Note: Descriptions are shown in the official language in which they were submitted.
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AGENT CAPABLE OF MODULATING A GLYCOSPHINGOLIPID-ASSOCIATED ACTIVITY THUS
AFFECTING AN
IMMUNE DISORDER
The present invention relates to a medicament.
In particular, the present invention relates to a medicament useful in the
treatment of
immune disorders. In particular, these disorders include inflammatory
conditions,
autoimmune diseases, allergic conditions, the treatment of cancers, including
human
leukaemias of a T cell origin and as agents in the prevention of human
transplantation
rejection and graft versus host disease (GVHD).
More in particular, in one aspect the present invention relates to an
immunomodulatory
agent.
More in particular, the present invention relates to such an agent' optionally
co-
administered with a specific antigen for use in the treatment.of mammalian
particularly
human immune disorders. These disorders include inflammatory conditions,
autoimmune diseases, allergic conditions, the treatment of cancers, including
human
leukaemias of a T cell origin and as agents in the prevention of human
transplantation
rejection and graft versus host disease (GVHD).
Autoimmunity is the term used to describe the mechanism by which the body
generates an immune response to self antigens. It has been disclosed in the
art that
agents which have GM-I binding activity, or which have an effect on GM-I
mediated
intracellular signalling events but no GM-1 binding activity, are useful as
therapeutic
agents for, inter alia, autoimmune diseases. W095/10301 disclosed agents which
consisted of a mucosa-binding molecule linked to a specific tolerogen.
However, in
W097/02045 we disclosed that in fact linkage between a GM-1 binding agent and
the
tolerogen was not required. Particular examples of GM-1 binding agents
provided in
W097/02045 were cholera toxin (Ctx), the B subunit of cholera toxin (CtxB),
the
E.coli heat labile toxin (Etx) and the B subunit of the E.coli heat labile
toxin (EtxB).
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The term "allergy or hypersensitivity" is used to describe an adaptive immune
response which occurs in an exaggerated or inappropriate form. Allergic or
hypersensitivity reactions are the result of normally beneficial immune
responses
acting inappropriately to foreign antigens (usually environmental
macromolecules) and
sometimes cause inflammatory reactions and tissue damage. In these situations,
a
normally harmless environmental stimulus, called an "allergen", triggers an
immune
response which upon re-exposure, is re-activated to generate pathological
damage.
Allergies or hypersensitivities are distinguished into four types of
reactions. The first
three are antibody-mediated, the fourth is mediated mainly by T cells and
macrophages.
In Type I Immediate Hypersensitivity/Atopic Allergy, the principal immune
response
to the allergen involves the production of IgE antibodies. Such disorders are
by far the
most prevalent in humans and are seen as principal targets for new therapeutic
approaches. Although these diseases are not exclusively IgE mediated, IgE
binds to
cells within the tissues such as mast cells and basophils and the cross-
linking of IgE on
the cells surfaces by allergen invokes the release of many inflammatory
mediators.
Typical examples of such diseases include asthma, allergic cough, allergic
rhinitis and
2o conjunctivitis, atopic eczema and dermatitis, urticaria, hives, insect bite
allergy, dietary
and certain drug allergies. In many cases, the particular allergens are known.
By way
of example, the principal allergen in asthma is DerPl from house dust mite but
other
triggers of asthma such as pet dander antigens also exist.
Type II or antibody dependent cytotoxic hypersensitivity occurs when
antibodies of a
different type, usually IgG and IgM, binds to either self antigen or foreign
antigen on
cells and leads to phagocytosis, killer cell activity or complement mediated
lysis.
These types of allergies are relatively unusual but can include some allergies
to drugs.
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Type III hypersensitivity develops when immune complexes are formed in large
quantites or cannot be cleared adequately by the reticuloendothelial system.
The
immune complexes usually result from the deposition of antibody, usually IgM
or IgG,
allergen complexes at these sites. In normal circumstances, antibody binds to
allergen
and is cleared by a variety of tissue cells. However, a number of factors may
influence
the persistence of the immune complexes and where they remain in the blood for
prolonged periods, they can lodge and establish inflarnmation in the kidneys,
skin
(where they cause rashes) and joints (where they can cause a type of arthritis
other than
rheumatoid arthritis).
Type IV or delayed type hypersensitivity (DTH) does not involve antibody but
instead
the prolonged activation of T lymphocytes. These T cells are capable of
secreting
soluble factors causing tissue damage and enhancing the recruitment and
activation of
other cell types to the tissues. Incoming cells themselves contribute to the
inflammation and tissue damage. DTH is most seriously manifested when antigens
(for example those associated with mycobacteria tuberculosis) are trapped in a
macrophage and cannot be cleared. T cells are then stimulated to elaborate
cytokines
which mediate a range of inflammatory responses. DTH reactions are less common
than Type I reactions but are seen in graft rejection and allergic contact
dermatitis
which is generally manifested as a contact sensitivity (allergy usually
involving skin
rash) to environmental "contact allergens" such as heavy metals.
Researchers have shown that a state of immunological unresponsiveness, also
known
as "immunological or oral tolerance", can be induced by the oral
administration of
dietary protein antigens (Weiner, HL, Immunol Today 1997 18: 335-343; Sun et
al
1994 ibic~. The inhalation of antigens can also induce a state of specific
immunological unresponsiveness or "nasal tolerance". Thus, systemic
immunological
tolerance can be induced when antigen is administered at mucosal sites (such
as orally
or nasally). WO 95/01301 discloses an immunological tolerance-inducing agent
comprising a mucosa-binding agent linked to a specific tolerogen. W095/10301
also
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includes mention of the treatment of allergy using a mucosa binding agent
coupled to
an allergen. Other researchers such as Tamara et al (1997 Vaccine 15: 225-229)
have
taken directly the protocol of WO 95/10301 and tested its efficacy in
preventing
allergy in a marine model of Type I allergy. They reported a significant
lowering of
IgE levels which are a strong predictor of efficacy and they cite data,
following
administration of EtxB coupled to ovalbumin (the results were not included),
which
shows that EtxB was not effective once IgE levels are established. It has also
been
shown that orally administered Ctx and Etx can act on several humoral and
cellular
immune responses not only at the gastrointestinal tract, but also in distant
mucosal
1o effector sites such as the respiratory tract. These data suggest that these
mucosal
adjuvants have a potential use in oral immunisation strategies to improve the
local
immune responses in remote mucosal tissues, in accordance with the concept of
a
corlmon mucosal immune system (Bienenstock J 1974 The physiology of the local
immune system and the gastrointestinal tract. In: Progress in Immunology II,
vol 4:
clinical aspects, LL. Brent, J.Holborrow, Eds. Amsterdam, North Holland, pp197-
207;
Ruedl et al 1996 ibid; Umesaki 1992 ibid; Czerkinsky and Holmgren (1994 Cell
Mol
Biol 40: 37-44).
The induction of immunological tolerance may include a number of different
mechanisms which may be summarised as follows:
(i) a process whereby antigen reactive cells are removed through triggering
them to commit suicide (apoptosis);
(ii) an induction of anergy or the long term inactivation of the antigen
reactive
cells;
(iii) immune deviation of the antigen reactive cells away from the production
of pathological responses;
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(iv) suppression of the antigen reactive cells or their regulation by specific
factors or regulatory cells
In the treatment of allergy, it is possible that the treatment of any of these
mechanisms
5 may be useful. However, while the deletion of antigen reactive cells and/or
the
induction of anergy are useful strategies once the precise allergens are
known,
invoking these mechanims will usually silence only those cells which respond
to the
allergen which was given in the treatment regime. On the other hand, the
implementation of immune deviation or suppression strategies has the advantage
of
l0 potential regulation of responses to antigens which are involved in the
condition but
were not part of the treatment. This phenomenon, known as "bystander
suppression"
allows the "spread" of tolerance to other antigens (such as allergens) in the
target
tissues through the possible secretion of non-antigen specific suppressor
molecules in
that tissue as a result of the interaction between the antigen specific cells
and the
specific immunising antigen. In this way, as Iong as at least one of the
antigens
involved in the disorder is known, the condition may be treated even if there
are other
antigens implicated as well. Thus, the goal of a good treatment is the
induction of a
specific immune deviation or suppression.
Oral administration of antigens - such as allergens and autoantigens - has
long been
recognised as a method to prevent peripheral T cell responses and, in the case
of
autoantigens, has also been shown to prevent or delay the onset of several
experimental autoimmune diseases including experimental allergic
encephalomyelitis
(EAE). Major problems recognised with such strategies are that it usually
requires
feeding of large, if not massive, doses of autoantigens and it is generally
less efficient
in an immune as opposed to a naive host. The latter problem has limited the
therapeutic potential of this strategy. However, it has now been shown by Sun
et al
(1994 Proc Natl Acad Sci 91: 10795-10799) that oral administration of minute
amounts of prototype particulate and soluble protein antigens conjugated to
cholera
toxin B subunit (CtxB), the nontoxic receptor-binding moiety of cholera toxin,
can
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readily induce tolerance in the peripheral T-cell compartment and is effective
not only
in naive but also in systemically sensitised animals. In addition, oral
administration of
minute amounts of an autoantigen, myelin basic protein (MBP) coupled to CtxB
can
prevent EAE in Lewis rats (Sun et al 1996 Proc Natl Acad Sci 93: 7196-7201).
Other
researchers have also shown that feeding even a single dose of minute amounts
(microgram) of antigens conjugated to the receptor binding nontoxic B subunit
moiety
of cholera toxin (CtxB) can markedly suppress systemic T cell mediated
inflammatory
reactions in naive as well as in experimental animals (Bergerot et al 1997
Proc Natl
Acad Sci 94: 4610-4614).
to Nashar and co-workers (Proc Natl Acad Sci 1996 93: 223-226; Int Immunol
1996 8:
731-736; Immunol 1997 91: 572-578) have demonstrated that the administration
of
EtxB and other homologues can modulate the immune response away from the
production of Thl cytokines such as IFNy and interleukin 2 (IL-2) and towards
the
secretion of Th2 cytokines such as IL-4, IL-10 and IL-13. IFNy is the
classical Thl
cytokine, IL-4 is the classical Th2 cytokine. This "immune deviation" is the
basis of
the disclosure in WO 97/02045.
Earlier, we surprisingly found that agents having GM1 binding activity exert
immunomodulatory effects useful in the treatment of allergic conditions and/or
2o hypersensitivity conditions (UK patent application No. 9800487.2).
GM1 is a member of family of ganglioside receptors comprising sialic acid
containing
glycolipids (also called glycosphingolipids) which are formed by a hydrophobic
portion, the ceramide and a hydrophilic part, that is the oligosaccharide
chain. Within
cells, gangliosides are usually associated with plasma membranes, where they
can act
as receptors for a variety of molecules and have been shown to take part in
cell-to-cell
interaction and in signal transduction.
Glycosphingolipids, other than GM1 gangliosides, are also capable of acting as
plasma
3o membrane receptors.
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One member of this glycosphingolipid family which acts as a plasma membrane
receptor is globotriaosylceramide, (Gb3), which is optionally represented as
Gal al-4Gal (31-4Glc ail-1 Cer
or
Gal al-4Ga1 (3 1-4Glc-Cer
io Gb3 mediates the internalisation of a family of E. coli derived toxins
called verotoxins
(VT), into susceptible cells by capping and receptor mediated endocytosis
(RNIE)
(Khine and Lingwood 1994 J Cell Physiol 161: 319-332). The action of the
verotoxins
(VT) in causing human vascular disease is dependent on the specific
recognition of
receptors on target endothelial cells (Nyholin et al 1996 Chem Biol 3: 263-
275). Gb3
has been shown to be the most effective receptor for VT-1 in vitro and to be a
functional plasma membrane receptor which mediates cytopathology for most
sensitive
cells (Boyd et al 1994 Eur J Biochem 223: 873-878).
E. coli verotoxin has been characterized as having an "A" subunit of
approximately
31,000 daltons and five "B" subunits each having an approximate molecular
weight of
5,500 daltons. The A subunit possesses the biological activity of the toxin
which is
involved in inhibiting protein synthesis, whereas the B subunits are presumed
to
mediate specific binding and receptor-mediated uptake of the toxin.
The verotoxins are closely related to Shiga toxin and have been widely
referred to as
Shiga-like toxins (SLTs) as they mediate the same enterotoxic, cytotoxic, and
neurotoxic effects of Shiga toxin, produced by the Shigella dysenteriae type
1. It has
been shown that the antigenically distinct Shiga like toxins (SLTs) SLT-1 and
SLT-II
use Gb3 as functional receptors (Samuel et al 1990 Infect Immun 58: 611-618)
whereas the SLT II variants may use different receptors.
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The B subunits of verotoxins, VTI and VT2 demonstrate terminal Gal a 1-4 Gal-
dependent binding. The binding B oligomers of SLTI, SLTII and SLTIIvh
recognize
host cell glycolipid receptors containing at minimum the disaccharide subunit
aGal(1-4)(3Ga1 at the non-reducing terminus. SLTIIvp has been shown to bind to
the
receptors containing this subunit but not necessarily at the non-reducing end
(Samuel,
J. E., et al Infect Immun (1990) 58:611-618; Boyd, B., et al Nephron (1989)
51:207-210; DeGrandis, S., et al J Biol Chem (1989) 264:12520-12525; Waddell,
T.,
et al Biochem Biophys Res Chem (1988) 152:674-679; Lingwood, C. A., et al J
Biol
Chem (1987) 262:8834-8839; Waddell, T., et al Proc Natl Acad Sci USA (1990)
87:7898-7901; Cohen, A., et al J Biol Chem (1987) 262:17088-17091; Jacewicz,
M.,
et al J. Exp Med (1986) 163:1391-1404; Lindberg, A. A., et al J Biol Chem
(1987)
262:1779-1785).
The carbohydrate recognition domain, also known as the P (k) trisaccharide,
of the Gb3 receptor for SLT-I (St Hilaire et al 1994 Biochem 33: 14452-14463)
is
represented as
methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D-glucopyranoside}
2o The terms "VT producing E coli (VTEC)" (Lingwood 1996 Trends Microbiol 4:
147-
153) or "Shiga like toxin producing E coli (SLT-EC)" (Newburg et al 1993 J
Infect
Dis 168: 476-479) are widely used and interchangeable. Recently, a new unified
nomenclature for the verotoxin, Shiga-like toxin and Shiga toxin family was
proposed
with the designation Stx being used for the entire family. The nomenclature
Vtx shall
be used throughout the text of the present invention.
Although workers know that the Gb3 receptor acts as a functional receptor for
verotoxins, the possibility that receptor binding modulates the immune
response has
not yet been explored.
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The present invention now seeks to provide new ways of treating disorders such
as
allergic conditions or hypersensitivity conditions, autoimmune and
inflammatory
diseases, cancers such as human leukaemias of a T cell origin, transplantation
rejection
and graft versus host disease (GVHD), all collectively called "immune
disorders".
According to a first aspect of the present invention, there is provided the
use of an
agent in the manufacture of a medicament to affect an immune disorder; wherein
the
agent is capable of modulating a glycosphingolipid associated activity; and
wherein
the modulation of the glycosphingolipid associated activity affects an immune
i0 disorder.
According to a second aspect of the present invention, there is provided the
use of an
agent in the manufacture of a medicament to affect an immune disorder; wherein
the
agent is capable of modulating a globotriaosylceramide (Gb3) associated
activity; and
wherein the modulation of the Gb3 associated activity affects an immune
disorder.
Preferably, the agent is capable in vivo of modulating lymphocyte populations.
Preferably, the agent is capable of acting as a vaccine adjuvant.
Preferably, the agent is capable of promoting the antigenicity of a protein.
According to a fourth aspect of the present invention, there is provided an
assay
method for identifying an agent according to the present invention capable of
affecting
an immune disorder; wherein the assay method comprises: (a) contacting an
agent with
a glycosphingolipid; (b) determining whether the agent modulates a
glycosphingolipid
associated activity; such that the modulation of the glycosphingolipid
associated
activity is indicative that the agent may be capable of affecting an immune
disorder.
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According to a fifth aspect of the present invention, there is provided an
assay method
according to the present invention wherein the assay is an assay to screen for
an agent
useful in the prevention and/or treatment of an immune disorder.
5 According to a sixth aspect of the present invention, there is provided a
process
comprising the steps of (a) perfornling the assay according to the present
invention; (b)
identifying one or more agents capable of modulating a glycosphingolipid
associated
activity; and (c) preparing a quantity of those one or more agents.
l0 According to a seventh aspect of the present invention, there is provided a
process
comprising the steps of (a) performing the assay according to the present
invention; (b)
identifying one or more agents capable of modulating a glycosphingolipid
associated
activity; and (c) preparing a pharmaceutical composition comprising one or
more
identified agents.
According to an eighth aspect of the present invention, there is provided a
process
comprising the steps of (a) performing the assay according to the present
invention; (b)
identifying one or more agents capable of modulating a glycosphingolipid
associated
activity; and (c) modifying one or more identified agents capable of
modulating a
glycosphingolipid associated activity; and (d) preparing a pharmaceutical
composition
comprising those one or more modified agents.
According to a ninth aspect of the present invention, there is provided an
agent
identified by the process of the present invention.
Preferably the agent identified had not previously been known to affect an
immune
disorder through modulation of a glycosphingolipid associated activity.
According to a tenth aspect of the present invention, there is provided a
method of
3o affecting an immune disorder with one or more agents; wherein the agent is
capable of
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modulating a glycosphingolipid associated activity in an in vitro assay
method; and
wherein the in vitro assay method is the assay method defined in the present
invention.
Preferably there is provided a method of affecting in vivo an immune disorder
with one
or more agents; wherein the agent is capable of modulating a glycosphingolipid
associated activity in an in vitro assay method; and wherein the in vitro
assay method is
the assay method defined in the present invention.
According to a eleventh aspect of the present invention, there is provided an
agent
according to the present invention for use as a pharmaceutical.
According to a twelfth aspect of the present invention, there is provided the
use of an
agent according to the present invention in the manufacture of a medicament to
affect
an immune disorder.
According to a thirteenth aspect of the present invention, there is provided a
pharmaceutical composition comprising or prepared from an agent according to
the
present invention.
Preferably, the agent capable of modulating a glycosphingolipid associated
activity is a
Gb3 binding agent.
Preferably the agent is a verotoxin.
Preferably the agent is VtxB.
In a particularly preferred embodiment the agent is the wild type VtxB.
In one embodiment, the agent has a pentameric structure.
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Prefefably the agents) is/are non-toxic.
Alternatively, preferably the agent is either a mutant of VtxB which is
capable of
modulating a glycosphingolipid associated activity or other equivalent
proteins thereof.
Preferably the agent is VtxB and mutants thereof which are capable of
modulating a
glycosphingolipid associated activity.
Preferably the glycosphingolipid is Gb3.
In one embodiment, the agent capable of modulating a glycosphingolipid
associated
activity is capable of cross-linking Gb3 receptors.
Preferably VtxB is a suitable example of an agent which is capable of cross-
linking
Gb3.
Preferably good cross-linking is achieved by virtue of its pentameric form.
Preferably the medicament is used for the treatment or prophylaxis of an
immune
2o disorder.
Preferably the medicament includes one or more antigens which are optionally
co-
administered with the agent.
In one preferred embodiment, the agent is coupled to an antigen.
In one preferred embodiment, the agent is uncoupled to an antigen.
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Preferably the agent may be administered to a mammal with or without ~6-
administration of an antigen.
Preferably the mammal is a human - e.g. a human patient.
In accordance with the present invention we have surprisingly found that the B
subunit
of E.coli verotoxin (VtxB) induces a similiar modulatory effect on CD8+ T
cells to
that of CtxB and EtxB. This effect is very surprising as VtxB binds to an
alternative
receptor (the glycolipid receptor Gb3) which is different from that utilised
by EtxB and
io CtxB and is was not expected that VtxB would induce similiar effects to
that of CtxB
and EtxB.
In particular, we have surprisingly found that the use of agents having Gb3
associated
activity, when given alone or when co-administered with suitable antigens,
exert
immunomodulatory effects useful in the treatment of immune disorders.
We have also surprisingly found that a verotoxin administered on its own and
without
adjuvant can serve to immunomodulate the immune system. Thus, VtxB can serve
as
a vaccine adjuvant which is capable of promoting the antigenicity of a
protein. In
2o addition, immune disorders may be treated with an agent, such as VtxB,
capable of
modulating a glycosphingolipid associated activity (for example, a Gb3
associated
activity) for instance, which is not coupled with an antigen.
Significantly, the linkage of the components was not found to be necessary.
Furthermore, our findings indicate that the mechanisms of protection against
immune
disorders may include, though not be limited to an immunomodulation of the
immune
system.
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Thus, the present invention is advantageous because immune disorders can be
treated
with an agent capable of modulating a glycosphingolipid associated activity.
The
agent may be coupled or uncoupled to an antigen.
The term "glycosphingolipid" as used with respect to the present invention
include
glycosphingolipids capable of acting as receptors for VtxB or VtxB-like agents
as well
as active fragments thereof. For the purposes of this invention; the
glycosphingolipid
is not GM1.
1o The term "glycosphingolipid associated activity" includes any one or more
of
modulating or immunomodulating a glycosphingolipid receptor, modulating any
signalling event prior to, during or subsequent to glycosphingolipid binding.
The term "Vtx" refers to the verotoxin and VtxB refers to the B subunit of the
vero
toxin. In other texts, these may sometimes be identified as VT or VTB
respectively.
Alternatively, the toxin can be referred to as a Shiga-like toxin (SLT) or
Stx. The
verotoxin can be made synthetically or isolated from natural sources.
Alternatively, it
can be obtained from commercial sources.
2o As used herein, "shiga-like toxin" or "SLT" refers to group of toxins
produced by
enterohemorrhagic E. coli that resemble the Shigella-produced shiga toxins as
is
commonly understood in the art. These toxins comprise an enzymatically active
A
subunit and a multimeric receptor binding B subunit. Such SLTs include SLTI
and
the various grouped toxins designated in the art SLTII.
The term "GM1 binding agent" includes any agent which acts as an
immunomodulator
through interacting with a GM 1 ganglioside receptor.
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The term "Etx" herein means the E. coli heat labile enterotoxin and EtxB is
the B
subunit of Etx. In other texts, these may sometimes be identified as LT or Lt
and LTB
or LtB respectively.
5 The term "adjuvant" includes a substance that enhances an immune response to
an
antigen.
The term "vaccine adjuvant" includes an agent which is delivered with an
unrelated
antigen, such that the agent is capable of facilitating an immune response to
the
1o unrelated antigen. In this way, the agent acts as a so-called vaccine
adjuvant. The
term "vaccine adjuvant" includes the term "mucosal adjuvant".
The term "mucosal adjuvant" includes an agent which is delivered mucosally
with an
unrelated antigen, such that the agent is capable of facilitating a mucosal
immune
15 response to the unrelated antigen. In this way, the agent acts as a so-
called mucosal
adjuvant.
A "vaccine carrier" includes a carrier of relevant antigens (Szostak et al
1996 J
Biotechnol 44: 161-170)
The term "mucosal surfaces" includes but is not limited to oral, sublingual,
nasal,
vaginal, rectal, salivary, intestinal and conjunctiva) surfaces.
The term "mucosal membrane" and/or "mucosal tissue" includes but is not
limited to
the intestine, the lung, the mouth, the genital tract, the nose and the eye.
The term "mucosal immunogen" includes an agent administerable by a mucosal
route
that has the capability to evoke cell mediated immune reactions and/or delayed
type
hypersensitivity reactions.
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The term "immunological or oral tolerance" means a reduction in immunological
reactivity of a host towards a specific antigen(s). Immunological or oral
tolerance may
not mean a complete suppression of the immune response to a particular antigen
but it
is a form or tolerance also known as "immune deviation" or "split tolerance".
The term "immune deviation" or "split tolerance" can be used to describe the
likely
preservation of local IgA responses with the retention of some IgG responses
but with
the down regulation of delayed hypersensitivity and IgE responses.
The term "tolerance" means a state of specific immunoIogical unresponsiveness.
The term "immune disorder" includes conditions such as allergic conditions or
hypersensitivity conditions, autoimmune and inflammatory diseases, cancers
such as
human leukaemias of a T cell origin, transplantation rejection and graft
versus host
disease (GVHD).
The term "autoimmunity" is used to describe the mechanism by which the body
generates an immune response to self antigens.
The term "agent capable of modulating a glycosphingolipid associated activity"
can be
used to describe any agent which acts as an immunomodulator through
interacting with
a glycosphingolipid.
The term "immunomodulator" includes any agent that alters the extent of the
immune
response to an antigen, by altering the antigenicity of the antigen or by
altering in a
nonspecific manner the specific reactivity or the nonspecific effector
associated
mechanisms of the host.
The term "administered" includes delivery by viral or non-viral techniques.
Viral
delivery mechanisms include but are not limited to adenoviral vectors, adeno-
associated
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viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral
vectors, and
baculoviral vectors. Non-viral delivery mechanisms include lipid mediated
transfection,
liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and
combinations thereof. The routes for such delivery mechanisms include hut are
not
limited to mucosal, nasal, oral, parenteral (such as intravenous (iv),
intramuscular (im),
or subcutaneous (sc) route), gastrointestinal, topical, or sublingual routes.
The term "co-administered" means that the site and time of administration of
each of
the agent and the antigen are such that the necessary modulation of the immune
system
1o is achieved. Thus, whilst the agent and the antigen may be administered at
the same
moment in time and at the same site, there may be advantages in administering
the
agent at a different time and to a different site from the antigen. The agent
and antigen
may even be delivered in the same delivery vehicle (such as a MacrosolTM-see
W095/13795 and W096/14871).
The term "administered" includes but is not limited to delivery by a mucosal
route, for
example, as a nasal spray or aerosol for inhalation or as an ingestable
solution; a
parenteral route where delivery is by an injectable form, such as, for
example, an
intravenous, intramuscular or subcutaneous route.
The term "systemic immunisation" means the introduction of an antigen into a
non-
mucosal tissue such as the skin or the blood.
The term "self antigens" means components derived from host tissues.
The term "target interaction components" includes but is not limited to an
agent
capable of modulating a glycosphingolipid associated activity, a
glycosphingolipid
and/or an antigen.
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18
The team "uncoupled" - which is synonymous with the term "unlinked" - means
that
the agent is not coupled to the antigen.
The term "coupled" - which is synonymous with the term "linked" - means the
linkage
of the agent with the antigen - which includes but is not limited to direct
linkage (such
as by a covalent bond) or indirect linkage such as by the provision of
suitable spacer
groups.
However, in accordance with the present invention, the agent and/or antigen
can be
1 o coupled to another entity.
The term "affect" includes modulation, such as treatment, prevention,
suppression,
alleviation, restoration or other alteration of pre-existing condition and/or
to potentially
affect a future condition, as well as any combination thereof.
An "antigen" means an agent which, when introduced into an immunocompetent
animal, stimulates the production of a specif c antibody or antibodies that
can combine
with the agent. The antigen may be a pure substance, a mixture of substance or
soluble
or particulate material (including cells or cell fragments). In this sense,
the term
includes any suitable antigenic determinant, auto-antigen, self antigen,
tolerogen,
allergen, hapten, and immunogen, or parts thereof, as well as any combination
thereof,
and these terms are used interchangeably throughout the text.
A "tolerogen" means a tolerated antigen.
A "hapten" means a small molecule which can act as an epitope but is incapable
by
itself of eliciting an antibody response.
Examples of antigens include but are not limited to: insulin; myelin basic
protein;
3o antibody or antibody fragments; gamma globulins; transplantation antigens
or cells
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19
expressing said transplantation antigens, such as red blood cells, platelets
and
lymphocytes; bacterial toxins; and/or synthetic peptides and/or corresponding
variants,
homologues, derivatives or fragments thereof of the above mentioned antigens.
An "allergen" includes any antigen that stimulates an allergic reaction,
inducing a
Type I hypersensitivity reaction.
Examples of common allergen sources are outlined in the Table below.
Group E:ampler of Allergens
Airborne
grass pollens ragweed, rye, couch, wild
oat, timothy,
Bermuda, Kentucky blue, mugwort
tree pollens alder, birch, hazel, beech,
Cupressae, oak,olive
moulds Aspergillus spp., Cladosporium
spp., Alternaria
spp., Basidospores, Ascomycetes
cereal grains wheat, rye, oat
animal dander and cat, dog, horse, rabbit, guinea
urine pig, hamster
bird feathers budgerigar, parrot, pigeon,
duck, chicken
house dust mite Dermatophagoides pteronyssinus,
D.farinae,
Euroglyphus maynei
insects cocla~oach, fly, locust, midge
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Oral
seafood, legumes, peanuts,
nuts, cereals, dairy
foods products, eggs, fruits, tomatoes,
mushrooms,
alcoholic beverages, coffee,
chocolate
Wigs penicillins, sulphonamides
and other antibiotics,
sulphasalazine, carbamazepine
Injected
bee and wasp stings, ant and
mosquito bites
insects blood products, sera, vaccines,
contrast media,
drugs (including anti-asthma
drugs and
Wigs antibiotics)
The term "allergic condition" includes but is not limited to asthma, allergic
cough,
allergic rhinitis and conjunctivitis, atopic eczema and dermatitis, uticaria,
hives, insect
5 bite allergy, dietary allergy (peanut, fish milk, wheat etc) and drug
allergies.
The term "hypersensitivity condition" includes but is not limited to
conditions such as
contact hypersensitivity induced by plant poison ivy.
to The teen "agent" includes entities capable of modulating a
glycosphingolipid
associated activity. The agent can be one or more of an inorganic or organic
chemical,
as well as combinations thereof. By way of example the agent can be a
polypeptide as
well as a variantlhomologue/derivative/fragment thereof so long as they retain
the
required immunomodulatory activity. It also includes mimics and equivalents
and
15 mutants thereof. Other agents for the prevention and/or treatment of immune
disorders
include antibodies to the target interaction components. Such antibodies
include, but
are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab
fragments,
fragments produced by a Fab expression Library and specifically designed
humanised
monoclonal antibodies.
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Agents capable of modulating a glycosphingolipid associated activity may be
designed
and produced as outlined above, by methods which are known in the art. By way
of
example, when the agent of the invention is a protein such as the VtxB subunit
it may
be produced, for use in all aspects of this invention by a method in which the
gene or
genes coding for the specific polypeptide chain (or chains) from which the
protein is
formed, is inserted into a suitable vector and then used to transfect a
suitable host. For
example, the gene coding for the polypeptide chain from which the VtxB
assemble
may be inserted into suitable expression vectors (such as plasmids) which are
then
used to transfect host cells, such as Vibrio sp.60. The protein is purified
and isolated
io in a manner known per se. Mutant genes expressing active mutant VtxB
protein may
then be produced by known methods from the wild type gene.
In E. coli, verotoxins are encoded by one or more bacteriophages and,
furthermore,
individual strains may produce either one or both VT1 and VT2. Both the
natural and
the recombinant forms of E. coli verotoxin have been isolated. One such
recombinant
cloned toxin is pJLB28 which expresses both the A and B subunits (Huang, A. et
al
(1986) J. Bacteriol. 166, 375-379).
Where a target interaction component is a protein, procedures well known in
the art
2o may be used for the production of antibodies to that component.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, etc.
may be immunized by injection with the target interaction component or any
derivative
or homologue thereof or oligopeptide which retains immunogenic properties.
Depending on the host species, various adjuvants may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's,
mineral gels such as aluminium hydroxide, and surface active substances such
as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and
Corynebacterium
parvum are potentially useful human adjuvants.
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Where a target interaction component is a protein, monoclonal antibodies to
that
component may be prepared using any technique which provides for the
production of
antibody molecules by continuous cell lines in culture. These include, but are
not
limited to, the hybridoma technique originally described by Koehler and
Milstein
(1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al
(1983) immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030)
and
the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and
Cancer
Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the
production of "chimeric antibodies", the splicing of mouse antibody genes to
human
1o antibody genes to obtain a molecule with appropriate antigen specificity
and biological
activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855;
Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-
4.54).
Alternatively, techniques described for the production of single chain
antibodies (US
Patent No. 4,946,779) can be adapted to pmduce target interaction component
specific
single chain antibodies.
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening recombinant immunoglobulin libraries or panels of
highly
specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad
Sci 86:
3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
Antibody fragments which. contain specific binding sites for a target
interaction
components may also be generated. For example, such fragments include, but are
not
limited to, the F(ab')2 fragments which can be produced by pepsin digestion of
the
antibody molecule and the Fab fragments which can be generated by reducing the
disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression
libraries may
be constructed to allow rapid and easy identification of monoclonal Fab
fragments
with the desired specificity (Ruse WD et al (1989) Science 256:1275-128 1).
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The target interaction components of the present invention or a derivative or
homologue thereof and/or a cell line that expresses the target interaction
components of
the present invention or a derivative or homologue thereof may be used to
screen for
antibodies, peptides, or other agent, such as organic or inorganic molecules,
that act as
modulators of the target interaction, thereby identifying a therapeutic agent
capable of
modulating the target interaction. For example, antibodies capable of
modulating the
target interaction may be identified.
Alternatively, screening of peptide libraries or organic libraries made by
combinatorial
to chemistry with recombinantly expressed target interaction components or a
derivative
or homologue thereof or cell lines expressing the target interaction
components or a
derivative or homologue thereof may be useful for identification of
therapeutic agents
that function by modulating the target interaction. Synthetic compounds,
natural
products, and other sources of potentially biologically active materials can
be screened
in a number of ways deemed to be routine to those of skill in the art.
A target interaction component polypeptide, its immunogenic fragments or
oligopeptides thereof can be used for screening therapeutic compounds in any
of a
variety of drug screening techniques. The polypeptide employed in such a test
may be
2o free in solution, affixed to a solid support, borne on a cell surface, or
located
intracellularly. The abolition of activity or the formation of binding
complexes
between the target interaction component and the agent being tested may be
measured.
Alternatively, phage display can be employed in the identification of
candidate agents
which affect the target interaction components.
Phage display is a protocol of molecular screening which utilises recombinant
bacteriophage. The technology involves transforming bacteriophage with a gene
that
encodes an appropriate ligand (in this case a candidate agent) capable of
reacting with
a target interaction component (or a derivative or homologue thereof) or the
nucleotide
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24
sequence (or a derivative or homologue thereof) encoding same. The transformed
bacteriophage (which preferably is tethered to a solid support) expresses the
appropriate Iigand (such as the candidate agent) and displays it on their
phage coat.
The entity or entities (such as cells) bearing the target molecules which
recognises the
candidate agent are isolated and amplified. The successful candidate agents
are then
characterised. Phage display has advantages over standard affinity ligand
screening
technologies. The phage surface displays the candidate agent in a three
dimensional
configuration, more closely resembling its naturally occuring conformation.
This
allows for more specific and higher affinity binding for screening purposes.
Accordingly, the present invention provides a method for screening a plurality
of
agents for specific binding affinity with the target interaction component or
a
derivative or homologue thereof comprising providing a plurality of agents;
combining
the target interaction components or a derivative or homologue thereof with
each of a
plurality of agents for a time sufficient to allow binding under suitable
conditions; and
detecting binding of the target interaction components, or a derivative or
homologue
thereof to each of the plurality of agents, thereby identifying the agent or
agents which
specifically bind the target interaction components. In such an assay, the
plurality of
agents may be produced by combinatorial chemistry techniques known to those of
skill
in the art.
Another technique for screening provides for high throughput screening of
agents
having suitable.binding affinity to the target interaction component
polypeptides and is
based upon the method described in detail in WO 84/03564. In summary, large
numbers of different small peptide test compounds are synthesized on a solid
substrate,
such as plastic pins or some other surface. The peptide test agents are
reacted with the
target interaction component fragments and washed. A bound target interaction
component is then detected - such as by appropriately adapting methods well
known in
the art. A purified target interaction component can also be coated directly
onto plates
for use in the aforementioned drug screening techniques. Alternatively, non-
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neutralizing antibodies can be used to capture the peptide and immobilize it
on a solid
support.
The present invention also provides a pharmaceutical composition comprising
5 administering a therapeutically effective amount of an agent capable of
modulating a
glycosphingolipid associated activity and a pharmaceutically acceptable
carrier,
diluent, excipient or adjuvant.
The pharmaceutical compositions may be for human or animal usage and will
typically
1o comprise any one or more of a pharmaceutically acceptable diluent, carrier,
excipient
or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be
selected
with regard to the intended route of administration and standard
pharmaceutical
practice. The pharmaceutical compositions may comprise as - or in addition to -
the
carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending
agent(s),
15 coating agent(s), solubilising agent(s).
The pharmaceutical composition may be formulated together with an appropriate
antigen.
2o Alternatively, a kit may be provided comprising separate compositions for
each of the
therapeutic agent and the antigen.
In some embodiments of the present invention, the pharmaceutical compositions
will
comprise one or more of an agent that has been screened by an assay of the
present
25 invention; wherein the agent is capable of modulating a glycosphingolipid
associated
activity.
The present invention also relates to pharmaceutical compositions comprising
effective
amounts of antigen in admixture with a pharmaceutically acceptable diluent,
carrier,
3o excipient or adjuvant (including combinations thereofj.
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The present invention also provides a method of treating a mammal, such as a
human
patient, comprising administering to said mammal an effective amount of the
pharmaceutical composition of the present invention.
The present invention relates to pharmaceutical compositions which may
comprise all
or portions of the target interaction components alone or in combination with
at least
one other agent, such as a stabilizing compound, and may be administered in
any
sterile, biocompatible pharmaceutical carrier, including, but not limited to,
saline,
buffered saline, dextrose, and water.
There may be different composition/formulation requirements dependent on the
different delivery systems.
The pharmaceutical composition of the present invention may be formulated to
be
delivered by a mucosal route, for example, as a nasal spray or aerosol for
inhalation or
ingestable solution, or parenterally in which the composition is formulated by
an
injectable form, for delivery, by, for example, an intravenous, intramuscular
or
subcutaneous route. Alternatively, the formulation may be designed to be
delivered by
both routes.
Where the agent is to be delivered mucosally through the gastrointestinal
mucosa, it is
preferably stable during transit though the gastrointestinal tract; for
example, it should
be resistant to proteolytic degradation, stable at acid pH and resistant to
the detergent
effects of bile.
Typically, a physician will determine the actual dosage which will be most
suitable for
a subject and it will vary with the age, weight and response ~of the
particular subject.
While a single dose of the agent and the antigenic determinant may be
satisfactory,
multiple doses are contemplated within the scope of the invention.
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Where appropriate, the pharmaceutical compositions can be administered by
inhalation, in the form of a suppository or pessary, topically in the form of
a lotion,
solution, cream, ointment or dusting powder, by use of a skin patch, orally in
the form
of tablets containing excipients such as starch or lactose, or in capsules or
ovules either
alone or in admixture with excipients, or in the form of elixirs, solutions or
suspensions containing flavouring or colouring agents, or they can be injected
parenterally, for example intracavernosally, intravenously, intramuscularly or
subcutaneously. For parenteral administration, the compositions may be best
used in
the form of a sterile aqueous solution which may contain other substances, for
example
1o enough salts or monosaccharides to make the solution isotonic with blood.
For buccal
or sublingual administration the compositions may be administered in the form
of
tablets or lozenges which can be formulated in a conventional manner.
There may be different delivery requirements dependent on the different
composition/formulation systems.
Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia
viruses,
or from various bacterial plasmids, may be used for delivery of the agent to
the
targeted tissue and/or cell population. Methods which are well known to those
skilled
in the art can be used to construct recombinant vectors containing the agent.
Alternatively, the agent can be delivered to target cells in liposomes.
By way of example, the controlled release of vaccine antigens on mucosal
surfaces
using biodegradable polymer microspheres may help to target antigens and
reduce the
numbers of doses required for primary immunisation (Gupta and Siber 1995
Vaccine
13: 1263-1276)
Encapsulation of vaccines in biodegradable microspheres provides excellent
mucosal
immunogens. Recombinant Norwallc Virus-like (rNV) particles may also be used
for
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28
the oral delivery of rnucosal immunogens (Ball et al 1996 Arch Virol Suppl 12:
243-
249)
Viral Like Particles (VLPs) have been utilised as vaccine delivery system for
multiple
immunogens including B and T cell epitopes (Roy 1996 Intervirology 39: 62-71).
One preferred method of oral delivery uses formations as described in
W095/13795,
W096/17593 and W096/17594. These formulations allow macromolecules such as
proteins to be solubilised in "oils" for oral delivery. Such formulations
therefore allow
to delivery of the macromolecules to mucosal surfaces in the gut.
In a further approach, again when the therapeutic agent is a protein, it is
possible to
deliver such proteins by means of a bacterial delivery system such as that
described in
WO 93/17117. This system utilises the bacterium Lactococcus lactis to deliver
proteins, for instance orally or indeed by other mucosol routes such as
nasally.
In summary, the present invention provides the use of an agent in the
manufacture of a
medicament to affect an immune disorder; wherein the agent is capable of
modulating
a glycosphingolipid associated activity.
In another broad aspect, the present invention provides an agent capable of
modulating
a glycosphingolipid associated activity and which is potently immunogenic.
Other aspects of the present invention are now presented below by way of
numbered
paragraphs, which include:
1. The use of a Gb3 binding agent, or an agent having an effect on Gb3
intracellular signalling events, but no Gb3 binding activity, in the
preparation of a
medicament for the treatment or prophylaxis of inflammatory conditions,
autoimmune
3o diseases, transplant rejection, graft versus host disease, immune disorders
or cancer.
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2. The use as claimed in paragraph 1 wherein the agent is VtxB.
3. The use as claimed in paragraph 1 or paragraph 2 wherein the medicament
also
comprises one or more antigens/allergens.
4. A pharmaceutical formulation comprising a Gb3 binding agent, or an agent
having an effect on Gb3 intracellular signalling events, but no Gb3 binding
activity,
optionally together with one or more pharmaceutically acceptable carriers,
diluents or
excipients.
5. A pharmaceutical formulation as claimed in paragraph 4 wherein the agent is
VtxB.
6. A pharmaceutical formulation as claimed in paragraph 5 which also comprises
one or more allergens/antigens.
7. A pharmaceutical formulation as claimed in paragraph 5 or paragraph 6 which
is a vaccine formulation.
8. A method for the treatment or prophylaxis of an inflammatory condition, an
autoimmune disease, transplant rejection, GVHD, allergic, or other
hypersensitive
conditions or cancer which comprises administering to a subject an effective
amount of
a Gb3 binding agent, or an agent having an effect on Gb3 intracellular
signalling
events, but no Gb3 binding activity.
9. A method of vaccinating a subject against, for example an immune disorder,
which comprises administering to a subject an effective amount of a Gb3
binding
agent, or an agent having an effect on Gb3 intracellular signalling events,
but no Gb3
binding activity.
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10. A product comprising an agent having Gb3 binding activity, or an agent
having
an effect on Gb3 mediated intracellular signalling events, but no Gb3 binding
activity,
in the preparation of a medicament to treat an inflammatory condition, an
autoimmune
disease, transplant rejection, CVHD, an immune disorder or cancer, and at
least one
5 antigen/allergen as a combined preparation for simultaneous, separate or
sequential
use.
The present invention will now be further described by example in which
reference is
made to the following Figures:
Figure 1 shows the serum anti-VtxB antibody responses in 1VIH mice following
subcutaneous immunisation with VtxB; and
Figure 2 shows the upregulation in IL-2R (CD25) expression induced by binding
of
15 EtxB (A), CtxB (B) and VtxB (C) to lymphocyte populations.
In slightly more detail, the serum anti-VtxB antibody titre in column 1 of
Figure 1 was
obtained in 1VIH mice immunised with lOp,g VtxB in complete Freund's adjuvant
on
day 1 and lOpg VtxB in incomplete Freund's adjuvant on Days 8 and 15 whereas
the
20 serum anti-VtxB antibody titre in column 2 was obtained in N»i mice
immunised with
lOp,g of VtxB without complete or incomplete Freund's adjuvant on days 1, 8
and 15.
Mice were bled on day 28. Antibody titres to VtxB were determined on VtxB-
coated
microtitre plates.
25 The upregulation of CD25 expression on CD8+ T cells induced by CtxB, EtxB
and
VtxB in Figure 2 is represented by increased binding of CD25 antibodies
labelled with
FITC and detected as a shift in the intensity of FITC fluorescence by FACs
analysis
The dotted lines represent levels of CD25 expression in unstimulated cells.
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EXAMPLES
Example 1. Serum anti-VtxB antibody responses in NI~I mice following
subcutaneous immunisation with VtxB.
NIH mice were immunised (s.c.) on days 1, 8 and 15 with 10~g of VtxB in
Freund's
adjuvant, lOp.g of VtxB alone or PBS (control). Mice were bled on day 28.
Antibody
titres to VtxB were determined on VtxB-coated microtitre plates.
The results in Figure 1 demonstrate that the serum anti-VtxB antibody titre in
NIH
mice treated with VtxB without adjuvant were almost twice that seen in NIH
mice
treated with VtxB with adjuvant. These results indicate that (i) in the
absence of
adjuvant, VtxB is potently immunogenic (ii) VtxB administered on its own and
without adjuvant can serve to immunomodulate the immune system and (iii) that
(i)
and (ii) above indicate that VtxB is capable of acting as a vaccine adjuvant.
Example 2. Examination of CD25 (IL-2R) cell surface expression on VtxB
treated lymphocyte populations by FRCS analysis.
2o NIH female mice ranging between the ages of 6 to 8 weeks were sacrificed
and
Mesenteric lymph node tissue was subsequently removed into Hanks balanced salt
solution (HBSS without Calcium and Magnesium). Lymphocytes were then dispersed
into the solution and away from fibrous tissue through gently pressing the
tissue
through a wire mesh.
Following 3 washes in HBSS, CD8+T cells were purified by separation in a
magnetic
MACS column. Briefly, 100 million cells were resuspended in 300~t1 MACS buffer
{PBS/0.5% BSA/O.SmM EDTA). For positive selection 201 of MACS anti-CD8
antibody was added to cell suspension and the mixture was incubated on ice for
a
3o period of 30 minutes to allow time for effective antibody interaction.
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Following incubations cells were subsequently washed in cold MACS buffer and
passed through a pre-washed VS+ selection column (MACS). The CD8+ T cells
bound to the column and then eluted by the force of a plunger, washed and
cultured for
20 hours at a concentration of 2 million cell/ml in the presence of either no
antigen or
40~,g/ml EtxB, VtxB or CtxB.
After the period of stimulation, cells were washed and resuspended in.I-
IBSS/2%
sodium azide at a concentration of 2 million/ml. Phycoerythrin (PE) conjugated
anti-
CD8 and biotin labelled anti CD25 were incubated on ice with the cellular
suspensions
l0 at dilutions of 1 in 250 and 1 in 125 respectively for 45 minutes.
Streptavidin
conjugated FITC was then added to cell samples following a brief spin and
incubated
for a further 45 minutes.
Following antibody incubations, cell samples were washed twice in cold HBSS
followed by once in ISOTON solution. Levels of FITC and PE fluorescence as a
representation of CD25 and CD8 expression respectively were analysed by a FACS
machine (Hewlettt Packard).
The results demonstrate the surprising finding that the B subunit of E.coli
verotoxin
(VtxB) induces a similiar modulatory effect on CD8+ T cells, in terms of
upregulation
of IL-2R expression, to that of CtxB and EtxB. This effect is very surprising
as VtxB
binds to an alternative receptor (the glycolipid receptor Gb3) which is
different from
that utilised by EtxB and CtxB and is was not expected that VtxB would induce
similiar effects to that of CtxB and EtxB.
Screens for Agents capable of modulating glycosphingolipid associated activity
Agents capable of modulating glycosphingolipid associated activity are tested
by any
one of a variety of methods.
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33
Examples of such methods include, but are not limited to the following
methods:
1. Binding to a glycosphingolipid, such as Gb3, is determined by using
purified Gb3
to coat microtiter plates. Following blocking of further non-specific protein
binding to
the plate, the agent under investigation is applied to the plate and allowed
to interact
prior to washing and detection with specific antibodies to said agent.
Conjugation of
the antibodies either directly or indirectly to an enzyme or radiolabel allows
subsequent quantification of binding either using colormetric or radioactivity
based
methods (ELISA or RIA respectively).
to
2. The pentasaccharide moiety of a glycosphingolipid, such as Gb3, is bound to
a
suitable column matrix in order to allow standard affinity chromatography to
be
performed. Removal of known compounds applied to the column from the diluent
are
used as evidence for binding activity. Alternatively, where mixtures of
compounds are
applied to the column, elution and subsequent analysis allows the properties
of the
agent capable of modulating glycosphingolipid associated activity to be
determined.
Protein analysis includes peptide sequencing and tryptic digest mapping
followed by
comparisons with available databases. If eluted proteins cannot be identified
in this
2o way, then standard biochemical analysis, such as, for example, mass
determination by
laser desorption mass spectrometry is used to further characterise the
compound. Non-
proteins eluted from Gb3-affinity columns are analysed by HPLC and mass
spectrometry of single homogenous peaks.
3. The ability to bind to glycosphingolipid, such as Gb3, and the precise
affinity of the
interaction may be determined using plasmon surface resonance as previously
reported
by (Kuziemko et al (1996) Biochem 35:6375-6384).
Other screens for agents capable of activating B cells, CD4+ T cells,
depleting CD8+ T
3o cells and inducing alterations in lymphocyte nuclear morphology
characteristics of
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34
cells undergoing apoptosis are outlined in W097/02045 and are incorporated
herein by
reference.
Evaluation of identified agents
The identification of agents capable of modulating a glycosphingolipid
associated
activity such that the modulation of the glycosphingolipid associated activity
affects an
autoimmune condition is determined as follows:
1o Animal models of an autoimmune disease can be used to assess the efficacy
of an
agent capable of modulating a glycosphingolipid associated activity to prevent
autoimmune disease.
An example of the use of an animal model of autoimmune disease is the
induction of
arthritis in DBA/1 mice. Evaluation of agents is conducted by injecting groups
of
DBA/1 mice with bovine collagen in Complete Freund's Adjuvant (CFA) by
intradermal (id) injection in the dank in the presence or absence of the agent
capable of
modulating a glycosphingolipid associated activity in order to assess
prevention of
disease development.
Such mice are given a second injection of collagen in Incomplete Freund's
Adjuvant
(IFA) in the presence/absence of the agent capable of modulating a
glycosphingolipid
associated activity. All animals are assessed for severity of disease on day
45 by
measuring hind limb ankle thickness or scoring each hind limb digit for
swelling.
The identification of agents capable of modulating a glycosphingolipid
associated
activity such that the modulation of the glycosphingolipid associated activity
affects an
allergic condition and/or a hypersensitivity condition is determined as
follows:
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Laboratory animals are stimulated to produce antigen-specific IgE by methods
well
known in the art. By way of example, mice are challenged with alum
precipitated
soluble protein antigen (e.g. ovalbumin or allergens known to be involved in
human
allergic diseases such as ragweed or house dust mite antigens) either
subcutaneously or
5 intraperitoneally.
In the unmanipulated animal, this procedure routinely leads to the production
of
antigen-specific IgE which is easily detected in the serum, by standard
ELISAs, using
the antigen to coat suitable microtiter plates. Serum from the immunised mice
is
1o applied to the plates after non-specific protein binding has been blocked
and the
presence of IgE is determined using widely available labelled antibodies
specific for
marine IgE.
In order to screen agents for their capability to prevent or treat allergy,
agents capable
~5 of modulating glycosphingolipid associated activity are administered to
mice either in
the presence or absence of the challenge antigen at a range of doses, and by a
variety of
routes. Although the oral route is the preferred method of administration,
delivery can
be by other mucosal surfaces or parenterally. The frequency of such
administration as
well as the timing of repetitive dosing is also investigated. Such
intervention strategies
20 are utilised either prior to the IgE inducing antigen challenge
(prophylaxis) or after the
IgE inducing antigen challenge (treahnent). Antigen challenge can be either
with (i)
the antigen used as part of the prophylactic or treatment protocol; (ii) an
unrelated
antigen or (iii} a mixture of the challenge and unrelated antigen in order to
test the
specificity of the response and the induction of bystander suppression
respectively.
Efficacy is determined in a variety of ways and is manifested as a number of
different
outcomes.
1. Antigen-specific IgE levels. Measurement of serum IgE by specific ELISA (as
3o described) is used to determine whether prophylactic or treatment protocols
are
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36
capable of reducing levels of serum antigen-specific IgE. Other methods known
in the
art for the determination of IgE response are used either as alternatives to
ELISA or in
order to provide complementary data. Such methods include the so-called
"IJssing
Chamber test" or "passive cutaneous anaphylaxis" assay. A reduction in
specific IgE,
as determined by any of these assays, is a strong marker of potential clinical
efficacy.
2. Antigen specific T-cell reactivity. The responses of T-cells, derived from
secondary Lymphoid organs of the treated animals to the challenge antigen, is
investigated using established methodology. Cell suspensions are prepared and
cultured, in the presence or absence of the challenge antigen. At appropriate
time
intervals after the initiation of the cultures, samples are assessed for cell
proliferation
and cytokine production.
Cytokines are measured by specific capture ELISA, by intracellular staining
followed
by cytometric analysis, by RT-PCR or by other established procedures.
Comparison of
cell proliferation and cytokine production, in the presence of antigen as
opposed to its
absence, provides in each case a measure of that part of the response which is
specific
to the challenge antigen. Evidence of efficacy of prophylactic or treatment
protocols is
demonstrated by a reduction in the production of Th2 associated cytokines (in
2o particular IL-4) or by an increased expression of cytokines which are
involved in
down-regulating the allergic response (for example, IL-10 or TGF(3).
3. IgG and IgA levels. Protocols which do not reduce the levels of antigen
specific
IgE can still be considered as potentially effective in the event that they
are also able to
enhance the production of other non-allergy associated antibody isotopes. Thus
investigation of serum and mucosal secretions from animals which have been
either
untreated or given agents under investigation as part of prophylactic or
treatment
protocols for the presence of IgG and IgA are also carried out. Standard
antigen
specific ELISA assays (as described) utilising detecting antibodies specific
for IgG and
specific subclass thereof, and IgA are used for this purpose. Enhanced
production of
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37
secreted or serum IgG or IgA antibodies indicate efficacy since such
antibodies can be
expected to prevent an allergen from cross-linking IgE bound to mast cells,
basophils
and eosinophils or limit the uptake of antigen across the mucosal epithelium
and hence
prevent the subsequent allergic inflammatory response.
In one aspect of the present invention, a glycosphingolipid binding assay for
the
detection of glycosphingolipid binding agents' such as verotoxins has been
developed
according to the teachings as set out in US5164298. This assay is based on the
immobilization of deacylated globotriaosyl ceramide in rnicrotitre wells.
In one preferred embodiment of this invention, deacylated Gb3 is bound to a
microtitre
plate for use in an ELISA for the detection of a Gb3 binding agent such as
verotoxin.
The verotoxin present in verotoxin containing samples, or verotoxin positive
controls
will bind to the deacylated Gb3 which has been bound to the plate.
i5
The glycolipid-bound toxin is visualized by use of a polyclonal rabbit
antiserum and
an immunoperoxidase indicator system. Other indicator systems well known to
those
skilled in the art of ELISA would also be suitable.
2o Those skilled in the art of ELISA would also know that a deacylated Gb3,
having a
free amino group, could also be covalently bound to another protein either
directly or
through the incorporation of a spacer arm. This second protein could then be
used in
the primary binding step in the assay. Similarly, instead of attaching the
glycosphingolipid to a protein as an assay component, it could be covalently
bound
25 directly to a solid phase support as an assay component or alternatively,
the assay
component may be a liposome which contains the glycosphingolipid receptor.
Such
solid phase supports include microtitre plates, test tubes, glass beads,
nitrocellulose
and latex particles. The plates or test tubes may be of glass or a plastic
such as
polyvinyl chloride, polystyrene or latex.
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38
The principle of using a glycosphingolipid in a receptor-based assay for
verotoxin
could be applied to any of the well known assay technologies, including
radioimmunoassay, cell-binding cytotoxicity assays, thin layer chromatography
assays
and agglutination assays. The principle could also be used in a fluorescence
based
receptor assay for verotoxin using toxin sensitive target cells as the
receptor bearing
vehicle.
Enzyme Linked Immunosorbent Assays (ELISAs)
Binding of VtxB to Gb3 or deacylated Gb3 can also be examined using an
adaptation
of the GMI ELISA reported by Amin, T., & Hirst, T.R (1994 Prot. Express. and
Purif.
5, I98-204) where GMI is replaced by Gb3 or deacylated Gb3 respectively.
Sera and gut secretions are examined for the presence of anti-B subunit IgG
and IgA
antibodies by ELISAs in which samples are applied to microtitre plates
(lmmulon I,
Dynateck, USA) coated with S~,g/ml of VtxB in PBS. Anti-B subunits IgA
antibodies
in gut secretion supernatants are extrapolated finm a standard curve made by
coating 2
rows of wells on each plate with 1 ~g/ml rabbit anti-mouse IgA (a chain
specific;
Zymed Lab, USA) in PBS followed by addition of 1 ~,g/ml of mouse myeloma IgA
2o (MOPC 315, Sigma, USA). To measure total IgA, wells are coated with rabbit
anti-
mouse IgA followed by addition of gut secretion supernatants. All samples are
serially
diluted. Goat anti-mouse IgG (Fc fragment specific; Jackson Lab., USA) or goat
anti-
mouse IgA (a chain specific; Sigma) peroxidase conjugate are diluted and added
to all
wells. The anti-B subunit IgG titer, giving an A4so"m >_ 0.2, is determined.
The IgA
anti-B subunit response for the VtxB subunit in gut secretions is calculated
as "IgA
specific activity" [mean IgA anti-B subunit (pg/ml) /total IgA (~,g/ml)].
A known ELISA method for measuring cytokine levels of IL-2, IL-4, IL-5, IL-10
and
IFN-y is used. Briefly, microtiter plates are coated with rat antibodies to
mouse IL-2,
3o IL-4, IL-5, IL-10 and IFI~-y. Plates are blocked with 2% (w/v) bovine serum
albumin.
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39
Supernatants from culture medium are added to wells and diluted down. One row
on
each plate for each cytokine contains a standard amount of recombinant
cytokines.
Plates are then incubated with O.S~g/ml of biotinylated anti-cytokine
monoclonal
antibodies followed by addition of avidine-peroxidase and 3,3', S,5' -
Tetramethylbenzidene (TMB) substrate and read at A~spnm~
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods and
system
of the invention will be apparent to those skilled in the art without
departing from the
1o scope and spirit of the invention. Although the invention has been
described in
connection with specific preferred embodiments, it should be understood that
the
invention as claimed should not be unduly limited to such specific
embodiments.
Indeed, various modifications of the described modes for carrying out the
invention
which are obvious to those skilled in molecular biology or related fields are
intended to
be within the scope of the following claims.
