Note: Descriptions are shown in the official language in which they were submitted.
W093/19096 PCT/GB93/00597
, ~.
~1 3~7 ~1~
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:
WO93/19096 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
WO93/19096 PCT/GB93/00597
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).
WO93/19096 PCT/GR93/00597
~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
.
WO93/19096 PCT/GB93/00597
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
26
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,
WO93/19096 PCT/GB93/00597
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.
WO93/19096 PCT/GB93/00597
.~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~
WO93/19096 PCT/GB93/00597
; . 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
WO93/19096 ~ t 3 ~ 7 ~ PCT/GB93/00597
37
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
W093/19096 PCT/GB93/OOS97
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.
