U.S. patent application number 09/867914 was filed with the patent office on 2001-11-01 for therapeutic agents.
Invention is credited to Hirst, Timothy Raymond, Nashar, Toufic Osman, Williams, Neil Andrew.
Application Number | 20010036917 09/867914 |
Document ID | / |
Family ID | 26307329 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036917 |
Kind Code |
A1 |
Williams, Neil Andrew ; et
al. |
November 1, 2001 |
Therapeutic agents
Abstract
A method for treating diabetes in a mammalian subject in need of
same wherein the method comprises: administering to the mammalian
subject an agent capable of modulating a ganglioside GM-1 (GM-1)
associated activity in an amount effect to treat the disease;
wherein the agent, when in vivo, has an effect on GM-1 mediated
intracellular signalling events; and wherein if the agent is for
co-administration with an antigen, then the agent and the antigen
are not so linked to form a single active agent.
Inventors: |
Williams, Neil Andrew;
(Axbridge, GB) ; Hirst, Timothy Raymond;
(Clevedon, GB) ; Nashar, Toufic Osman; (Bristol,
GB) |
Correspondence
Address: |
MARY M. KRINSKY, Ph. D., J.D.
PATENT ATTORNEY
79 TRUMBULL STREET
NEW HAVEN
CT
06511
US
|
Family ID: |
26307329 |
Appl. No.: |
09/867914 |
Filed: |
May 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09867914 |
May 30, 2001 |
|
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08999458 |
Dec 29, 1997 |
|
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Current U.S.
Class: |
424/185.1 ;
514/12.2; 514/54; 514/6.9; 514/7.3; 514/8.9 |
Current CPC
Class: |
A61K 39/0258 20130101;
A61K 39/00 20130101; G01N 33/505 20130101; Y10S 514/885 20130101;
Y10S 530/868 20130101; G01N 33/6863 20130101; G01N 33/564 20130101;
C07K 14/245 20130101; A61K 39/39 20130101; A61P 19/02 20180101;
A61K 39/0005 20130101; G01N 2800/02 20130101; A61K 45/06 20130101;
A61P 29/00 20180101; A61K 38/28 20130101; A61K 2039/55544
20130101 |
Class at
Publication: |
514/3 ;
514/54 |
International
Class: |
A61K 038/28; A61K
031/726 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 1995 |
GB |
9513733.7 |
Claims
1. A method for treating diabetes in a mammalian subject in need of
same wherein the method comprises: administering to the mammalian
subject an agent capable of modulating a ganglioside GM-1 (GM-1)
associated activity in an amount effect to treat the disease;
wherein the agent, when in vivo, has an effect on GM-1 mediated
intracellular signalling events; and wherein if the agent is for
co-administration with an antigen, then the agent and the antigen
are not so linked to form a single active agent.
2. A method according to claim 1 wherein the effect on GM-1
mediated intracellular signalling events is due to an agent having
GM-1 binding activity.
3. A method according to claim 1 wherein the agent is an
immunomodulator.
4. A method according to claim 3 wherein the agent is selected from
the group consisting of Ctx, Etx, the B subunit of Ctx and the B
subunit of Etx and mutants and derivatives thereof.
5. A method according to claim 1 wherein the agent is for
co-administration with an antigen selected from the group
consisting of self or cross-reacting antigen, alloantigen and
xenoantigen.
6. A method according to claim 1 wherein the agent is for
co-administration with an auto antigen.
7. A method according to claim 5 wherein the autoantigen is
insulin.
8. A method according to claim 1 wherein the agent, when in vivo,
is capable of modulating lymphocyte populations.
9. A method according to claim 1 wherein the agent, when in vivo,
is capable of acting as a vaccine adjuvant.
10. A pharmaceutical composition comprising an agent as defined in
claim 1 and a pharmaceutically acceptable carrier(s), diluent(s),
excipient(s) or adjuvant of any combination thereof.
11. A composition according to claim 10 wherein the composition is
for nasal administration.
12. A composition according to claim 10 wherein the composition is
for oral administration.
13. A method for modulating an immune response in a mammalian
subject in need of prevention against or treatment of diabetes
wherein the method comprises administering to the mammalian subject
an effective amount of an agent as defined in any one of the
preceding claims wherein if the agent is for co-administration with
an antigen, then the agent and the antigen are not so linked to
form a single active agent.
14. A method according to claim 13 wherein the modulation of the
immune response is determined by measuring a change in at least one
parameter selected from the group consisting of: a change in Th2
asociated cytokine levels, a change in antigen specific T-cell
reactivity, a change in Th1 associated cytokine levels and any
combination thereof.
15. A method according to claim 14 wherein the agent decreases the
production of Th1 associated cytokines.
16. A method according to claim 15 wherein the Th1 associated
cytokine is IFN.gamma..
17. A method according to claim 14 wherein the agent increases the
production of Th2 associated cytokines.
18. A method according to claim 17 wherein the Th2 associated
cytokine is selected from the group consisting of IL-4 and IL-10
cytokines and any combination thereof.
19. A method according to claim 13 wherein the agent is capable of
preventing the onset of insulin dependent diabetes mellitus
(IDDM).
20. A method according to claim 19 wherein the agent, when in vivo,
is co-administered with an autoantigen.
21. A method according to claim 20 wherein the autoantigen is
selected from the group consisting of GAD, GAD65, IAA and
insulin.
22. A method according to claim 20 wherein the agent is EtxB.
23. A method according to claim 13 wherein the agent is capable of
treating pancreatic islet inflammation or for reducing the
incidence of IDDM.
24. A method according to claim 23 wherein the agent is EtxB.
25. An assay method for identifying an agent useful in the
prevention against and/or treatment of diabetes wherein the assay
method comprises: (i) contacting a test agent with a ganglioside
receptor wherein the agent is not linked to an antigen (ii)
determining whether the agent modulates a ganglioside associated
activity by measuring a change in at least one parameter selected
from the group consisting of: a change in specific T cell
reactivity, a change in Th1 associated cytokine levels; a change in
Th2 associated cytokine levels and any combination thereof; and
(iii) identifying the useful agent by observation of modulation of
ganglioside associated activity.
26. A method according to claim 25 wherein the agent binds to GM-1
ganglioside receptors.
27. A method according to claim 26 wherein the agent is selected
from the group consisting of Ctx, Etx, CtxB, EtxB and mutants or
derivatives thereof that bind to GM-1.
28. A method according to claim 25 wherein the agent has an effect
on GM1 mediated intracellular signalling events but no GM1 binding
activity.
29. A method according to claim 25 wherein the agent is capable of
promoting an immune deviation away from Th1 associated cytokines
and towards Th2 associated cytokines.
30. A method according to claim 25 wherein the agent is capable of
promoting a suppression of a Th1 response.
31. The use of an agent in the preparation of a medicament to
prevent and/or treat a diabetic condition; wherein the agent is
capable of modulating a ganglioside GM-1 (GM-1) associated
activity; wherein if the agent is for co-administration with an
antigen, then the agent and the antigen are not so linked to form a
single active agent; and wherein the modulation of the ganglioside
associated activity affects the diabetic condition.
32. The use according to claim 31 wherein the modulation of the
GM-1 associated activity is determined by measuring a change in at
least one parameter selected from the group consisting of: a change
in Th2 asociated cytokine levels, a change in antigen specific
T-cell reactivity, a change in Th1 associated cytokine levels and
any combination thereof.
33. The use according to claim 32 wherein the agent decreases the
production of Th1 associated cytokines.
34. The use according to claim 33 wherein the Th1 associated
cytokine is IFN.gamma..
35. The use according to claim 32 wherein the agent increases the
production of Th2 associated cytokines.
36. The use according to claim 35 wherein the Th2 associated
cytokine is selected from the group consisting of IL-4 and IL-10
cytokines.
37. The use according to claim 31 wherein the agent is capable of
preventing the onset of insulin dependent diabetes mellitus
(IDDM).
38. The use according to claim 37 wherein the agent is
co-administered with an auto antigen.
39. The use according to claim 38 wherein the autoantigen is
selected from the group consisting of GAD, GAD65, IAA and
insulin.
40. The use according to claim 39 or claim 40 wherein the agent is
EtxB.
41. The use according to claim 39 wherein the agent is capable of
treating pancreatic islet inflammation or reducing the incidence of
IDDM.
42. The use according to claim 41 wherein the agent is EtxB.
43. A method according to claim 14 wherein the agent causes a
change in cytokines associated with T regulatory cell.
44. A method according to claim 43 wherein the cytokines are
selected from the group consisting of IL-10 and TGF.beta. and any
combination thereof.
45. The use according to claim 32 wherein the agent causes a change
in cytokines associated with T regulatory cell.
46. The use according to claim 45 wherein the cytokines are
selected from the group consisting of IL-10 and TGF.beta. and any
combination thereof.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of co-pending
U.S. application Ser. No. 08/999,458, filed Dec. 29, 1997, which
claimed priority benefit of GB 9513733.7, filed Jul. 5, 1995.
PCT/GB/96/01614, filed internationally 5 July 1996, which
designated the U.S., is a related case that also claimed priority
benefit of GB 9513733.7.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to therapeutic agents for use in the
treatment of mammalian, particularly human, autoimmune diseases.
The invention also relates to therapeutic agents useful in the
treatment of human leukaemias of a T cell origin, as so-called
"vaccine carriers", and as agents for use in the prevention of
human transplantation rejection and graft versus host disease
(GVHD).
[0004] 2. Description of the Related Art
[0005] In an article entitled "Morphologic and Functional
Alterations of Mucosal T Cells by Cholera Toxin and its B subunit"
by Charles O. Elson et al., The Journal of Immunology, 1995, 154;
1032-1040 it is disclosed that the cholera toxin (Ctx) and the CtxB
subunit inhibit CD8.sup.+ and CD4.sup.+ T cells.
[0006] Reference is also made to the paper entitled "Prevention of
Acute Graft-Versus-Host Disease by Treatment with a Novel
Immunosuppressant" by B. Yankelevich et al., The Journal of
Immunology, 1995, 154: 3611-3617. This identifies CtxB as an agent
for use in bone marrow transplantation for the prevention of acute
graft-versus-host disease (GVHD).
[0007] WO 95/10301 discloses an immunological tolerance-inducing
agent comprising a mucosa-binding molecule linked to a specific
tolerogen.
[0008] As used herein, the term "Ctx" refers to the cholera toxin
and "CtxB" to the B subunit of the cholera toxin. In other texts,
these may sometimes be identified as "CT" or "Ct" and "CTB" or
"CtB" respectively. 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.
[0009] E. coli Enterotoxin.
[0010] As mentioned above, the term Etx refers to the heat labile
E. coli enterotoxin which is a member of a family of toxins that
includes cholera toxin and which cause diarrhoeal diseases in
humans and domestic animals. Such toxins are comprised of two
components, the so called A and B-subunits. The ability to cause
diarrhoea resides with properties of the A-subunit, which is an
enzyme capable of increasing the concentration of the biochemical
second messenger cyclic AMP within epithelial cells lining the
intestine. The rise in cyclic AMP levels leads to the loss of ions
from the cells and the consequent loss of water which causes
diarrhoea. The B-subunit moiety has evolved in order to deliver the
A-subunit into epithelial cells by a process which involves it
binding to a membrane located glycolipid receptor, GM1. The
B-subunit is itself not toxic, and is unable to cause
diarrhoea.
[0011] EtxB.
[0012] The B-subunit is composed of five individual polypeptides
bound tightly together into a doughnut ring like structure. Each
polypeptide contains a site for interaction with GM1, and thus
exposure of cells to EtxB causes cross-linking of GM1 at the cell
surface. The overall size of EtxB is 60 kilodaltons, with each of
the five polypeptides being composed of 103 amino acids. Its
exceptional stability results from the very close association of
interfaces between adjacent polypeptides which form the B-subunit
pentamer. Thus EtxB is stable as a pentamer under conditions which
would lead to disruption of normal protein structure. This
stability is reflected by the observation that the pentamer remains
intact at 84.degree. C., between pH 2 and pH 11, and in the
presence of ionic detergents and proteolytic enzymes. This makes
EtxB one of the most stable protein complexes known and may
facilitate ease of use as a component in human medicines.
[0013] The key finding that EtxB can alter immune responses has
come from investigations of its binding to GM1 on cells other than
those of the intestinal epithelium. GM1 is found on all cells of
the immune system, and its cross-linking by EtxB triggers signals
which can alter their activity. Administration of EtxB either into
the nose, by mouth, or by injection, can alter the local
environment within which immune responses are triggered. This
facilitates the production of high levels of antibodies against
antigens of infectious agents which are mixed with EtxB, and can
cause the down-regulation of the damaging inflammatory responses
which are associated with autoimmunity. Thus, EtxB may be used:
alone in the treatment of autoimmune disease (as an
immunotherapeutic), or in combination with vaccine antigens (where
it acts as an adjuvant). The ability to act as an adjuvant
following mucosal delivery makes the B-subunit almost unique since
most infectious agents gain access to the body through these
surfaces, and injected vaccines do not stimulate strong responses
at such sites.
[0014] The Immunological Mechanisms Underlying the use of the
B-Subunit.
[0015] The B-subunits ability to modulate the immune response is
dependent on its capacity to modulate the activity of T-cells,
B-cells and populations of antigen presenting cells. Each of these
cell types plays a critical role in the development of the immune
response. In the normal response to a foreign organism, antigens
are internalised by antigen presenting cells, of which dendritic
cells are the most important. These cells are specialised in
breaking down proteins into short amino acid sequences (peptides)
which associate with major histocompatibility complex (MHC)
molecules which are then transported to the cell surface.
[0016] Foreign peptides bound to class II MHC molecules are
recognised by T-helper cells (CD4+ T-cells) which are activated as
a result and begin to divide, differentiate and secrete
hormone-like messengers called cytokines. The T-helper cells then
co-ordinate and maintain the immune response. Subsequent responses
can involve the activation of i) B-cells which mature into plasma
cells capable of producing antibodies, ii) macrophages and
neutrophils which enter the sites of infection and ingest foreign
material leading to its destruction, and iii) other types of T-cell
(CD8+ T-cells) which can recognise virally infected cells of the
body and kill them.
[0017] Most normal immune responses will involve activation of all
of these components to some extent, however, it is clear that
certain factors can affect which particular components are
dominant. Further, in certain circumstances it is clearly
beneficial to be able to tailor which type of response is elicited.
For example, in preventing infection at mucosal surfaces, it is
desirable to stimulate a strong antibody response, but avoid the
activation of macrophages and neutrophils which can themselves
cause inflammation and tissue damage.
[0018] In order to co-ordinate different types of immune response,
humans have evolved the capacity to sense the nature of the foreign
challenge and alter the T-helper cell response accordingly. Thus,
T-helper cells can be distinguished as being either T-helper 1
(Th1) or T-helper 2 (Th2) cells. Th1 cells secrete cytokines
including gamma interferon (IFN.gamma.) and interleukin (IL)-2
which activate macrophages, neutrophils and CD8+ T-cells and which
lead to the production of antibodies which promote inflammation. In
contrast, Th2 cells secrete IL-4, IL-5, IL-6 and IL-10,
downregulate Th1 responses and promote the production of antibodies
which are secreted at mucosal surfaces, or which do not trigger
inflammation. In addition to the cross-regulation of Th1 responses
by Th2 cells and vice versa, it is also clear that other CD4+
T-cell populations are induced which down-regulate both types of
response (T-regulatory cells). In, for example, an immune response
to a virus which infects the eye, it is desirable to trigger a
strong Th2 response which can arm the local tissue with antibodies
to block the infection, while avoiding stimulating Th1 responses
which will themselves cause damage to such a delicate tissue.
[0019] Autoimmune disease results when the bodies own processes of
regulation breakdown. In these cases components of the body are
mistakingly identified as `foreign` and an immune response is
mounted which attacks the individuals own tissues. In the majority
of examples of autoimmune disease, the immune response is driven by
Th1 cells which cause macrophages and neutrophils to enter and
disrupt the tissue. For example, in the case of rheumatoid
arthritis an immune response to joint-associated antigens leads to
the chronic infiltration of neutrophils and macrophages which cause
cartilage and bone degradation, pain, swelling and loss of
function. Further, type 1 diabetes results from a similar process
leading to the destruction of insulin producing islets within the
pancreas. The precise reasons for the loss of regulation within the
immune systems of certain individuals are not clear, but certainly
involve complex genetic and environmental factors.
[0020] The B-subunit influences the processes involved in antigen
recognition by T-helper cells. In doing so it can promote the
activation of Th2 and T-regulatory cells, while at the same time
suppressing the activation of Th1 cells. Consequently, EtxB may be
used to treat Th1-driven autoimmune diseases, or may be added to
antigens derived from infectious agents to trigger production of
large quantities of protective mucosal and serum Th2-associated
antibodies. Importantly, EtxB does not promote the production of
IgE antibodies which are the cause of allergy.
[0021] The B-Subunit Alters the Balance between Th1 and Th2 Immune
Responses.
[0022] It is clear that the local microenvironment in which antigen
is presented to CD4+ T-cells determines the nature of the
subsequent response (FIG. 12). Certain factors can promote the
differentiation of T-helper cells into Th1 cells, while others
cause differentiation into Th2 cells or T-regulatory cells.
Evidence indicates that EtxB influences many of these events. The
differentiation of T-helper cells into Th1 cells is promoted by
cytokines produced by antigen presenting cells themselves (in
particular, dendritic cell and macrophage derived IL-12) as well as
CD8+ T-cells (producing gamma interferon).
[0023] The present inventors have established that EtxB inhibits
the production of IL-12 by antigen presenting cells (FIG. 13) and
removes CD8+ T-cells by causing them to die by apoptosis (FIG. 14).
Therefore, EtxB interferes with the two major factors which promote
the development of pro-inflammatory Th1 responses. The
differentiation of T-helper cells into Th2 cells is promoted by
their interaction with B-cells during the activation process, and
by the secretion of IL-10 from antigen presenting cells. IL-10 is
also thought to play a critical role in the generation and
activities of T-regulatory cells. The present inventors have shown
that EtxB activates B-cells leading to their enhanced interaction
with T-helper cells (FIG. 15), and causes the production of IL-10
by antigen presenting cells (FIG. 16). Thus, EtxB enhances the
presence of the two major factors which promote the activation of
Th2 cells and creates conditions which favour the induction of
regulatory cell populations.
[0024] Taken together, the activities of EtxB allow it to shift the
balance of the immune response suppressing the Th1 arm while
promoting the Th2 and T-regulatory arms (FIG. 12). Thus, EtxB can
turn off the damaging inflammation in autoimmunity, and can trigger
the production of non-inflammatory antibodies in the serum and at
mucosal surfaces.
[0025] The B-Subunit can be Used to Prevent or Treat Autoimmune
Disease.
[0026] The ability of EtxB to intervene in the processes underlying
autoimmune disease has been tested in animal models of arthritis.
Usefulness in the treatment or prevention of arthritis has been
established using a widely recognised mouse model in which disease
is induced in male DBA/1 mice by the injection of type II collagen
in complete Freunds adjuvant (collagen-induced arthritis). Mixing a
joint antigen in this way with an adjuvant which triggers strong
Th1 responses leads to a strong pro-inflammatory immune response
against collagen. This response is characterised by the appearance
of anti-collagen antibodies and demonstrable T-cell reactions. By
day 20 after induction, joint swelling becomes apparent and its
incidence and severity continues to increase until approximately
day 45 at which point 70-80% of animals have some swelling, usually
involving the hind ankle and knee joints. Damage is also
occasionally noted in the forelimbs. Like rheumatoid arthritis in
humans, the model is characterised by joint swelling and
histological signs of macrophage and neutrophil infiltration into
the joint space. In addition, cartilage and bone destruction are a
common feature along with the formation of a pannus. Assessment of
the immune response to collagen in mice with arthritis reveals the
presence of pro-inflammatory antibodies as well as high levels of
the Th1 cytokine, gamma interferon, in cultures of lymph node
T-cells.
[0027] Collagen induced arthritis (CIA) can be prevented by
treatment with EtxB alone. When EtxB is given to mice intranasally,
orally or by injection prior to the collagen injection, it can
block the induction of disease as revealed by a reduction in
clinical joint swelling (FIG. 17) and histological damage (FIG.
18). In the experiments shown, EtxB was administered into the nose
or by mouth on four occasions daily up to the day of collagen
injection. Injected EtxB was given on a single occasion on the day
of collagen challenge. In each case the dose of EtxB used was 100
.mu.g. Investigations have shown that in the case of nasal delivery
identical levels of disease protection can be achieved with 10
.mu.g, or 1 .mu.g. The delivery of EtxB led to a dramatic reduction
in pro-inflammatory anti-collagen antibody levels and led to a
shift in the nature of the T-helper cell response away from the
production of gamma interferon.
[0028] The B-Subunit can be used as a Potent Mucosal Vaccine
Adjuvant.
[0029] The need to potentiate immune responses to vaccine antigens
is widely recognised. At present the only adjuvant licensed for
human use is alum, which is given by injection and fails to elicit
significant mucosal antibody production. Given that the majority of
infectious disease causing organisms enter across mucosal surfaces,
it is clear that an effective mucosal adjuvant will have widespread
applications. The present inventors have shown that EtxB is a
highly potent adjuvant which can stimulate strong immunity to
foreign antigens after mucosal administration. Important work has
been carried out by the present inventors (see WO 99/58145) using a
mouse model of herpes simplex virus type 1 (HSV-1) infection of the
eye.
[0030] Herpes simplex virus type 1 infection usually occurs in
early childhood and affects over 90% of adults. The initial
infection is usually unnoticed, although occasionally can cause
severe disease. However, following initial infection the virus
resides, dormant, in the nervous system which supplies the face and
eyes. In some people HSV can reactivate and leads to disease in any
of these areas. On the skin HSV-1 causes `cold sores` which are
usually self-limiting, although unpleasant. However, when virus
enters the eye, it causes ulcers on the surface of the eye which
can result in blindness and the need for corneal transplantation.
HSV-1 is the major cause of post-infection blindness in the
developed world. A close relative of HSV-1, HSV-2, is associated
with similar, although more severe, infections of the genital
tract.
[0031] When a glycoprotein mixture from HSV-1 is given to alone to
mice intranasally, it fails to induce an immune response to HSV. By
contrast, the addition of EtxB to this glycoprotein mixture
potentiates a very strong response involving the secretion of large
amounts of anti-HSV antibody into the serum and at mucosal surfaces
(FIG. 21). The antibody response is clearly evident both at the eye
as well as at distant sites including the vagina. The underlying
T-helper cell response to HSV is associated with the production of
predominantly Th2 cytokines. The immune response induced using EtxB
as a mucosal adjuvant can protect mice against a severe ocular
HSV-1 infection (FIG. 22a). In the model, the vaccine, when given
prior to challenge with HSV-1 reduces the severity of symptoms in
the eye and prevents the high levels of viral spread to the central
nervous system (a rare complication in humans). These findings
demonstrate that EtxB can be used as an adjuvant for a prophylactic
vaccine against HSV-1.
[0032] In further studies, the present inventors have shown that
EtxB can be used to develop a therapeutic vaccine against HSV-1. In
these experiments mice were infected with HSV-1 and left in order
to allow the virus to become dormant in the nervous system prior to
vaccination. Intranasal administration of HSV-1 glycoproteins with
EtxB altered the nature of the existing response to HSV-1 in these
animals such that they showed markedly reduced levels of disease
following reactivation of the virus using a physiological stimulus
to the eye (FIG. 22b). This additional finding highlights the
potential of EtxB as a critical component allowing therapeutic
vaccination. These findings demonstrate that intranasal
administration of a vaccine containing EtxB as an adjuvant induces
immunity at local and distant mucosal sites. A similar intranasal
vaccine using glycoproteins from HSV-2 may be effective in
stimulating protective immunity against genital infection. Further,
the profile of the immune response stimulated using EtxB is of
clear relevance to protection against a wide range of infectious
agents. Indeed, experiments have shown that EtxB may be used to
stimulate immunity against a broad range of other antigens.
[0033] The B-Subunit can be used to Target the Delivery of Peptides
into Cells.
[0034] The effective induction of cytotoxic T-cell responses
requires the entry of antigens into the cytosol of antigen
presenting cells. While some externally added soluble antigen may
enter this compartment, targeted delivery into the cytosol should
augment the induction of this component of the immune response.
Cytotoxic T-cell responses are particularly important in
facilitating the removal of infectious agents which reside within
cells of the body, such as viruses and certain bacteria. Thus, in
some vaccines the ability to augment the cytotoxic T-cell response
as well as stimulate high levels of antibodies would be beneficial.
To achieve this, an efficient delivery system which results in the
targeting of antigens into the cytosol is required. The B-subunit
exhibits characteristics which indicate that it may function in
this way.
[0035] Cross-linking of GM1 by the B-subunit is followed by its
internalisation into vessicles within the cell. This capacity to
enter cells has been used to deliver attached peptides into the
cytosol. The present inventors have demonstrated that peptides
ranging from nine to twenty seven amino acids in length can be
genetically or chemically conjugated to the B-subunit without
interfering with its stability or ability to bind to GM1. Addition
of such conjugates to cells results in the liberation of the
attached peptides within a vessicular compartment and their
subsequent delivery to the cytosol. Delivery of EtxB in this way
may lead to the presentation of the peptides to stimulate the
activation of cytotoxic T-cells.
[0036] The B-Subunit is a Lead Compound to the Development of Small
Molecule Mimetics.
[0037] All of the described effects of EtxB are dependent on its
ability to bind to the cell surface. The present inventors have
demonstrated in WO 00/14114 that a mutant B-subunit that is unable
to bind to the cell surface is completely ineffective. A refinement
of this approach has identified a loop with the B-subunit which is
responsible for triggering the effects on immune cells.
Surprisingly, this loop is not directly involved in allowing
binding to GM1, indicating a critical role for interaction with a
secondary receptor at the cell surface. A synthetic peptide
corresponding to this loop exhibits a similar capacity to modulate
T-cell function in vitro. This observation indicates that the loop
represents a lead compound for small molecule mimetic chemistry
which may allow the development of higher affinity analogues for
use as immunotherapeutics and vaccine adjuvants.
[0038] Diabetes.
[0039] Insulin dependent diabetes mellitus (IDDM) is an autoimmune
disease resulting form the T-cell dependent destruction of
insulin-producing cells from the pancreas Langerhans islets (1). It
affects about 4 million people in Europe and North America alone
and usually appears before the age of 30. There is no cure.
Sufferers must give themselves daily insulin injections to control
their blood glucose levels. It is unclear what triggers the immune
system's attack on the islet cells because the regulation of the
auto-aggressive immune response is complex, resulting from the
interaction between several T cell subsets and their activation of
mononuclear phagocytes. Islet destruction, both in humans and
rodents, is attributed to the existence of auto-reactive CD4+T
cells that recognise islet antigens and belong to the Th1 subset
(i.e. secrete inflammatory cytokines such as IFN.gamma.) (2). Such
cells could be isolated from diabetic rodent spleens or pancreas
inflammatory infiltrates and transferred the disease to syngenic
receipents (3).
[0040] The present invention seeks to provide an improved mechanism
for preventing and treating IDDM.
SUMMARY OF THE INVENTION
[0041] The effectiveness of the B-subunit in treating autoimmune
diabetes has been established using the NOD mouse model in which
disease arises spontaneously at between 14 and 25 weeks of age. As
in human type I diabetes, disease in the NOD is influenced by
complex genetic and environmental factors which allow the
development if an immune response to several pancreatic antigens.
The mice develop a non-specific insulitis (immune infiltration of
the pancreas) at between 6 and 8 weeks of age, and this leads to
progressive islet destruction such that 70 to 80% become diabetic.
Diabetes is readily demonstrated by the presence of high glucose
levels in the urine or blood.
[0042] The present invention demonstrates the surprising finding
that:
[0043] (i) when a sub-optimal insulin administration protocol was
used (6.times.10 ug doses on alternate days over 2 weeks in NOD
mice at 6 weeks of age) NOD mice were not protected from the
development of IDDM;
[0044] (ii) when similar doses of EtxB, were administered i.n.
according to the same schedule, this did not prevent the
development of IDDM in the NOD mice. However, when EtxB was admixed
insulin according to the same schedule, this lead to a decreased
incidence of IDDM. The prevention of IDDM following administration
of EtxB admixed with insulin at 6 weeks, was associated with a
clear Th1 to Th2 switch in the cytokine profile of pancreatic T
cells responding to TCR engagement.
[0045] In addition, the present invention also demonstrates the
surprising finding that the late administration of EtxB alone to
NOD mice revealed a marked difference in T cell cytokine profiles
from those reported above. By way of explanation, the late
administration of EtxB to NOD mice at 10-12 weeks of age led to a
dramatic reduction in the incidence of IDDM. This dramatic
reduction in the incidence of IDDM was also associated with the
production of Th1 -associated .gamma.IFN as was seen with earlier
treatment with EtxB+insulin, but this was not associated with a
concomitant rise in Th2 cytokine secretion. Instead, the levels of
IL-4 were unchanged and IL-10 was not detected either in lymph node
cell cultures from treated or untreated animals. These findings
suggested that the mechanisms of IDDM prevention by EtxB alone and
EtxB+insulin may be different. In this respect, the early
administration of EtxB admixed with insulin to NOD mice at 6 weeks
of age may result in the EtxB acting as an `adjuvant` promoting
immune deviation away from Th1 associated cytokine responses and
towards Th2 associated cytokine responses. In contrast, the late
administration of EtxB alone (in the absence of an autoantigen such
as insulin) may effectively block diabetes but pancreatic
inflammation needs to be established in order for it to do so. In
contrast to the T cell cytokine profile associated with early
administration of EtxB admixed with insulin, the reduction in the
incidence of IDDM by the administration of EtxB alone is associated
with the suppression of a Th1 cytokine associated response without
promotion of a Th2 cytokine associated response.
[0046] In one aspect, the present invention provides a
pharmaceutical composition, which comprises an agent of the present
invention and a pharmaceutically acceptable carrier, diluent or
excipient (including combinations thereof).
[0047] The pharmaceutical compositions may be for human or animal
usage in human and veterinary medicine and will typically comprise
any one or more of a pharmaceutically acceptable diluent, carrier,
or excipient. Acceptable carriers or diluents for therapeutic use
are well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985). 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).
[0048] Preservatives, stabilizers, dyes and even flavouring agents
may be provided in the pharmaceutical composition. Examples of
preservatives include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
[0049] There may be different composition/formulation requirements
dependent on the different delivery systems. By way of example, the
pharmaceutical composition of the present invention may be
formulated to be delivered using a mini-pump or 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 injiectable form, for delivery, by, for example,
an intravenous, intramuscular or subcutaneous route. Alternatively,
the formulation may be designed to be delivered by both routes.
[0050] Where the pharmaceutical composition is to be delivered
mucosally through the gastrointestinal mucosa, it should be able to
remain 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.
[0051] 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
chalk, 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 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.
[0052] Typically, a physician will determine the actual dosage
which will be most suitable for an individual subject and it will
vary with the age, weight and response of the particular patient
and severity of the condition. The dosages below are exemplary of
the average case. There can, of course, be individual instances
where higher or lower dosage ranges are merited.
[0053] The compositions (or component parts thereof) of the present
invention may be administered orally. In addition or in the
alternative the compositions (or component parts thereof) of the
present invention may be administered by direct injection. In
addition or in the alternative the compositions (or component parts
thereof) of the present invention may be administered topically. In
addition or in the alternative the compositions (or component parts
thereof) of the present invention may be administered by
inhalation. In addition or in the alternative the compositions (or
component parts thereof) of the present invention may also be
administered by one or more of: parenteral, mucosal, intramuscular,
intravenous, subcutaneous, intraocular or transdermal
administration means, and are formulated for such
administration.
[0054] By way of further example, the pharmaceutical composition of
the present invention may be administered in accordance with a
regimen of 1 to 10 times per day, such as once or twice per day.
The specific dose level and frequency of dosage for any particular
patient may be varied and will depend upon a variety of factors
including the activity of the specific compound employed, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity
of the particular condition, and the host undergoing therapy.
[0055] Hence, the pharmaceutical composition of the present
invention may be administered by one or more of the following
routes: oral administration, injection (such as direct injection),
topical, inhalation, parenteral administration, mucosal
administration, intramuscular administration, intravenous
administration, subcutaneous administration, intraocular
administration or transdermal administration.
[0056] The invention is illustrated by way of examples in which
reference is made to the following Figures.
DESCRIPTION OF THE FIGURES
[0057] FIG. 1 represents an analysis of physico-chemical properties
of EtxB and a mutant form of EtxB, (EtxB(G33D)).
[0058] (A) SDS-PAGE analysis of EtxB or EtxB (G33D): 5 .mu.g of
each protein were analysed under reducing conditions in the
presence of .beta.-mercaptoethanol with or without prior heating.
Lane 1, wild type EtxB, unheated. Lane 2, EtxB (G33D), unheated.
Lane 3; wild type EtxB, heated at 95.degree. C. Lane 4, EtxB
(G33D), heated at 95.degree. C. Molecular weight standards (BioRad)
are shown on the left-hand side of the panel.
[0059] (B) Determination of apparent molecular mass of EtxB and
EtxB (G33D) by gel filtration chromatography: standard curve
(circles) was generated using, from top to bottom: bovine serum
albumin (66 kDa), hen egg albumin (45 kDa), bovine erythrocyte
carbonic anhydrase (29 kDa) and horse heart cytochrome c (12.4
kDa); EtxB and EtxB (G33D) eluted with apparent molecular masses of
36 kDa and 38 kDa respectively; Ve-elution volume of the protein,
Vo-void volume of the gel filtration column.
[0060] (C) ELISA for comparative binding of EtxB and EtxB (G33D) to
ganglioside GM1: plates were coated with GM1, blocked and incubated
with 1 .mu.g/ml of either EtxB or EtxB (G33D) diluted serially (3
fold) from 1 .mu.g/ml.
[0061] FIG. 2 illustrates that receptor binding by EtxB is
essential for its potent immunogenicity in vivo. BALB/c mice (4 in
each group) were either injected s.c. with EtxB or EtxB (G33D) in
PBS or given the proteins orally in bicarbonate buffer. Sera were
analysed 10 days following two s.c. injections (A) or 1 week
following 3 oral doses (B), and gut secretions were analysed 1 week
following 3 oral doses (C). No reaction was detected in samples
from control mice (not shown). Results are expressed as mean IgG
antibody titre in serum, while IgA in gut secretion is expressed as
`specific activity` as described below.
[0062] FIG. 3 illustrates the kinetics of lymphocyte proliferation.
Mice were injected i.p. with 30 .mu.g of EtxB (G33D) in complete
Freund's adjuvant. MLNs were isolated 10 days later and cells were
incubated in the absence of antigen (open square) or in the
presence of 80 g/ml EtxB (filled triangles), EtxB (G33D) (open
triangles) or their disassembled monomeric forms of EtxB (filled
circles) and EtxB (G33D) (open circles) generated by heating at 95
C. The last 6 hour on each sampling day cells were pulsed with 1
.mu.Ci of (.sup.3H) Thd. Data represents mean cpm and SEM of
triplicate wells.
[0063] FIG. 4 illustrates that EtxB causes increased activation of
B cells. Mice were immunized with EtxB (G33D) in CFA. Cells were
isolated from mesenteric lymph nodes (MLN) 10 days later and
incubated in the presence of 80 .mu.g/ml of either EtxB or EtxB
(G33D) or a mixture of 40 .mu.g/ml of each protein. Cells were
labelled with biotinylated anti-CD25 (7D4) and Phycoerythrin (PE)
anti-B220 (Ra3-6D2). Streptavidin FITC was used as a secondary
antibody conjugate. Controls for the antibodies were also included
(not shown). Dual flow cytometric analysis was performed on day 4
of proliferation.
[0064] FIG. 5 illustrates that EtxB causes increased activation of
CD4+T cells and depletion of CD8*T cells. The immunization
procedure, cell isolation and the in vitro challenge are as
described in the legend of FIG. 4. To detect CD25, biotinylated
anti-CD25 (7D4) and Streptavidin FITC were used. To detect CD4 and
CD8, FITC labelled anti-CD4 (RNRM4-5) and FITC labelled
anti-CD8.alpha. (53-6.7) were used. Appropriate controls for the
antibodies were included (not shown).
[0065] FIG. 6 shows the selective depletion of OVA-responsive
CD8.sup.+ T cells by EtxB. Cultures of cells from MLN taken from
OVA-primed mice were established for 5 days, in the absence of
antigen or in the presence of OVA+EtxB, OVA+EtxB(G33D) or OVA alone
at 100 .mu.g OVA and 40 .mu.g/ml each of EtxB or EtxB(G33D) or 100
.mu.g OVA alone. Cells were labelled with the following rat
antibodies: FITC-anti-CD4 or FITC-anti-CD8 and both with
biotin-anti-CD25 (IL-2R.alpha.) followed by
Streptavidin-phycoerythrin. Non-stained cells or cells stained with
the second antibody alone were also included as controls. Cells
were analysed by FACS (Becton Dickinson). The higher increase in
the proportion of total cells which are CD25+ in cultures
containing EtxB compared with other treatments is due to the
presence of higher proportion of B cells expressing this marker
(not shown). The scale of fluorescence intensity is log.
[0066] FIG. 7 shows that receptor binding by EtxB induces
alterations in lymphocyte nuclear morphology characteristic of
cells undergoing apoptosis. Mesenteric lymph node cells (MLNC)
comprising >90% CD3.sup.+ T cells and depleted of macrophages
were incubated for 18 h with either 80 .mu.g/ml EtxB or with 80
.mu.g/ml EtxB(G33D) and stained with acridine orange. Cells were
examined under conventional or confocal fluorescence microscopy
(Leica TCS 4D). A representative microscopic field (x 540) for each
treatment is shown [EtxB, left hand panel; EtxB (G33D), right hand
panel]. Cells which were incubated in the absence of antigen gave
similar results to those treated with EtxB(G33D) (not shown).
[0067] FIG. 8 shows EtxB receptor-mediated apoptosis of CD8.sup.+ T
cells as measured by cell cycle analysis. The proportion of
CD4.sup.+ and CD8.sup.+ SPLTC in the sub-G.sub.0/G.sub.1 stage of
the cell cycle was determined by flow cytometric analysis of the
DNA content following staining with propidium iodide. SPLTC were
isolated from the spleen by negative selection as described above.
The cells were treated for 18 h with: (a) no antigen, (b) 80
.mu.g/ml EtxB(G33D) or (c) 80 .mu.g/ml EtxB and then stained with
FITC-rat anti-CD4 or FITC-rat anti-CD8.alpha.. The cells were
subsequently stained with propidium iodide. The proportion of cells
co-stained with propidium iodide was determined by gating on cells
stained with either anti-CD4 or anti-CD8 antibodies. This
experiment has been carried out on cells, results of which are also
reported in FIG. 7 and Table 3.
[0068] FIGS. 9a and 9b show the results of experiments conducted to
show that GM-1 binding by EtxB inhibits the development of collagen
induced arthritis in an animal model.
[0069] FIG. 10 shows the results of an experiment conducted to
illustrate that EtxB but not EtxB(G33D) induces apoptosis in a
population of normal human peripheral blood mononuclear cells.
[0070] FIG. 11 shows the results of an experiment which shows that
cross-linking of GM1 leads to apoptosis in a proportion of murine
CTLL cells.
[0071] FIG. 12 shows the mechanism of action of EtxB. By
interacting with a number of cells critical in the development of
the immune response, EtxB is able to alter the nature of the
response. The induction of apoptosis in CD8.sup.+ T-cells, and the
suppression of IL-12 production by antigen presenting cells inhibit
the activation of pro-inflammtory responses. The induction of IL-10
secretion by antigen presenting cells and the activation of B-cells
promotes the differentiation of T-cells to Th2 and T regulatory
cells. Both cell types contribute to the suppression of the Th1
response. Th2 cells promote the production of non-complement fixing
antibodies and mucosal IgA, the latter may be further aided by
T-regulatory cell production of TGF. Importantly, EtxB does not
stimulate the production of Th2 associated IgE.
[0072] FIG. 13 shows the_effect of LPS and IFN.gamma. driven
cytokine production by CD14+ cells. Peripheral blood CD14+
monocytes were cultured in the presence of increasing doses of EtxB
overnight prior to the addition of IL-12 inducing
LPS+gamma-interferon. IL-12 levels were measured at 48 hours.
[0073] FIG. 14 shows that EtxB causes CD8+T cell apoptosis.
Survival of CD8+ or CD4+'T-cells was assessed after 96 hours in
culture with either EtxB or PBS alone using dual colour flow
cytometry. CD8+ cells were clearly depleted from cultures
containing EtxB. Direct assessment of DNA content in purified
CD8+T-cells using propidium iodide after 24 hours in culture
reveals the increased presence of apoptotic cells (sub-diploid DNA)
following EtxB treatment.
[0074] FIG. 15 shows enhanced activation of B-cells. Flow cytometry
reveals that culture of lymph node cells with EtxB, but not a
mutant B-subunit for 48 hours, causes increased expression of
molecules involved in B-cell activation and T-cell interaction.
[0075] FIG. 16 shows stimulation of cytokine production by CD 14+
cells. Culture of human CD14+ monocytes with increasing
concentrations of EtxB leads to the dose dependent secretion of
IL-10, but does not stimulate IL-12 production. Cytokines are
measured by cELISA.
[0076] FIG. 17 shows mucosal or parenteral delivery of EtxB can
prevent collagen induced arthritis in the DBA/1 mouse. EtxB or PBS
was given at the times indicated to DBA/1 mice by the routes shown,
and in PBS. On day 0 animals were challenged with a sub-cutaneous
injection of type II collagen in CFA. Arthritis was scored on an 8
point scale by inspection of ankle joints.
[0077] FIG. 18 shows that_EtxB protects mice from histological
damage in arthritis. Knee joints are shown from untreated (top) or
treated (bottom; 100 mg EtxB s.c.) mice and stained. Examination of
the untreated joint reveals clear granulomatous inflammation and
disruption of the cartilage and bone. There are no abnormalities in
the treated mouse joint.
[0078] FIG. 19 shows Table 4.
[0079] FIG. 20 shows that the B-subunit can prevent diabetes in the
NOD mouse. NOD mice, which spontaneously develop diabetes, were
given 10 .mu.g of the B-subunit (EtxB) or PBS alone (positive
control) on 6 occasions intranasally. Diabetes was assessed by
measurement of urinary glucose levels at the times indicated.
[0080] FIG. 21 shows that a mixture of EtxB and HSV-1 glycoproteins
stimulates strong systemic and mucosal antibody titres after
intranasal administration. HSV-1 glycoproteins (10 .mu.g) were
given to female NIH mice (n=10) admixed with increasing
concentrations of EtxB intranasally on three occasions at 7 day
intervals. Three weeks after the final vaccination, anti-HSV
antibody levels were measured by specific ELISA. Results are shown
as either the percentage of the response stimulated by live virus
infection (serum IgG) or endpoint titres (mucosal antibodies).
[0081] FIG. 22a shows that intranasal vaccination of NIH mice with
EtxB and HSV-1 proteins protects against primary infection.
Intranasal vaccination of NIH mice (n=15) with 20 mg of EtxB+10
.mu.g HSV-1 proteins protects against ocular scarification with
live HSV-1. Disease was assessed by slit lamp examination of the
eye, the eye lid and surrounding skin.
[0082] FIG. 22b shows that intranasal vaccination with EtxB and
HSV-1 proteins protects against recurrent infection. NIH mice
(n=15) were given live HSV-1 by ocular scarification, and left to
become latently infected. Mice were then intranasally vaccinated
with EtxB+HSV-1 proteins and disease was triggered 6 weeks later by
UV-treatment of the eye. Disease was assessed as described in FIG.
22a.
[0083] FIG. 23 shows the effect of insulin, EtxB and EtxB+insulin
on IDDM. Six week old NOD mice receiving admixed insulin+EtxB but
not either of these alone have a reduced incidence of IDDM. The 4
week old female NOD mice (n=12) were treated i.n. 6 times over 2
weeks (4 to 6 weeks age) with insulin 10 ug/dose, EtxB 10 ug/dose,
insulin+EtxB 10 ug+10 .mu.g/dose or left untreated, then diabetes
mellitus incidence was assessed until they were 30wk old.
[0084] FIG. 24 shows cytokine secretion levels by pancreatic lymph
note (PLN) lymphocytes isolated from NOD mice. Cytokine secretion
by pancreatic lymph node (PLN) lymphocytes isolated from NOD mice.
Female NOD mice (n=3/group, 4 weeks old) were treated i.n. with
insulin (10 ug/dose.times.6 over 2 weeks), EtxB or a mixture of
insulin+EtxB. 3 days after the last dose, PLN were collected and
lymphocytes were plated on anti-CD3 (clone 7D6) coated plates for
48 h (1 million/ml/well in MEM+10% FCS). Subsequently, cytokine
secretion was measured in a 24 h cELISA. Data represent average
+/-standard deviation of triplicates.
[0085] FIG. 25 shows the EtxB+insulin prevents Langerhans islet
destructive infiltration. Insulin+EtxB i.n. treatment prevents
Langerhans islet destructive infiltration. 4-week old NOD female
mice were treated with insulin or EtxB or insulin admixed with EtxB
in PBS (6 doses over 2 weeks, 10 ug/dose of each), then the
pancreas were collected, stained with hematoxylin and eosin and
islet infiltration was assessed by double-blind scoring. The means
and standard deviations are shown in the figure for untreated
(diamonds) and treated mice (circles).
[0086] FIG. 26 shows the effect of EtxB+insulin on 6 week old NOD
female mice. 6 week old NOD female mice were treated with
insulin+EtxB (10 ug+10 ug), 6 doses over 2 weeks. Then splenocytes
from treated (circles) or untreated controls (squares) were
collected, CD4+ cells were separated using MACS and injected i.v.
mixed with equal numbers (5.times.10.sup.6) splenocytes from
diabetic NOD mice to 7.5 Gy-irradiated 8 week old receipents.
Diabetes mellitus was assessed by measuring glucosuria every other
day over the following 50 days.
[0087] FIG. 27 shows that 10 week old NOD mice receiving EtxB alone
have a reduced incidence of IDDM. 10 week old NOD mice receiving
EtxB alone have a reduced incidence of IDDM. Ten week old female
NOD mice (n=10) were treated i.n. 6 times over 2 weeks with EtxB
(.quadrature.) or left untreated (.smallcircle.), then diabetes
mellitus incidence was assessed until they were 30 wk old.
[0088] FIG. 28 shows cytokine secretion levels by pancreatic lymph
node (PLN) lymphocytes from NOD mice. Cytokine secretion by
pancreatic lymph node (PLN) lymphocytes isolated from NOD mice.
Female NOD mice (n=4/group, 12 weeks old) were treated i.n. with
EtxB (10 .mu.g/dose) or PBS alone. 7 days after the last dose, PLN
were collected and lymphocytes were plated on anti-CD3 (clone 7D6)
coated plates for 48 h (1 million/ml/well in MEM+10% FCS).
Subsequently, cytokine secretion was measured in a 24 h cELISA.
Data represent average +/-standard deviation of triplicates.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The basis for all aspects of the present invention is the
finding that EtxB (the pure B-subunit of the E. coli heat labile
enterotoxin) 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.sup.+ T cells and an associated
activation of B cells. These effects are absent when a mutant EtxB
protein lacking GM1 binding activity is employed.
[0090] Aspects of the present invention are presented in the
accompanying claims and in the following description and drawings.
These aspects are presented under separate section headings.
However, it is to be understood that the teachings under each
section are not necessarily limited to that particular section
heading.
[0091] As used herein, 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. The term "agent" includes but is not limited to
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
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
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.
[0092] As used herein, the term "agent having GM1 binding activity"
includes any agent which acts as an immunomodulator through
interacting with a GM1 ganglioside receptor.
[0093] As used herein, 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.
[0094] 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. The ganglioside
can be made synthetically or isolated from natural sources.
Alternatively, it can be obtained from commercial sources.
[0095] As used herein, 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.
[0096] As used herein, the term "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, cross-reacting
antigen, alloantigen, tolerogen, allergen, hapten, and immunogen,
or parts thereof, as well as any combination thereof, and these
terms are used interchangeably throughout the text.
[0097] As used herein, the term "antigenic determinant" refers to
any specific chemical structure within, and generally small in
relation to, an antigen molecule that is recognisable by a
combining site of an antibody or T-cell or B-cell receptor, and at
which combination takes place. It determines the specificity of the
antibody-antigen reaction. Several different antigenic determinants
may be carried by a single molecule of antigen.
[0098] As used herein, the term "autoantigen" means an antigen that
is a normal constituent of an individual and has the capacity to
produce an immune response in the same individual in certain
circumstances. An example of an autoantigen of the present
invention is insulin. As used herein, the term "autoantigen"
includes but is not limited to GAD, GAD65, IAA (islet associated
antigen).
[0099] As used herein, the term "self antigen" means any
potentially antigenic molecule originating from an individual that
is recognised as nonforeign by the individual's immune system and
towards which immunological tolerance is normally shown.
[0100] As used herein, the term "autoimmunity" is used to describe
the mechanism by which the body generates an immune response to
self-antigens.
[0101] As used herein, the term "cross-reacting antigen" means an
antigen that it able to react with an antibody produced in response
to another antigen. This may be because the two antigens share the
same determinants or carry determinants that are sufficiently alike
stereochemically to enable the antibody to react with both of
them.
[0102] As used herein, the term "alloantigen" means an antigen that
is part of an animal's self-recognition system, such as the major
histocompatability complex molecules. When injected into another
animal, they trigger an immune response aimed at eliminating them.
As used herein, the term "tolerogen" means a tolerated antigen.
[0103] As used herein, the term "allergen" includes any antigen
that stimulates an allergic reaction, inducing a Type I
hypersensitivity reaction. Examples of common allergen sources are
disclosed in WO 99/38350.
[0104] As used herein, the "hapten" means a small molecule which
can act as an epitope but is incapable by itself of eliciting an
antibody response.
[0105] As used herein, the term "immunogen" includes any substance
that, when introduced into the body, elicits humoural or
cell-mediated immunity, but not immunological tolerance.
[0106] As used herein, the term "tolerance" means a state of
specific immunological unresponsiveness.
[0107] As used herein, 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".
[0108] As used herein, 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.
[0109] As used herein, 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.
[0110] As used herein, the term "xenoantigen" means an antigen that
is foreign to a particular animal.
[0111] As used herein, 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 a
macrosol)--but with the proviso that the agent and the antigen are
unlinked.
[0112] As used herein, the term "effect" 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. As
used herein, the term "linked"--which is synonymous with the term
"coupled"--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.
[0113] As used herein, the term "not so linked"--which is
synonymous with the term "uncoupled"--means that the agent is not
coupled to the antigen. However, in accordance with the present
invention, the agent and/or antigen can be coupled to another
entity.
[0114] As used herein, 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.
[0115] As used herein, 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.
[0116] As used herein, 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.
[0117] As used herein, the term "systemic immunisation" means the
introduction of an antigen into a non-mucosal tissue such as the
skin or the blood.
[0118] As used herein, the term "administered" also includes but is
not limited to delivery by viral or non-viral techniques. Viral
delivery mechanisms include but are not limited to adenoviral
vectors, adeno-associated 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 but are not limited to mucosal, nasal, oral,
parenteral, gastrointestinal, topical, or sublingual routes.
[0119] As used herein, the term "adjuvant" includes a substance
that enhances an immune response to an antigen.
[0120] As used herein, 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
unrelated antigen. In this way, the agent acts as a so-called
vaccine adjuvant. The term "vaccine adjuvant" includes the term
"mucosal adjuvant".
[0121] As used herein, 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.
[0122] As used herein, the term "mucosal surfaces" includes but is
not limited to oral, sublingual, intranasal, vaginal, rectal,
salivary, intestinal and conjunctival surfaces.
[0123] As used herein, 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.
[0124] As used herein, the "vaccine carrier" includes a carrier of
relevant antigens (Szostak et al 1996 J Biotechnol 44:
161-170).
[0125] It is to be appreciated that all references herein to
"treatment" include one or more of curative, palliative and
prophylactic treatment. In particular, the term "treatment"
includes but is not limited to pre-diabetic treatment and
post-diabetic treatment. By way of example, a subject in a
pre-diabetic state may be treated to prevent the onset and/or
progression of diabetes.
[0126] Preferably, the term treatment includes at least curative
treatment and/or palliative treatment.
[0127] The treatment may be for treating conditions associated with
diabetes.
[0128] As used herein, the term "diabetes" refers to a disorder in
which the level of blood glucose is persistently above the normal
range. Type I diabetes mellitus, the so-called juvenile type, often
manifests itself in children and young adults. There is a marked
failure to release insulin from the beta cells of the islets of
Langerhans in the pancreas and frequently an almost complete
absence of insulin in the beta cells. The only effective treatment
is administration of insulin, hence the frequent designation of
this form as insulin-dependent diabetes mellitus (IDDM). As used
herein, the terms diabetes, Type I diabetes mellitus and IDDM are
used interchangeably.
[0129] As with the term "treatment", the term "therapy" includes
curative effects, alleviation effects, and prophylactic
effects.
[0130] The therapy may be on humans or animals.
[0131] The therapy may be for treating conditions associated with
diabetes.
[0132] Autoimmune Disease
[0133] Autoimmunity is the term used to describe the mechanism by
which the body generates an immune response to self-antigens.
[0134] In accordance with a first aspect of the invention, there is
provided:
[0135] (i) an agent having GM-1 binding activity, other than Ctx or
Etx, or the B subunits of Ctx and Etx; or
[0136] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0137] for use as an agent in the treatment or the prevention of an
autoimmune disease.
[0138] Agents in accordance with the present invention have been
found to modulate lymphocyte populations leading to the induction
of apoptosis in CD8.sup.+ T cells, the enhanced activation of
CD4.sup.+ cells and polyclonal activation of B cells. These events
are likely to shift the immune response towards induction of Th2
associated cytokines. Such responses to self or cross-reacting
antigens are understood to mediate protection for certain
autoimmune diseases.
[0139] In a first embodiment of this first aspect of the present
invention, the agent is used in a method of treating an autoimmune
disease which is in progress. In this embodiment, the agent is
administered to a patient with or without co-administration of a
self or cross-reacting antigen. Administration of the agent in
accordance with this embodiment of the first aspect of the
invention modulates the nature of the immune response towards the
self-antigen away from the activation of disease-causing
inflammation and hence protects against autoimmune disease.
[0140] In a second embodiment of this first aspect of the present
invention, the agent is used in a method for the "vaccination" of a
mammalian subject against an autoimmune disease, in which the agent
is co-administered with the self or cross-reacting antigenic
determinant (or a combination of different self or cross-reacting
antigenic determinants) associated with said disease. In such a
manner, the subject's immune response to the self-antigen or
cross-reacting antigen is switched away from the activation of
pathogenesis, which therefore protects against a future autoimmune
response to the self-antigen.
[0141] In this first aspect of the invention, the therapeutic agent
and the self or cross-reacting antigenic determinant are, or may
be, co-administered to the subject. By this we mean that the site
and time of administration of each of the therapeutic agent and the
antigenic determinant are such that the necessary modulation of the
immune system is achieved. Thus, whilst the therapeutic agent and
the antigenic determinant may be administered at the same moment in
time and at the same site, there may be advantages in administering
the therapeutic agent at a different time and to a different site
from the antigenic determinant. Whilst single doses of the
therapeutic agent and the antigenic determinant may be
satisfactory, multiple doses are contemplated within the scope of
this aspect of the invention.
[0142] In this second embodiment of the first aspect of the
invention, the therapeutic agent and the antigenic determinant may
be linked, for example covalently linked, to form a single active
agent, although separate administration, in which the therapeutic
agent and the antigenic determinant are not so linked is preferred
because it enables separate administration of the different
moieties.
[0143] Specific autoimmune diseases which may be treated in
accordance with this aspect of the present invention are the
autoimmune diseases where pathology is associated with
cell-mediated immunity, such as rheumatoid arthritis, multiple
sclerosis and diabetes.
[0144] Additionally, under this first aspect of the present
invention, there is provided the use of Ctx, Etx or the B subunit
of Ctx or Etx, for the manufacture of a medicament for use as an
agent for the prevention of an autoimmune disease.
[0145] Also provided is a pharmaceutical composition for the
treatment of a human autoimmune disease comprising
[0146] (i) an agent having GM-I binding activity; or
[0147] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0148] and a pharmaceutically acceptable carrier or diluent
therefor.
[0149] The pharmaceutical composition of this aspect of the
invention may be formulated to be delivered by a mucosal route, for
example as a nasal spray, or parenterally in which the composition
is formulated in an injectable form, for delivery by, for example,
an intravenous, intramuscular or subcutaneous route.
[0150] The pharmaceutical composition may be formulated together
with the appropriate self or cross-reacting antigen. Alternatively,
a kit may be provided comprising separate compositions for each of
the therapeutic agent and the antigenic determinant.
[0151] Specific therapeutic agents which may be used in this aspect
of the invention are EtxB and CtxB or mutants thereof retaining GM1
binding activity.
[0152] The agents for use in the first aspect of the present
invention should preferably be substantially non-toxic, although
some degree of toxicity may be tolerated in a severe therapy of
this kind.
[0153] This first aspect of the invention extends to cover the use
of all agents having GM1 binding activity, for use in the treatment
of mammalian autoimmune disease, as well as those agents having an
effect on GM-1 mediated intracellular signalling events, and which
therefore mimic GM-1 binding agents.
[0154] Thus, this first aspect of the present invention is not
limited to the use of EtxB protein as a therapeutic agent in the
treatment of a human autoimmune disease. However, the use of the
EtxB protein (which is a pentamer of five identical subunits) for
such a treatment represents a preferred embodiment of the present
invention. In addition to the wild type EtxB, this preferred aspect
of the invention also extends to mutants of EtxB which have GM-1
binding activity as well as to other equivalent proteins, such as
the cholera toxin B subunit (CtxB) and mutants thereof which have
GM1 binding activity.
[0155] Other therapeutic agents for the treatment of autoimmune
disease in accordance with the first aspect of this invention are
humanised monoclonal antibodies, which bind GM1. Methods known in
the art for identifying and preparing such agents are well
known.
[0156] T-lymphocyte Leukaemias
[0157] According to a second aspect of this invention, there is
provided:
[0158] (i) an agent having GM-1 binding activity, other than Ctx or
Etx, or the B subunits of Ctx and Etx; or
[0159] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0160] for use in the treatment of human leukaemias of a T cell
origin, such as human leukaemias of a CD8 T cell origin.
[0161] The agents for use in the second aspect of the present
invention should preferably be substantially non-toxic, although
some degree of toxicity may be tolerated in a severe therapy of
this kind.
[0162] Additionally, under this second aspect of the present
invention, there is provided the use of Ctx or Etx, or the B
subunits of Ctx and Etx for the manufacture of a medicament for
treatment of human leukaemias of a T cell origin, such as human
leukaemias of a CD8 T cell origin.
[0163] Also provided is a pharmaceutical composition for the
treatment of human leukaemias of a T cell origin comprising
[0164] (i) an agent having GM-1 binding activity; or
[0165] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0166] and a pharmaceutically acceptable carrier or diluent
therefor.
[0167] The pharmaceutical composition of this aspect of the
invention may be formulated to be delivered by a mucosal route, for
example as a nasal spray, or parenterally in which the composition
is formulated in an injectable form, for delivery by, for example,
an intravenous, intramuscular or subcutaneous route.
[0168] This second aspect of the invention extends to cover the use
of all agents having GM1 binding activity, for use in the treatment
of human leukaemias of a T cell origin, as well as those agents
having an effect on GM-1 mediated intracellular signalling events,
and which therefore mimic GM-1 binding agents.
[0169] Thus, this second aspect of the present invention is not
limited to the use of EtxB protein as therapeutic agents in the
treatment of human T cell leukaemias. However, the use of the EtxB
protein for such a treatment represents a preferred embodiment of
the present invention. In addition to the wild type EtxB, this
preferred aspect of the invention also extends to mutants of EtxB
which have GM-1 binding activity as well as to other equivalent
proteins, such as the cholera toxin B subunit (CtxB) and mutants
thereof which have GM1 binding activity.
[0170] Other alternative therapeutic agents for the treatment of
these diseases in accordance with this aspect of the invention are
humanised monoclonal antibodies, which bind GM1. Methods known in
the art for identifying and preparing such agents are well
known.
[0171] Transplant Rejection and GVHD
[0172] In accordance with a third aspect of this invention, there
is provided:
[0173] (i) an agent having GM-1 binding activity, other than Ctx or
Etx, or the B subunits of Ctx and Etx; or
[0174] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0175] for use as a therapeutic agent for the prevention/treatment
of transplant rejection or GVHD.
[0176] Additionally, under this third aspect of the present
invention, there is provided the use of Ctx or Etx or the B subunit
of Etx or Ctx for the manufacture of a medicament for the
prevention of transplant rejection or GVHD.
[0177] In preferred embodiments of this aspect of the invention,
the therapeutic agents described may be used in the prevention of
solid organ transplant rejection, either allogeneic or xenogeneic.
They may also be employed in the prevention of acute graft versus
host disease (GVHD), for example during bone marrow transplantation
procedure.
[0178] In embodiments of this aspect of the invention where the
patient is treated prior to transplantation, the therapeutic agent
would be co-administered with alloantigen or xenoantigen. In
embodiments in which the patient is treated after transplantation,
the therapeutic agent is employed without co-administration of
antigen.
[0179] In the embodiment of this aspect of the invention, where the
therapeutic agent and allo- or xeno-antigenic determinant are
co-administered to the subject, we mean that the site and time of
administration of each of the therapeutic agent and the antigenic
determinant are such that the necessary modulation of the immune
system is achieved. Thus, whilst the therapeutic agent and the
antigenic determinant may be administered at the same moment in
time and at the same site, there may be advantages in administering
the therapeutic agent at a different time and to a different site
from the antigenic determinant. Furthermore, the therapeutic agent
and the antigenic determinant may be covalently linked to form a
single active agent, although separate administration, in which the
therapeutic agent and the antigenic determinant are not so linked
is preferred because it enables separate administration of the
different moieties.
[0180] Whilst single doses of the therapeutic agent and the
antigenic determinant may be satisfactory, multiple doses are
contemplated within the scope of this aspect of the invention.
[0181] In this aspect of the invention, where the agent is being
used in the prevention of GVHD, the agent would normally be applied
direct to the cells, for example bone marrow cells, to be
transplanted.
[0182] The agent is preferably substantially non-toxic, although
some degree of toxicity may be tolerated in severe therapies of
this kind.
[0183] Also provided is a pharmaceutical composition for use in the
treatment of transplant rejection, comprising
[0184] (i) an agent having GM-1 binding activity; or
[0185] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0186] and a pharmaceutically acceptable carrier or diluent
therefor.
[0187] The pharmaceutical composition of this aspect of the
invention may be formulated to be delivered by a mucosal route, for
example as a nasal spray, or parenterally in which the composition
is formulated in an injectable form, for delivery by, for example,
an intravenous, intramuscular or subcutaneous route.
[0188] The pharmaceutical composition may be formulated together
with the appropriate allo- or xeno-antigeneic determinant.
Alternatively, a kit may be provided comprising separate
compositions for each of the therapeutic agent and the antigenic
determinant.
[0189] This third aspect of the invention extends to cover the use
of all agents having GM1 binding activity, for use in the
prevention/treatment of transplant rejection or GVHD, as well as
those agents having an effect on GM-1 mediated intracellular
signalling events, and which therefore mimic GM-1 binding
agents.
[0190] Thus, this third aspect of the invention is not limited to
the use of EtxB protein as a therapeutic agent in the treatment of
a transplant rejection. However, the use of the EtxB protein (which
is a pentamer of five identical subunits) for such a treatment
represents a preferred embodiment of the present invention. In
addition to the wild type EtxB, this preferred aspect of the
invention also extends to mutants of EtxB which have GM-1 binding
activity as well as to other equivalent proteins, such as the
cholera toxin B subunit (CtxB) and mutants thereof which have GM1
binding activity.
[0191] Other alternative therapeutic agents for the treatment of
transplant rejection in accordance with the invention are humanised
monoclonal antibodies, which bind GM1. Methods known in the art for
identifying and preparing such agents are well known.
[0192] Vaccination
[0193] CtxB and EtxB have already been suggested as so-called
"vaccine carriers". It has now been discovered that the basis for
this effect, in part, is the ability of EtxB to modulate lymphocyte
populations (as discussed above) by binding to the GM-1
receptor.
[0194] Thus, in accordance with a fourth aspect of the present
invention, there is provided:
[0195] (i) an agent having GM-1 binding activity, other than Etx or
Ctx or the B subunits of Etx or Ctx; or
[0196] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0197] for use in the vaccination of a mammalian subject.
[0198] The agent is capable of modulating the immune response when
delivered together with an unrelated foreign antigenic determinant.
Where the agent is delivered parenterally, such immunomodulation is
in terms of the immune response being "directed" in a particular
desired direction. Where the agent is delivered mucosally with an
unrelated antigen, as a so-called "mucosal adjuvant", the agent is
capable of facilitating a mucosal immune response to the unrelated
antigen. The antigen and agent may be delivered together as
separate moieties, or may be linked together, for example by a
covalent linkage.
[0199] The agent is preferably non-toxic. In addition, where the
agent is to be delivered mucosally through the gastrointestinal
mucosa, it should be able to remain stable during transit through
the gastrointestinal tract; for example, it should be resistant to
proteolytic degradation, stable at acid pH and resistant to the
detergent effects of bile. Also provided is a pharmaceutical
composition for use in the vaccination of a mammalian subject,
comprising
[0200] (i) an agent having GM-1 binding activity; or
[0201] (ii) an agent having an effect on GM-1 mediated
intracellular signalling events, but no GM-1 binding activity;
[0202] and a pharmaceutically acceptable carrier or diluent
therefor.
[0203] The pharmaceutical composition of this aspect of the
invention may be formulated to be delivered by a mucosal route, for
example as a nasal spray, or parenterally in which the composition
is formulated in an injectable form, for delivery by, for example,
an intravenous, intramuscular or subcutaneous route.
[0204] The pharmaceutical composition may be formulated together
with the appropriate antigenic determinant. Alternatively, a kit
may be provided comprising separate compositions for each of the
therapeutic agent and the antigenic determinant.
[0205] This fourth aspect of the invention extends to cover the use
of all agents having GM1 binding activity, as immunomodulators, as
well as those agents having an effect on GM-1 mediated
intracellular signalling events, and which therefore mimic GM-1
binding agents.
[0206] Thus, this fourth aspect of the invention is not limited to
the use of EtxB protein as an immunomodulator. However, the use of
the EtxB protein (which is a pentamer of five identical subunits)
in such a way represents one embodiment of the present invention.
In addition to the wild type EtxB, this preferred aspect of the
invention also extends to mutants of EtxB which have GM-1 binding
activity as well as to other equivalent proteins, such as the
cholera toxin B subunit (CtxB) and mutants thereof which have GM1
binding activity.
[0207] Other alternative therapeutic agents for use as an
immunomodulator in accordance with this aspect of the invention are
humanised monoclonal antibodies, which bind GM1. Methods known in
the art for identifying and preparing such agents are well
known.
[0208] When the therapeutic 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 EtxB assemble may
be inserted into, for example, plasmid pMMB68, which is then used
to transfect host cells, such as Vibrio sp.60. The protein is
purified and isolated in a manner known pr se.
[0209] Mutant genes expressing active mutant EtxB protein may then
be produced by known methods from the wild type gene.
[0210] As previously stated, agents having GM-1 binding activity,
such as specifically designed humanised monoclonal antibodies, may
be designed and produced as outlined above, by methods which are
known in the art.
[0211] In all aspects of the invention, the agent having GM1
binding activity may also be capable of cross-linking GM1
receptors. EtxB is one such agent which is capable of cross-linking
GM1 receptors by virtue of its pentameric form.
EXAMPLES
[0212] The following examples are presented to further illustrate
and explain the present invention and should not be taken as
limiting in any regard.
Example 1
[0213] This example illustrates the requirement for GM-1 binding to
induce differential effects on lymphocyte populations
[0214] Materials and Methods
[0215] Generation of a receptor-Binding Mutant of EtxB
[0216] A Gly-33 to Asp substitution was introduced into the
receptor binding site of human EtxB using plasmid pTRH29, a
derivative of the phagemid vector pBluescript IIKS+, that contains
the genes for the A- and B-subunits of Etx (Yu, J., Webb, H. &
Hirst, T. R. (1992), Molec. Microbiol. 6, 1949-1958). Mutagenesis
was performed with an in vitro oligonucleotide-directed mutagenesis
kit (Amersham International) using single-stranded pTRH29 as a
template and a synthetic oligonucleotide
(5'-TCTCTTTTATCTGCCATCG-3') (from the Microanalytical Facility,
IAPGR, Cambridge Research Station, UK) as the mutagenic primer. The
correct Gly to Asp substitution was confirmed by dideoxy sequencing
using Sequenase II (United States Biochemical Corp.) and the
resultant plasmid was designated pTRH56. The mutant etxB gene from
pTRH56 was excised, using EcoRI and SpeI restriction enzymes, and
inserted into pMMB68 (Sandkvist, M., Hirst, T. R. &
Bagdasarian, M. (1987) J. Bacteriol. 169, 4570-4576) to yield a
broad host range expression vector, pTRH64 expressing
EtxB(G33D).
[0217] Antigens
[0218] Wild-type EtxB and EtxB(G33D) were purified from culture
supernatants of Vibrio sp.60 (pMMB68) and Vibrio sp.60 (pTRH64),
respectively, using a modification of the method reported by Amin
and Hirst (Amin, T., & Hirst, T. R. (1994) Prot. Express. and
Purif. 5, 198-204). Briefly, proteins were purified by
diafiltration and hydrophobic interaction chromatography and
concentrated by anion-exchange chromatography. The protein
solutions were desalted on a PD10 column (Pharmacia, UK)
equilibrated with phosphate buffered saline (PBS; 1 OmM sodium
phosphate, 150 mM NaCl, pH7.4) and stored at -30.degree. C.
[0219] The purity of EtxB and EtxB(G33D) were confirmed by SDS
polyacrylamide gel electrophoresis. The molecular mass of the
individual monomers were confirmed by laser desorption mass
spectrometry (Protein Science Facility, University of Kent).
[0220] Apparent molecular masses of EtxB and EtxB(G33D) were
determined by gel filtration chromatography using a SMART system
(Pharmacia). Proteins were eluted from a Superdex 75 PC 3.2/30
column in PBS, pH7.5.
[0221] Irreversible denaturation of B subunit pentamers, for use in
lymphocyte proliferation assays (see below), was achieved by
heating the proteins at 95.degree. C. for 5 min.
[0222] Animals, Sample Collection and Immunization Protocols
[0223] BALB/c mice (H-2.sup.d; high responder to EtxB) of 7-12
weeks of age were purchased from Charles River Laboratories and
maintained at the University of Kent animal house. Antibody
responses to EtxB or EtxB(G33D) were measured after s.c. injection
of mice with 30 g of protein in PBS, followed by boosting 10 days
later. Another group of mice were given the same protein dose
orally in sodium bicarbonate (50 .mu.g/ml) on 3 occasions, and at
one week intervals. Control mice were given PBS. Blood was
collected 10 days following the last s.c. injection or one week
following the last oral feeding. Gut secretions from live mice were
isolated in a protease inhibitor solution as previously described
(Elson, C. O., Ealding, W. & Lefkowitz, J. (1984) J Immunol.
Meth. 67, 101-108), one week following the last feeding. Samples
were then sonicated and clarified by centrifugation
(13,226.times.g, 10 min, at 4.degree. C.).
[0224] For the proliferative assays, mice were injected i.p. with
30 g ofEtxB or EtxB(G33D) in complete Freund's adjuvant (CFA) and
the mesenteric lymph nodes isolated 10 days later. Control
unimmunized mice were also included and their lymph nodes isolated
in a similar manner.
[0225] Enzyme Linked Immunosorbent Assays (ELISAs)
[0226] Binding of EtxB or EtxB(G33D) to GM1 was examined by a
GM1-ELISA (Amin, T., & Hirst, T. R. (1994) Prot. Express. and
Purif. 5, 198-204).
[0227] Sera and gut secretions were examined for the presence of
anti-B subunit IgG and IgA antibodies by ELISA's in which samples
were applied to microtitre plates (Immulon I, Dynateck, USA) coated
with 5 g/ml of eitherEtxB or EtxB (G33D) in PBS. Anti-B subunits
IgA antibodies in gut secretion supernatants were extrapolated from
a standard curve made by coating 2 rows of wells on each plate with
1 .mu.g/ml rabbit anti-mouse IgA (.alpha. 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
were coated with rabbit anti-mouse IgA followed by addition of gut
secretion supernatants. All samples were serially diluted. Goat
anti-mouse IgG (Fc fragment specific; Jackson Lab., USA) or goat
anti-mouse IgA (a chain specific; Sigma) peroxidase conjugate were
diluted and added to all wells. The anti-B subunit IgG titer,
giving an A.sub.450 nm of 0.2, was determined. The IgA anti-B
subunit response for each of EtxB and EtxB (G33D) in gut secretions
was calculated as "IgA specific activity" [mean IgA anti-B subunit
(.mu.g/ml)/total IgA (.mu.g/ml)].
[0228] An ELISA method for measuring cytokine levels of IL-2, IL-4,
IL-5, IL-10 and IFN- was used, as described previously (Harper, H.
M., PhD thesis, Univeristy of Bristol (1995)). Briefly, microtiter
plates were coated with rat antibodies to mouse IL-2, IL-4, IL-5,
IL-10 and IFN-.gamma.. Plates were blocked with 2% (w/v) bovine
serum albumin. Supernatants from culture medium were added to wells
and diluted down. One row on each plate for each cytokine contained
a standard amount of recombinant cytokines. Plates were then
incubated with 0.5 .mu.g/ml of biotinylated anti-cytokine
monoclonal antibodies followed by addition of avidine-peroxidase
and 3,3',5,5'-Tetramethylbenzidene (TMB) substrate and read at
A.sub.405nm.
[0229] Lymphocyte Proliferation Assay
[0230] Mice were sacrificed by cervical dislocation, mesenteric
lymph nodes were excised aseptically and minced through a stainless
steel mesh into Hank's balanced salt solution (HBSS) (Flow
Laboratories, Irvine, Renfrewshire, UK.). Cells were washed by
centrifugation (500.times.g, 10 min, 4.degree. C.) in HBSS and
resuspended in modified Eagle's medium (Flow) to which 20 mM Hepes
(Flow), 100 IU Penicillin, 100 g/ml Streptomycin, 4 mM L-glutamine
(Flow) and 2-mercaptoethanol had been added (complete medium).
Fresh autologous normal mouse serum from unimmunized mice was added
to a final concentration of 0.5% (v/v). Cultures contained
2.times.10.sup.6 viable cells/ml in either 2 ml volumes in 24-well
plates or 8 ml volumes in 25 cm.sup.3 flasks (Nunc A/S, Roskide,
Denmark) and were established in the presence and absence of
antigens as indicated in the figures legend. Cultures were
incubated at 37 .mu.C in a humidified atmosphere of 5% CO.sub.2 and
95% air for 6 days. At desired timepoints, 0.1 ml samples were
removed from the cultures and transferred to 96 well U-bottomed
plates (Nunc) and pulsed with 1 .mu.Ci/well of [.sup.3H]-Thd
(Amersham, U.K) for 6 h before harvesting (Mach III harvesting 96
Tomtec, Orange, Conn. USA) and counting by standard liquid
scintillation 1450 Micro .beta. plus, LKB-Wallac, Turku, Finland).
Similarly, 0.5 ml of supernatant was sampled from cultures for
cytokine analysis. Cells were pelleted and the supernatants stored
at -68.degree. C. until analysed.
[0231] Phenotypic Analysis of Cultured Cells
[0232] Cultured cells harvested on day 4 of culture were washed and
viable cells recovered at the interface of a HBSS/18% metrizamide
(Nyegaard and Co., Oslo, Norway) gradient following centrifugation
at 500.times.g for 15 min at 20.degree. C. Cells were washed twice
and resuspended in HBSS containing 0.2% sodium azide (Sigma) and
10% normal rat serum. The following rat antibodies (Pharmingen, San
Diego, USA) were used: fluorescein isothiocyanate (FITC) labelled
anti-CD4 (RNRM4-5), FITC labelled anti-CD8 (53-6.7),
biotin-labelled anti-CD25 (7D4) and Phycoerythrin (PE) labelled
anti-B220 (RA3-6D2). Additionally, for the biotin-labelled
antibodies Streptavidin-PE or Streptavidin-FITC (Serotech, UK) were
used. All antibodies were diluted in HBSS containing azide and used
at predetermined concentrations. 200 .mu.l of 2.times.10.sup.6
cells and 200 l of each the antibodies were mixed and incubated on
ice for 30 min. When Streptavidin -PE or FITC secondary antibodies
were required cells were incubated with these antibodies for
additional 30 min. Appropriate controls for FITC and PE antibodies
were also included. Cells were washed with HBSS and then analysed
by 2 flow cytometery (Becton Dickinson).
[0233] Results
[0234] Generation and Characterization of a Receptor Binding Mutant
of EtxB
[0235] A Gly to Asp substitution was introduced into the B subunit
of E. coli heat-labile enterotoxin by oligonulceotide-directed
mutagenesis of EtxB, in order to generate a mutant B subunit
defective in receptor recognition. The mutant protein, designated
EtxB(G33D), and wild type EtxB were purified to homogeneity (see
Materials and Methods). The molecular mass of purified EtxB and
EtxB(G33D) were determined by laser desorption mass spectrometry.
Masses were within 20 Da of the theoretical masses of 11702 and
11760 Da for monomeric EtxB and EtxB (G33D), respectively. When
analysed by SDS-PAGE without prior heating, both wild-type EtxB and
EtxB(G33D) migrated as discrete stable oligomers, with apparent
molecular weights of 42 kDa and 56 kDa (FIG. 1A, lane 1 and lane 2,
respectively). The observed electrophoretic mobility and
SDS-stability of EtxB is a characteristic property of the B subunit
pentamer (see Sandkvist, M., Hirst, T. R. & Bagdasarian, M.
(1987) J. Bacteriol. 169, 4570-4576). The slower electrophoretic
mobility of oligomeric EtxB(G33D) is not due to a difference in the
number of constituent B subunit monomers, since both pentameric
EtxB and EtxB(G33D) exhibited similar retention times when analysed
by high resolution gel filtration chromatography. Thus, the
discrepancy in the electrophoretic mobility of the EtxB(G33D)
oligomer with respect to wild-type EtxB, is likely to be due to the
introduced negatively charged Asp residue causing a reduction in
SDS binding and a subsequent slower migration.
[0236] EtxB and EtxB(G33D) were also compared for their stability
in low pH buffers, resistance to 1.0 mg/ml of either trypsin or
proteinase K, and relative reactivity towards a panel of anti-B
subunit monoclonal and polyclonal antibodies. In each of these
tests EtxB(G33D) exhibited identical properties to wild-type EtxB.
It is therefore concluded that a Gly to Asp substitution at residue
33 in EtxB does not alter the oligomeric configuration, SDS, pH or
protease stability, or antibody reactivity compared with wild-type
EtxB.
[0237] The ability of EtxB(G33D) to bind to its receptor GM1, was
evaluated using a GM1-ELISA (FIG. 1C). This showed a highly
significant reduction in the ability of the mutant to bind GM1
compared with the wild type protein (>99% reduction in the
A.sub.450 nm reading). Furthermore, in contrast to wild type EtxB,
EtxB(G33D) failed to bind to CHO cells when examined by
immunofluorescence. It is concluded that EtxB(G33D) is defective in
its capacity to bind GM1 ganglioside, in vitro and in situ.
[0238] The Potent Immunogenicity of EtxB in vivo is Dependent on
Receptor Binding
[0239] The importance of receptor binding in the immunogenicity of
EtxB was evaluated in mice following either oral delivery or s.c.
injection of EtxB or EtxB(G33D) in PBS. Oral delivery of EtxB
resulted in detection of a high IgG antibody titer in serum and IgA
antibody activity in gut secretions (FIG. 2). In contrast, a
similar regime of oral immunization with EtxB(G33D) failed to
generate any detectable antibody activity. EtxB(G33D) did induce a
serum antibody response following s.c. injection, although the
response was considerably lower in comparison to the antibody
response to wild type EtxB, with >160 fold reduction in the mean
antibody titer, 1050 versus 171000, respectively. It is concluded
that receptor binding by EtxB is essential for its potent
immunogenicity in vivo.
[0240] Receptor Binding does not Influence the Extent of Lymphocyte
Proliferation in the Presence of EtxB or EtxB(G33D)
[0241] The effect of EtxB or EtxB(G33D) on lymphocyte proliferation
in vitro was examined. Lymphocytes were isolated from the popliteal
and mesenteric lymph nodes (MLN) of mice immunized with either EtxB
or EtxB(G33D) and stimulated in vitro with either protein, or with
a heat-denatured preparation of EtxB or EtxB(G33D). The
proliferative response of lymphocytes derived from the popliteal or
MLN was similar. In each case, proliferation to each of the protein
preparations increased with increasing B subunit concentration. A
representative set of data from an experiment using MLN is shown in
Table 1. The magnitude of the response to wild type and mutant
pentamers was comparable as was that in the presence of
heat-denatured wild type and mutant monomers. FIG. 3 shows the
kinetics of the proliferative responses obtained in the presence of
80 .mu.g/ml of each of the protein preparations. Reactivity was
dependent on the presence of antigen, and followed a similar
pattern in the presence of each protein. Reactivity was evident on
day 3 of culture with incorporation of [.sup.3H]-Thd reaching a
peak on day 4 and waining thereafter. The minor differences in the
timing of peak responses apparent in FIG. 3 were not observed in
repeat experiments, showing that the anamnestic characteristics of
the responses to the EtxB and EtxB(G33D) are comparable. It is
concluded that the level of stimulation in the presence of the
native proteins is not likely to be influenced by receptor binding
or the introduced mutation.
[0242] Toxin Receptor Binding Causes Immunomodulation of B Cells
and T Cell Subsets
[0243] To examine if receptor binding by EtxB exerts any effect on
the populations of lymphoid cells in vitro, lymphocytes were
isolated from the MLN of mice primed i.p. with EtxB(G33D) and then
stimulated with either EtxB or EtxB(G33D) or a mixture of both.
Additionally, a parallel experiment using MLN-derived lymphocytes
from mice injected with EtxB was undertaken and resulted in
essentially identical findings to those obtained from EtxB(G33D)
primed mice.
[0244] (i) EtxB Causes Increased Activation of B Cells
[0245] The effect of EtxB on B cells were examined by expression of
the activation marker CD25 (IL-2R.alpha.) in association with the B
cell marker B220 (CD45R). As shown in FIG. 4 the number of B cells
in cultures stimulated with EtxB was 62.9% of total cells, of which
a high proportion (28.4%) expressed the cell activation marker
CD25. In contrast, the proportion of B cells after stimulation in
the presence of EtxB(G33D) was less than half of that of the wild
type (22.26%) and fewer were activated (5.6%). To establish whether
the effects exerted by EtxB were dominant, cells were incubated in
the presence of an equimolar concentration of EtxB and EtxB(G33D).
The flow cytometric data was similar to that obtained following
stimulation in the presence of wild type EtxB alone (with 60.6% B
cells, of which 26% were activated). It is concluded that the
receptor binding property of EtxB mediates an increased activation
of B cells in vitro.
[0246] (ii) EtxB Causes Increased Activation of CD4.sup.+ T Cells
and Complete Depletion of CD8.sup.+ T Cells.
[0247] To examine the influence of B subunit receptor binding on T
cells, lymphocytes were labelled with antibodies to CD4 or to CD8
in association with antibodies to CD25 (FIG. 5). Additionally, some
cells were separately labelled with antibodies to the CD3 marker
(not shown). The proportion of T cells expressing the CD4 marker
when stimulated in the presence of EtxB was 36.7%, of which a high
proportion (32.7%) were activated. In contrast, no detectable CD8+T
cells were present in the culture containing EtxB.
[0248] By comparison, both CD4.sup.+ and CD8.sup.+ T cells were
present in the culture stimulated in the presence of EtxB(G33D).
Such cultures contained a large proportion of CD4.sup.+ T cells
(66.6%), but only 12% of these were activated. The proportion of
CD8.sup.+ T cells detected in the presence of EtxB(G33D) was 11.7%
of the total number of cells, but very few of these were activated
which is indicated by the absence of the CD25 marker. Additionally,
in the presence of a mixture consisting of an equimolar
concentration of EtxB and EtxB(G33D) the pattern of responding
cells was similar to that in the presence of wild type EtxB alone;
with 41.68% CD4.sup.+ T cells (of which 28.6% were CD25+) and no
detectable CD8.sup.+ T cells (FIG. 5). In all these analyses, the
proportion of cells staining with CD3 was approximately equal to
the sum of those expressing CD4 and CD8 markers. These data
demonstrate that the increase in activation of B and CD4.sup.+ T
cells and the selective depletion of CD8.sup.+ T cells are mediated
by toxin receptor occupancy.
[0249] Production of Cytokines
[0250] To assess whether the effect of EtxB on lymphocyte
populations could be dependent on a change in cytokine production,
cell cultures were incubated with either EtxB or EtxB(G33D) and
supernatants removed on days 2, 3, 4, 5 and 6 for analysis. The
results from samples collected on day 5 are shown in Table 2 when
the maximum concentration of cytokines was detected. Both
IFN-.gamma. and IL-2 were detected in the supernatants from
cultures stimulated in the presence of EtxB or EtxB(G33D), although
the relative levels of these cytokines varied. The medium from
cells incubated with wild type EtxB, contained a 3-fold higher
concentration of IL-2 and a 1.5 fold lower level of IFN- compared
with supernatants from cultures stimulated in the presence of
EtxB(G33D). Despite the finding that other proliferating T cell
cultures responding to other antigens yielded high levels of IL-4,
IL-5 and IL-10 none of these cytokines were detected in cultures
stimulated with EtxB or EtxB(G33D). The increase in the level of
IL-2 and decrease in the level of IFN-.gamma. following stimulation
with EtxB, compared with EtxB(G33D), most likely reflects the
activation status of B and CD4.sup.+ T cells. Nonetheless, the
results indicate that the profound effect of wild type EtxB on the
CD8.sup.+ T cell population is unlikely to be mediated by a major
shift in the cytokine profile, as a consequence of receptor
occupancy.
[0251] Discussion
[0252] These investigations show that the introduction of a single
point mutation (G33D) in the receptor binding site of EtxB caused a
significant loss in the ability to bind GM1. Importantly, the
mutant EtxB(G33D), exhibited identical physico-chemical properties
to the wild type EtxB with respect to conformation, as revealed by
gel chromatography, stability in SDS, acid and proteases. When the
specific antibody responses were measured following immunization
with either EtxB or EtxB(G33D), dramatic differences were noted.
Subcutaneous injection with EtxB(G33D) in mice resulted in a highly
significant drop in the antibody titer compared with wild type (ca
>160 folds) while no antibody response was detected following
oral administration. It is possible that these differences result
from the disruption of a dominant epitope involved either in the
recognition of the molecule by antibody, or the stimulation of
effective T cell help for antibody production. However, it is
noteworthy that the Gly to Asp substitution had no effect on the
recognition of the B subunit by a panel of specific polyclonal and
monoclonal antibodies. Further, the proliferative responses
obtained when EtxB or EtxB(G33D) were added to cultures were
comparable regardless of which of the proteins were used for in
vivo priming; demonstrating that the T cell reactivity was not
specific to either molecule. It is therefore concluded that
receptor binding by EtxB is essential for its potent immunogenicity
in vivo.
[0253] The importance of receptor binding in the potent
immunogenicity of EtxB may be explained in a number of ways.
Firstly, binding of the B subunit of Etx and Ctx to GM1 may
increase the efficiency of uptake of these proteins, raising the
local protein concentration available to the immune system. Other
classes of proteins which are able to bind mucosal surfaces are
found to be effective immunogens (De Aizpura, H. J. &
Russell-Jones, G. J. (1988) J. Exp. Med. 167, 440-451). The
observed differences in the immunogenicity of EtxB and its mutant
following oral administration may indeed be due to efficient uptake
of EtxB from the lumen of the gut. However, the dramatic
differences noted after parenteral immunization (where antigen is
delivered locally at high concentration) are suggestive of other
effects. For example, binding of EtxB to GM1 may affect the
efficiency of antigen presenting cell activity. Such binding could
cause activation of class II-bearing cells, particularly with
respect to their expression of essential co-stimulatory molecules,
such as B7, which is associated with their acquiring enhanced
antigen presenting activity (Jenkins, M. K. & Johnson, J. G.
(1993) Curr. Opin. Immunol. 5, 361-367). Alteratively, receptor
binding may have direct effects on sub-populations of lymphocytes.
A number of observations from this study provide strong evidence
that this is indeed the case.
[0254] The in vitro studies demonstrated that EtxB was able to
induce the proliferation of primed lymph node cells. This property
was not dependent on receptor binding, since responses with similar
anamnestic characteristics were obtained using either wild type
EtxB, EtxB(G33D) or heat-denatured monomeric forms of these
proteins which cannot bind GM1. These observations are interesting
in themselves since it has been widely reported that commercial
preparations of Ctx and CtxB or purified recombinant CtxB are
strongly inhibitory of lymphocyte proliferation in vitro. The
apparent discrepancy may have arisen from the fact that previous
experiments had been conducted on purified lymphocytes and had
largely used mitogen stimulated lymphocyte cultures (which are not
clonally restricted responses), where a different mechanism may be
involved. Consistent with this was our observation that the
proliferation of Con A-stimulated lymphocytes was indeed inhibited
by EtxB. However, the analyses of cell populations in cultures of
primed lymph node cells stimulated with either EtxB or EtxB(G33D)
revealed important differences with respect to B cells as well as
CD4 and CD8-bearing T-cells.
[0255] B cells were detected after 4 days of culture in the
presence of either EtxB or EtxB(G33D). However, by comparison with
EtxB(G33D), the relative proportion of B cells present in cultures
with EtxB was increased by approximately 100%. This increase was
associated with the expression of CD25 on a very high proportion of
the B cells. In the experiment shown, the responding lymphocytes
were primed with EtxB(G33D) in vivo. Similar experiments with cells
from EtxB immunized mice revealed comparable results. Thus,
irrespective of any in vivo effects associated with receptor
binding, cultures in the presence of EtxB contained a larger
proportion of B cells compared with those stimulated with
EtxB(G33D). These effects on B cells also appear not to be
dependent, at least in part, on regulation by T cells, in vitro, as
the results do not suggest a major shift in the profile of the
detected cytokines. Therefore, in vitro, receptor binding by EtxB
appears to be associated with a direct effect on B cells, resulting
in proportional expansion of this population as well as their
activation. It is also noteworthy that CtxB has been shown to
increase expression of MHC class II on virgin B cells, a property
which was not exhibited by a GM1 binding mutant CtxB (G33E)
(Francis, M. L., Ryan, J., Jobling, M. G., Holmes R. K., Moss, J,
& Mond J. J. (1992) J. Immmunol. 148, 1999-2005). The results
in these experiments suggest the presence of direct mitogenic
effects by EtxB on antigen-primed B cells and demonstrate that such
effects are mediated by receptor binding.
[0256] In addition to the effects of EtxB on B cells in culture,
flow cytometric analyses reveal that this toxoid caused the
complete depletion of any detectable CD8.sup.+ cells. Once again,
this effect was shown to be dependent on receptor binding, since
this population of T cells were not depleted in cultures containing
EtxB(G33D). Further, complete depletion of CD8.sup.+ cells in
cultures containing EtxB was observed, from mice immunized with
wild type EtxB. There are three possible mechanisms by which such
an effect may be mediated. 1) It is known that binding of Ctx or
CtxB to GM1 on rat MLN cells induces patch and cap formation (Craig
S. W. and Cuatrecasas P., (1975) Proc. Natl. Acad. Sci. USA,
Vol.72, pages 3844-3848). It is possible that in this process
EtxB-GM1 complexes and other molecules, including CD8, are
internalized. Such a process would prevent flow cytometric
detection of these cells using CD8 as a marker, and may result in
their death due to the associated loss of the surface TCR complex.
Although the latter may account for the absence of CD8.sup.+ T
cells in the culture, others found no loss of the TCR complex from
the surface of human Jarkat T cell line when CtxB was used
(Imboden, J. B., Shoback, D. M., Pattison, G, & Stobo, J. D.
(1986) Proc. Natl. Acad. Sci. USA 83, 5673-5677). Absence of
effects as a result of capping is supported by the finding that CD3
and CD4 markers were not affected. 2) An alterative mechanism would
involve effects exerted by cytokines in culture. In this study,
both IL-2 and IFN- were detected. The results, however do not
suggest a major shift in the cytokine profile which would explain
such a dramatic effect on CD8.sup.+ T cells. 3) Absence of
CD8.sup.+ T cells may be due to active induction of apoptosis.
Death of lymphocytes by apoptosis may involve capping as described
above, or could be mediated in the absence of capping by effects on
the signalling events in the cell. Activation-induced programmed
death is dependent on Ca.sup.2+ and involves phosphatases and
kinases. Binding of CtxB to lymphocytes has been shown to inhibit
protein kinase C-dependent proliferation and induced a pronounced
increase in intra-cellular Ca.sup.2+, events which were not
associated with an increase in CAMP level. The ability of EtxB to
deplete CD8.sup.+, but not CD4.sup.+ T cells could be due to
differential effects of signals associated with the CD4/CD8-TCR
complex, resulting from crosslinking GM1 on the surface of these
subset of lymphocytes. This could be as a result of differential
binding of the toxoid on the membrane as reported for CtxB or
alternatively to the differential signalling mechanisms in
CD4.sup.+ and CD8.sup.+ T cells.
[0257] In the complete absence of detectable CD8.sup.+ T cells,
EtxB increased the proportion of CD4.sup.+ T cells which were
activated, by comparison with the receptor binding mutant. The
essential requirement for CD4.sup.+ T cells in response to Ctx has
been demonstrated in vivo. The reason for the increased activation
of this T cell subset is, however, unclear. It is noteworthy that
CtxB has been shown to stimulate DNA synthesis and cell division in
quiescent non-transformed mouse 3T3 cells. A selective mitogenic
effect on CD4.sup.+ T cells was also found in the presence of plant
lectins which bind to Gal.beta.-1-3-3GalNAc, the same component
that EtxB binds to in GM1. The possibility can not be ruled out
that EtxB mediates a GM1-binding dependent direct effect on
CD4.sup.+ T cells, causing their activation. However, it is also
possible that the increased activation of CD4.sup.+ T cells in
cultures containing EtxB is a consequence of those changes to the B
cell and CD8.sup.+ T cells populations described. B cell activation
is known to be associated with an enhancement of their competency
as antigen presenting cells for CD4.sup.+ T cells. Further,
CD8.sup.+ T cells are widely associated with a regulatory role in
immune reactivity both in vivo and in vitro. Their removal from T
cell proliferative cultures has been associated with prolonged and
enhanced levels of CD4.sup.+ T cell division.
[0258] Taken together, the potent immunogenicity of EtxB in vivo,
as shown in this study, can be suggested to occur as a result of
its ability to increase activation of B cells exerted by growth
regulating effects following binding to GM1. Activation of
CD4.sup.+ T cells and the ability of EtxB to increase production of
IL-2 in culture in vitro may provide the necessary signal for
further expansion of B cell clones. Depletion of CD8.sup.+ T cells
by EtxB in vitro in this study may also provide another mechanism
of immunopotentiation in vivo following systemic or oral delivery,
particularly in the light of the involvement of this subset of
cells in suppression of the immune response and in oral tolerance.
In this regard, both Ctx and Etx have been shown to abrogate oral
tolerance to cofed soluble proteins and other studies implicated
Ctx and CtxB depletion effects on intra-epithelial lymphocytes in
the gut or in the dome of Peyer's patch to explain this mechanism.
CtxB-inhibitory effects on CD8.sup.+ T cells in vitro has also been
shown to prevent graft versus host reaction.
[0259] In conclusion, it has been demonstrated that the presence of
potent immunomodulatory effects by EtxB on the antibody response in
vivo, and on populations of lymphocytes in vitro. Furthermore, it
has been demonstrated that these effects are mediated by receptor
binding. Our findings are also pertinent to an understanding of the
ability of Etx and Ctx to act as potent adjuvants and as potential
protein carriers for other antigens and suggest that such
properties rely on the capacity of these toxoids to bind
ganglioside receptors on the surface of lymphoid cells.
Example 2
[0260] This example illustrates that the effects on CD8 cells are
irrespective of antigen recognition and are mediated by
apoptosis.
[0261] Recombinant preparations of EtxB and EtxB(G33D) were
prepared as in Example 1. Both proteins were well characterised
with respect to binding to GM1, binding to a panel of monoclonal
and polyclonal antibodies and various other physico-chemical
properties. Ovalbumin (OVA) was purchased from Sigma (Poole, UK).
Mesenteric lymph nodes (MLN) were isolated from BALB/c mice [high
responder strain to EtxB (Nashar, T. O. and Hirst, T. R. 1995.
Immunoregulatory role of H-2 and intra-H-2 alleles on antibody
responses to recombinant preparations of B-subunits of Escherichia
coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB).
Vaccine 13:803.)] 8-10 weeks old. Mice were injected i.p. with 200
g of OVA (Sigma) emulsified in incomplete Freund s adjuvant
(Sigma). MLN were removed 10 days after injection, minced through a
stainless steel mesh into HBSS (Flow, Irvine, UK). The recovered
cells were washed in HBSS by centrifugation (500 g, 10 min,
4.degree. C.) and resuspended in modified Eagle s medium (Flow)
containing 20 mM HEPES, 100 IU penicillin, 100 .mu.g/ml
streptomycin, 4 mM L-glutamine and 5.times.10.sup.-5 M
2-mercaptoethanol (complete medium) to which 0.5% (v/v) of fresh
autologous mouse serum was added. Cultures contained
2.times.10.sup.6 viable cells/ml in 2 ml volumes in 24-well plates
(Nunc, Roskide, Denmark) and were established in the presence of
100 .mu.g/ml OVA (dialysed extensively in complete medium), either
alone or with 40 g/ml of EtxB or EtxB(G33D). Cultures were
incubated at 37 C in 5% CO.sub.2 and 95% air for 5 days. At desired
time points, 0.1 ml samples were removed from the cultures and
transferred to 96 well U-bottomed plates (Nunc) and pulsed with 1
Ci/we 11 of [.sup.3H]thymidine (Amersham, UK) for 6 h before
harvesting (Mach III harvesting 96; Tomtec, Orange, Conn.) and
counting by standard liquid scintillation (1450 Micro b plus;
LKB-Wallac, Turku, Finland). For flow cytometric analysis (Becton
Dickinson, Erenbodegem-Aalst, Belgium) of T cells, cells were
stained with the following rat antibodies (PharMingen, Cambridge,
UK): FITC labelled anti-CD4 (RNRM4-5) or FITC-anti-CD8.alpha.
(53-6.7) and with biotin-labelled anti-CD25 (IL-2R.alpha.) (7D4)
followed by Streptavidin-phycoerythrin. Additionally, for the
biotin-labelled antibodies FITC-labelled Streptavidin was used.
FACS analysis of recovered cells was performed on the peak day of
proliferation (day 4), as determined by [.sup.3H]thymidine
incorporation.
[0262] For apoptosis assays, fresh MLN cells (MLNC) and splenic T
cells (SPLTC) were isolated from BALB/c mice, 8-10 weeks old. MLNC
comprising >90% CD3.sup.+ T cells, as determined by flow
cytometric analysis, were incubated for 2 h in petri dishes
(Costar, Cambridge, Mass.) in complete medium containing 10% FCS,
at 37.degree. C. in 5% CO.sub.2 and 95% air to remove adherent
cells. The non-adherent fraction was subsequently pipetted off,
pelleted and washed twice in HBSS before use. SPLTC were purified
by negative selection using glass beads coated with normal mouse
serum followed by rabbit anti-mouse .gamma.-globulins as described
(Wigzell, H. 1976. Specific affinity fractionation of lymphocytes
using glass or plastic bead columns. Scand. J. Immunol. 5:(suppl.5)
23.). The selected population of T cells were >90% CD3.sup.+ as
determined by flow cytometric analysis.
[0263] CD4.sup.+ and CD8.sup.+ T cells were separated as follows:
non-adherent MLNC were labelled with rat phycoerythrin-anti-mouse
CD4 (4708-02) or FITC-anti-mouse CD8 (53-6.7) (PharMingen) and were
then incubated with MACS colloidal super-paramagnetic microbeads
conjugated with goat anti-rat IgG (H+L) F(ab').sub.2 (PharMingen),
according to the manufacturer s instructions. These were applied to
mini-MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) in
order to separate both positively (>99% pure) and negatively
(>90% pure) selected populations of CD4 and CD8+ T cells, as
determined by flow cytometric analysis.
[0264] Two methods were used for quantification of apoptosis: i)
staining DNA with acridine orange to examine nuclear morphology
and, ii) cell cycle analysis following staining DNA with propidium
iodide and with either anti-CD4 or anti-CD8 antibodies. Cultures of
2.times.10.sup.6/ml MLNC, SPLTC and fractionated MLNC were
established in complete medium containing 10% FCS, in the absence
or presence of 80 .mu.g/ml of either EtxB or EtxB(G33D) and
examined from 4 to 18 h. Following incubation, cells were pelleted,
washed with HBSS and stained with 5 g/mlacridine orange (Sigma).
Thymocytes were isolated and treated in the absence or in the
presence of 10.sup.-7 M dexamethasone and used as a positive
control for cells undergoing apoptosis. Nuclear morphological
changes in lymphocytes were examined by conventional or confocal
fluorescence microscopy (Leica TCS 4D). The proportion of CD4.sup.+
and CD8.sup.+ SPLTC in the sub-G.sub.0/G.sub.1 stage of the cell
cycle was determined by flow cytometric analysis of the DNA content
following staining with propidium iodide as described (O'Connor, P.
M., Jackman, J., Jondle, D., Bhatia, K., Magrath, I. and Kohn, K.
W. 1993. Role of p53 tumor suppressor gene in cell cycle arrest and
radiosensitivity of Burkitt's lymphoma cell lines. Cancer.Res.
53:4776.). Cells isolated from 18 h cultures of SPLTC incubated
alone or with 40 g/m 1 EtxB or EtxB(G33D) were stained with FITC
rat anti-CD4 or FITC-anti-CD8.alpha.. Stained cells were adjusted
to 1.times.10.sup.6/ml in cold HBSS containing 20 mM HEPES and 0.5
mM EDTA and were fixed with cold ethanol added dropwise. Then, 50
g/m l propidium iodide and 40 g/mlribonuclease A (DNase free) were
added, and the cells incubated for 1 h at room temperature. The
relative intensity of DNA staining with propidium iodide in CD4 and
CD8.sup.+ T cells was determined by gating on cells co-stained with
each mAB.
[0265] In Example 1, the observation that CD8.sup.+ T cells are
completely depleted from cultures of lymph node cells proliferating
in response to EtxB suggested that EtxB exerts a polyclonal effect
on this T cell subset. To investigate whether such effects are
dependent on the activation of EtxB responsive cells, cultures were
established from OVA-primed mice and stimulated with OVA alone or
with OVA plus either EtxB or the mutant EtxB(G33D). Similar peak
levels of proliferation (day 4 of culture in each case) were
achieved in the presence of OVA alone, OVA plus EtxB or OVA plus
EtxB(G33D) (9734.+-.347, 12,031.+-.135 and 9305.+-.290 c.p.m.
respectively). However, there was a dramatic difference in the
distribution of T cell subsets in these cultures after 4 days (FIG.
6). All cultures contained CD4.sup.+ T cells of which similar
proportions co-expressed the activation marker CD25. However,
CD8.sup.+ T cells were undetectable in cultures incubated with OVA
plus EtxB, but were clearly present (although not activated as
assessed by CD25 expression) in cultures with OVA plus EtxB(G33D)
or OVA alone. This establishes that EtxB induces depletion of
CD8.sup.+ T cells responding to an antigen other than EtxB.
Moreover, the absence of such a response to EtxB(G33D) indicates
that depletion is triggered following toxoid receptor interaction.
It was also noted that the presence of wild-type EtxB caused a
significant increase in the proportion of B cells of which a large
number were CD25.sup.+ (not shown) as had previously been found for
EtxB responsive cultures (Example 1). It is therefore concluded
that receptor occupancy by EtxB exerts profound immunomodulatory
effects on lymphocytes irrespective of their antigen
specificity.
[0266] The possibility that CD8.sup.+ T cells undergo apoptosis
when cultured in the presence of EtxB was investigated. MLNC or
purified SPLTC, from unprimed mice, were incubated with EtxB or
EtxB(G33D) and changes in cell nuclear morphology after staining
with acridine orange were recorded over a period of 4 to 18 h
(Table 3 and FIG. 7). Cell morphological changes were characterized
by the presence of condensation of chromatin resulting in the
lobular appearence of the nucleus (FIG. 7). Other cell features
such as blebbing of the plasma membrane and the presence of
apoptotic bodies were also observed. These morphological changes
occurred in approximately one third of each of the cell
preparations treated with EtxB, whereas a much lower incidence was
observed in cells cultured with EtxB(G33D) or without exogenous
antigen (Table 3). Since CD8.sup.+ T cells accounted for
.about.35-40% of the MLNC and SPLTC preparations, depletion of
these cells could account for the observed apoptosis. To establish
if this was the case, populations of purified CD8 and CD4.sup.+ T
cells were cultured for 18 h in the presence of antigens (Table 3).
Similar percentages of morphological changes were induced in
negatively selected populations of CD4.sup.+ T cells (containing
>90% CD4-bearing cells) on treatment with either EtxB,
EtxB(G33D) or no antigen, indicating that binding of EtxB to its
receptor does not trigger apoptosis in this T cell subset. In
contrast, >70% of the negatively selected CD8.sup.+ T cells
(>90% pure) exhibited morphological changes when cultured with
wild-type EtxB; while incubation with either no antigen or
EtxB(G33D) caused changes in only 11-19% of this T cell population,
respectively. Further, the presence of low numbers of contaminating
cells in the purified populations used (.about.10% in each case)
could not account for the observed effects since more highly
purified populations containing >99% of CD8 or CD4.sup.+ T-cells
(isolated by positive selection) responded to EtxB in a similar
manner (60% were apoptotic in the presence of EtxB, compared with
7% for both no antigen and EtxB(G33D) treatments) (Table 3).
Apoptosis was detected in 40% and 98% of thymocytes after 18 h
incubation in the absence or in the presence of dexamethasone
respectively.
[0267] To demonstrate that the morphological changes observed in
our cultures were consistent with the induction of apoptosis, the
appearance of subdiploid DNA in cultures of SPLTC treated for 18 h
with EtxB was evaluated. Cells were subjected to flow cytomeric
analysis after co-staining with propidium iodide and either
anti-CD8 or anti-CD4 antibodies (FIG. 8). Approximately 48% of the
CD8.sup.+ T cells from cultures incubated with EtxB fell below the
diploid G.sub.0/G.sub.1 peak of propidium iodide staining,
indicating that they were undergoing apoptosis (O'Connor, P. M. et
al, supra). A small proportion of cells expressing CD4, in cultures
with EtxB, also exhibited sub-G.sub.0/G.sub.1 levels of DNA
(.about.11%; which may result from the death of such a high
proportion of CD8.sup.+ T cells). In contrast, the majority of CD4
or CD8.sup.+ T cells cultured without antigen or in the presence of
EtxB(G33D) were in G.sub.0/G.sub.1 phase of the cell cycle, with
<5% exhibiting apoptosis. We conclude that the observed nuclear
morphological changes, and the presence of sub-G.sub.0/G.sub.1
levels of DNA, in a substantial proportion of CD8.sup.+ T cells
treated with EtxB demonstrate a selective apoptosis triggered by
the cholera-like enterotoxoid. The failure of the receptor binding
mutant, EtxB(G33D), to induce similar effects demonstrates that the
induction of CD8.sup.+ T cell apoptosis is linked to its ability to
bind to GMI ganglioside.
Example 3
[0268] Groups of 8 male DBA/1 mice were either unchallenged (group
A) or were each injected with 100 g of bovine collagen in CFA on
day 0 byintra-dermal (i.d.) injection into the flank. Collagen
injected mice were either left unprotected (group B; positive
control) or attempts were made to prevent disease development by
the administration i.d. at an adjacent site to collagen challenge
of; 100 g ofEtxB in IFA on day 0 (group C), 100 g of EtxB in IFA on
day 14 (group D), or 100 gEtxB(G33D) in IFA on day 0 (group E). All
animals, except those in group A, received a boosting dose of
collagen in IFA i.d. on day 21, and disease severity was assessed
on day 45 by measuring hind limb ankle thickness (experiment A) or
scoring each hind limb digit for swelling (scale 0-3 where
0=normal, and 3=maximal swelling; experiment B).
[0269] The results obtained are illustrated in FIGS. 9a and 9b.
These show that EtxB, but not EtxB(G33D), dramatically protects
mice from the development of collagen-induced arthritis.
Example 4A
[0270] Two separate human buffy coat samples (obtained from normal
human blood donors) were used as a source of mononuclear cells.
Cells were isolated over Ficoll-paque and washed extensively before
culture in the absence of antigen or with 80 g/ml of either EtxB or
EtxB(G33D) as indicated. Prior to culture the cell populations
comprised 24% CD8+, 27% CD4+ and 27% CD8+, 22.9% CD4+ for each
sample respectively. After culture for 18 hours the appearance of
apoptotic cells was assessed in samples of cells stained with
acridine orange (as detailed under Example 2). The results obtained
are shown in FIG. 10; these illustrate that EtxB but not EtxB(G33D)
induces apoptosis in a population of normal human peripheral blood
mononuclear cells.
Example 4B
[0271] The murine T cell line, CTLL-2, was cultured to confluence
and then the cells washed before being reseeded at 1.times.10.sup.6
cells/ml in the absence of antigen or with 80 g/ml of either EtxB
or EtxB(G33D) as indicated. After 18 hours samples were removed and
the percentage of cells showing signs of apoptosis was assessed
using acridine orange (detailed under Example 2). The results
obtained are illustrated in FIG. 11. They show that cross-linking
of GM1 leads to apoptosis in a proportion of murine CTLL cells.
1TABLE 1 Lymphocyte proliferation in the presence of EtxB or EtxB
(G33D) Dose .mu.g/ml EtxB EtxB(G33D) EtxB* EtxB(G33D)* 0 117.9
146.8 (3.5) 124.5 116.1 (6.35) (7.9) (14.6) 5 4928 2860 2424 1431.5
(37.5) (98.7) (3.8) (88.3) 10 6978 3681 2518 4231 (30.6) (4.6)
(21.6) (96.4) 20 7084 6912 (47.3) 4394 5075 (100) (42.1) (24.8) 40
8844 (26) 8586 (143.7) 7431 4368.5 (118.9) (45.3) 80 10246 12510
(121.8) 7986 7276 (369.5) (30.7) (210.3) 160 11311 13525 (352.7) --
-- (247)
[0272] Mice were injected i.p. with 30 .mu.g of EtxB (G33D) in
complete Freund's adjuvant (CFA). Mesenteric lymph nodes were
isolated 10 days later. Cells were isolated and incubated for 4
days in the presence of EtxB, EtxB (G33D) or disassembled monomeric
forms of these proteins (*), generated by heating at 95.degree. C.
Proliferation was determined by addition of 1 .mu.Ci of (.sup.3H)
dThd for the last 6 hours on day 4. Data represents mean cpm and
SEM of triplicate wells. Cells isolated from unimmunized mice gave
<1500 cpm (dose 160 .mu.g/ml).
2TABLE 2 Cytokine analysis in the presence of EtxB or EtxB (G33D)
Protein IL-2 (pg/ml) IFN- (pg/ml) EtxB 318 2700 EtxB (G33D) 67
4068
[0273] Mice were injected with EtxB (G33D) in CFA and mesenteric
lymph nodes cells were isolated 10 days later. Cells were then
incubated in vitro with either EtxB or EtxB(G33D) and samples of
supernatants analysed for cytokine content on day 5 of cellular
proliferation.
3TABLE 3 EtxB-receptor mediated apoptosis in fractionated
lymphocyes. Cells Time (h) No antigen EtxB(G33D) EtxB MLNC 4
0.sup.a (8) 2 (0) 1 (3) 18 8 (10) 5 (18) 29 (35) SPLTC 4 3 (7) 2
(6) 3 (5) 18 17 (5) 16.5 (12) 31 (32) Negative selection CD4 18 5
(37) 6 (31) 9 (35) CD8 18 18 (11) 19 (15) 76 (73) Positive
selection CD4 18 6 4 6 CD8 18 7 7 60
[0274] Nuclear morphological changes in fractionated CD4 and
CD8.sup.+ T cells after 4 or 18 h incubation in the absence of
antigen, or with 80 .mu.g/ml of EtxB or EtxB(G33D) were examined by
fluorescence microscopy following staining with acridine orange.
Whole MLN were depleted of adherant cells. SPLTC were isolated by
negative selection in glass beads column coated with mouse
.gamma.-globulins and rabbit anti-mouse as a secondary antibody.
Fractionated SPLTC were obtained following labelling with rat
phycoerythrin-anti-mouse CD4 or FITC-anti-mouse CD8.alpha. which
were then incubated with MACS colloidal super-paramagnetic
microbeads conjugated with goat anti-rat IgG (H+L) F(ab).sub.2.
These were separated using mini-MACS columns to obtain both the
positively (>99% pure) and negatively (>90% pure) selected
fractions of CD4 and CD8.sup.+ T cells. Nuclear morphological
changes were examined from 4 to 18 h in a random sample of 200
cells per treatment as described in the legend to FIG. 7. Maximum
percentage of apoptotic cells occurred after 18 h. The data in
brackets when indicated represent results from another separate
experiment. Data for MLN and SPLTC are representative of a total of
four experiments.
Examples 8-12
[0275] Relating to Diabetes
[0276] Insulin dependent diabetes mellitus (IDDM) is an autoimmune
disease resulting form the T-cell dependent destruction of
insulin-producing cells from the pancreas Langerhans islets (1). It
affects about 4 million people in Europe and North America alone
and usually appears before the age of 30. There is no cure.
Sufferers must give themselves daily insulin injections to control
their blood glucose levels. It is unclear what triggers the immune
system's attack on the islet cells because the regulation of the
auto-aggressive immune response is complex, resulting from the
interaction between several T cell subsets and their activation of
mononuclear phagocytes. Islet destruction, both in humans and
rodents, is attributed to the existence of auto-reactive CD4+T
cells that recognise islet antigens and belong to the Th1 subset
(i.e. secrete inflammatory cytokines such as IFN.gamma.) (2). Such
cells could be isolated from diabetic rodent spleens or pancreas
inflammatory infiltrates and transferred the disease to syngenic
receipents (3).
[0277] Research Design and Methods
[0278] Mice.
[0279] NOD mice were purchased from Jackson Laboratories (Bar
Harbor, USA) and were subsequently bred in our animal facilities
under sterile conditions. Spontaneous diabetes in female mice from
our colony appears around 12 weeks of age and reaches 80-90% by 30
weeks. Diabetes was characterised by weight loss and glucosuria
levels above 111 mmol/l on three consecutive measurements one week
apart. Glucosuria was measured using Diastix(.RTM. strips from
Bayer (Newbury, UK).
[0280] Proteins.
[0281] EtxB was produced by expression in a marine Vibrio followed
by purification from the culture supernatant. Insulin, purified
from porcine pancreas (Sigma, Poole, UK) was dissolved in
phosphate-buffered saline (pH 7.4) and admixed with EtxB
immediately before administration.
[0282] Nasal Administration Protocol.
[0283] Female NOD mice were given 6 doses of the treatments
described on alternate days, over two weeks. Briefly, light
anaesthesia was induced by inhalation using Halothane-RM*
(Rhone-Merieux, Harlow, LK), the 201 dose was placed on the tip of
the nose and was taken up with the respiratory movements.
[0284] Cytokine Secretion Assessment.
[0285] IFN.gamma., IL-4 and IL-10 secretion by activated cells from
the pancreatic lymph nodes was measured by cellular sandwich
ELISAs. Briefly, a single cell suspension
(2.times.10.sup.6/ml/well) from the pancreatic lymph nodes was
cultured for 48h on mouse anti-CD3-coated (clone 7D6, 10 .mu.g/ml)
24-well plates. MaxiSorp.TM. ELISA plates from Nunc (Roskilde,
Denmark) were coated overnight at 4.degree. C. with capture
antibody, then lymphocyte suspension was transferred to the ELISA
plate and further incubated for 24 h. The plates were then washed
and captured cytokines were detected using biotinylated
anti-cytokine detection antibodies and the streptavidin-peroxidase
detection system (3).
[0286] Diabetes Adoptive Transfer.
[0287] 5 6-week old female NOD mice were given 6 doses of 10 ug
insulin+10 ug EtxB in 20 ul PBS or PBS only (on alternate days,
over two weeks). One day after the last treatment, spleens were
collected, pooled and CD4+ T lymphocytes were isolated using CD4
(L3T4) MicroBeads from Miltenyi Biotec (Bisly, UK) according to the
manufacturer's recommendations. 5.times.10.sup.6 CD4+ purified T
cells from either Insulin+EtxB or PBS only-treated mice were then
mixed with an equal number of spleen cells from diabetic NOD female
mice and injected intravenously to 8 week-old, 7,5
Gy-pre-irradiated female NOD mice. Glucosuria was then assessed
every other day using Diastix.RTM. strips and mice were considered
as diabetic after four consecutive measurements showed glucosuria
levels above 111 mmol/l.
[0288] Insulitis Assessment.
[0289] Insulitis was assessed by histology. Pancreases were
collected, fixed in neutral buffered formalin, embedded in
paraffin, cut and stained with hematoxylin and eosin. Slides were
viewed by light microscopy and 10-30 islets from at least two
sections 100 um apart from each mouse were scored in a double
blinded fashion. Based on the severity of insulitis, each islet was
scored from 1 to 5 as follows: 1=free of insulitis;
2=peri-insulitis; 3=moderate insulitis (less than 50% of the islet
is infiltrated); 4=severe insulitis (more than 50% of the islet is
infiltrated) and 5=complete islet destruction with very few islet
cells visible.
Example 5
[0290] Repeated nasal or oral administration of relatively high
doses of insulin was shown to induce a regulatory CD4+ T cell
population that prevents diabetes mellitus (4).
[0291] Results.
[0292] Surprisingly, the present invention demonstrates that:
[0293] (i) when a sub-optimal insulin administration protocol was
used (6.times.10 ug doses on alternate days over 2 weeks) NOD mice
were not protected;
[0294] (ii) when similar doses of EtxB, were administered i.n.
according to the same schedule, this did not prevent the
development of IDDM in the NOD mice;
[0295] (iii) however, admixed insulin+EtxB did lead to a decreased
incidence of IDDM (FIG. 23).
[0296] Summary.
[0297] Intranasal administration of admixed insulin and EtxB to 6
week old NOD mice prevented the onset of diabetes mellitus.
Example 6
[0298] Islet-reactive T cells localise preferentially in the
pancreatic lymph nodes (PLN), so autoreactive immune responses and
their type are best investigated by assessing the cytokine
secretion of PLN lymphocytes. We collected PLN from the NOD mice
after the described treatment with insulin or EtxB or an admixture
of these and investigated cytokine secretion from lymphocytes after
activation with anti-CD3 as described (5).
[0299] Results.
[0300] We found a decrease of IFN.gamma. secretion and an increase
of IL-4 and IL-10 in the insulin+EtxB-treated mice when compared
with those left untreated or treated with either insulin or EtxB
alone (FIG. 24) while TGF.beta. secretion was not influenced by any
treatment (data not shown).
[0301] Summary.
[0302] Pancreatic lymph node cells from NOD mice protected against
diabetes mellitus by insulin+EtxB treatment secrete less IFN.gamma.
and more IL-4 and IL-10 than the unprotected ones.
Example 7
[0303] In order to assess the effect of insulin+EtxB-treatment upon
islet infiltration, we treated the mice as previously described,
then collected the pancreas when mice (n=3 per time point) were 6,
12, 18 and 24 weeks of age.
[0304] Results.
[0305] Histological examination of hematoxylin-eosin-stained
sections showed a lower degree of inflammatory infiltrate in the
insulin+EtxB-treated mice when compared with those left untreated
or treated with either insulin or EtxB alone (FIG. 25).
[0306] Summary.
[0307] Protection against diabetes mellitus is associated with a
lower degree of insulitis.
Example 8
[0308] In order to investigate whether the effect of insulin+EtxB
leads to a long-lasting protection mediated by regulatory cells, we
co-transferred splenocytes from recently diabetic mice with equal
numbers of CD4+ cells from mice that were either treated with
insulin+EtxB or left untreated to 7.5 Gy-irradiated mice.
[0309] Results.
[0310] CD4+ T cells from the untreated mice could not prevent the
rapid development of IDDM induced by the autoreactive cells from
the recently diabetic ones. Conversely, insulin+EtxB leads to the
development of a CD4+ regulatory cell population that prevents the
development of IDDM. Long-term assessment data (FIG. 24) supports
this finding, suggesting that protection is long-term and does not
represent a mere delay of the disease.
[0311] Summary.
[0312] Nasal treatment with insulin+EtxB generates regulatory CD4+
cells that transfer protection.
Example 9
[0313] Thirty days after cell transfer (when all the mice that
received splenocytes from diabetic mice+CD4+ cells from untreated
mice have developed IDDM), their pancreases were collected and
their insulitis degree was assessed by histology. PLN were also
collected and cytokine secretion from activated lymphocytes was
assessed.
[0314] Results.
[0315] The data obtained (Table 4-FIG. 29) shows that the
transferred regulatory cells from the insulin+EtxB-treated mice
lead to a decrease of IFN.gamma. secretion and an increase of IL-4
and IL-10 and a decrease of insulitis grade.
[0316] Summary.
[0317] Transferred protection is associated with a lower degree of
insulitis and a Th2-skewed cytokine secretion profile.
Example 10
[0318] The finding that EtxB was not able to prevent diabetes in
the NOD mouse when given in the absence of added insulin at 6 weeks
of age, contrasted with our findings from models of arthritis. In
CIA, as little as 1 .mu.g of EtxB given on four occasions was
sufficient to block the progression of disease. We hypothesised
that the timing of administration may be key to the differences
observed between the models. It is conceivable that EtxB triggers
the activation of regulatory cell populations which are only active
over a relatively short period of time. In order to test this, we
delayed treatment of NOD mice until they had reached 10-12 weeks of
age. By this time, peri-insulitis is well established and the
immune response to islet antigens has been induced.
[0319] Results.
[0320] Our findings showed that 6 treatments with 10 .mu.g EtxB
i.n. (a dose which was ineffective when given at 6 weeks of age)
was now able to dramatically reduce the incidence of IDDM (FIG.
27). This suggests that EtxB can effectively block diabetes when
given in the absence of autoantigen, but that pancreatic
inflammation needs to be established in order for it to do so.
[0321] Summary.
[0322] Once islet inflammation is already established, EtxB is able
to prevent IDDM in the NOD mouse when given alone.
Example 11
[0323] The prevention of IDDM following administration of EtxB
admixed with insulin at 6 weeks, was associated with a clear Th1 to
Th2 switch in the cytokine profile of pancreatic T cells responding
to TCR engagement. In order to determine whether prevention of IDDM
following the late administration of EtxB alone was associated with
a similar shift, we carried out an identical assessment of
pancreatic lymph node cell function.
[0324] Results.
[0325] The results revealed a marked difference from those reported
above. Whereas the late administration of EtxB led to a dramatic
reduction in the production of Th1-associated .gamma.IFN as was
seen with earlier treatment with EtxB+insulin, this was not
associated with a concomitant rise in Th2 cytokine secretion.
Instead, the levels of IL-4 were unchanged and IL-10 was not
detected either in lymph node cell cultures from treated or
untreated animals (FIG. 28). This findings suggests that the
mechanisms of IDDM prevention by EtxB alone and EtxB+insulin may be
different. In the latter case EtxB is acting as an `adjuvant`
promoting immune deviation away from Th1 and toward Th2. In the
former case, the Th1 response is being suppressed without promotion
of a Th2 response.
[0326] Summary.
[0327] Altered pancreatic lymph node cytokine production in
response to late administration of EtxB alone.
Example 12
[0328] The effectiveness of the B-subunit in treating autoimmune
diabetes has been established using the NOD mouse model in which
disease arises spontaneously at between 14 and 25 weeks of age. As
in human type I diabetes, disease in the NOD is influenced by
complex genetic and environmental factors which allow the
development if an immune response to several pancreatic antigens.
The mice develop a non-specific insulitis (immune infiltration of
the pancreas) at between 6 and 8 weeks of age, and this leads to
progressive islet destruction such that 70 to 80% become diabetic.
Diabetes is readily demonstrated by the presence of high glucose
levels in the urine or blood.
[0329] Results
[0330] The present inventors have demonstrated that injection of
the B-subunit into NOD mice during the time at which insulitis is
becoming established can block the progression to diabetes,
preserving insulin levels and reducing islet destruction (FIG.
20).
[0331] Summary
[0332] The present invention demonstrates that as a result of its
ability to bind to receptors on mammalian cells, EtxB is able to
modulate the function of a variety of cells involved in the
induction and maintenance of immune responses. This activity allows
it to subtly alter local micro-environmental conditions which can
dramatically change the nature of immunity, and forms the basis of
its usefulness in treating autoimmune diseases, such as diabetes
and infectious diseases. The use of the B subunit is advantageous
because it is a protein which is readily produced in batch
fermentation cultures of bacteria, is exceptionally thermo-stable
and acid stable and can withstand lyophilisation and long term
storage. In addition, the use of pure recombinantly produced
B-subunit, which is devoid of A-subunit is also advantageous
because it has been shown to be completely unable to cause any
toxic effect in humans.
[0333] Given the effectiveness of the B-subunit in treating both
arthritis and diabetes, EtxB may prove to be similarly effective in
blocking other Th1 mediated autoimmune diseases such as multiple
sclerosis and inflammatory bowel disease. The disease protection
experiments described have been carried out in animal models to
date. These models are well characterised and closely mimic the
human counterpart diseases. In addition, a large body of data
indicates that human immune cells are modulated by EtxB in vitro in
ways identical to those noted in the animal experiments. Taken
together these data provide a firm basis for the use of EtxB in
treating human disease.
[0334] 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 chemistry or biology or related fields
are intended to be covered by the present invention. All
publications mentioned in the this specification are hereby
incorporated herein in their entireties by reference.
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[0340] The above description is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
invention, and is not intended to detail all thos obvious
modifications and variations of which will become apparent to the
skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention, which is
defined by the following claims. The claims are intended to cover
the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context
specifically indicates the contrary.
* * * * *