U.S. patent application number 14/628717 was filed with the patent office on 2015-06-18 for agent for treating allergic or hypersensitivity condition.
The applicant listed for this patent is Trident Pharmaceuticals, Inc.. Invention is credited to John Bienenstock, Timothy Raymond Hirst, Neil Andrew Williams.
Application Number | 20150165010 14/628717 |
Document ID | / |
Family ID | 10825085 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150165010 |
Kind Code |
A1 |
Williams; Neil Andrew ; et
al. |
June 18, 2015 |
Agent for Treating Allergic or Hypersensitivity Condition
Abstract
The use of an agent in the manufacture of a medicament to affect
an allergic condition and/or a hypersensitivity condition is
described. The agent is capable of modulating a ganglioside
associated activity. The agent is not coupled to an antigen. The
modulation of the ganglioside associated activity affects an
allergic condition and/or a hypersensitivity condition.
Inventors: |
Williams; Neil Andrew;
(Cheddar, GB) ; Hirst; Timothy Raymond; (Manuka,
AU) ; Bienenstock; John; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trident Pharmaceuticals, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
10825085 |
Appl. No.: |
14/628717 |
Filed: |
February 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13745082 |
Jan 18, 2013 |
8992940 |
|
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14628717 |
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11691798 |
Mar 27, 2007 |
8357372 |
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13745082 |
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09600060 |
Jul 10, 2000 |
7208155 |
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PCT/GB1999/000070 |
Jan 8, 1999 |
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11691798 |
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Current U.S.
Class: |
424/257.1 ;
435/7.92; 436/501; 530/350 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 11/02 20180101; A61P 37/04 20180101; G01N 33/5041 20130101;
A61K 2039/577 20130101; A61P 17/02 20180101; A61P 27/14 20180101;
G01N 2333/705 20130101; A61P 27/02 20180101; G01N 33/68 20130101;
A61P 43/00 20180101; A61K 39/0258 20130101; A61K 38/164 20130101;
G01N 2500/02 20130101; A61P 37/02 20180101; A61P 11/06 20180101;
A61P 17/04 20180101; C07K 14/245 20130101; A61K 38/45 20130101 |
International
Class: |
A61K 39/108 20060101
A61K039/108; G01N 33/50 20060101 G01N033/50; C07K 14/245 20060101
C07K014/245 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 9, 1998 |
GB |
9800487.2 |
Claims
1. The use of an agent in the manufacture of a medicament to affect
an allergic condition and/or a hypersensitivity condition; wherein
the agent is capable of modulating a ganglioside associated
activity; wherein the agent is not coupled to an antigen; and
wherein the modulation of the ganglioside associated activity
affects an allergic condition and/or a hypersensitivity
condition.
2. The use of an agent in the manufacture of a medicament to affect
an allergic condition and/or a hypersensitivity condition; wherein
the agent is capable of modulating a GM1 associated activity;
wherein the agent is not coupled to an antigen; and wherein the
modulation of the GM1 associated activity affects an allergic
condition and/or a hypersensitivity condition.
3. The use of an agent according to claim 1, wherein the agent is
capable of blocking an IgE mediated response.
4. An assay method for identifying an agent according to claim 1
that is capable of affecting an allergic condition and/or a
hypersensitivity condition; wherein the assay method comprises: (a)
contacting an agent with a ganglioside receptor; (b) determining
whether the agent modulates a ganglioside associated activity; such
that the modulation of the ganglioside associated activity is
indicative that the agent may be capable of affecting an allergic
condition and/or a hypersensitivity condition; and wherein the
agent is not coupled to an antigen.
5. An assay method according to claim 4, wherein the assay is to
screen for an agent useful in the prevention and/or treatment of an
allergic condition and/or a hypersensitivity condition.
6. A process comprising the steps of: (a) performing the assay
according to claim 4; (b) identifying one or more agents capable of
modulating a ganglioside associated activity; and (c) preparing a
quantity of those one or more agents.
7. A process comprising the steps of: (a) performing the assay
according to claim 4; (b) identifying one or more agents capable of
modulating a ganglioside associated activity; and (c) preparing a
pharmaceutical composition comprising those one or more identified
agents.
8. A process comprising the steps of: (a) performing the assay
according to claim 4; (b) identifying one or more agents capable of
modulating a ganglioside associated activity; and (c) modifying one
or more identified agents that modulates a ganglioside associated
activity; and (d) preparing a pharmaceutical composition comprising
those one or more modified agents.
9. An agent identified by the process of claim 6.
10. An agent according to claim 9, wherein the agent is a GM1
binding agent.
11. An agent according to claim 10, wherein the agent is EtxB.
12. An agent according to claim 9, wherein the agent had not
previously been known to affect an allergic condition and/or a
hypersensitivity condition through modulation of a ganglioside
associated activity.
13. A ganglioside according to claim 4, wherein the ganglioside is
a GM1 ganglioside.
14. A method of affecting an allergic condition or a
hypersensitivity condition with one or more agents; wherein the
agent is capable of modulating a ganglioside associated activity in
an in vitro assay method; and wherein the in vitro assay method is
the assay method defined in claim 4.
15. Use of an agent according to claim 4, in the manufacture of a
medicament to affect an allergic condition and/or a
hypersensitivity condition.
16. An agent prepared by a process according to claim 6 for use as
a pharmaceutical.
17. A pharmaceutical composition comprising or prepared from an
agent according to claim 9.
18. Use of an agent in the preparation of a pharmaceutical
composition according to claim 7, for the treatment of an allergic
condition and/or a hypersensitivity condition.
19. An agent capable of modulating a ganglioside associated
activity substantially as described herein.
Description
RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 13/745,082, filed Jan. 18, 2013, which is a
continuation of U.S. patent application Ser. No. 11/691,798 (now
U.S. Pat. No.: 8,357,372), filed on Mar. 27, 2007, which claims
benefit of U.S. patent application No. 09/600,060 (U.S. Pat. No.
7,208,155; now abandoned), filed on Jul. 10, 2000, which is a
national stage application of International Application No.
PCT/GB99/00070, filed on Jan. 8, 1999, which claims the benefit of
GB 9800487.2, filed on Jan. 9, 1998, the disclosure of each is
incorporated by reference in its entirety.
DESCRIPTION OF THE INVENTION
[0002] The present invention relates to a medicament. In
particular, the present invention relates to a medicament useful to
affect an allergic condition and/or a hypersensitivity condition.
More in particular, in one aspect the present invention relates to
an immunological tolerance inducing 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, allergic and other hypersensitivity
diseases.
[0003] When an adaptive immune response occurs in an exaggerated or
inappropriate form, the term allergy or hypersensitivity is
applied. 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, and the fourth is mediated
mainly by T cells and macrophages.
[0004] 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.
[0005] Typical examples of such diseases include asthma, allergic
cough, allergic rhinitis and 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 DerP1
from house dust mite but other triggers of asthma such as pet
dander antigens also exist.
[0006] 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.
[0007] Type III hypersensitivity develops when immune complexes are
formed in large quantities 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 inflammation in the kidneys, skin
(where they cause rashes) and joints (where they can cause a type
of arthritis other than rheumatoid arthritis).
[0008] 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.
[0009] 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 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).
[0010] Escherichia coli (E. coli) heat labile enterotoxin (Etx) and
its closely related homologue, cholera toxin (Ctx), are examples of
protein toxins which bind to glycolipid receptors on host cell
surfaces. Each toxin consists of six noncovalently linked
polypeptide chains, including a single A subunit (27 kDa) and five
identical B subunits (11.6 kDa) which principally bind to GM1
ganglioside receptors found on the surfaces of mammalian cells
(Nashar et al 1996 Proc Nati Acad Sci 93: 226-230). The A subunit
is responsible for toxicity possessing adenosine diphosphate (ADP)
ADP-ribosyltransferase activity, whereas the B subunits (EtxB and
CtxB) are non-toxic oligomers which bind and cross-link a
ubiquitous cell surface glycolipid ganglioside, called GM1, thus
facilitating A subunit entry into the cell.
[0011] The GM1 ganglioside receptor is a member of family of
gangliosides 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. Gangliosides are defined as any ceramide
oligosaccharide carrying, in addition to other sugar residues, one
or more sialic residues (Oxford Dictionary of biochemistry and
molecular biology. Oxford University Press. 1997. Eds Smith A D,
Datta S P, Howard Smith G, Campbell P N, Bentley R and McKenzie H
A). Although first described in neural tissue, several studies have
shown that gangliosides are almost ubiquitous molecules expressed
in all vertebrate tissues. Within cells, gangliosides are usually
associated with plasma membranes, where they may act as receptors
for a variety of molecules and take part in cell-to-cell
interaction and in signal transduction. In addition, gangliosides
are expressed in cytosol membranes like those of secretory granules
of some endocrine cells such as the pancreatic islets and adrenal
medulla.
[0012] Gangliosides contain in their oligosaccharide head groups
one or more residues of a sialic acid which gives the polar head of
the gangliosides a net negative charge at pH 7.0. The sialic acid
usually found in human gangliosides is N-acetylneuraminic acid.
Over 20 different types of gangliosides have been identified,
differing in the number and relative positions of the hexose and
sialic residues which form the basis of their classification.
Nearly all of the known gangliosides have a glucose residue in
glycosidic linkage with ceramide, residues of D-galactose and
N-acetyl-D-galactosamine are also present.
[0013] In the ganglioside nomenclature of gangliosides, devised by
Svennerholm (Biochemistry Lehninger 2nd Ed 1975 Worth Publishers
Inc p 294-295), the subscript letters indicate the number of sialic
groups. M is monosialo, D is disialo and T is trisialo.
[0014] One of the best studied members of the ganglioside family is
the monosialosylganglioside, GM1, which has been shown to be the
natural receptor for the cholera toxin. Soluble ganglioside GM1
binds to the toxin with high affinity and inactivates it
(Svennerholm 1976 Adv Exp Med Biol 71: 191-204).
[0015] The chemical formula for GM1 can be represented as:
Gal.beta.3GalNAc.beta.4(NeuAc alpha 3)Gal.beta.4Glc.beta.1Cer
where Glc is D-glucose, Gal is D-galactose, GalNAc is
N-acetyl-D-galactosamine; NeuAc is N-acetylneuraminic acid, Cer is
ceramide.
[0016] The chemical formula for GM1 can also be represented as
galactosyl-N-acetylgalactosaminyl{sialosyl}lactosyl ceramide
or
galactosyl-N-acetyl-galactosaminyl-(sialyl)-galactosylglusosylceramide
[0017] The x-ray crystal structures of Etx bound to lactose (Sixma
et al 1992 Nature (London) 355: 561-564) and CtxB bound to the
pentasaccharide of GM1 (Merritt et al 1994 Protein Sci 3: 166-175)
have revealed that CtxB and EtxB bind to the terminal galactose and
sialic acid moieties of GM1 which can be represented as
Gal.beta.-1-3-3GalNAc
and that such binding does not induce any striking changes in B
subunit conformation.
[0018] Furthermore the cholera toxin has been shown to demonstrate
an absolute requirement for terminal galactose and internal sialic
acid residues (as in GM1) with tolerance for substitution with a
second internal sialic acid (as in GD1b).
[0019] Etx, like Ctx also probably binds to the terminal sugar
sequence
Gal.beta.1-3GalNAc.beta.1-4(NeuAc alpha 2-3)Gal
where GalNAc is the N-acetylgalactosamine and NeuAc is
N-acetylneuraminic acid.
[0020] In addition to binding to GM1, EtxB binds weakly to other
gangliosides, including non-galactose containing GM2 and asialo-GM1
as well as galactoproteins (Nashar et al Immunology 1997 91:
572-578). Other researchers have shown that EtxB is capable of
binding to GM1 and tolerated removal or extension of the internal
sialic acid residue (as in asialo-GM1 and GD1b respectively) but
not substitution of the terminal galactose of GM1 (Umesaki and
Setoyama 1992 Immunology 75: 386-388).
[0021] In contrast to the poor immunogenicity of the A subunit
alone, both EtxB and CtxB are exceptionally potent immunogens and
their respective holotoxins, Etx and Ctx, are known to be
exceptionally potent adjuvants when given orally in combination
with unrelated antigens (Ruedl et al 1996 Vaccine 14: 792-798;
Nasbar et al 1993 Vaccine 11: 235; Nashar and Hirst 1995 Vaccine
13: 803; Bison and Balding 1984 J Immunol 133: 2892; Lycke and
Holmgren 1986 Immunology 59: 301). Because of their remarkable
immunogenicity, both EtxB and CtxB have been used as carriers for
other epitopes and antigens (Nashar et al 1993 ibid) and have been
used as components of vaccines against cholera and E. coli
diarrhoea (Jetborn et al 1992 Vaccine 10: 130). &p The ability
of the B subunit of Ctx and Etx to interact with receptors present
on mammalian cells has been shown to exert modulatory effects on
the function of those cells. It is known that cells of the immune
system are differentially affected following such interaction. In
particular, WO 95/020045 discloses that EtxB binds to GM1
ganglioside receptors which are found on the surfaces of mammalian
cells and that this binding induces differential effects on
lymphocyte populations including a specific depletion of CD8+ T
cells and an associated activation of B cells. These effects are
absent when a mutant EtxB protein lacking GM1 binding activity is
employed. These observations have led to the use of agents capable
of binding to GM1 in the prevention and treatment of autoimmune
disease, transplant rejection and graft versus host disease (GVHD).
These studies suggest that agents that bind to GM1 or mimic binding
to GM1 promote the induction of immunological tolerance.
[0022] 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. (Sun et al 1994 ibid; Sun et al 1996 ibid; Bergerot et al
1997 ibid). 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 orally or nasally by a mucosal route. WO 95/01301
discloses an immunological tolerance-inducing agent comprising a
mucosa-binding agent linked to a specific tolerogen, WO95/10301
also includes mention of the treatment of allergy using a mucosa
binding agent coupled to an allergen. Other researchers such as
Tamura 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 murine 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 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 common 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, I. L. 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).
[0023] The induction of immunological tolerance may include a
number of different mechanisms which may be summarised as follows:
[0024] (i) a process whereby antigen reactive cells are removed
through triggering them to commit suicide (apoptosis); [0025] (ii)
an induction of anergy or the long term inactivation of the antigen
reactive cells; [0026] (iii) immune deviation of the antigen
reactive cells away from the production of pathological responses;
[0027] (iv) suppression of the antigen reactive cells or their
regulation by specific factors or regulatory cells
[0028] In the treatment of allergy, it is possible that the
induction of any of these mechanisms 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 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 either
the possible secretion of non-antigen specific suppressor molecules
or through suppressive cellular interactions in that tissue as a
result of the interaction between the antigen specific cells and
the specific immunising antigen. In this way, as long 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.
[0029] 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 Th1
cytokines such as IFN.gamma. and interleukin 2 (IL-2) and towards
the secretion of Th2 cytokines such as IL-4, IL-10 and IL-13.
IFN.gamma. is the classical Th1 cytokine, IL-4 is the classical Th2
cytokine. This "immune deviation" is the basis of the disclosure in
WO 97/02045 and has been shown to be effective in the treatment of
autoimmune diseases. The experimental results in WO 97/02045 would
suggest that GM1 binding agents would not find use in the treatment
of allergic conditions and/or hypersensitivity conditions since
such conditions involve IgE, the production of which is generally
accepted to be promoted by IL-4 and down regulated by
IFN.gamma..
[0030] The present invention now seeks to provide new ways of
treating allergic conditions and/or hypersensitivity conditions
through the induction of a specific immune deviation or
suppression.
[0031] 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 allergic condition and/or a hypersensitivity
condition; wherein the agent is capable of modulating a ganglioside
associated activity; wherein the agent is not coupled to an
antigen; and wherein the modulation of the ganglioside associated
activity affects an allergic condition and/or a hypersensitivity
condition.
[0032] 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 allergic condition and/or a hypersensitivity
condition; wherein the agent is capable of modulating a GM1
associated activity; wherein the agent is not coupled to an
antigen; and wherein the modulation of the GM1 associated activity
affects an allergic condition and/or a hypersensitivity
condition.
[0033] According to a third aspect of the present invention, there
is provided an agent according to the present invention capable of
blocking an IgE mediated response.
[0034] Preferably, the agent is capable in vivo of blocking an IgE
mediated response.
[0035] 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 allergic condition
and/or a hypersensitivity condition; wherein the assay method
comprises: (a) contacting an agent with a ganglioside; (b)
determining whether the agent modulates a ganglioside associated
activity; such that the modulation of the ganglioside associated
activity is indicative that the agent may be capable of affecting
an allergic condition and/or a hypersensitivity condition; and
wherein the agent is not coupled to an antigen.
[0036] 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 allergic condition and/or a
hypersensitivity condition.
[0037] According to a sixth 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 ganglioside associated
activity; and (c) preparing a quantity of those one or more
agents.
[0038] 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 ganglioside associated
activity; and (c) preparing a pharmaceutical composition comprising
those one or more identified agents.
[0039] 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 ganglioside associated
activity; and (c) modifying one or more identified agents capable
of modulating a ganglioside associated activity; and (d) preparing
a pharmaceutical composition comprising those one or more modified
agents.
[0040] According to a ninth aspect of the present invention, there
is provided an agent identified by the process of the present
invention.
[0041] Preferably the agent identified had not previously been
known to affect an allergic condition and/or a hypersensitivity
condition through modulation of a ganglioside associated
activity.
[0042] According to a tenth aspect of the present invention, there
is provided a method of affecting an allergic condition and/or a
hypersensitivity condition with one or more agents; wherein the
agent is capable of modulating a ganglioside associated activity in
an in vitro assay method; and wherein the in vitro assay method is
the assay method defined in the present invention.
[0043] Preferably there is provided a method of affecting in vivo
an allergic condition and/or a hypersensitivity condition with one
or more agents; wherein the agent is capable of modulating a
ganglioside associated activity in an in vitro assay method; and
wherein the in vitro assay method is the assay method defined in
the present invention.
[0044] According to a eleventh aspect of the present invention,
there is provided an agent according to the present invention for
use as a pharmaceutical.
[0045] 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 allergic
condition and/or a hypersensitivity condition.
[0046] 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.
[0047] Preferably the agent is a GM1 binding agent.
[0048] Preferably, the agent capable of modulating a ganglioside
associated activity is selected from a group consisting of Ctx,
Etx, CtxB and EtxB.
[0049] Preferably, the agent capable of modulating a ganglioside
associated activity is capable of blocking an IgE mediated response
in a subject with an allergic condition and/or a hypersensitivity
condition.
[0050] Preferably the subject is a human--e.g. a human patient.
[0051] Preferably the agent is EtxB.
[0052] In a particularly preferred embodiment the agent is the wild
type EtxB.
[0053] Alternatively, preferably the agent is either a mutant of
EtxB which is capable of modulating a ganglioside associated
activity or other equivalent proteins thereof.
[0054] Preferably the agent(s) is/are non-toxic.
[0055] Preferably the agent is CtxB and mutants thereof which are
capable of modulating a ganglioside associated activity.
[0056] Preferably the ganglioside is a GM1 ganglioside
receptor.
[0057] Preferably the agent capable of modulating a ganglioside
associated activity is capable of cross-linking GM1 ganglioside
receptors.
[0058] Preferably EtxB is one such agent which is capable of
cross-linking GM1 ganglioside receptors by virtue of its pentameric
form.
[0059] Preferably the medicament is used for the treatment or
prophylaxis of a Type I allergic and/or a Type IV hypersensitivity
condition such as contact hypersensitivity.
[0060] Preferably the medicament includes one or more antigens
which are optionally co-administered with antigen.
[0061] Preferably the agent may be administered to a mammal with or
without co-administration of an antigen.
[0062] Preferably the mammal is a human--e.g. a human patient.
[0063] In accordance with the present invention we have
surprisingly found that the use of agents capable of modulating a
ganglioside associated activity, when given alone or when
co-administered with suitable antigens, can be used as an effective
treatment for allergic and/or hypersensitivity conditions. Previous
workers have either not attempted to find a mechanism (Sun et al
1996 ibid) or have argued that agents capable of modulating a
ganglioside associated activity, such as EtxB and CtxB, cause a Th1
to Th2 switch in the immune response to antigen (WO97/02045). Since
allergic conditions are known in the art to be promoted by Th2
responses, then the previous findings suggest that such agents
would either be ineffective in treating allergies or may even
worsen them.
[0064] We have surprisingly found that while EtxB and CtxB promote
some aspects of Th2-associated responses, in some cases, they may
not stimulate the production of the key factor in triggering
allergy, IgE. Thus allergic conditions and/or hypersensitivity
conditions can be treated with an agent capable of modulating a
ganglioside associated activity, for instance, which is not coupled
with an antigen.
[0065] Significantly, the linkage of the components was not found
to be necessary. Furthermore, our findings indicate that the
mechanisms of protection against allergic conditions and/or
hypersensitivity conditions may include, though not be limited to
either the suppression of antigen specific IgE secretion and/or the
upregulated production of non-inflammatory antigen specific
antibody isotypes (particularly IgG and IgA).
[0066] Thus, the present invention is advantageous because allergic
conditions and/or hypersensitivity conditions can be treated with
an agent capable of modulating a ganglioside associated activity
which is optionally co-administered with an antigen.
[0067] The term "ganglioside" as used with respect to the present
invention include its normal definition in the art (such as that
defined above) as well as active fragments thereof.
[0068] The ganglioside can be made synthetically or isolated from
natural sources. Alternatively, it can be obtained from commercial
sources.
[0069] The term "ganglioside associated activity" includes any one
or more of modulating or immunomodulating a ganglioside receptor,
modulating any signalling event prior to, during or subsequent to
ganglioside receptor binding.
[0070] The term "Ctx" refers to the cholera toxin and CtxB refers
to the B subunit of the cholera toxin. In other texts, these may
sometimes be identified as CT or Ct or CTB or CtB respectively.
[0071] 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.
[0072] The term "adjuvant" includes a substance that enhances an
immune response to an antigen.
[0073] 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 response to the
unrelated antigen. In this way, the agent acts as a so-called
mucosal adjuvant.
[0074] The term "mucosal surfaces" includes but is not limited to
oral, sublingual, intranasal, vaginal, rectal, salivary, intestinal
and conjunctival surfaces.
[0075] 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.
[0076] A "vaccine carrier" includes a carrier of relevant antigens
(Szostak et al 1996 J Biotechnol 44: 161-170).
[0077] The term "mucosal immunogen" includes an agent
administerable by a mucosal route that has the capability to evoke
local and/or systemic antibody production and/or cell mediated
immune reactions and/or delayed type hypersensitivity
reactions.
[0078] A "hapten" means a small molecule which can act as an
epitope but is incapable by itself of eliciting an antibody
response.
[0079] The term "immunological or oral tolerance" means a reduction
in immunological reactivity of a host towards a specific tolerated
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".
[0080] 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/or IgE responses.
[0081] The term "tolerance" means a state of specific immunological
unresponsiveness.
[0082] A "tolerogen" means a tolerated antigen.
[0083] The term "autoimmunity" is used to describe the process by
which the body generates an immune response to self-antigens.
[0084] The term "agent capable of modulating a ganglioside
associated activity" can be used to describe any agent which acts
as an immunomodulator through interacting with a ganglioside.
[0085] The term "GM1 binding agent" includes any agent which acts
as an immunomodulator through interacting with a GM1 ganglioside
receptor.
[0086] 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.
[0087] The term "administered" includes delivery by viral or
non-viral techniques. Viral delivery mechanisms include but are not
limited to adenoviral vectors, adeno-associated viral (AAV)
vectors, 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 but are
not limited to mucosal, nasal, oral, parenteral, gastrointestinal,
topical, or sublingual routes.
[0088] 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 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 Macrosol.TM.--see WO95/13795 and
WO96/14871)--but with the proviso that the agent and the antigen
are uncoupled.
[0089] 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.
[0090] The term "systemic immunisation" means the introduction of
an antigen into a non-mucosal tissue such as the skin or the
blood.
[0091] The term "self antigens" means components derived from host
tissues.
[0092] The term "target interaction components" includes but is not
limited to an agent capable of modulating a ganglioside associated
activity, a ganglioside and/or an antigen.
[0093] 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 an ionic
or covalent bond) or indirect linkage by the provision of suitable
spacer groups.
[0094] The term "uncoupled"--which is synonymous with the term
"unlinked"--means that the agent is not coupled to the antigen.
[0095] However, in accordance with the present invention, the agent
and/or antigen can be coupled to another entity.
[0096] 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.
[0097] An "antigen" means an agent which, when introduced into an
immunocompetent animal, stimulates the production of a specific
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.
[0098] An "allergen" includes any antigen that stimulates an
allergic reaction, inducing a Type I hypersensitivity reaction.
[0099] Examples of common allergen sources are outlined in the
Table below.
TABLE-US-00001 Group Examples 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 urine cat, dog, horse, rabbit, guinea pig, hamster bird
feathers budgerigar, parrot, pigeon, duck, chicken house dust mite
Dermatophagoides pteronyssinus, D. farinae, Euroglyphus maynei
insects cockroach, fly, locust, midge Oral foods seafood, legumes,
peanuts, nuts, cereals, dairy products, eggs, fruits, tomatoes,
mushrooms, alcoholic beverages, coffee, chocolate drugs
penicillins, sulphonamides and other antibiotics, sulphasalazine,
carbamazepine Injected insects bee and wasp stings, ant and
mosquito bites drugs blood products, sera, vaccines, contrast
media, drugs (including anti-asthma drugs and antibiotics)
[0100] The term "allergic condition" includes but is not limited to
asthma, allergic cough, allergic rhinitis and conjunctivitis,
atopic eczema and dermatitis, uticaria, hives, insect bite allergy,
dietary allergy (peanut, fish milk, wheat etc) and drug
allergies.
[0101] The term "hypersensitivity condition" includes but is not
limited to conditions such as contact hypersensitivity induced by
plant poison ivy.
[0102] The term "agent" includes entities capable of modulating a
ganglioside 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
variant/homologue/derivative/fragment thereof so long as they
retain the required immunomodulatory activity. It also includes
mimics and equivalents and mutants thereof. Other agents for the
treatment of allergic conditions or hypersensitivity conditions
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.
[0103] Agents capable of modulating a ganglioside 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 EtxB subunit or the CtxB
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 EtxB assemble may be inserted
into, for example, plasmid pMM68, which is then used to transfect
host cells, such as Vibrio sp. 60. The protein is purified and
isolated in a manner known per se. Mutant genes expressing active
mutant EtxB protein may then be produced by known methods from the
wild type gene.
[0104] Where a target interaction component is a protein,
procedures well known in the art may be used for the production of
antibodies to that component.
[0105] 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, and dinitrophenol.
BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are
potentially useful human adjuvants.
[0106] 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 Nati 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 antibody genes to obtain a
molecule with appropriate antigen specificity and biological
activity can be used (Morrison et al (1984) Proc Nati Acad Sci
81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et
al (1985) Nature 314:452-454). Alternatively, techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,779) can be adapted to produce target interaction component
specific single chain antibodies.
[0107] 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 Nati Acad Sci
86: 3833-3837), and Winter G and Milstein C (1991; Nature
349:293-299).
[0108] 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').sub.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').sub.2 fragments. Alternatively,
Fab expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse W D et al (1989) Science 256:1275-128 1).
[0109] 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.
[0110] Alternatively, screening of peptide libraries or organic
libraries made by combinatorial 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.
[0111] 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 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.
[0112] Alternatively, phage display can be employed in the
identification of candidate agents which affect the target
interaction components.
[0113] 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 sequence (or a derivative or homologue thereof)
encoding same. The transformed bacteriophage (which preferably is
tethered to a solid support) expresses the appropriate ligand (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.
[0114] 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.
[0115] Another technique for screening provides for high throughput
screening of agents having suitable binding affinity to the target
interaction components 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-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0116] The present invention also provides a pharmaceutical
composition for treating a subject in need of same comprising
administering a therapeutically effective amount of an agent
capable of modulating a ganglioside associated activity and a
pharmaceutically acceptable carrier, diluent, excipient or
adjuvant.
[0117] The pharmaceutical compositions may be for human or animal
usage and will typically 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), coating agent(s), solubilising agent(s).
[0118] The pharmaceutical composition may be formulated together
with an appropriate antigen.
[0119] Alternatively, a kit may be provided comprising separate
compositions for each of the therapeutic agent and the antigen.
[0120] 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 invention;
wherein the agent is capable of modulating a ganglioside associated
activity.
[0121] The present invention also relates to pharmaceutical
compositions comprising effective amounts of antigen in admixture
with a pharmaceutically acceptable diluent, carrier, excipient or
adjuvant (including combinations thereof).
[0122] The present invention also provides a method of treating a
subject in need of same comprising administering to said subject an
effective amount of the pharmaceutical composition of the present
invention.
[0123] 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.
[0124] There may be different composition/formulation requirements
dependent on the different delivery systems.
[0125] 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.
[0126] Where the agent is delivered mucosally through the
gastrointestinal mucosa, it is preferably stable during transit
though the gastrointestinal tract; for example, it is preferably
resistant to proteolytic degradation, stable at acid pH and
resistant to the detergent effects of bile.
[0127] 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.
[0128] 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 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.
[0129] There may be different delivery requirements dependent on
the different composition/formulation systems.
[0130] 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.
[0131] By way of example, the controlled release of 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).
[0132] Encapsulation of vaccines in biodegradable microspheres
provides excellent mucosal to immunogens. Recombinant Norwalk
Virus-like (rNV) particles may also be used for mucosal antigen
delivery (Ball et al 1996 Arch Virol Suppl 12: 243-249).
[0133] 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).
[0134] One preferred method of oral delivery uses formations as
described in WO95/13795, WO96/17593 and WO96/17594. These
formulations allow macromolecules such as proteins to be
solubilised in "oils" for oral delivery. Such formulations
therefore allow delivery of the macromolecules to mucosal surfaces
in the gut.
[0135] 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.
[0136] In summary, the present invention provides the use of an
agent in the manufacture of a medicament to affect an allergic
condition and/or a hypersensitive condition; wherein the agent is
capable of modulating a ganglioside associated activity wherein the
agent is not coupled with an antigen.
[0137] In another broad aspect, the present invention provides an
immunological tolerance inducing agent comprising an agent capable
of modulating a ganglioside associated activity which is not
coupled to an antigen.
[0138] Other aspects of the present invention are now presented
below by way of numbered paragraphs, which include:
[0139] 1. The use of an agent having GM1 binding activity, or an
agent having an effect on GM1 mediated intracellular signalling
events, but no GM1 binding activity, in the preparation of a
medicament to treat an allergic or other hypersensitive condition,
with the proviso that said agent is not coupled with an allergen
and/or an antigen.
[0140] 2. The use as defined in paragraph 1 wherein the agent is a
GM1 binding agent such as Ctx, Etx, CtxB or EtxB or a mutant form
or derivative thereof.
[0141] 3. The use as defined in paragraph 1 and paragraph 2 wherein
the medicament is for the prophylaxis or treatment of asthma,
allergic cough, allergic rhinitis, conjunctivitis, atopic eczema,
dermatitis, uticaria, hives, insect bite allergy, dietary allergy
(peanut, fish, milk, wheat etc), drug allergies or contact and
other hypersensitivities.
[0142] 4. A method for the treatment or prophylaxis of an allergic
or other hypersensitive condition which comprises administering to
a subject an effective amount of an agent having GM1 binding
activity, or an agent having an effect on GM1 mediated
intracellular signalling events, but no GM1 binding activity, with
the proviso that said agent is not coupled with an allergen and/or
an antigen.
[0143] 5. A method as defined in paragraph 4 wherein the agent is a
GM1 binding agent such as Ctx, Etx, CtxB or EtxB or a mutant form
or derivative thereof.
[0144] 6. A method as defined in paragraph 4 or paragraph 5 wherein
the method is for the prophylaxis or treatment of asthma, allergic
cough, allergic rhinitis, conjunctivitis, atopic eczema,
dermatitis, uticaria, hives, insect bite allergy, dietary allergy
(peanut, fish, milk, wheat etc), drug allergies or contact
hypersensitivity.
[0145] 7. A pharmaceutical composition for the treatment of a human
allergic and/or hypersensitivity disease comprising [0146] (i) an
agent having GM1 binding activity; or [0147] (ii) an agent having
an effect on GM1 mediated intracellular signalling events, but no
GM1 binding activity; with the proviso that the agent is not
coupled with an allergen/antigen; and a pharmaceutically acceptable
carrier or diluent therefor.
[0148] 8. A product comprising an agent having GM1 binding
activity, or an agent having an effect on GM1 mediated
intracellular signalling events, but no GM1 binding activity, in
the preparation of a medicament to treat an allergic or other
hypersensitive condition, with the proviso that said agent is not
coupled with an allergen and/or an antigen, and at least one
antigen/allergen as a combined preparation for simultaneous,
separate or sequential use.
[0149] The present invention will now be described only by way of
example.
EXAMPLES
[0150] Screens for Agents Capable of Modulating Ganglioside
Associated Activity
[0151] Agents capable of modulating ganglioside associated activity
are tested by any one of a variety of methods.
[0152] Examples of such methods include, but are not limited to the
following methods:
[0153] 1. Binding to a ganglioside receptor, such as GM1, is
determined by using purified GM1 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).
[0154] 2. The pentasaccharide moiety of a ganglioside, such as GM1,
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 ganglioside
associated activity to be determined.
[0155] Protein analysis includes peptide sequencing and tryptic
digest mapping followed by comparisons with available databases. If
eluted proteins cannot be identified in this 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 GM1-affinity columns are
analysed by HPLC and mass spectrometry of single homogenous
peaks.
[0156] 3. The ability to bind to gangliosides, such as GM1, and the
precise affinity of the interaction may be determined using plasmon
surface resonance as previously reported [Kuziemko et al (1996)
Biochem 35:6375-6384].
[0157] Evaluation of Identified Agents
[0158] The identification of agents capable of modulating
ganglioside associated activity such that the modulation of the
ganglioside associated activity affects an allergic condition
and/or a hypersensitivity condition is determined as follows:
[0159] 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 intraperitoneally.
[0160] 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
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 murine IgE.
[0161] In order to screen agents for their capability to prevent or
treat allergy, agents capable of modulating ganglioside 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 are utilised either prior to the IgE inducing antigen
challenge (prophylaxis) or after the IgE inducing antigen challenge
(treatment). 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.
[0162] Efficacy is determined in a variety of ways and is
manifested as a number of different outcomes.
[0163] 1. Antigen-specific IgE levels. Measurement of serum IgE by
specific ELISA (as described) is used to determine whether
prophylactic or treatment protocols are 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 "Ussing 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.
[0164] 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.
[0165] 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 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.beta.).
[0166] 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 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 cosinophils or limit the uptake of
antigen across the mucosal epithelium and hence prevent the
subsequent allergic inflammatory response.
[0167] Enzyme Linked Immunosorbent Assays (ELISAs)
[0168] Binding of EtxB or EtxB (G33D) to GM1 is examined by a
GM1-ELISA (Amin, T., & Hirst, T. R. (1994) Prot. Express. and
Purif. 5, 198-204).
[0169] 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 (Immulon I, Dynateck, USA) coated
with 5 .mu.g/ml of either EtxB or EtxB (G33D) in PBS. Anti-B
subunits IgA antibodies in gut secretion supernatants are
extrapolated from a standard curve made by coating 2 rows of wells
on each plate with 1 .mu.g/ml rabbit anti-mouse IgA (a chain
specific; Zymed Lab, USA) in PBS followed by addition of 1 .mu.g/ml
of mouse myeloma IgA (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 A.sub.450 nm.gtoreq.0.2, is determined. The IgA anti-B
subunit response for each of EtxB and EtxB (G33D) in gut secretions
is calculated as "IgA specific activity" [mean IgA anti-B subunit
(.mu.g/ml)/total IgA (.mu.g/ml)].
[0170] A known ELISA method for measuring cytokine levels of IL-2,
IL-4, IL-5, IL-10 and IFN-.gamma. is used. Briefly, microliter
plates are coated with rat antibodies to mouse IL-2, IL-4, IL-5,
IL-10 and IFN-.gamma.. Plates are blocked with 2% (w/v) bovine
serum albumin. 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 0.5 .mu.g/ml of biotinylated anti-cytokine
monoclonal antibodies followed by addition of avidin-peroxidase and
3,3', 5,5'-Tetramethylbenzidene (TMB) substrate and read at
A.sub.450 nm.
[0171] 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 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.
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