desulphation/re-N-acetylation of HS, abolished the high affinity interaction (results not shown). `

The problem of identifying the bFGF binding domains WO93/19096 ~ t 3 2 7 ~. O PCT/GBg3/00597 in HS was addressed using the enzymes heparinase and heparitinase which selectively cleave the polysaccharide in different structural domains. As already mentioned, heparinase acts in the N-sulphated regions and specific~
ally cleaves disaccharides that contain 2-O-sulphated iduronate i.e. Glc~SO3(+6S)-al,4-IdoA(2S ?, the ma~or products being oligosaccharides of 9-lO kDa, whiIe in contrast heparitinase cleaves GlcA-containing di-saccharides, principally GlcNAc-al,4-GlcA present in :
regions of low sulphation, but does not attac~ N-sulphated sequences of the type ~GlcNSO3(+6S)-al,4-IdoA(+2S)]n.

Heparinase scission of HS resulted in products with si~nificantly reduced affinities for bFGF, elution occurring in the range 0.25 - 0.75M NaCl-(see Fig. lB).
The effects of heparitinase digestion were even more marked with the majority of the material either failing to blnd to the column or eluting at 0.25 - O.75M NaCl (se~
Flg.lC). However, a minor populatton of oligosaccharides in the heparitinase digest dtsplayed an affinity for bFGF
that was comparable to the intact HS (elutlng in the range l.0 to l.5M NaCl~. Gel filtration size exclusian chromat-ography on Bio-Gel P-6 showed that these high affinlty products comprised two oligosaccharide fractions pre-dominantly dpl2 and dpl4 in size (see, Fig. lD), equivalentto six and seven disaccharide units. These are the largest fragments present in signi f icant quantities in heparitinase digests of human skin flbroblast HS. The foregoing data indicated that extended N-sulphated sequences in HS contain the highest affinity binding site for bFGF, and that IdoA(2S) residues make an important contribution to the interaction.
I

Specificity of binding of oligosaccharides To investigate the specificity of oligosaccharide :
interaction in more detail and the structural features involved, quantities of heparitinase-resistant oligo- :~-saccharides were prepared directly from heparitinase .

WO93/19096 ~ 3 22 PCT/GB93/005~7 digests of HS using Bio-Gel P-6 gel filtration size exclusion chromatography. Selected components of size dpl2 and dpl4 (12.5% of total product) were pooled and then fractionated a~ain by bFGF affinity chromatography.
Three major fractions were identified which eluted at 0.7SM, 1.0M and 1.25M NaCl and were designated oligo-L
(low), oligo-M (medium) and oligo-H (high) affinity oligo-saccharides. Re-application of the fractions to the -column confirmed their different affinities for bFGF (see Flg. 2). Oligosaccharides of size dpl4 were mainly present in the oligo-H fraction whereas the oligo-M and oligo-L fractions were predominantly dpl2 (see Fig 3).

Heparinase treatment caused a marked reduct:ion in binding of these oligosacc~harides to bFGF (see Fiy. 2), and the extent of depolymerisation (Fig. 3) correlated `
closely with loss of affinity. The presence of major products dp4 and dp6 in size (Fig. 3) was indicative of ~;
cleavage of internal linkages.
Disaccharide compos~tion of oligo~accharides The disaccharide composition of the H, M and L
oligosaccharides (see Table 1) was assessed by polysaccharide lyase depolymerisation and strong anion exchange HPLC as hereinbefore descri.bed. The calculated molar ratios are shown in Table 2. The most striking aspect of the analyses was the high content of disulphated disacchar~des of the type nHexA(2S)-al,4-GlcNS03, particularly in oligo-H and oligo-M (approximately 74% and 60% respectively of disaccharide units). The heparinase sensitivity of these fractions (Flgs. 2 and 3) indicated that the majority of the 2-sulphated HexA residues were originally IdoA(2S) before cleavage of their glycosidic bonds and becoming unsaturated. Since the content of IdoA(2S)-disaccharides in the native HS was approximately 10-12%, the results indicated an enrichment of these residues of approximately seven-fold in oligo-H and six-fold in oligo M. Overall the concentration of nHexA(2S)-w093/lgo96 ~ ~ ~3~7~ PCT/GB93/00597 ` ? 23 al,4-GlcNS03 strongly correlated with the differing bFGF
affinities of the H, M and L oligosaccharides (see Table 1 and Fig. 2). In contrast there was a marked inverse correlation of binding strength with the content of the 6-0-sulphated derivatives nHexA-al,4-GlcNAc(6S) and nHexA-al,4-GlcNS03(6S). These accounted for 26% of di-saccharides in oligo-L but were minor components in oligo-H (Tables 1 and 2). The amount of N-acetylated dlsaccharide nHexA-al,4-GlcNAc was similar in each of the oligosaccharide preparations and corresponded to approximately one per fragment (Tables 1 and 2).

Deaminitive scission with low pH nitrous acid resulted almost exclusively in disaccharide products with the oligo-H and oligo-M fractions (see Fig. 4A and 4B: 99%
and 95% respectively), whereas both disaccharides (76%) and tetrasaccharides (24%) were major products from the ollgo-~ fraction (Flg. 4C). Thus, virtually all the ~nternal hexosaminidic linkages within the oligo-saccharides in the H and M frac~ions involved GlcNS03resldues and the N-acetylated unlt would clearly be at the reducing end of the fragment (see below). This is in contrast to oligo-L for wh~ch the results indicated the presence of an internal N-acetylglucosamine in a larse proportion (60-70%) of the oligosaccharides. The dl-saccharides released from the H, M and L fractions by n~trous acid were also examined by strong anion exchange in order to establish the iden~ity of the constituen~
uronic acid residues. Oligo-H yielded 71~ of labelled product eluting in the position of the standard IdoA(2S)-aManR; the remaining labelled product eluted as an unsulphated peak, corresponding to nGlcA-aManR and IdoA-GlcNAc (results not shown). Oligo-M and oligo-L yielded 61% and 37% respectively as IdoA(2S)-aManR; thus, the content of the disaccharide IdoA(2S)-aManR in each of the fractions correlated well with that of nHexA(2S)-GlcNS03 established by lyase depolymerisation (Table 1).

~3 ~ 24 ~

, Table 1 Disaccharide composition of HS oli~osaccharides with d~fferin~ affinities for bFGF

HS oligosaccharides (dpl2-14) with low (oligo-L), ~.
medium (oligo-M~ and hi~h (oligo-H) affinity for bFGF were prepared as described in connection with Fi~. 2.
Disaccharide composition was analysed by strong anion exchange HPLC as described.~ ~
_ _ -Disaccharide Oligo-L Oligo-M Oligo-H

nHexA-GlcNAc 13.6 16.0 11.3 nHexA-GlcNAc(6S) 12.4 2.6 0.9 nHexA-GlcNS03 27.3 11.3 7.5 nHexA-GlcNSO3(6S) 14.0 4.5 1.4 nHexA(2S)-GlcNSO3 31.0 59.8 74.2 nHexA(2S)-GlcNSO3(6S) 0.6 2.2 1.0 Disaccharide yield % 98.9 96.4 96.3 .

25 ~jl 3~7~

.

Table 2 Stoichiometry of constituent disaccharides of HS
oligosaccharides with differing affinities for bFGF

This table shows the average relative molar ratios of the constituent disaccharides of the HS oligo-saccharides (based on the disaccharide composition data of Table l) and the predominant average size of these oligo-saccharides.

15 Disaccharide Oligo-L Oligo-M Oligo-H

, nHexA-GlcNAc 0.82 1.00 0.82 nHexA-GlcNAc(6S) 0.75 0.16 0.07 nHexA-GlcNS03 1.~5 0.70 0.55 nHexA-GlcNS03(6S) 0.85 0.28 0.10 nHexA(2S)-GlcNSO3 1.88 3.72 5.39 n~exA(2S)-GlcNSO3(6S) 0.04 0~14 0.07 25 Total Disaccharide (moles/mole oligosaccharide) 6 6 7 A novel enzyme, heparitinase IV (from Seikagaku Kogyo Co.), was also used to characterise the sequence of these oligosaccharides. This enzyme has a similar linkage specificity to heparinase ~i.e. GlcNS03(+6S)-a(1-4)-IdoA(2S)], but is much more efficient at cleaving smallsubstrates (such as tetrasaccharides and hexasaccharides) which contain susceptible linkages.

wo 93/1~3 ~ PCT/GB93/00597 Treatment of Oligo-H with the enzyme heparitinase IV
resulted in a high degree of depolymerisation to give di-saccharide and tetrasaccharide products (69% and 31~ of 3H
label respectively, as shown in Figure 5). These results indicated a ratio of 4.5:1 for the number of disaccharides to tetrasaccharides, in good agreement with the expected ratio (5:1) based on the predominant sequence proposed for Oligo-H.

10Treatment of oligo-M with heparitinase IV also resulted in a high degree of depolymerisation giving mainly disaccharide and tetrasaccharide products, but also some hexasaccharides (51%, 36% and 13% of 3H label respectively - see also Figure 5). These results indicate 15a molar ratio of 4:1.4:0.3 for di:tetra:hexasaccharides, in reasonable agreement with the expected ration (4:1:0) based on the predominant sequence proposed for Oligo-M.

The experimental work described above showed that a sulphated oligosaccharide fraction (oligo-H) in fibroblast ~S composed of a sequence of seven dlsaccharides bound particularly strongly to bFGF. The dominant structural unit in the oligosaccharide was IdoA(2S)-al,4-GlcNSO3 (74%
of disaccharide~; Table 1) and both the 2-O-sulphate and the N-sulphate groups appeared to be essential for binding acti~it Analysis of the disaccharide composition following deaminitive scission csnfirmed that the identity of the uronic acid moiety of thi disaccharide was IdoA(2S) and not GlcA(2S). The only other disaccharides present in approximately stoichiometric amounts were nHexA-1,4-GlcNS03 and nHexA-al,4-GlcNAc. Because oligo-H
was a product of heparitlnase digestion it was deduced that the sequence of the principal or most predominant oligosaccharide component or components is:
nGlcA-~1,4-GlcNS03-al,4-[IdoA(2S)-al,4-GlcNS03~5-al,4-IdoA-1,4-GlcR
or, alternatively, 27~ 3 2 7 '^` ~
nGlcA~ 4-[GlcNso3-al~4-IdoA(2s)]6-al~4-GlcR
where R is generally NAc but may be NSO3 These structures have subsequently been confirmed, although further minor variations can occur by the occasional presence of 6-O-sulphate groups on any of the amino sugars (e.g. 1% of disaccharides contain GlcNAc(6S);
see Table 1) without significantly affecting the specific binding affinity. Polysaccharide lyase depolymerisation of these sequences would produce a disaccharide composition for oligo-H which closely matches that shown in Table 1, and the fact that the GlcNAc residue is placed at the reducing end of the sequence (position 14) correlates with the fact that essentially all the internal linkages were sensitive to deaminitive scission (Fig. 4A), indicating the presence of a single contiguous sequence of N-sulphated disaccharides. If the GlcNAc was locatad elsewhere in the sequence, even at position 2, deaminitive sclssion would also produce a significant fraction of tetrasaccharide products in add~tion to disaccharides (e.g. as observed with oligo-L, Fig. 4C).

In the case of oligo-M, it has been established that the principal or predominant oligosaccharide chains have a sequence nGlcA-GlcNSO3(~6S)- r IdoA(2S)-GlcNSO3]4-IdoA-GlcR('6S) where R is generally NAc but may be NSO3 It is believed that this is the first time an extended conti~uous sequence of IdoA(2S)-al,4-GlcNSO3 units has ~een identified in HS. The surprisingly low content of 6-O-sulphate groups clearly distinguishes this oligosaccharide from typical N-sulphated sequences in heparin in which the GlcNSO3 residues are frequently sulphated at C-6 i~e. rIdoA(2S)-al,4-GlcNSO3(6S)~n. The oligo-H sequence identified here may not necessarily represent the minimal sequence for optimal binding to bFGF, ~ut it seems noteworthy that full activation of bFGF

WO93/19096 ~ ~ 28 PCT/GB93/00597 (measured by its ability to bind to the flg receptor) requires heparin fragments of about the same size as oligo-H, i.e. dpl4-dpl6. The related cytokine acidic FGF
is also strongly activated by heparln oligosaccharides in this size range and has also been found to bind to oligo-saccharides of the kind herein identified.

An indication of the structural requirements for optimum high affinity interactions with bFGF can be obtained by comparing the composition of oligo-H with the oligosaccharides of medium and low affinity for bFGF
which, as oligosaccharide products resistant to heparitin-ase digestion, con~ain the same basic disaccharide repeat of IdoA-GlcNSO3. Oligo-H and oligo-M have similar degrees .:
of sulphation (l.6 an~ l.5 sulphates/disaccharide respectively) but oligo-M contains approximately 60% of disaccharides in the form o~ IdoA(2S)-al,4-GlcNSO3 compared to 74~ in oligo-H. About 10% of amino sugars in `.
oligo-M are 6-0-sulphated (Table l) which in terms of ~0 overall sulphation largely offsets the lower concentrat-ions of IdoA(2S). The only other detectable difference between the two fractions is in size, ol~go-M containing ~.
predominantly six disacchar~des compared to seven in oligo-H (Fig. 3). It is believed that the combined effects of fragment size and enric~ent of IdoA(2S) are the key properties that facili~ te a stronger interaction of oligo-H with bFGF. The importance of IdoA(2S) is emphasised by the analytlcal data on the low affinity fragment oligo-L (size dpl2) in which only 31~ of 30 disaccharides contain this component and there appear to -be no more than two of ~he basic IdoA-GlcNSO3 disacchar~de repeat units which may or may not be contiguous within the sequence. However, oligo-L is still quite highly sulphated (l.3 sulphates~disaccharide) because of the higher content of GlcNSO3(6S) and GlcNAc(6S) (Table l), and has some specific bFGF-binding activity so that the main oligosaccharide components thereof, having the sequences hereinbefore specified, may have some utility.

W093/1~96 ~ 3 ~1 7 ~ PCT/GB93/OOS97 The data obtained provide some revealing insights into the structural heterogeneity of HS. The differential O-sulphation of the large N-sulphated oligosaccharides probably reflects a complex mechanism of HS biosynthesis in which the 2- and 6- sulphotransferases may be regulated independently. Although a specific sequence consisting of GlcNS03 and IdoA(2S) appears to be designed for strong binding to bFGF, it is believed that sequences with d~fferent sulphation patterns, especially those with mixed lO 2- and 6-sulphate isomers, may interact with other HS- `
b~nding proteins and other members of the FGF family may bind preferentially with HS sequences which are slightly different to those preferentially recognized by bFGF.
Thus, it will be appreciated that while retain~ng the same lS basic characteristics, there is scope for some structural variations. In particular, even for bFGF (or aFGF) a h~gher proportion of 6-sulphated glucosamine residues is probably unlikely to be seriously detrimental to bindlng affinity, and even sequences similar to oligo-M and oligo-L can provlde oligosaccharides having a useful degree ofspec~fic binding affin~ty although not as high as for oligo-H.

The identification of speclfic bind~ng sequences in glycosaminoglycans (GAGs) is central to an understanding of their biologi~al functions. The sequences of t~e ant~thrombin-III binding region in heparin was a major advance in this field ~L~ndahl, 1984). The interaction is ~
specific, requiring a d~stinct sugar sequence and sulphat- `
30 ion pattern, rather than being determined ma~nly by ;
relatlvely unspecific electrostatic forces. Antithrombin-III is ~activated by heparin in a manner analogous to HS/heparin activation of bFGF. Thus, specific inter-actions with GAGs can convert proteins from latent to active form~, and the inventors hereof have obtained evidence indicating that the oligosaccharides of the present invention can also be effective in activating ~ ;
FGF's such as bFGF. This activation ability, however, WO93/19096~ 30 PCT/GB93/OOSg7 does not appear to be present, at least to a significant extent, in oligosaccharides composed of less that six disaccharide units. When a protein ligand for HS or a similar GAG is known, the method herein disclosed in accordance with the present invention, wherein specific polysaccharide scission and size fractionation is followed by af f inity chromatography, should also prove useful for isolating and characterising the protein-binding domains in these other cases.
Biological Activity In connection with the biological activity of heparitinase-resistant oligosaccharides, further studies have been carried out on the relationship between oligo-saccharide affinity and act~vation of aFGF and bFGF, usinga HS-dependent bioassay of FGF-stimulated mitogenesis.
This assay depended on the fact that 3T3 fibroblasts grown in the presence of the chemical sodium chlorate (which supresses polysaccharide sulphation) do not respond to aFGF or bFGF, but responsiveness (measured by incorpor-ation of 3H-thymidine) is restored by addltion of HS or heparin (as little as l-lOng~ml) to the culture med~um, thus allowing testing of the ability of exogenous HS
oligosaccharides to activate FGFs. A number of oligo-saccharides with a range of structures and affinities forFGFs have been studied using this assay, in particular heparitinase-resistan~ oligos of size dp6, 8, lO, 12, 14 and 16 and larger (from porcine mucosal HS). Preliminary results for ~oth bFGF and aFGF are shown in Figure 7, and indicate that oligosaccharides dpl2 or larger are active, whereas those dplO or smaller are inactive. This suggests that only the larger oligosaccharides of a particular structure excised from HS by heparitinase (and presumably of similar structure to those herein described) are capable of full biological activation of FGFs, and that smaller structures, even if they have some degree of blnding affinity, do not activate these growth factors.

.~31 ~ t ~
Preparation ~ .
In practice, the oligosaccharides of this invention ;~
may be conveniently prepared from purified heparan sulphate, native or recombinant, using the gel filtration chromatography and FGF-affinity chromatography techniques herein described in relation to the investigative experimental work, although generally the heparan sulphate may not need to be radiolabelled for purely preparative purposes. Oligosaccharides derived by heparitinase scission can readily be monitored by virtue of the unsaturated terminal uronic acid residue which absorbs strongly in the ultraviolet range (maximum at 232nm).
` ' .

A specific example of the preferred method of carrying out of the inventlon and of preparing oligo-saccharide products having a relatively high affinlty for bFGF in accordance therewith will now be described in more deta~l, starting with the preliminary purification of the 20 heparan sulphate source mater$al. :.

EXAMPLE `~

25 Prelimlnarv Purificatlon of HS `
GQnerally applicable procedures for the extraction .
and purlfication of PGs and GACs have been described in two detailed reviews whlch cover methods for both connective tissue and cultured cells ~Heinegard & -Sommarin, 1987, Methods ~n Enzymology, 144, 319-372;
Yanagashita et al, 1987, Methods in Enzymology, 138, 279-289), but a preferred procedure for the purpose of this example is now described for the purification of HS from skin fibroblast cells grown in culture.
Confluent cultures of fibroblasts are maintained at 37~C (C02/air, 1:19) in Eagle's minimal essential medium supplemented with 15% (v/v? donor-calf serum, 2mM-glut- `
amine, lmM-sodium pyruvate, non-essential amino acids, :
;

W093/19096 ~ PCT/GB93/~597 c~ ~3 32 ~? ~
penicillin (100 units/ml) and streptomycin (100~g/ml).

Cells can be harvested at confluence, after biosynthetic radiolabelling if necessary ~by incubating for 72 hours with Na35SO4 (e.g. at 10-50~Ci/ml) and/or ~3H~glucosamine ~e.g. at 10-20~Ci/ml)]. HS can be extracted from both the medium and the cell layer. The medium is removed and the cell layers washed twice with warm (37C) phosphate-buffered saline (PBS). These `
combined solutions are centrifuged (200xg, 10 min) to pellet cells and other debris and the result~ng supernatant constitutes the medium extract. HS is efficiently extracted from the cell layers by treatment with 0.05~ (w/v) trypsin in PBS at 37C for 30 min. The resulting cell suspension is centrifuged as above, and the supernatant removed carefully. After washing the pellet twice with ~BS the combined supernatants constitute the cell layer trypsin extract.

~he crude soluble extracts are subjected to initial pur~fication by anion exchange chromatography. Samples in PBS are loaded onto a DEAE-Sephacel column (lcm x 5cm) and washed with 0.3M NaCl in 20mM phosphate buffer, pH 6.8, to elute contaminating proteins and hyaluronic acid. PGs and 25 GAGs which rema~n bound are eluted with a gradient of 0.3-l.OM NaCl in 20mM phosphate bufffer. Fractions corresponding to HS (typically eluting at approximately 0.53M NaCl) are collected, pooled, desalted on a Sephadex G-25 column (2.5cm x 40cm) with distilled water as the 30 eluant, and freeze dried. Traces of contaminating GAGs (e.g. chondroitin and dermatan sulphate) can be removed by treatment with 1 unit of chrondroitinase ABC for 3-4 at 37-C. Protein cores of H5PGs can then be removed by add~ng Pronase (5mg/ml final concentration) and calcium 35 acetate (5mM final concentration) to the Chondroitinase ABC digest and digesting for 24 hours at 37C. HS chains are recovered by step elution from DEAE-Sephacel with lM
NaCl after eluting contaminants with 0.3 NaCl. The 7~

; . 33 :.
fractions containing HS are then heated at 100C for 10 .
minutes, followed by either dialysis against distilled water (using Spectrapor 7 high purity dialysis membrane) or by desalting on a Sephadex G-2S coiumn as above,

5 followed by freeze drying.

DePolYmerisation of HS to selectivelY produce sulphated oligosaccharides:_ 10Biosynthetically 3~ and/or 35so4 labelled HS chains purified as above are treated with heparitinase, i.e. `
heparitinase I tEC 4.2.2.8) from Seikagaku Kogyo Co, Tokyo, Japan, to provide cleavage within regions of relatively low sulphation while leaving intact the! more highly sulphated domains ri~h in N- and 0-sulphate groups and iduronate residues. More specifically, a sample of freeze dried HS (7x106 dpm 3H) is treated with haparitln-ase I (5 milli-units) in 200~1 of lOOmM Na acetate, pH
7.0, conta~ning 0.2mM Ca acetate, at 37C, for 16 hours, followed by addition of a further aliquot of 5 milli-units of the heparitinasP I and incubation for 1 hour at 37C.
Digestion is ~ypically complete in 3-4 hours, but should normally be continued for 16 hours in order to ensure comple~e cleavage of all heparitinase-susceptible 25 linkages. Progress of the reaction can be monitored in `:
the case of unlabelled HS by measuring the increase in absorbance at 282nm due ~o formation of 4,5-unsaturated hexuronate residues a~ the non-reducing ends of dlgestion . :~
products. Digestion is terminated by heating at 100C for ~min.

An alternative chemical method for selective preparation of sulphated domains from HS is to specifically de-N-acetylate the polysaccharide, followed by specific cleavage at the resulting N-unsubstituted glucosamine residues. The methodological details have been described in detail previously (Shaklee and Conrad :.
( 1984 ), Biochem. J. 217, 187-197; Guo and Conrad (1989~, :

W093/19096 ~ PCT/GB93/00597 C~ 3 .~ 34 ,~
Analytical Biochemistry, 176, 96-104). Briefly, de-N-acetylation is carried out by hydrazinolysis by heating the sample at 96C in 70% (w/v) aqueous hydrazine containing 1% (w/v) hydrazine sulphate, for approximately 4 hours. After drying in a stream of air the mixture is neutralized by addition of SOOmM sulphuric acid. The sample is then subjected to deaminitive cleavage at pH 4.0 to specifically cleave at the resulting N-unsubstituted glucosamine residues. This method generates oligo-saccharides which differ from those prepared by heparitin-ase treatment in that they terminate in intact hexuronate residues at their non-reducing ends and in 2,5-anhydro-mannose residues at thelr reduclng ends. The latter residues are normally converted to their 2,5-annhydro-D-mann~tol derivatives by reduction with NaBH4 Radiolabel can be introduced into the oligosaccharides at this stage, if required, by using NaB3H4 as the reducing agent.
..
Fractlonation of oligosaccharides bY ~el filtration:
The oligosaccharide products of the heparitinase (or chemical) treatment method are partially resolved on the bas~s of size by gel filtrat~on chromatography, the result being individual peaks consisting of complex mixtures of oligosaccharides composed of defined numbers of di-sacchar~de units, ranging in size from dlsaccharides upwards, each differing from the next by an increase in slze of one compl~te disaccharide unit. For analytical purposes (e.g. sample loads up to approximately lOmg~
columns (1 x 120cm or 1 x 240cm) packed with Bio-Gel P6 or 3~ Bio-Gel PlO (commercially available from Biorad Ltd.) are suitable, but for the separation of larger quantities the column diameter can be increased appropriately to allow scaling-up. Bio-Gel PlO is particularly suitable for separation of oligosaccharides larger than dplO in size.
The sample is loaded on to the top of the gel and eluted with 500mM NH4HC03 at a flow rate of 4ml/hour. Fractions of lml are collected and a small aliquot taken from each for liquid scintillation counting if the HS has been W093/19096 ~ 3 .2 7 ~ ~ PCT/GB93/00597 ~ 35 ~
labelled. Alternatively, unlabelled HS oligosacaharides ;
can be detected by measuring the absorbance at 232 nm, either of the individual fractions or continuously with a W monitor. Fractions corresponding to oligosaccharide peaks of defined sizes (determined by previous calibration with standards) are pooled and freeze dried. Figure 8 shows a typical result for gel filtration of Bio-Gel P6 of the heparitinase digest of 3H-labelled fibroblast HS
described abo~e.
Fractionation of oligosaccharides bY bFGF affinitY
chromatographY: ;
Individual peaks containing mixtures of oligo-saccharides composed of defined numbers of disaccharide ;
lS units are further fractionated on the basis of their -affinity for bFGF. A description of the preparation of an analytical scale affinity matrix containing bFGF
immobilized on Affi-Gel lO has been described earlier.
For use on a preparative scale, a similar procedure will ~`
20 be followed but the guantity of bFGF coupled and the -amount of gel will be determined by the sample capacity -~
required. The following description is of the use of bFGF-Affi-Gel lO matrix for the separation of 3H-labelled HS oligosaccharides from the fibroblast HS of this 25 particular example.

Approximately lml of bFGF-Affi-Gel lO affinity matrix is packed into a glass column (bed dimensions 6mm x -~5mm). Samples are loaded onto the column in lOmM Tris-30 HCl, pH 6.5, at a flow rate of 0.25 ml/min. Unboundmaterial is eluted by collecting five lml fractions.
Bound material is eluted with a gradient of sodium chloride (0-2M NaCl in column buffer) at a flow rate of 0.5 ml/min. This can be conveniently achieved by a 35 discontinuous step gradient (e.g. increasing concentration `~
of NaCl by steps of 2SOmM NaCl or other suitable increment~. Five lml fractions are collected at each concentration. Alternatively, a linear- continuous '~
:

WO 93/lgO96 ~ PCr/GB93/00597 ~,3 3 -~ 36 gradient ( e. g. with a total volume of 50ml ) may be used to elute bound fragments, and lml fractions collected. A
small aliquot is taken from each fraction for liquid scint~llation counting (or detection by W absorbance).

Figure 6 shows a typical result of the bFGF affinity chromatography of heparitinase-resistant oligosaccharides peaks of different sizes (dp2-dpl4) prepared by Bio-Gel P6 gel filtration. Selected fractions containing oligo-saccharides having the same affinities for bFGF arepooled, desalted either by dialysis against distilled water using Spectrapor 7 1000-Mr cut-off membrane (Pierce Ltd) and/or by again using gel filtration on a Bio-Gel ~2 column (l.Scm x 30cm) eluted with 500mM NH4HC03 at a flow rate of lOml/hour, and freeze dried. The fractions of particular interest are those for dp=12 and dp=14.

Additional Purification:
Additional purification of the oligosaccharidas with selected specific affinities for bFGF c~n be achieved by further steps of gel filtration chromatography and bFGF-a~finity chromatography. This was carried out for example in the preliminary experimental work in respect of which Figure 2 illustrates the bFGF-affinity profiles of oligo-saccharides (dpl2 and dpl4) selected for low (750mMfraction), medium (lOOOmM fraction) and high (>1250mM
fraction) a~finity for bFGF by an bFGF-affinity step following a second application to the affinity column. In additlon, it is also possible i~ de~ired to further purify the oligosaccharides to apparent homogeneity by applicat-ion of two addltional techniques: strong anion exchange (SAX) HPLC ~hich separates mainly according to anionic charge properties, and gradient polyacrylamide gel electrophoresis (PAGE) which separates predominantly according to molecular size.

Thus, to obtain increased purity of binding oligo-saccharides by ~AX HPLC chromatography, separations are

6 ~ t 3 ~ 7 ~ PCT/GB93/00597 made on ProPac PA1 columns (from Dionex Ltd), either an analytical column (4 x 250mm) or alternatively a semi~
preparative column (9 x 2SOmm). After equilibration in --mobile phase (double distilled water adjusted to pH 3.5 S with HCl) at lml/min samples are injected and oligo- -~
saccharides eluted with a linear gradient of sodium -chloride (0-2M over 180 minutes) in the same mobile phase.
The eluant is monitored in-line for W absorbance (A282) for detection of unlabelled oligosaccharides, and/or for ~;
10 radioactivity (e.g. using an in-line monitor such as a ~;
Radiomatic Flo-one/Beta A-200 detector, Canberra Packard --~
Ltd).

Gradient PAGE methods have been described in ~etail in previous publications (e g. Turnbull, J.E. & Gallagher, J.T. (1988) ~iochem. J. 251, 5~7-608; Turn~ull, J.E. & ;-Gallagher, J.T. (1990) Biochem. J. 265, 715-724; Turnbull et al ( 1993), "Approaches to the structural analysis of GAGS" in Ext~acellular Matrix Macromolecules: A Pract cal .:
~pproach, Oxford University Press; Turnbull (1993), ~Oligosaccharide mapping and sequence analysis of GAGs~, ~n Methods in Molecular B~ology: Memb~ane Methods, Humans Press, Chapter 24). This methodology provides a very powerful technique for resolving complex mixtures of large ollgosaccharides into single apparently homogeneous species~ and it can be adapted to preparatlve scale for the separation of large quantities of oli~osaccharides, elther by eluting directly from the gel using approprlate apparatus or by electrotransfer from the gel onto a positively-charged nylon membrane, followed by recovery from the membrane by elution with salt as described in the above references.

Various combinations of these techniques described can thus enable a very high degree of final purification of homogeneous preparations of well defined oligo-saccharide products in accordance with the invention which have specific sequences and defined affinities for growth 3 l.q ` 38 factors such as bFGF.

Inso~ar as the basic features of oligosaccharide . sequences have been identified and characterised that give rlse to a specific FGF binding affinity, whlch ~n the case of oligo-H can be of the same order as in heparan sulphate, it will be appreciated that thls knowledge can also enable such oligosaccharides and analogues thereof having like binding affinities now to be made or 1o constructed synthetically and they may be " tailor made" to suit requirements using conventional synthetlc methods.
For example, insofar as these compounds can be regarded as being built up of three types o~ main disaccharide unlts, 15 precursors of these units d~signated ~ , ~ and ~ , -which are to be arranged as ~ ~ , may be separately synthesised w~th O-acetyl or O-methylchloro-acetonyl (OMCA) protected terminal groups, e.g.
20 MCA~AC, ~AC and MCA ~ . The MCAO and OAC
groups can then be converted selectively to -OH groups,-e.g. by pyr~dine and hydrazlne respectively, to enable `
firstly the required number of B units to be coupled together followed by the select~ve coupling of the required term~nal units to build up the chain, and the structure produced can then be subjected to deacylation, O-sulphation, hydrogenolysis and N-sulphation as necessary .
to glve the flnal product. For a general review to synthetic methods, reference may be made to "Heparin", edited by D.A. Lane and V. Lindahl, page 5l onwards.

`-WO93/19096 3~1 ~ f~J 7 vi ~3 PCT/~B93/00597 T~ERAPEUTIC USES

In general, for therapeutic use of the oligosaccharide products of the present invention and administration to mammals in need of treatment, an effective growth factor binding amount of the active oligosaccharide, which may be in the form of a pharmaceutically acceptable salt, will be made up as a pharmaceutical formulation ready for sdministration in any suitable manner, for example orally, lO parenterally (including subcutaneously, intramuscularly -and intravenously), or topically, or in a slow-release dlspensing device for implantation. Such formulations may be presented in unit dosage form and may comprise a pharmaceutical composition, prepared by any of the methods 15 well known in the art of pharmacy, in which the active ~
oligosaccharide component or components is in intimate -association or admixture with at least one other ingredient providing a compatible pharmaceutically acceptable carrier, diluent or excipient. Alternatively, such formulations may comprise a protective envelope of ccmpatible or relatively inert pharmaceutically acceptabla material within which is contained the active oligosaccharide component or compone.nts with or without a~sociation or admixture with any other ingredients.
It may be noted that for pharmaceutical use, it may be preferable for the oligosaccharides of the present invention to be in the form in which their non-reducing ~
ends are unsaturated, as obtained by heparitinase scission, since there is some evidence that this form may be more resistant to bio-transformation which could reduce efficiency. However, this may not be essential for all applications.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, W003/t90~6 ~ ~ PCT/GB93/00597 with capsules being a preferred type of formulation for providing the most effective means of oral delivery. For parenteral administration the formulations may comprise s~erile liquid preparations of a predetermined amount of the active oligosaccharide component contained in sealed ampoules ready for use.

The amount of the oligosacch~ride products of the invention, and dosing regimen required for effective therapeutic use will of course vary~ and will be ul~imately at the discretlon of the medical or veterinary~ practition-er treating the mammal in each particular case. The factors to be considered by such a practitioner, e~.g. a phys~cian, include not only the particular disorder being treated (and whether growth factor stimulation or growth factor inhibition is required) but also the route of admini~tration and type of pharmaceutical formulation;
the mammal's body weisht; surface area, age and ~eneral conditlon. However, a suitable e~fective bFGF inhibitory dose, ~.g. for ant~tumour treatment, might perhaps be in the range of about 1,0 to about 75 mg/kg bodyweight, preferably in the range of about 5 to 40 mg/kg with most suit~ble doses being for example in the range of 10 to 30 mg/kg. In daily treatment for example, the total daily dose may be given as a sinyle dose, multiple doses, e.g.
two to slx tlmes per day, or by intxavenous infusion for any se~ected duratlon. For example, ~sr a 75 kg mammal, the dose range could perhaps be about 75 to 500 mg per day, and a typical dose would commonly be a~out 100 mg per day. If discrete multiple doses are indicated, treatment might typically be 50mg of an oligosaccharide produc~, as h~reinbefore defined, givan 4 times per day in the form of a tablet, capsule, liquid (e.g. syrup) or in;ection.

- 35 As previously indicated, in some cases where th~
treatment required consists in administering a growth factor such as bFGF, for example to promote tissue repair as in wound healing applications, the active oligo-WO93/19096 ~.t 3 2 7 ~ PCT/GB93/00597 .. 41 ..
saccharide component may be co-administered with the growth factor.

Apart from their use in conjunction with the administration of growth factors in wo~nd healing applications and in other medical applications where it is desired to increase or st$mulate growth factor acti~ity, e.g. bone healing, nerve regeneration, duodenal or venous ulcers, vari~us ocular and retinal disorders, 10 atherosclerosis, degenerative muscle disorders, ischaemia, ..
or for protecting tissues against serious damage during ~.
radiation treatment, ~he medical uses of the .;
ol~gosaccharide compounds or products of the p:resent invention will probably be most frequently targett:ed to the inhibition of grow~h factor activity, and pharmaceutical formulations or compositions containing these oligosaccharides are expected to be especially.
useful, as previously indicated, for trea~ing conditions that arise, or are aggravated, as a result of activlty of .`
growth factors promoting harmful growth or cell proliferation, e.g. conditions, such as diabet~c rettnopathy, capsular opacification, proliferative vitreoretlnopathy, tumour angiogenesis, cancer cell growth and metastasis, rheumatoid arthritis, mild muscular dystrophy, Alzheimer disaase, various viral infections (e.g. Herpes Simplex type 1), or :restenosis following angioplasty and other forms of chronic inflammat~on.

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 compounds, herein dlsclosed either explicltly or implicitly 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.

Claims (39)

1. An oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's), characterised in that it is obtainable by depolymerisation of heparan sulphate with heparitinase and it consists essentially of non-heparin oligosaccharide chains which are substantially homogeneous with respect to FGF binding affinity and which contain at least six disaccharide units including a contiguous sequence of sulphated disaccharide units that are each composed of an N-sulphated glucosamine residue (?6S) and a 2-O-sulphated iduronic acid residue, and in that the content (if any) of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 is less than 20%.

2. An oligosaccharide product as claimed in Claim 1 in which each of said sulphated disaccharide units is IdoA(2S)-.alpha.1,4-GlcNSO3.

3. An oligosaccharide product as claimed in Claim 1 or 2 in which said oligosaccharide chains consist of a sequence of less than ten disaccharide units.

4. An oligosaccharide product as claimed in any of Claims 1 to 3, further characterised in that it is substantially completely resistant to depolymerisation by heparitinasc but not by heparinase.

5. An oligosaccharide product as claimed in any of Claims 1 to 4 in which at least the predominating majority of said oligosaccharide chains are all of the same length.

6. An oligosaccharide product as claimed in any of the preceding claims in which substantially all said oligosaccharide chains consist of a sequence of six disaccharide units in all.

7. An oligosaccharide product as claimed in any of the preceding claims in which said oligosaccharide chains include a contiguous sequence of at least four said sulphated disaccharide units.

8. An oligosaccharide product as claimed in any of Claims 1 to 4 in which substantially all the oligosaccharide chains consist of a sequence of seven disaccharide units of which at least five are included in said contiguous sequence of sulphated disaccharide units.

9. An oligosaccharide product as claimed in any of the preceding claims in which the content (if any) of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 is less than 20% of the total number of glucosamine residues.

10. An oligosaccharide product as claimed in Claim 9 in which the content (if any) of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 is less than 5% of the total number of glucosamine residues.

11. An oligosaccharide product as claimed in any of Claims 1 to 10 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 heparitinase 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 FGF growth factor as the immobilised ligand in order to separate out the FGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient, advantageously a serially stepped gradient, to fractionate said fragments in respect of FGF
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.

12. A non-heparin oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's), characterised in that (a) it is composed predominantly of a molecular species:

in which X is ?HexA-GlcNSO3(?6S), Y is IdoA(2S)-GlcNSO3(?6S), Z is IdoA-GlcR(?6S) or IdoA(2S)-GlcR(?6S) where R is NSO3 or NAc, and n is in the range 4 to 7 (b) the number, if any, of monosaccharide residues having a 6-O-sulphate group is less than 20% of the total number of monosaccharide residues;
(c) it is obtainable by a process comprising the steps of digesting a heparan sulphate with heparitinase 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 chromatography, collecting a fraction or fractions containing oligosaccharide chains having a particular size selected within the range of 12 to 18 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using an immobilised FGF ligand and recovering the more strongly FGF-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.

13. An oligosaccharide product as claimed in Claim 12, wherein Y is exclusively IdoA(2S)-GlcNSO3.

14. An oligosaccharide product as claimed in Claim 12 or 13, wherein n is 5 or 6.

15. An oligosaccharide product as claimed in Claim 14 wherein said molecular species consists of a total of seven disaccharide units in all.

16. An oligosaccharide product as claimed in Claim 12 or 13 wherein n is 4 and the total number of disaccharide units in said molecular species is 6.

17. An oligosaccharide product as claimed in any of Claims 12 to 16 in which the content, if any, of monosaccharide residues having a 6-O-sulphate group is less than 5% of the total number of monosaccharide residues.

18. An oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's) that is substantially all composed of non-heparin oligosaccharide chains which are fourteen monosaccharide residues in length and which contain an internal contiguous sequence of 5 or 6 disaccharide units each consisting of an IdoA(2S) residue linked to a GlcNSO3(?6S) residue, with less than 20% of the glucosamine residues (terminal or internal) being 6-O-sulphated.

19. An oligosaccharide product as claimed in Claim 18 wherein the oligosaccharide chains have sequences selected from (?)GlcA-GlcNSO3-[IdoA(2S)-GlcNSO3]5-IdoA-GlcR
and (?)GlcA-[GlcNSO3-IdoA(2S)]6-GlcR
where R is NSO3 or NAc.

20. An oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's) that is substantially all composed of non-heparin oligosaccharide chains which are twelve monosaccharide residues in length and which contain an internal contiguous sequence of 4 disaccharide units each consisting of an IdoA(2S) residue linked to GlcNSO3(?6S) residue, with less than 20% of the glucosamine residues (terminal or internal) being 6-O-sulphated.

21. An oligosaccharide product as claimed in Claim 20 wherein the predominant oligosaccharide chain sequence is (?)GlcA-GlcNSO3(?6S)-[IdoA(2S)-GlcNSO3]4-IdoA-GlcR(?6S) where R is NSO3 or NAc.

22. An oligosaccharide product having a relatively high specific binding affinity for basic fibroblast growth factor (bFGF) and consisting essentially of non-heparin oligosaccharide chains having a disaccharide sequence ?GlcA-.beta.1,4-GlcNSO3-.alpha.1,4-[IdoA(2S)-.alpha.1,4-GlcNSO3]5-.alpha.1,4-IdoA-.alpha.1,4-GlcR
or ?GlcA-[GlcNSO3-IdoA(2S)]6-GlcR
where R is NSO3 or NAc or minor variants thereof which have at least the same relatively high specific binding affinity for bFGF.

23. A method of isolating from a glycosaminoglycan such as heparan sulphate an oligosaccharide product as claimed in any of the preceding claims composed of small oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for a fibroblast growth factor (FGF) that itself binds to said glycosaminoglycan or to the corresponding proteoglycan in multicellular biological systems, said method comprising the steps of (a) preparing an affinity chromatographic matrix or substrate incorporating a sample of said fibroblast growth factor as the affinity ligand immobilised thereon;
(b) treating said glycosaminoglycan 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 four to nine in order to extract from the latter and retain on said matrix or substrate size selected oligo-saccharide fragments of the glycosaminoglycan that have at least some binding affinity for the immobilised said fibroblast growth factor;
(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).

24. A method as claimed in Claim 23, wherein the glycos-aminoglycan is heparan sulphate derived from heparan sulphate proteoglycan of mammalian cells and the fibroblast growth factor is a cytokine that when activated under physiological conditions stimulates mammalian cells through binding interaction with signal transducing receptors on the surface of said cells.

25. A method as claimed in Claim 24 in which the selective scission reagent is heparitinase and the heparan sulphate is partially depolymerised to the fullest extent by digesting therewith until cleavage of the heparitinase sensitive linkages is complete.

26. A method as claimed in Claim 24 wherein the selective scission reagent is nitrous acid which, after prior treatment of the heparan sulphate with an N-deacetylating agent, is reacted at about pH 4 with the polysaccharide to cleave it at the free amino groups therein.

27. A method as claimed in any of Claims 23 to 26 wherein the fibroblast growth factor is basic fibroblast growth factor (bFGF).

28. A method as claimed in any of Claims 23 to 27, wherein the fractions collected from the size fractionation stage are those that appear to contain oligosaccharides composed of seven disaccharide units.

29. A method as claimed in any of Claims 23 to 27 wherein the fractions collected from the size fractionation stage are those that appear to contain oligosaccharides composed of six disaccharide units.

30. An oligosaccharide product as claimed in any one of Claims 1 to 22 for therapeutic use as an active FGF-activity stimulating agent for promoting healing or tissue repair in treating mammals in need of such treatment, for example in conditions such as wound healing, bone healing, nerve regeneration, duodenal or venous ulcers, various ocular and retinal disorders, atherosclerosis, degenerative muscle disorders, ischaemia, or for protecting tissues against serious damage during radiation treatment.

31. A medical composition comprising the oligosaccharide product of Claim 30 in association with an exogenous FGF
growth factor for co-administration therewith in carrying out the treatment therein referred to.

32. An oligosaccharide product as claimed in any one of Claims 1 to 22 for therapeutic use as an active FGF-activity inhibiting agent for controlling or reducing cell growth or proliferation in treating mammals in need of such treatment, for example in connection with conditions such as diabetic retinopathy, capsular opacification, proliferative vitreoretinopathy, tumour angiogenesis, cancer cell growth and metastasis, rheumatoid arthritis, mild muscular dystrophy, Alzheimer disease, various viral infections (e.g. Herpes Simplex type 1), or restenosis following angioplasty.

33. A pharmaceutical formulation or composition for medical use comprising a therapeutically effective non-toxic amount of an FGF-activity modulating agent comprising an oligosaccharide product as claimed in any of Claims 1 to 22 or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier or vehicle.

34. Use of an oligosaccharide product as claimed in any of Claims 1 to 22, for the manufacture of a medical preparation for use in the treatment of diabetic retinopathy, capsular opacification, proliferative vitreoretinopathy, tumour angiogenesis, cancer cell growth and metastasis, rheumatoid arthritis, mild muscular dystrophy, Alzheimer disease, various viral infections (e.g. Herpes Simplex type 1), or restenosis following angioplasty or for use in promoting repair of damaged tissues in conditions such as wound healing, bone healing, nerve regeneration, duodenal ulcers, various ocular and retinal disorders, atherosclerosis, degenerative muscle disorders, ischaemia, or for protecting tissues against serious damage during radiation treatment.

35. An oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's), consisting essentially of oligosaccharide chains which are substantially homogeneous with respect to FGF binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of sulphated disacchaxide units that are each composed of an N-sulphated glucosamine residue (?6S) and a 2-O-sulphated iduronic acid residue with less than 5% of said glucosamine residues having a 6-O-sulphated group.

36. A pharmaceutical composition or formulation for use in controlling the activity of fibroblast growth factors in mammals for promoting tissue repair or for inhibiting cell growth or proliferation in the treatment of disorders resulting therefrom, said composition or formulation comprising a therapeutically useful and therapeutic amount of an essentially pure oligosaccharide product as claimed in Claim 35.

37. A method for treating a mammal to promote healing or tissue repair in the case of wounds, duodenal or venous ulcers, various ocular and retinal disorders, atherosclerosis, degenerative muscle disorders, or ischaemia, for promoting bone healing or nerve regeneration, or for protecting tissues against serious damage during radiation treatment, which comprises administering to a mammal in need of such treatment an effective amount of an essentially pure oligosaccharide product as claimed in Claim 35 or in any of Claims 1 to 22.

38. A method as claimed in Claim 37 in which the oligosaccharide product is co-administered with a preparation of bFGF.

39. A method of treating diabetic retinopathy, capsular opacification, proliferative vitreoretinopathy, tumour angiogenesis, cancer cell growth and metastasis, rheumatoid arthritis, mild muscular dystrophy, Alzheimer disease, various viral infections (e.g. Herpes Simplex type 1), or restenosis following angioplasty in mammals by inhibiting FGF growth factor activity, which method comprises administering to a mammal in need of such treatment an effective amount of an essentially pure oligosaccharide product as claimed in Claim 35 or in any of Claims 1 to 22.