Note: Descriptions are shown in the official language in which they were submitted.
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Materials and Methods Relating to Identifvina
InositolnhosohoQlvcan Mimetics
Field of the Invention
The present invention relates to materials and methods
for screening for inositolphosphoglycan (IPG) mimetics,
and in particular to methods employing antibodies capable
of specifically binding to inositolphosphoglycans (IPGs).
Hackcrround of the Invention
Many of the actions of growth factors on cells are
thought to be mediated by a family of inositol
phosphoglycan (IPG) second messengers (Rademacher et al,
1994). It is thought that the source of IPGs is a "free"
form of glycosyl phosphatidylinositol (GPI) situated in
cell membranes. IPGs are thought to be released by the
action of phosphatidylinositol-specific phospholipases
following ligation of growth factors to receptors on the
cell surface. There is evidence that IPGs mediate the
action of a large number of growth factors including
insulin, nerve growth factor, hepatocyte growth factor,
insulin-like growth factor I (IGF-I), fibroblast growth
factor, transforming growth factor (3, the action of IL-2
on B-cells and T-cells, ACTH signalling of adrenocortical
cells, IgE, FSH and hCG stimulation of granulosa cells,
thyrotropin stimulation of thyroid cells, cell
proliferation in the early developing ear and rat mammary
gland.
Soluble IPG fractions have been obtained from a variety
of animal tissues including rat tissues (liver, kidney,
muscle brain, adipose, heart) and bovine liver. IPG
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2
biological activity has also been detected in malaria
parasitized red blood cells (RHC) and mycobacteria. We
have divided the family of IPG second messengers into
distinct A and P-type subfamilies on the basis of their
biological activities. In the rat, release of the A and
P-type mediators has been shown to be tissue-specific
(Kunjara et al, 1995).
There are some references in the prior art to the
production of polyclonal antibodies having anti-IPG
specificity (see Romero et al, 1990; Huang et al, 1993;
Nestler et al, 1991; Represa et al, 1991). In these
papers, a single polyclonal antibody is described which
was raised against the GPI-anchor of the variant surface
protein (VSG) of Trypanosoma brucei. The authors were
forced to use this cross-reacting antigen for obtaining
anti-IPG antibodies as soluble IPGs had not been purified
and characterised, and so were not available for raising
specific anti-IPG antibodies directly. The polyclonal
antibodies produced using this immunisation protocol
react also with the GPI-anchor moieties of GPI-anchor
containing proteins. The papers report that this
polyclonal antibody inhibits the action of insulin on
human placental steroidogenesis, inhibits the action of
insulin on muscle cells, inhibits the action of insulin
on rat diaphragm and inhibits the action of nerve growth
factor on chick cochleovestibular ganglia (CVG).
Summary of the Invention
Broadly, the present invention relates to the use of
polyclonal or monoclonal antibodies specific for
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inositolphosphoglycans (IPGs) in identifying compounds
having one or more of the biological activities of a P or
A-type IPG.
we have previously found and isolated families of P and
A-type IPGs, and shown that some of their activities are
tissue specific. This tissue specificity could be
advantageous in employing the IPGs as pharmaceuticals as
they are likely to have very selective biological
properties when administered in vivo. However, in
instances where the IPGs themselves are difficult to
isolate from natural sources in large quantities or are
difficult to synthesise, it may instead be desirable to
use a mimetic compound having the same or a similar
activity that is easier to make or obtain. The closely
related biological activities of the IPGs then become a
problem in screening for such candidate mimetic
compounds, as many conventional approaches to screening
will not be able to differentiate the subtle differences
in biological activity of different IPGs.
Hy way of example, some mimetic compounds have already
been found and are described in W099/38516. This
application discloses that compound C3, 1D-6-O-(2-amino-
2-deoxy-a-D-glucopyranosyl)-myo-inositol 1,2-(cyclic
phosphate), promotes neuron proliferation ~s do A-type
IPGs, and compound C4, iD-6-0-(2-amino-2-deoxy-a-D-
glucopyranosyl)-chino-inositol 1-phosphate. promotes
neurite growth as do P-type IPGs.
The present invention approaches the problem of screening
AMENDED SHEET
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4
for IPG mimetics by taking advantage of the generation of
a range of anti-IPG antibodies specific for a variety of
epitopes on the /PGs. Accordingly, it is possible to use
these antibodies to characterise the IPGs in the
families, providing a "fingerprint~~ or binding profile
that can distinguish a given IPG from other family
members. This opens up the possibility of using the
antibodies and the binding profiles to screen for IPG
mimetics having an activity of a specific IPG, e.g. the
activity of P-type IPG from placenta. This is especially
useful as it simplifies the screening of candidate
compounds, identifying lead compounds that are likely to
have a specific IPG biological activity. The method has
the further advantages in that it is not necessary to
structurally characterise the IPGs or the candidate
compounds, provided that the /PGs are isolated in a
sufficiently pure form to raise the group of antibodies,
and that it is amenable to high throughput screening
(HTS). The method could also be used to provide a
stringent selection of reactive clones against a specific
IPGs.
Accordingly, in a first aspect, the present invention
provides a method for determining a binding profile for
an inositolphosphoglycan (IPG) or a candidate mimetic
compound, the method comprising contacting the IPG or
candidate mimetic compound with an anti-IPG antibody in a
binding assay and determining the binding of the IPG or
candidate mimetic compound to the antibody to provide the
binding profile.
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In some embodiments, the binding profile comprises the
results of binding assays in which more than one IPG or
candidate mimetic compound is contacted with a given
anti-IPG antibody. Alternatively or additionally, the
5 binding profile comprises the results of binding assays
in which more than one anti-IPG antibody is contacted
with the IPGs or candidate mimetic compounds.
In a further aspect, the present invention provides a
method for identifying inositolphosphoglycan (IPG)
mimetics, the method comprising:
(a) contacting a P or A-type IPG with an anti-IPG
antibody in a binding assay and determining the binding
of the IPG to the antibody to provide a binding profile
for the IPG;
(b) contacting one or more candidate mimetic
compounds with the anti-IPG antibody in a binding assay
and determining the binding of the candidate mimetic
compounds to the antibody to provide binding profiles for
the candidate mimetic compounds: and,
(c) comparing the binding profiles of the IPG and
candidate mimetic compounds to identify for candidate
mimetic compounds likely to have the IPG activity.
In a further aspect, the present invention provides a
method for screening for inositolphosphoglycan (IPG)
mimetics, the method comprising having contacted a P or
A-type IPG with an anti-IPG antibody in a binding assay
to determine a binding profile for the IPG, the further
steps of:
(a) contacting one or more candidate mimetic
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compounds with the anti-IPG antibody in a binding assay
and determining the binding of the candidate mimetic
compounds to the antibody to provide the binding profiles
for the candidate mimetic compounds; and,
(b) comparing the binding profiles of the IPG and
candidate mimetic compounds to identify candidate mimetic
compounds likely to have given IPG activity.
In the above methods, preferably, the method further
comprises the step of testing a candidate mimetic
compound identified in the method to determine whether it
has a desired IPG activity.
Thus, in these aspects of the invention the exposure of
one or more antibodies to individual P or A-type IPGs
provides binding profiles) which can be used as a tool
to screen a library of candidate compounds to identify
candidate compounds likely to have given IPG activity.
In the present invention, a "binding profile~~ is defined
as the responses) obtained when an IPG or a candidate
mimetic compound is used in one or more binding assays
employing one or more anti-IPG antibodies as binding
agent(s). Such responses may be recorded simply as
positive or negative, or the magnitude of the response
may be determined, either to provide a value
representative of the response or determine whether,
within the limits of experimental error, the response is
above a threshold value. In general, the binding profile
will depend on the binding agent (anti-IPG antibody) or
developing agent used and/or the conditions used to carry
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out the assay. The discrimination between different IPGs
and candidate compounds that the binding profile provides
will generally increase as the number of individual
assays comprising the profile increases and as the
precision of the determination of the response in the
assays increases.
In the present invention, the candidate compounds
selected in the screen may be those have identical or
near identical binding profiles to the given IPG, or in a
less stringent screen, candidate compounds having similar
binding profiles could be selected (e.g. preferably to
within 20~, or more preferably to within 10~, or still
more preferably to within 5~ of the value obtained in a
binding assay using the IPG). Further, the selectivity
of the screening method can be changed by altering the
stringency in the determination of the binding profiles
of the IPGs and/or in the stringency of the conditions
used to screen for mimetics. This could be done by using
a competition assay versus a direct binding assay. The
latter gives no information on the binding constant,
whereas a competition assay provides access to numerical
binding constants.
Thus, the present invention provides a test method which
establishes common or similar structural motifs in IPGs
and candidate IPG mimetic compounds, identifying mimetic
compounds likely to have an IPG activity or activities in
common or similar to the IPG.
The compounds selected can then be subjected to further
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testing, providing lead compounds for further development
as pharmaceuticals.
In these aspects, where the candidate compounds are
present in a library or mixture of compounds, the method
may include the additional step of isolating compounds
found to have the same or similar binding profiles to the
IPG. This approach can help to increase the throughput
of screening assay, allowing large numbers of candidate
compounds to be screened.
Alternatively or additionally, the binding profiles of
more than one IPG can be determined and these profiles
used to screen simultaneously for mimetics for these
IPGs.
Exemplary protocols for raising anti-IPG antibodies are
set out below. Examples of antibodies that could be
employed in the method include those produced by
hybridoma cell lines 2F7, 2D1 and 5H6 deposited at
European Collection of Cell Cultures (ECACC) under
accession numbers 98051201, 98031212 and 98030901. Based
on the teaching herein, the skilled person could readily
prepare further antibodies having the same or different
binding specificities for use in the present method.
The inventors have surprisingly found that despite the
fact that IPGs are small molecules and carbohydrates, it
is possible to raise antibodies to them. Further, the
fact that conjugation of the IPGs to a carrier is not
required means that epitopes on the IPGs are not blocked
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9
or altered by the conjugation reaction, allowing
antibodies to be raised to a full complement of epitopes
on the IPGs. The method is applicable to the production
of both polyclonal and monoclonal antibodies, as is
demonstrated by the examples below. Preferably, the
animal is immunised via an intraperitoneal route using a
soluble form of the IPG.
In a further aspect, the present invention provides a
candidate mimetic compound as identifiable by the above
methods.
In a further aspect, the present invention provides an
inositolphosphoglycan or candidate IPG mimetic compound
binding profile as obtainable by the above methods.
Conveniently, the antibodies are immobilised on a solid
support so that they can be exposed to liquid samples
containing the IPGs or candidate mimetics. Preferably,
the different antibodies are immobilised at discrete
locations on the support (e.g. individual wells on a
multiwell plate or as microspots) so that they can be
distinguished from each other.
In a further aspect. the present invention provides a
solid support having immobilised thereon a plurality of
different anti-IPG antibodies, the individual antibodies
being immobilised at discrete locations on the support.
Preferably, the method employs at least 5 different anti-
IPG antibodies, more preferably at least 10 antibodies,
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more preferably at least 15 antibodies and more
preferably at least 20 antibodies. The use of larger
numbers of antibodies that are specific for a range of
epitopes of the IPG family members helps to ensure that
5 the antibody binding profile obtained when the antibodies
bind to the IPG family member can be distinguished from
the profile obtained using other IPG family members.
Embodiments of the present invention will now be
10 described by way of example and not by limitation with
reference to the accompanying drawings.
Brief Description of the Drawincs
Figure 1 shows a dose-response curve of absorbance
plotted against IPG concentration using anti-IPG
antibodies in a Sandwich ELISA test.
Figure 2 shows the results of experiments comparing the
binding specificity of monoclonal antibody 5H6, a prior
art polyclonal antibody raised against the GPI anchor of
VSG and a polyclonal antibody raised against the cross
reactive determinant (CRD) common to GPI anchored
proteins.
Figure 3 shows the results of a PDH phosphatase assay
demonstrating that monoclonal antibody 5H6 is capable of
inhibiting the action of P-type TPG from rat liver.
Figure 4 shows binding profiles for 8 candidate compounds
obtained using monoclonal antibody 2D1 as the binding
agent and two polyclonal antibodies as developing agents.
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Figure 5 shows binding profiles for 8 candidate compounds
obtained using monoclonal antibody 5H6 as the binding
agent and two polyclonal antibodies as developing agents.
Figure 6 shows binding profiles for 8 candidate compounds
obtained using monoclonal antibody A2 as the binding
agent and two polyclonal antibodies as developing agents.
Detailed Description
/PGs and 2PG Analocues
Studies have shown that A-type mediators modulate the
activity of a number of insulin-dependent enzymes such as
cAMP dependent protein kinase (inhibits), adenylate
cyclase (inhibits) and cAMP phospho-diesterases
(stimulates). In contrast, P-type mediators modulate the
activity of insulin-dependent enzymes such as pyruvate
dehydrogenase phosphatase (stimulates), glycogen synthase
phosphatase (stimulates) and cAMP dependent protein
kinase (inhibits). The A-type mediators mimic the
lipogenic activity of insulin on adipocytes, whereas the
P-type mediators mimic the glycogenic activity of insulin
on muscle. Both A and P-type mediators are mitogenic
when added to fibroblasts in serum free media. The
ability of the mediators to stimulate fibroblast
proliferation is enhanced if the cells are transfected
with the EGF-receptor. A-type mediators can stimulate
cell proliferation in the CVG.
Soluble IPG fractions having A-type and P-type activity
have been obtained from a variety of animal tissues
including rat tissues (liver, kidney, muscle brain,
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adipose, heart) and bovine liver. A and P-type IPG
biological activity has also been detected in human liver
and placenta, malaria parasitized RHC and mycobacteria.
The ability of a polyclonal anti-inositolglycan antibody
to inhibit insulin action on human placental
cytotrophoblasts and HC3H1 myocytes or bovine-derived IPG
action on rat diaphragm and chick cochleovestibular
ganglia suggests cross-species conservation of many
structural features. However, it is important to note
that although some prior art references include reports
of A and P-type IPG activity in some biological
fractions, the purification or characterisation of the
agents responsible for the activity was not disclosed
until it was reported in the references below.
A-type substances are cyclitol-containing carbohydrates,
also containing Znz' ion and optionally phosphate and
having the properties of regulating lipogenic activity
and inhibiting cAMP dependent protein kinase. They ma
y also inhibit adenylate cyclase, be mitogenic when added
to EGF-transfected fibroblasts in serum free medium, and
stimulate lipogenesis in adipocytes.
P-type substances are cyclitol-containing carbohydrates,
also containing Mnz+ and/or Znz' ions and optionally
phosphate and having the properties of regulating
glycogen metabolism and activating pyruvate dehydrogenase
phosphatase. They may also stimulate the activity of
glycogen synthase phosphatase, be mitogenic when added to
fibroblasts in serum free medium, and stimulate pyruvate
dehydrogenase phosphatase.
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Methods for obtaining A-type and P-type IPGs are set out
in detail in Caro et al, 1997 and in W098/01116 and
W098/01117. Methods for obtaining the free GPI
precursors of the A and P-type IPGs are set out below.
Glycolipids were partially purified from rat liver
membranes following the method described in Mato et al
(1987), with minor modifications (Varela-Nieto et al,
1993). The membrane fraction was obtained by sequential
centrifugation from 300 g of material. Total lipids were
obtained by chloroform/methanol extraction followed by
the removal of nonpolar lipids. GPI was separated from
other phospholipids by sequential acid/base silica gel
G60 t.l.c. (see below). Lipids were extracted from the
t.l.c. plate with methanol, dried, applied to a Sep-Pak
C18 cartridge, eluted with methanol, and dried. GPI was
spotted onto the origin of a silica gel G60 t.l.c. plate
which was developed twice in an acidic solvent system
[chloroform: acetone: methanol: acetic acid: water
(50:20:10:10:5, by volume)] and in a basic solvent system
[chloroform: methanol: ammonium hydroxide: water
(45:45:3:5:10, by volume)]. Alternatively, GPI molecules
were further resolved by double-dimension t.l.c. as
described in Clemente et al (1995) and Avila et al (1992)
or by using high-performance (HP)-t.l.c. plates developed
in chloroform: methanol: ammonium hydroxide: water
(40:45:3:5:15, by volume) as described in Gaulton (1991).
Lipid migration was calibrated in parallel with
phospholipid standards that were detected by staining
with iodine. In order to generate free IPGs from the
purified GPI, the GPI was incubated with 1 unit of PI-PLC
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14
from Bacillus thuringiensis for 3 hours according to the
manufacturer's instructions. At the end of the
incubation, the reaction was terminated by loading the
sample onto a Sep-Pak C18 cartridge. The water eluate
(5m1) was dried.
Antibodies
Antibodies capable of specifically binding to P and A-
type IPGs are disclosed herein and are used in assays
involving IPGs and candidate mimetic compounds. These
antibodies can be modified using techniques which are
standard in the art. Antibodies similar to those
exemplified for the first time here can also be produced
using known methods. These methods of producing
antibodies include immunising a mammal (e. g. mouse, rat,
rabbit, horse, goat, sheep or monkey) with the IPG or a
fragment thereof. Antibodies may be obtained from
immunised animals using any of a variety of techniques
known in the art, and screened, preferably using binding
of antibody to antigen of interest. Isolation of
antibodies and/or antibody-producing cells from an animal
may be accompanied by a step of sacrificing the animal.
As an alternative or supplement to immunising a mammal
with an IPG, an antibody specific for an IPG may be
obtained from a recombinantly produced library of
expressed immunoglobulin variable domains, e.g. using
lambda bacteriophage or filamentous bacteriophage which
display functional immunoglobulin binding domains on
their surfaces; for instance see W092/01047. The library
may be naive, that is constructed from sequences obtained
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from an organism which has not been immunised with any of
the TPGs (or fragments), or may be one constructed using
sequences obtained from an organism which has been
exposed to the antigen of interest.
5
The term °monoclonal antibody~~ as used herein refers to
an antibody obtained from a substantially homogenous
population of antibodies, i.e. the individual antibodies
comprising the population are identical apart from
10 possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies can be
produced by the method first described by Kohler and
Milstein, Nature, 256:495, 1975 or may be made by
recombinant methods, see Cabilly et al, US Patent No.
15 4,816,567, or Mage and Lamoyi in Monoclonal Antibody
Production Techniques and Applications, pages 79-97,
Marcel Dekker Inc, New York, 1987.
In the hybridoma method, a mouse or other appropriate
host animal is immunised with the antigen by
subcutaneous, intraperitoneal, or intramuscular routes to
elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the
IPG used for immunisation. Alternatively, lymphocytes
may be immunised in vitro. Lymphocytes then are fused
with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell, see
Goding, Monoclonal Antibodies: Principles and Practice,
pp. 59-103 (Academic Press, 1986). Immunisation with
soluble IPG and via the intraperitoneal route shown in
the examples was surprisingly effective in producing
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antibodies specific for IPGs.
The hybridoma cells thus prepared can be seeded and grown
in a suitable culture medium that preferably contains one
or more substances that inhibit the growth or survival of
the unfused, parental myeloma cells. For example, if the
parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient
cells.
Preferred myeloma cells are those that fuse efficiently,
support stable high level expression of antibody by the
selected antibody producing cells, and are sensitive to a
medium such as HAT medium.
Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed
against the IPGs. Preferably, the binding specificity is
determined by enzyme-linked immunoabsorbance assay
(ELISA). The monoclonal antibodies of the invention are
those that specifically bind to either or both P and A-
type IPGs.
The epitope bound by the antibodies can be mapped using
synthetic compounds known to bind to one of the deposited
antibodies, e.g. to see whether an antibody has
substantially the same or the same binding specificity as
the deposited monoclonal antibodies 2F7, 2D1 or 5H6. This
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can also be carried out in competitive studies to
determine whether a given anti-IPG antibody competes with
one of the deposited antibodies for a particular IPG
epitope. Thus, the present invention includes antibodies
which are capable of binding to an IPG epitope bound by
an exemplified antibody. Preferably, this epitope is
present on both rat liver A-type TPG and human placenta
P-type IPG. The exemplified monoclonal raised using rat
liver A-type IPGs are surprisingly effective in assays
for the diagnosis of pre-eclampsia, as shown in the
examples below.
In a preferred embodiment of the invention, the
monoclonal antibody will have an affinity which is
greater than micromolar or greater affinity as determined
(i.e > 10-6 mol), for example, by Scatchard analysis, see
Munson & Pollard, Anal. Biochem., 107:220, 1980.
After hybridoma cells are identified that produce
neutralising antibodies of the desired specificity and
affinity, the clones can be subcloned by limiting
dilution procedures and grown by standard methods.
Suitable culture media for this purpose include
Dulbecco~s Modified Eagle's Medium or RPM1-1640 medium.
In addition, the hybridoma cells may be grown in vivo as
ascites tumours in an animal.
The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites
fluid, or serum by conventional immunoglobulin
purification procedures such as, for example, protein A-
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Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
Nucleic acid encoding the monoclonal antibodies of the
invention is readily isolated and sequenced using
procedures well known in the art, e.g. by using
oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains
of murine antibodies. The hybridoma cells of the
invention are a preferred source of nucleic acid encoding
the antibodies or fragments thereof. Once isolated, the
nucleic acid is ligated into expression or cloning
vectors, which are then transfected into host cells,
which can be cultured so that the monoclonal antibodies
are produced in the recombinant host cell culture.
Hybridomas capable of producing antibody with desired
binding characteristics are within the scope of the
present invention, as are host cells containing nucleic
acid encoding antibodies (including antibody fragments)
and capable of their expression. The invention also
provides methods of production of the antibodies
including growing a cell capable of producing the
antibody under conditions in which the antibody is
produced, and preferably secreted.
Antibodies according to the present invention. may be
modified in a number of ways. Indeed the term ~~antibody'~
should be construed as covering any binding substance
having a binding domain with the required specificity.
Thus, the invention covers antibody fragments,
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derivatives, functional equivalents and homologues of
antibodies, including synthetic molecules and molecules
whose shape mimics that of an antibody enabling it to
bind an antigen or epitope, here a P or A-type
inositolphosphoglycan.
Examples of antibody fragments, capable of binding an
antigen or other binding partner; are the Fab fragment
consisting of the VL, VH, C1 and CH1 domains; the Fd
fragment consisting of the VH and CH1 domains; the Fv
fragment consisting of the VL and VH domains of a single
arm of an antibody: the dAb fragment which consists of a
VH domain; isolated CDR regions and F(ab~)2 fragments, a
bivalent fragment including two Fab fragments linked by a
disulphide bridge at the hinge region. Single chain Fv
fragments are also included.
A hybridoma producing a monoclonal antibody according to
the present invention may be subject to genetic mutation
or other changes. It will further be understood by those
skilled in the art that a monoclonal antibody can be
subjected to the techniques of recombinant DNA technology
to produce other antibodies, humanised antibodies or
chimeric molecules which retain the specificity of the
original antibody. Such techniques may involve
introducing DNA encoding the immunoglobulin variable
region, or the complementarity determining regions
(CDRs), of an antibody to the constant regions, or
constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP-A-184187, GB-A-
2188638 or EP-A-0239400. Cloning and expression of
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chimeric antibodies are described in EP-A-0120694 and EP-
A-0125023.
Imcnunoa s s avs
5 The antibodies described above can be employed in the
method aspects of the invention in a wide variety of
different assay formats. The antibodies can be used as
binding agents capable of specifically binding to P
and/or A-type IPGs or as developing agents for
10 determining the fraction of binding sites of a binding
agent occupied by analyte after exposure to a test
sample.
In some instances, employing the antibodies, particularly
15 as developing agents in assays, involves tagging them
with a label or reporter molecule which can directly or
indirectly generate detectable, and preferably
measurable, signals. The linkage of reporter molecules
may be directly or indirectly, covalently, e.g. via a
20 peptide bond or non-covalently. Linkage via a peptide
bond may be as a result of recombinant expression of a
gene fusion encoding antibody and reporter molecule. Any
method known in the art for separately conjugating the
antibody to the detectable moiety may be employed,
including those methods described by Hunter et al, Nature
144:945, 1962; David et al, Biochemistry 13:1014, 1974;
Pain et al, J. Immunol. Meth. 40:219, 1981; and Nygren, J
Histochem. and Cytochem. 30:407, 1982.
One favoured mode is by covalent linkage of each antibody
with an individual fluorochrome, phosphor or laser dye
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with spectrally isolated absorption or emission
characteristics. Suitable fluorochromes include
fluorescein, rhodamine, luciferin, phycoerythrin and
Texas Red. Suitable chromogenic dyes include
diaminobenzidine. Other detectable labels include
radioactive isotopic labels, such as 3H, 14C, 3zp~ 355 iz6l~
or 99mTC, and enzyme labels such as alkaline phosphatase,
~i-galactosidase or horseradish peroxidase, which catalyze
reactions leading to detectable reaction products and can
provide amplification of signal.
Other reporters include macromolecular colloidal
particles or particulate material such as latex beads
that are coloured, magnetic or paramagnetic, and
biologically or chemically active agents that can
directly or indirectly cause detectable signals to be
visually observed, electronically detected or otherwise
recorded. These molecules may be enzymes which catalyze
reactions that develop or change colours or cause changes
in electrical properties. for example. They may be
molecularly excitable, such that electronic transitions
between energy states result in characteristic spectral
absorptions or emissions. They may include chemical
entities used in conjunction with biosensors.
Alternatively or additionally, the antibodies can be
employed as binding agents, relying on the fact that the
antibodies are capable of specifically binding the IPGs
in preference to other substances present in the sample.
Conveniently, the binding agents) are immobilised on
solid support. e.g. at defined locations, to make them
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22
easy to manipulate during the assay. This can be
achieved using techniques well known in the art such as
physisorption or chemisorption, e.g. employing
biotin/avidin or biotin/streptavidin to chemically link
the antibodies to the solid support. Generally, the
sample is contacted with the binding agents) under
appropriate conditions so that P and A-type IPGs present
in the sample can bind to the binding agent(s). The
fractional occupancy of the binding sites of the binding
agents) can then be determined using a developing agent
or agents.
As mentioned above, the developing agents are labelled
(e.g. with radioactive, fluorescent or enzyme labels) so
that they can be detected using techniques well known in
the art. Thus, radioactive labels can be detected using
a scintillation counter or other radiation counting
device, fluorescent labels using a laser and confocal
microscope, and enzyme labels by the action of an enzyme
label on a substrate, typically to produce a colour
change. The developing agents) can be used in a
competitive method in which the developing agent competes
with the analyte for occupied binding sites of the
binding agent, or non-competitive method, in which the
labelled developing agent binds analyte bound by the
binding agent or to occupied binding sites. Both methods
provide an indication of the fraction of the binding
sites occupied by the analyte, and hence the
concentration of the analyte in the sample, e.g. by
comparison with standards obtained using samples
containing known concentrations of the analyte.
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The antibodies of the present invention may be employed
in any-known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays, see Zola, Monoclonal
Antibodies: A Manual of Techniques, pp 147-158 (CRC
Press, Inc, 1987).
Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or
epitope, of the IPG to be detected. In a sandwich assay,
the test sample analyte is bound by a first antibody
which is immobilised on a solid support, and thereafter a
second antibody binds to the analyte, thus forming an
insoluble three part complex. The second antibody may
itself be labelled with a detectable moiety (direct
sandwich assays) or may be measured using an anti-
immunoglobulin antibody that is labelled with a
detectable moiety (indirect sandwich assay). For
example, one type of sandwich assay is an ELISA assay, in
which case the detectable moiety is an enzyme.
There is also an increasing tendency in the diagnostic
field towards miniaturisation of such, e.g. making use of
binding agents, such as antibodies immobilised in small,
discrete locations (microspots) and/or as arrays on solid
supports or on diagnostic chips. In one embodiment, the
present invention provides a support having immobilised
at discrete locations thereon a plurality of anti-IPG
antibodies.
These approaches can be particularly valuable as they can
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24
provide great sensitivity (particularly through the use
of fluorescent labelled reagents), require only very
small amounts of the candidate compounds or IPGs being
tested and allow a variety of separate assays can be
carried out simultaneously. This latter advantage can be
useful in screening libraries of compounds produced by
combinantorial techniques. Examples of techniques
enabling this miniaturised technology are provided in
wo84/01031, wo88/1058, w089/01157, wo93/8472, wo95/18376/
w095/18377, W095/24649 and EP-A-0373203.
Affinity Purification
The antibodies of the invention also are useful as
affinity purification agents. In this process, the
antibodies capable of specifically binding P- and/or A-
type IPGs are immobilized on a suitable support, such as
Sephadex~ resin or filter paper, using methods well known
in the art. The immobilized antibody then is contacted
with a sample containing the IPGs to be purified, and
thereafter the support is washed with a suitable solvent
that will remove substantially all the material in the
sample except the IPGs, which is bound to the immobilised
antibody. Finally, the support is washed with another
suitable solvent, such as glycine buffer, pH 3-5, that
will release the IPGs from the antibody.
Mimetic Design
The designing of mimetics to a known pharmaceutically
active compound is a known approach to the development of
pharmaceuticals based on a ~~lead~~ compound. This might
be desirable where the active compound is difficult or
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WO 99/47926 PCT/GB99/00845
expensive to synthesise or where it is unsuitable for a
particular method of administration, e.g. peptides are
unsuitable active agents for oral compositions as they
tend to be quickly degraded by proteases in the
5 alimentary canal. Mimetic design, synthesis and testing
is generally used to avoid randomly screening large
number of molecules for a target property.
There are several steps commonly taken in the design of a
10 mimetic from a compound having a given target property.
Firstly, the particular parts of the compound that are
critical and/or important in determining the target
property are determined. These parts of the compound
constituting its active region are known as its
15 "pharmacophore".
Once the pharmacophore has been found, its structure is
modelled to according its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data
20 from a range of sources, eg spectroscopic techniques, X-
ray diffraction data and NMR. Computational analysis,
similarity mapping (which models the charge and/or volume
of a pharmacophore, rather than the bonding between
atoms? and other techniques can be used in this modelling
25 process. In a variant of this approach, the three-
dimensional structure of the ligand and its binding
partner are modelled. This can be especially useful
where the ligand and/or binding partner change
conformation on binding, allowing the model to take
account of this the design of the mimetic.
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A template molecule is then selected onto which chemical
groups-which mimic the pharmacophore can be grafted. The
template molecule and the chemical groups grafted on to
it can conveniently be selected so that the mimetic is
easy to synthesise, is likely to be pharmacologically
acceptable, and does not degrade in vivo, while retaining
the biological activity of the lead compound. The
mimetic or mimetics found by this approach can then be
screened to see whether they have the target property, or
to what extent they exhibit it. Further optimisation or
modification can then be carried out to arrive at one or
more final mimetics for in vivo or clinical testing.
Example 1
Production of Polyclonal and Monoclonal Antibodies
Against Inoaitolphosphoglycans (IPGs)
Inositolphosphoglycan (soluble form) obtained by PI-PLC
treatment of GPI purified from rat liver by sequential
Thin Layer Chromatography (TLC) was used to immunize New
Zealand rabbits and Balb/c mice as described below.
Alternatively, human IPGs could be obtained using the
methods described in Caro et al, 1997.
Rabbit Immunisation Procedure
Two New Zealand rabbits were anaesthetized and then
immunised with 750ug of IPG (soluble form) mixed in lml
of PBS with lml of complete Freund~s adjuvant (CFA). The
antigen-adjuvant emulsion was administered 1.5m1 by
intradertnal (id) injection and 0.5m1 by intramuscular
(im) injection.
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After one month, this protocol was repeated except that
incomplete Freund~s adjuvant (IFA) was used, and 1.5m1 by
administered by subcutaneous (sc) injection and 0.5m1 by
intramuscular (im) injection. This was repeated again on
days 60, 90, 120 and 150.
Mouse Immunisation Procedure
Four female Balb/c 6 weeks old mice were immunised with
60ug of IPG (soluble form) in 250u1 of PBS with 250u1 of
CFA. The antigen-adjuvant emulsion was injected by
intraperitoneal (ip) injection. After 21 days, the
injection was repeated except that IFA was used. On days
42 and 63 all the animals were injected ip with IFA. On
day 84, the best responder was injected 100u1 PBS
containing 60ug of IPG intravenous (iv) and 100u1 PHS
containing 60pg of IPG (ip). After 87 days, splenocytes
from best responder were fused to myleoma cells using
conventional techniques. Monitor test bleeds were
realized regularly.
Examt~le 2
Indirect ELISA Assay
The following indirect ELISA protocol is provides as an
example of a procedure for screening for the monoclonal
antibodies.
Add 100u1/well in all the steps.
(1) Add IPG diluted 1:800 in PBS in a F96 Polysorp Nunc-
Immuno plate. Incubate at least 7 days at 4°C.
(2) Wash with PBS three times.
(3) Add a blocking reagent for ELISA (Boehringer
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Mannheim) in distilled water (1:10 v/v) for 2 hours at
room temperature.
(4) Wash with PBS-Tween 20 (0.1%) three times.
(5) Add a purified monoclonal and/or a polyclonal
antibody (diluted between 1:12.5 and 1:3200 in PBS),
overnight at 4°C.
(6) Wash with PBS-Tween 20 (0.1%) three times.
(7) Add an anti-mouse IgM, biotinylated whole antibody
(from goat) (Amersham) in case of the previous addition
of a monoclonal antibody, or add an anti-rabbit Ig,
biotinylated species-specific whole antibody (from
donkey) (Amersham), in case of previous addition of a
polyclonal antibody, both of them diluted 1:1000 in PBS,
1 h 30 min at room temperature.
(8) Wash with PBS-Tween 20 (0.1%) three times.
(9) Add a streptavidin-biotinylated horseradish
peroxidase complex (Amersham) diluted 1:500 in PBS, 1 h
30 min at room temperature.
(10) Wash with PBS three times.
(il) Add 2,2-Azino-di-(3-ethylbenzthiazoline sulfonate
(6)) diammonium salt crystals (ABTS) to buffer for ARTS
(Boehringer Mannheim): Buffer for ARTS is added to
distilled water (1:10 v/v). lmg of ABTS is added to 1m1
of diluted buffer for ABTS.
(12) Read the absorbance in a Multiskan Plus P 2.01 using
a 405 nm filter in 5-15 min.
Comments:
Solvents used are phosphate-buffered saline (PBS) pH 7.2
or PBS-Tween 20 (0.1%) .
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Step 1: IPG (soluble form) is used in an indirect ELISA
(initial concentration approximately 60uM). Working
dilutions are normally between 1:400 and 1:800. Several
incubation times have been examined. The best and more
reproducible results have been obtained when incubation
time is at least seven days at 4°C, probably because
higher incubation times increase the number of molecules
of IPG that are bound to the solid phase.
Step 3: Blocking reagent for ELISA is a registered
product of Boehringer Mannheim. In order to prepare the
solution we must dissolve the content (27g) in 100 ml
redist. Water at room temperature while stirring for
approx 30 minutes. The concentrate is stable when stored
in aliquots at -20°C. For use, dilute one aliquot in a
ratio of 1:10 with redistilled water to yield a working
solution containing 1~ protein mixture (w/v) that was
obtained by proteolytic degradation of purified gelatin,
in 50mM Tris-HC1, 150mM NaCl, pH ca.7.4.
Step 5: A purified monoclonal and/or polyclonal antibody
are used in an indirect ELISA (initial concentration
approx 2mg/ml). Working dilutions are normally between
1:100 and 1:200, getting final concentrations of 20ug/ml
and l0ug/ml respectively). Incubation time for rapid
assays can be two hours at room temperature.
Step 7: The anti-rabbit and the anti-mouse antibodies are
used at 1:1000 dilution straight from the stock bottle.
The stock bottle is stored at 2--8°C. Under these
conditions the product is stable for at least six months,
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after which any remaining solution is discarded. iml of
biotinylated reagent is supplied in 0.1 M phosphate-
buffered saline of pH7.5 containing 1~ (w/v) bovine serum
albumin and 0.05 (w/v) sodium azide.
5
Step 9: Streptavidin-biotinylated/HRP is used at 1:500
dilution straight from the stock bottle. The stock
bottle is stored at 2-8°C. Under these conditions the
product is stable for at least three months, after which
10 any remaining solution is discarded. The complex is
supplied in 2m1 of phosphate-buffered saline pH7.5
containing 1~ (w/v) bovine serum albumin and an
antimicrobial agent.
15 Step 11: ABTS buffer is a registered product of
Boehringer Mannheim. The solution consists of sodium
perborate, citric acid and disodium hydrogen phosphate.
The solution is stored in aliquots of 1m1 at -20°C. For
use, dilute one aliquot in a ratio of 1:10 with
20 redistilled water.
Exaitmle 3
Sandwich ELISA Assay
The following ELISA protocol is provided as an example of
25 an assay for IPGs employing the deposited monoclonal
antibodies.
Add 100 ul/well in all the steps.
(1) Anti-IPG monoclonal antibody was diluted 1:100 in
30 PBS in added to a F96 Maxisorp Nunc-Immuno plate. The
plate was incubated for at least 2 days at 4°C.
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(2) The plate was washed with PBS three times.
(3) Blocking reagent for Elisa (Boehringer Mannheim) in
distilled water (1.5:10 v/v) was added for 2 hours at
room temperature.
(4) The plate was washed with PBS-Tween 20 (0.1%) three
times.
(5) Human serum diluted 1:4 in PHS was added to the
plate overnight at 4°C.
(6) The plate was wash with PHS-Tween 20 (0.1%) three
times.
(7) A purified polyclonal antibody (diluted 1:100 in
PBS) was added overnight at 4°C.
(8) The plate was washed with PBS-Tween 20 (0.1%) three
times.
(9) An anti-rabbit Ig, biotinylated species-specific
whole antibody (from donkey )(Amersham) was diluted
1:1000 in PBS, and added to the plate for 1 h 30 min at
room temperature.
(10) Wash with PBS-Tween 20 (0.1%) three times.
(11) Add a streptavidin-biotinylated horseradish
peroxidase complex (Amersham) diluted 1:500 in PBS, 1 h
mins at room temperature.
(12) The plate was washed with PBS three times.
(13) 2,2'-Azino-di-(3-ethylbenzthiazoline sulfonate (6))
25 diammonium salt crystals (ARTS) was made up in buffer for
ABTS (Boehringer Mannheim). The buffer for ABTS was
added to distilled water (1:10 v/v). lmg of ABTS was
added to 1ml of diluted buffer for ABTS.
(14) The absorbance was read in a Multiskan Plus P 2.01
30 using a 405 nm filter in 15-30 mins. -
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Comments:
The solvents used were phosphate-buffered saline (PBS)
pH7.2 or PBS-Tween 20 (0.1~).
In step (1): The examples employed purified monoclonal
antibody 2D1 in the Sandwich ELISA assays (initial
concentration 2.5mg/ml). Working dilutions varied
normally between 1:100 and 1:200, with final
concentrations of 25ug/ml and 12.5ug/ml, respectively.
The incubation time used in the assay can be varied. For
rapid assays, incubation time can vary between two hours
at room temperature or overnight at 4°C. However,
improved results are obtained when incubation time is at
least two days at 4°C. This improvement may be due to
the higher incubation times increasing the number of
molecules of monoclonal antibody that are bound to the
solid phase.
Step (3): Blocking reagent for ELISA is a registered
product of Boehringer Mannheim. In order to prepare the
solution, the content (27g) was dissolved in 100m1
redistilled water at room temperature while stirring for
approximately 30mins. The concentrate is stable when
stored in aliquots at -20°C. For use, aliquots were
diluted in a ratio of 1:10 with redistilled water to
yield a working solution containing 1~ protein mixture
(w/v) that was obtained by proteolytic degradation of
purified gelatin, in 50 mM Tris-HC1, 150 mm NaCl, pH ca.
7.4.
Step (5): Human serum was probed in several dilutions
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(1:1, 1:2, 1:4...). Working dilutions are normally
between 1:2 and 1:4. IPG in several dilutions was used
as a standard (positive control). Wells without serum
are used as negative control. The incubation time for
rapid assays was two hours at room temperature.
Step (7): A purified polyclonal antibody was used in
Sandwich ELISA (initial concentration 2 mg/ml). Working
dilutions were between 1:100 and 1:200, with final
concentrations of 20ug/ml and l0ug/ml respectively.
Step (9): The anti-rabbit was used at 1:1000 dilution
straight from the stock bottle, stored at 28°C. Under
these conditions the product is stable for at least six
months. iml of biotinylated reagent is supplied in 0.1 M
phosphate-buffered saline pH 7.5 containing 1~ (w/v)
bovine serum albumin and 0.05 (w/v) sodium azide.
Step i11): Streptavidin-biotinylated/HRP was used at
1:500 dilution straight from the stock bottle, stored at
2-8°C. Under these conditions the product is stable for
at least three months. The complex is supplied in 2m1 of
phosphate-buffered saline pH 7.5 containing 1~ (w/v)
bovine serum albumin and an antimicrobial agent.
Step (13): ABTS buffer is a registered product of
Boehringer Mannheim. The solution consists of sodium
perborate, citric acid and disodium hydrogen phosphate.
The solution is stored in aliquots of 1m1 at -20°C. For
use, one aliquot is diluted in a ratio of 1:10 with
redistilled water.
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34
Figure 1 shows a dose-response curve of assay response
(absorbance) plotted against IPG concentration,
demonstrating that the antibodies can be used to assay
for IPGs.
Example 4
Properties of Anti-IPG Antibody 5H6
As discussed above, a prior art polyclonal antibody
raised against the GPI-anchor of VSG was found to cross-
react with IPG containing fractions. Figure 2 shows that
a polyclonal antibody raised against IPGs cross reacted
with GPI-anchors. Thus, these results are consistent and
demonstrate that there is some cross reactivity between
the IPG and GPI anchors. In contrast, when the
reactivity of monoclonal antibody 5H6 towards soluble and
membrane bound VSG was tested, figure 2 (left hand
column) shows that it does not cross react in the same
way as the polyclonal antibody raised against IPGs
(middle column). In these experiments, a polyclonal
antibody raised against the common reactive determinant
(CRD) common to GPI anchored proteins was used as a
positive control (right hand column).
Thus, figure 2 shows that antibody 5H6 does not cross
react with either soluble or membrane bound VSG, whereas
both polyclonal antibodies react strongly. This property
distinguishes the antibodies of the invention from cross
reacting polyclonal antibodies. This in turn suggests
that polyclonal antibodies will not specifically detect
IPGs, but are a valuable reagent in ELISA sandwich assays
which require two anti-IPG antibodies, one of which is
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very specific (monoclonal) and the other of general
reactivity (polyclonal).
Figure 3 shows that monoclonal antibody 5H6 is capable of
5 inhibiting the action of P-type IPG from rat liver in a
P-type phosphatase assay. Thus, this and similar
antibodies could be used as P-type IPG antagonists, e.g.
in the treatment of conditions associated with elevated
levels of P-type IPGs such as pre-eclampsia.
Example 5
Use of Anti-IPG Antibodies to Screen for LPG Mimetics
By way of example, the following approach could be used
to screen for mimetics for a given IPG.
Firstly, a group of antibodies having different IPG
binding activities are used to provide binding profiles
for a given IPG, e.g. P-type IPG isolated from human
placenta, or P or A-type IPGs isolated from human liver.
Ideally, the number of antibodies used should be
sufficient to provide a unique binding profile, or the
conditions under which the binding profile is obtained
adjusted to assist in this aim. These results could be
used to define a table of binding profiles.
Tab- le 1~
Antibody P-type IPG A-type IPG p-t
ype IPG
Placenta Liver Liver
Ab, Yes Yes No
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36
Ab2 Yes Yes Yes
Abp No No Yes
Abp YeS NO NO
The table could record simply whether the antibodies
bound a given IPG, i.e. the epitope that the antibody was
specific for was present on the IPG, or whether the
binding was above a particular threshold level or whether
record the actual strength of the interaction between the
antibody and the IPG. Given the application of the
invention in screening large numbers of candidate
mimetics, it is desirable that the determination of
binding is as simple as possible.
Once the binding profile for a given IPG has been found,
the exercise could be repeated, to screen a library of
candidate compounds using the antibodies to determine
their binding profiles. A comparison of the binding
profiles of the IPGs and the candidate compounds would
then indicate candidate compounds likely to resemble the
given IPG, that consequently are likely to be useful
leads for pharmaceuticals.
Example 6
Assay Using Monoclonal Antibodies 2D1, 5H6 and A1 to
Obtain IPG Binding Profiles
An ELISA assay using monoclonal antibody 2D1 as a binding
agent and a labelled polyclonal developing antibodies
raised against rat liver A-type IPG and human placental
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37
P-type IPG was used to establish binding profiles for a
series of synthetic compounds SNK11, SNKB, SNK7, SNK6,
SNK12, SNK13, C4 and C3. Compound C3, iD-6-0-(2-amino-2-
deoxy-a-D-glucopyranosyl)-myo-inositol 1,2-(cyclic
phosphate), has been prepared previously, see Zapata et
al, 1994. Compound C4, 1D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-chiro-inositol 1-phosphate can be
synthesised as described in Jaramilio et al, 1994.
Figure 4 shows that candidate synthetic compounds C3, C4
and SNK12 showed appreciable binding to monoclonal 2D1
and the polyclonal developing agent raised against rat
liver A-type IPG.
Figure 5 shows a similar assay using monoclonal antibody
5HS, showing appreciable responses using the rat liver A-
type IPG and candidate compounds SNK6, SNK12, SNK13 and
C4. This assay also shows that weaker responses were
obtained using the placenta P-type developing agent with
compounds SNK6 and C4.
Figure 6 shows a further assay using anti-IPG monoclonal
antibody A1. This antibody reacted positively with all
of the candidate compounds using the rat liver A-type IPG
developing antibody, but illustrates that a binding
profile distinguishing candidate compounds or IPGs could
be based on the magnitude of the binding response as an
alternative or in addition to an assay for determining
whether or not binding takes place.
Similar profiles have been obtained using natural IPGs
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(data not shown). The comparison of the profiles
obtained for the candidate mimetics and the IPGs can
therefore be used to select mimetics likely to share an
IPG biological activity for further testing.
Thus, the assays illustrate that the binding profiles
provided by the present invention are dependent on the
binding agents and developing agents used, as well as the
identity of the IPGs or synthetic compounds. Further,
the profile can include either or both the existence of a
positive or negative response or the magnitude of it.
Table 2 collates the data from the assays using the rat
liver A-type developing agent to demonstrate that just 3
assays provides enough information to distinguish the
binding of 8 candidate compounds. Positive responses are
rated out of 5.
Table 2:
Compound/Ab 2D1 586 A1
SNK11 1/5 1/5 2/5
SNK8 - 1/5 1/5
SNK7 - - 5/5
SNK6 - 2/5 5/5
SNK12 1/5 1/5 3/5
SNK13 - 2/5 3/5
C4 2/5 3/5 2/5
C3 4/5 - 1/5
Taken together, these assays demonstrate that it is
possible to define a binding profile using different
anti-IPG antibodies as capture and developing agents for
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39
candidate compounds or a given IPG. The assays could be
repeated with further anti-IPG antibodies as binding and
developing agents, to develop more precise binding
profiles for different type of IPGs and candidate
synthetic compounds.
DeDOSits
The deposit of hybridomas 2F7, 2Dl and 5H6 in support of
this application was made at the European Collection of
Cell Cultures (ECACC) under the Budapest Treaty by
Rademacher Group Limited (RGL), The Windeyer Building, 46
Cleveland Street, London W1P 6DB, UK. The deposits have
been accorded accession numbers accession numbers
98051201, 98031212 and 98030901. RGL give their
unreserved and irrevocable consent to the materials being
made available to the public in accordance with
appropriate national laws governing the deposit of these
materials, such as Rules 28 and 28a EPC. The expert
solution under Rule 28(4) EPC is also hereby requested.
CA 02321734 2000-08-31
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References:
The reference mentioned herein are all incorporated by
reference in their entirety.
5 W098/01116 and W098/01117 (Rademacher Group Limited?.
Caro et al, Biochem. Molec. Med., 61:214-228, 1997.
Kunjara et al, In: Biopolymers and Hioproducts:
10 Structure, Function and Applications, Ed Svati et al,
301-305, 1995.
Rademacher et al, Brazilian J. Med. Biol. Res., 27:327-
341, 1994.
Nestler et al, Endocrinology, 129:2951-2956. 1991.
Romero et al, P.N.A.S., 87:1476-1480, 1990.
Huang et al, Endocrinology, 132:652-657, 1993.
Represa et al, P.N.A.S., 88:8016-8019, 1991.
Mato et al, J. Hiol. Chem., 262:2131-2137, 1987.
Clemente et al, Cell Signalling, 7:411-421, 1991.
Varela-Nieto et al, In Handbook of Endocrine Research
Techniques: Intracellular Mediators of Peptide Hormone
Action: Glycosylphospatidylinositol/Inositol
Phosphoglycan System, ed de Pablo et al, San Diego:
CA 02321734 2000-08-31
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41
Academic Press, 391-406, 1993.
Avila et al, Biochem. J., 282:681-686, 1992.
Gaulton, Diabetes. 40:1297-1304, 1991.
Zapata et al, Carbohydrate Res., 264;21-31, 1994.
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