Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-1-
A METHOD OF PROPHYLAXIS AND TREATMENT
FIELD OF THE INVENTION
The present invention relates generally to a method of prophylaxis and
treatment of
autoimmune disease conditions and agents useful for same. In a related
embodiment, the
present invention contemplates the use of mucosal antigens and/or agents
capable of
blocking or otherwise delaying cytotoxic T-lymphocyte (CTL) induction and/or
maturation
to prevent or at least reduce the likelihood or risk of CTL-mediated
autoimmune disease.
More particularly, the present invention contemplates mucosa-mediated
tolerance to
protect against or ameliorate the symptoms associated with autoimmune
pathology. Even
more particularly, the present invention provides a method for preventing
clinical insulin
dependent diabetes mellitus (>DDM) or preventing or reducing or ameliorating
the effects
of clinical >DDM by the aerosol administration of >DDM-associated autoantigens
to
mucosal surfaces or agents which block CTL induction and/or maturation.
BACKGROUND OF THE INVENTION
Reference to any prior art in this specification is not, and should not be
taken as, an
acknowledgment or any form of suggestion that this prior art forms part of the
common
general knowledge in Australia or any other country.
Bibliographic details of the publications numerically referred to in this
specification are
collected at the end of the description.
The increasing knowledge of the immune system in general and cellular immune
mechanisms in particular is greatly facilitating the design of therapeutic
agents and
alternative routes of their administration. One important area of research is
the mechanisms
underlying cellular immune hypo-responsiveness induced by particular antigens
in
autoimmune disease conditions.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-2-
An autoantigen can be assumed to be pathogenic if its administration modifies
the natural
history of autoimmune disease. Autoantigen-specific strategies of immune
tolerance
induction have been shown to favourably modify the natural history of
experimental
autoimmune disease in rodents (1-6). The presentation of soluble protein
antigen to
S mucosal surfaces, classically via the oral route, results in selective
suppression of antigen-
specific T cell-mediated, delayed-type hypersensitivity (DTH) and IgE
responses (1,7,8).
"Oral tolerance" has been associated with deviation of immunity away from T-
cell (Thl)
to antibody (Th2) responses, with the induction of regulatory T cells and, at
higher antigen
doses, with both T-cell anergy and T-cell deletion (1,9).
A particularly debilitating autoimmune condition is insulin-dependent diabetes
mellitus
(IDDM) which results from the selective destruction of insulin-producing (3
cells in the
islets of the pancreas, within an autoimmune inflammatory "insulitis" lesion
(10,11). The
primary role of autoreactive T cells in mediating (3-cell destruction has been
shown directly
in two spontaneous animal models of )DDM, the Bio-Breeding (BB) (12) rat and
the non-
obese diabetic (NOD) mouse (2). Target autoantigens that trigger or drive
immune
reactivity to (3 cells not only have diagnostic applications but are potential
agents for
specific immunotherapy (3-6). Several potentially pathogenic islet/(3-cell
autoantigens have
been identified by their reactivity with circulating antibodies or T cells in
rodents and
humans with sub-clinical or clinical >DDM, in particular insulin, glutamic
acid
decarboxylase (GAD) and tyrosine phosphatases of the IA-2 family (13).
However, insulin
and its precursor, pre-proinsulin, are the only IDDM autoantigens that are (3-
cell specific.
In humans, insulin autoantibodies (IAA) are a risk marker for the development
of clinical
IDDM (14) and have been detected before autoantibodies to other islet antigens
in the
offspring of diabetic mothers (15). Increased proliferation of peripheral
blood T cells to
human insulin can be demonstrated in up to half of sub-clinical and recently-
diagnosed
)DDM subjects (16), but responses are relatively low. This is possibly because
the
dominant human T-cell epitope is in proinsulin. A peptide that spans the
natural cleavage
site between the B chain of insulin and the connecting (C) peptide in
proinsulin was
reported to elicit T-cell proliferation in a majority of at-risk Il7DM
relatives (17). In the
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-3-
NOD mouse, IAA are reported to be a risk marker for the development of
diabetes (18)
and the majority of T-cell clones generated from the insulitis lesion reacted
to insulin B-
chain, amino acids 9-23 (19).
Several studies have evaluated mucosa-mediated tolerance to insulin in the NOD
mouse
model. For example, Zhang et al. (20) found that oral porcine insulin (1 mg
twice weekly)
delayed the onset and reduced the incidence of diabetes, and was associated
with splenic T
cells that partially blocked the transfer of diabetes to young, non-diabetic
mice by spleen
cells from diabetic mice. Subsequently, Bergerot et al. (21) reported that the
regulatory
cells induced by oral insulin were CD4+ T cells. However, in earlier studies
of oral
tolerance to guinea pig myelin basic protein (MBP) in the Lewis rat model of
experimental
autoimmune encephalomyelitis (EAE) (1), both CD4 and CD8 regulatory T cells
that
secrete IL-4, IL-10 and TGF-(3 were described.
There is a need to develop effective administration strategies for delivery of
antigens to
induce suppression of cell-mediated autoimmune conditions. The administration
strategies
must not only to be immunologically effective but also convenient, direct and
safe. In work
leading up to the present invention, the inventors investigated the aerosol
inhalation and
intranasal administration of insulin and its precursor (proinsulin) in an
animal model of
spontaneous IDDM, the non-obese diabetic (NOD) mouse, and showed that this was
effective in reducing pancreatic islet pathology and incidence of diabetes.
Furthermore,
aerosol insulin induced regulatory CD8y8 T cells which contributed to the
prevention of
diabetes. As an alternative strategy or a strategy which is capable of being
uses in
combination with the aforesaid approach, the present invention further
provides for
blocking or otherwise delaying CTL induction and/or maturation by, for
example, blocking
interaction between CD40 and CD40 ligand (CD40L).
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-4-
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word
"comprise",
or variations such as "comprises" or "comprising", will be understood to imply
the
inclusion of a stated element or integer or group of elements or integers but
not the
exclusion of any other element or integer or group of elements or integers.
One aspect of the present invention contemplates a method of inducing immune
tolerance,
including cytotoxic T-lymphocyte (CTL) tolerance, while substantially avoiding
CTL
immunity in response to a mucosal antigen, said method comprising selecting
said mucosal
antigen or modifying a mucosal antigen to disable the function of an MHC class
I
restricted epitope and then administering said selected or modified antigen
for a time and
under conditions sufficient to prevent or reduce CTL immunity to said mucosal
antigen.
Another aspect of the present invention contemplates a method of suppressing a
cell-
mediated autoimmune disease while substantially avoiding CTL immunity in
response to a
mucosal autoantigen, said method comprising selecting an autoantigen or
modifying said
mucosal autoantigen to disable the function of an MHC class I restricted
epitope and then
administering said selected or modified autoantigen for a time and under
conditions
sufficient to induce tolerance but prevent or reduce CTL immunity to said
autoantigen.
A further aspect of the present invention contemplates a method of suppressing
a cell-
mediated autoimmune disease in a subject, said method comprising the
administration as
an aerosol of an effective amount of an antigen associated with said
autoimmune disease
for a time and under conditions sufficient to prevent, reduce or otherwise
ameliorate
autoimmune pathology wherein said antigen substantially lacks a functional MHC
class I
interacting region.
Yet another aspect of the present invention provides a method of preventing,
reducing or
otherwise ameliorating an autoimmune disease condition in a subject, said
method
comprising the aerosol administration to said subject of an effective amount
of an antigen
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-5-
associated with said autoimmune disease for a time and under conditions
sufficient to
induce or stimulate immunoregulatory mechanisms which are protective against
cell-
mediated autoimmune pathology wherein said antigen substantially lacks a MHC
class I
interacting region.
Even yet another aspect of the present invention contemplates a method of
preventing,
reducing or otherwise ameliorating IDDM, slowly progressive (SP) IDDM or
gestational
type 1 diabetes in a subject, said method comprising the administration, as an
aerosol or
other functionally equivalent means, to said subject of an effective amount of
an
autoantigen associated with >DDM for a time and under conditions sufficient
for induction
of regulatory T cells and/or other suitable mechanisms sufficient to suppress
cell-mediated
autoimmune pathology associated with IDDM wherein said autoantigen
substantially lacks
a functional MHC class I interacting epitope.
Still another aspect of the present invention contemplates a method of
inducing,
suppressing or otherwise ameliorating or preventing IDDM in a subject, said
method
comprising administering proinsulin peptide truncated at its C-terminal
antigen end to
disable the function of any MHC class I restricted epitope for a time and
under conditions
sufficient to prevent or reduce CTL immunity and. otherwise induce immune
tolerance,
including CTL tolerance.
Still yet another aspect of the present invention provides a composition
comprising an
antigen associated with an autoimmune disease in an aerosol formulation
including one or
more pharmaceutically acceptable carriers and/or diluents.
Another aspect of the present invention contemplates a method of inducing CTL
tolerance
while substantially avoiding CTL immunity in response to a mucosal antigen,
said method
comprising administering to a subject a nucleic acid molecule or analogue
thereof
encoding said mucosal antigen but wherein said antigen substantially lacks a
functional
MHC class I restricted epitope for a time and under conditions sufficient to
prevent or
reduce induction of CTL immunity.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-6-
A further aspect of the present invention contemplates a method of preventing
or
suppressing IDDM while substantially avoiding CTL immunity, said method
comprising
administering to a subject a nucleic acid molecule or analogue thereof
encoding an )DDM-
S associated mucosal antigen which substantially lacks a functional MHC class
I restricted
epitope for a time and under conditions sufficient to prevent or reduce the
effects of
>DDM.
Another aspect of the present invention, therefore, contemplates a method of
inducing
tolerance to a mucosal antigen while substantially avoiding CTL immunity to
said antigen,
said method comprising administering said mucosal antigen or a nucleic acid
encoding
same for a time and under conditions sufficient to prevent or reduce CTL
immunity,
before, simultaneously or sequentially with the administration of an
antagonist of CTL
induction and/or maturation.
More particularly, the present invention contemplates a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
comprising administering said mucosal antigen or a nucleic acid encoding same
for a time
and under conditions sufficient to prevent or reduce CTL immunity, before,
simultaneously
or sequentially with the administration of an antagonist of CD40L-CD4
interaction.
Even another aspect of the present invention provides a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
comprising administering an agent for a time and under conditions sufficient
to block or
otherwise delay CTL induction and/or maturation.
More particularly, the present invention provides a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
comprising administering an agent for a time and under conditions sufficient
to block or
otherwise delay CTL induction and/or maturation wherein said agent blocks or
otherwise
disrupts CD40-CD40L interaction.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
Another aspect of the present invention contemplates a use of a mucosal
antigen with an
inactive MHC class I epitope in the manufacture of a medicament for the
treatment or
prophylaxis of a disease condition in a subject.
Yet another aspect of the present invention contemplates the use of an agent
in the
manufacture of a medicament for the treatment or prophylaxis of a disease
condition, said
agent capable of blocking CTL induction and/or maturation.
The present invention further provides for the use in combination of any of
the
methodologies contemplated above.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
_g_
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation showing that intranasal human insulin
(80 fig) at 56
days of age delays diabetes onset in female NOD mice.
Figure 2 is a graphical representation showing that intranasal human
proinsulin (40 pg) at
56 days of age delays diabetes onset.
Figure 3 is a graphical representation showing that intranasal human
proinsulin aa24-36
(40 pg at 56 days of age) delays diabetes onset.
Figure 4 is a graphical representation showing that aerosol insulin induces
CD8 T cells
that suppress transfer of diabetes. NOD male mice (n=16/group) aged 6-9 weeks
were
injected with pooled splenocytes from recently-diabetic 14-19 week old
females, together
with either unfractionated (A) or fractionated (B-E) splenocytes from aerosol
insulin- or
ovalbumin-treated NOD females, and their incidence of diabetes subsequently
monitored.
In the experiment shown, aerosol donor mice had been treated for 10
consecutive days and
then weekly from 49 days of age and were normoglycemic when sacrificed at 156
days of
age.
Figure 5 is a graphical representation showing that aerosol insulin induces
CD8 y8 T cells
that suppress transfer of diabetes. Young male NOD mice were co-injected with
"diabetic"
splenocytes (2 x 107) and total or fractionated splenic T cells from aerosol-
treated mice, as
in the legend to Figure 4. The numbers of fractionated cells injected were, in
A) ~10~ total
T cells and, from aerosol insulin mice, ~10~ y8-depleted T cells or 1.4 x 105
y8T cells and,
in B), from aerosol insulin mice, 107 total T cells, 2 x 106 CD8 T cells, 2 x
106 y8-
depleted CD8 T cells or 1.5 x 105 CD8 y8+ve T cells.
Figure 6 is a diagrammatical representation showing that adoptive transfer of
diabetes is
suppressed by CD8 y8 T cells induced by aerosol insulin: summary of 11
experiments.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
_g_
Figure 7 is a graphical representation showing that intranasal human
proinsulin aa24-36
(40 pg at 56 days of age) induces CD4 T cells that suppress adoptive transfer
of diabetes,
in NOD mice.
Figure 8 is a graphical representation showing that mouse proinsulin aa26-34
and to a
greater extent mouse proinsulin aa25-34 bind to the MHC class I molecule Kd.
In this
assay, expression of Kd on the surface of RMA-S cells is monitored by binding
of a
monoclonal anti-Ka antibody detected by fluorescence in a flow cytometer.
Addition of a
Kd-binding peptide to the cells stabilizes Kd and increases its expression on
the cell
surface, indicated by a right shift in the signal response. Isotype control =
control
monoclonal antibody; no peptide = constitutive Kd expression; HAP and LLO =
peptides
from 'flu hemagglutinin and Listeria known to bind to Kd and used as positive
controls.
Figure 9 is a graphical representation showing that mouse proinsulin aa25-34
induces Kd-
restricted cytotoxic T lymphocytes (CTL) in NOD mice. Six week old female mice
were
immunised subcutaneously with 50 pg of peptide in Complete Freund's Adjuvant.
After 14
days their spleens were removed and splenocytes re-stimulated in vitro with 10
pg/ml
peptide for 6 days. Splenocytes were then tested for CTL activity against 5'Cr
and peptide-
loaded RMA-S target cells.
Figures 10A and , lOB are diagrammatic representations showing that C-terminal
truncations enhance the effect of intranasal proinsulin B-C peptide to
suppress diabetes.
Figure 11 is a diagrammatic representation showing postulated mechanisms of (3-
cell
destruction in type 1 diabetes, including the role of CD40L-CD40 interaction
in activating
CD8 T cells to become CTLs.
Figure 12 is a graphical representation showing (A) systemic and (B) oral
priming of CTL
require CD40L signalling. (A) A single i.p. injection of 250 ~g of control mAb
6C8 (open
squares) or anti-CD40L mAb MRl (open circles) was given to C57B1/6 mice one
day
before challenge with 20 x 106 i.v. OVA-coated H-2Kbm-' splenocytes to prime
CTL. After
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-10-
14 days mice were killed and their splenocytes tested for CTL activity,
expressed as OVA-
specific lysis for representative individual mice. (B) The same doses of 6C8
or MR1 were
given to mice that were then fed 20 mg OVA on three alternate days. After 14
days,
without further priming, mice were killed and their splenocytes tested for CTL
activity,
this time expressed as lytic units per spleen for individual mice
(n=12/group).
Figure 13 is a graphical representation showing activation and expansion of
OVA-specific
CTL by oral OVA requires CD40L. C57B1/6 recipient mice congenic for Ly5.1 were
adoptively transferred with 3 x 10~ transgenic OT-I cells (Ly5.2) and then
given control
mAb 6C8 or anti-CD40L mAb MR1, 250 pg i.p. Mice from each treatment group were
then divided into two groups and fed either PBS or 20 mg OVA in PBS on three
alternate
days. mAb treatment was repeated before the third feeding. Mice were killed 14
days from
the start of feeding and the numbers and phenotype of OT-I cells in their
spleens analyzed
by flow cytometry. (A) Dot-plots of individual mice show CD44 (left) and CD62L
(L-
selectin) (right) expression on OT-I cells. The % of cells expressing a high
level of CD44
or a low level of CD62L is shown in the corresponding quadrant. The number of
OT-I cells
per spleen (B) CD44 expression (C) and % CD62L~° (D) OT-I cells in
individual recipient
mice treated with 6C8 or MRl and then fed PBS or OVA are shown for a single
experiment, but similar results were obtained three experiments.
Figure 14 is a graphical representation showing anti-CD40L treatment prevents
induction
of diabetes by oral OVA in RIP-OVA~° mice. RIP-OVA~° mice
bearing OT-I and OT-II
cells were injected with control mAb 6C8 or anti-CD40L mAb MR1, 250 ~g i.p. To
mimic
low-dose oral tolerance regimens, mice were then fed OVA, 0.5 mg on five
alternate days.
Blood glucose was measured 12 days after the start of feeding and values above
13 mmol/1
were considered diagnostic of diabetes. Data are pooled from two experiments.
Figure 15 is a graphical representation showing anti-CD40L treatment does not
limit oral
tolerance to systemic priming of CTL. CTL activity in response to i.v. priming
with OVA-
coated splenocytes (A) or to s.c. priming with OVA in CFA (B) is similarly
attenuated by
oral OVA in mice treated with control mAb 6C8 and anti-CD40L mAb MR1. Mice
were
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-11-
injected with 6C8 or MR1, 250 ~g i.p. and then fed either PBS (black squares)
or 20 mg
OVA in PBS (open circles) on three alternate days. After 14 days or 21 days
(not shown),
mice were primed systemically and seven days later killed and their
splenocytes recovered
for a standard in vitro S~Cr release assay of CTL activity. Primed splenocytes
as effectors
(E) were tested against SICr-loaded cells as targets (T). Each plot represents
an individual
mouse. CTL activity plots for individual mice were converted into lytic units
from four
experiments (C) after priming as in (A) and from two experiments (D) after
priming as in
(B), in which mice received either PBS or 20 mg oral OVA on three alternate
days (C) or
PBS or 0.5 mg oral OVA on five alternate days (D).
Figure 16 is a graphical representation showing anti-CD40L treatment does not
limit oral
tolerance to systemic priming of T-cell proliferation (A) and IFN-y (B)
responses, or
antibody production (C). Mice (n=3/group) were injected with control mAb 6C8
or anti-
CD40L mAb MR1 250 ~g i.p. They were then fed either PBS or 20 mg OVA in PBS on
three alternate days. After seven days, mice were immunized s.c. with OVA (0.1
mg) in
CFA in the base of tail. Ten days later, spleens and inguinal lymph nodes and
sera were
harvested for measurement of T-cell proliferation and cytokine production in
the absence
(solid) or presence (hatched) of O.lmg/ml OVA (mean and standard deviation
shown for
spleen), and anti-OVA antibodies, as described in Methods.
Figure 17 is a graphical representation showing the effect of treatment with
anti-CD40L
monoclonal antibody (MR-1) on diabetes incidence in NOD mice given aerosol
insulin.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-12-
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated on the surprising discovery that insulin
or its precursor
could be used to induce immune tolerance.
Accordingly, one aspect of the present invention contemplates a method of
inducing
immune tolerance while substantially avoiding CTL immunity in response to a
mucosal
antigen in a subject, said method comprising selecting said mucosal antigen or
modifying a
mucosal antigen to disable the function of an MHC class I restricted epitope
and then
administering said selected or modified antigen for a time and under
conditions sufficient
to prevent or reduce CTL immunity to said mucosal antigen.
Generally, the mucosal antigen is used for preventing a CTL-mediated
autoimmune
disease such as but not limited to diabetes and in particular, IDDM.
Accordingly, another aspect of the present invention contemplates a method of
suppressing
a cell-mediated autoimmune disease while substantially avoiding CTL immunity
in
response to a mucosal autoantigen, said method comprising selecting an
autoantigen or
modifying said mucosal autoantigen to disable the function of an MHC class I-
restricted
epitope and then administering said selected or modified autoantigen for a
time and under
conditions sufficient to induce tolerance but prevent or reduce CTL immunity
to said
autoantigen.
Administration of the autoantigen may be by DNA or polypeptide/peptide
delivery and
may be by any appropriate means but the preferred route of administration is
via mucosal
surfaces including via oral, nasal, pharyngeal, or bronchial passages and via
aerosol
including intranasal aerosol.
Yet another aspect of the present invention contemplates a method of
suppressing a cell-
mediated autoimmune disease in a subject, said method comprising the
administration as
an aerosol of an effective amount of an antigen associated with said
autoimmune disease
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-13-
for a time and under conditions sufficient to prevent, reduce or otherwise
ameliorate
autoimmune pathology wherein said antigen substantially lacks a functional MHC
class I
interacting region.
More particularly, the present invention provides a method of preventing,
reducing or
otherwise ameliorating an autoimmune disease condition in a subject, said
method
comprising the aerosol administration to said subject of an effective amount
of an antigen
associated with said autoimmune disease for a time and under conditions
sufficient to
induce or stimulate immunoregulatory mechanisms which are protective against
cell-
mediated autoimmune pathology wherein said antigen substantially lacks a MHC
class I
interacting region.
Reference hereinafter to "immunoregulatory mechanisms" should be understood as
a
reference to all mechanisms which regulate cell-mediated immune responses
including, but
not limited to, regulation of T-cell functional activity, for example,
regulation by one or
more of suppressor CD4 T cells, Thl, Th2 or CD8 T cells including yb T cells
(referred to
herein as "regulatory T cells"), or via regulation of cytokine production by
lymphoid,
myeloid or stromal cells.
The present invention is predicated in part on the recognition by the
inventors that some
mucosal autoantigens contain epitopes for MHC class I-restricted CTLs. As a
result,
administration of these antigens may result in CTL immunity and CTL tolerance.
Accordingly, the present invention requires the selection of. antigens which
lack or to
modify the antigens to remove functional MHC class I interacting epitopes.
In a particularly preferred embodiment, the MHC class I epitope is an MHC
class I (Ka)-
restricted epitope.
The present invention is hereinafter described with respect to preventing,
reducing or
otherwise ameliorating IDDM, slowly progressive IDDM (SPI17DM) also referred
to as
latent autoimmune diabetes in adults [LADA] and gestational diabetes due to
underlying
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-14-
IDDM. This is done, however, with the understanding that the present invention
extends to
a range of cell-mediated autoimmune conditions.
Accordingly, another aspect of the present invention contemplates a method of
preventing,
reducing or otherwise ameliorating IDDM, SPIDDM or gestational diabetes in a
subject,
said method comprising the administration, as an aerosol or other functionally
equivalent
means, to said subject of an effective amount of an autoantigen associated
with 117DM for
a time and under conditions sufficient for induction of regulatory T cells
and/or other
suitable mechanisms sufficient to suppress cell-mediated autoimmune pathology
associated with IDDM wherein said autoantigen substantially lacks a functional
MHC
class I interacting epitope.
Reference hereinafter to "IDDM" includes IDDM, SPIDDM and gestational IDDM.
The regulatory T cells induced will depend on the form of antigen and its
route of
administration. For example, when an undegraded, conformationally-intact
polypeptide or
whole protein molecule is administered (e.g. insulin), CD8 T cells and, more
particularly,
CD8y8 T cells are induced. Smaller peptides such as proinsulin peptides (e.g.
proinsulin
peptide 24-36) generally induce CD4 T cells and, more particularly, CD4a~3 T
cells.
Whole proteins may be degraded to peptides to generate predominantly CD4
regulatory T
cells, particularly if administration is via the oral route.
The absence of a functional MHC class I interacting epitope includes a single
or multiple
amino acid deletion encompassing all or part of the epitope region.
Alternatively, the
epitope may be blocked by other means such as by an antibody or other
molecular
interaction.
A particularly preferred form of administration is intranasal administration
via an aerosol
spray, drip or vapour.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-15-
The preferred antigen associated with IDDM used for intranasal administration
or other
route of administration is preproinsulin or proinsulin as well as insulin and
their immune
response stimulatory derivatives thereof such as but not limited to peptide
fragments of
proinsulin, preproinsulin or insulin, provided that such antigens lack or
substantially lack a
S functional MHC class I-associating region which is involved in inducing CTL
immunity.
Immune response stimulation preferably includes regulatory T cell stimulation.
However,
any islet antigen may be employed such as, but not limited to, glutamic acid
decarboxylase
(GAD) in its various isoforms (for example, GAD 65 and GAD 67) or derivatives
thereof
and tyrosine phosphatase IA-2 or derivatives thereof. The antigens may be from
human or
any non-human species such as mouse. The most preferred antigen is a
proinsulin peptide
modified to inactivate the MHC class I interacting region, defined by amino
acids 24 to 33.
The interacting region may be modified by generating peptides lacking one or
more key
MHC class I anchor residues or comprising modified residues such that MHC
class I
binding is reduced. Preferably, the proinsulin peptide undergoes a C-terminal
truncation to
inactivate the MHC class I epitope. As a result, induction of CTL immunity is
disassociated from induction of tolerance, including CTL tolerance.
Accordingly, another aspect of the present invention contemplates a method of
inducing,
suppressing or otherwise ameliorating or preventing IDDM in a subject, said
method
comprising administering proinsulin peptide truncated at its C-terminal
antigen end to
disable the function of any MHC class I restricted epitope for a time and
under conditions
sufficient to prevent or reduce CTL immunity and otherwise induce immune
tolerance,
including CTL tolerance.
As is discussed further below, the methods of the present invention may also
be used in
combination with a strategy to block CTL induction and/or maturation. In one
approach,
for example, the CD40-CD40 ligand (CD40L) interaction is blocked.
The term "derivatives" includes fragments, parts, portions, chemical
equivalents, mutants,
homologues and analogues of the antigens. Analogues may be derived from
natural
synthetic or recombinant sources and include fusion proteins. Chemical
equivalents of an
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-16-
antigen can act as a functional analog of an antigen. Chemical equivalents may
not
necessarily be derived from an antigen but may share certain conformational
similarities.
Alternatively, chemical equivalents may be specifically designed to mimic
certain
physiochemical properties of an antigen. Chemical equivalents may be
chemically
synthesised or may be detected following, for example, natural product
screenings.
A homologue of an antigen contemplated herein includes but is not necessarily
limited to
antigens derived from human or any non-human species such as mouse.
Derivatives include one or more insertions, deletions or substitutions of
amino acids.
Amino acid insertional derivatives include amino and/or carboxylic terminal
fusions as
well as intrasequence insertions of single or multiple amino acids.
Insertional amino acid
sequence variants are those in which one or more amino acid residues are
introduced into a
predetermined site in said peptide although random insertion is also possible
with suitable
screening of the resulting product. Deletional variants are characterised by
the removal of
one or more amino acids from the sequence. Substitional amino acid variants
are those in
which at least one residue in the sequence has been removed and a different
residue
inserted in its place. Additions to amino acid sequences include fusions with
other peptides
or polypeptides. It is possible, for example, that the subject preferred
peptides may be
substituted by other peptides or functional homologues or analogues. A hybrid
peptide may
comprise a combination of peptides.
The term "aerosol" is used in its most general sense to include any
formulation capable of
administration via nasal, pharyngeal, bronchial or oral passages. Aerosols
generally
comprise particles of liquid or solid suspended in a gas or vapour.
Conveniently, the
aerosol is a colloidal system such as a mist in which the dispersion medium is
a gas. The
method of administering the aerosol formulation may be by any means and may be
achieved using a hand pump, electric pump, pressurized dispenser, nasal drip
or other
convenient means. Furthermore, drop size may determine lung penetration and
the size of
the droplets may need to be manipulated to maximize efficacy of
administration. It should
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-17-
be understood that the method of the present invention extends to direct
application of said
formulations to intranasal surfaces.
In a particularly preferred embodiment, the aerosol is delivered at a rate of
from about 1 to
about 20 litres/min and preferably from about 2 to about 15 litres/min at a
droplet size of
from about 0.1 to about 10 ~m and more preferably from about 0.1 to about 6
Vim.
Conveniently, a stock solution of antigen is prepared at a concentration of
from about 0.5
to about 20 mg/ml or more preferably from about 1.0 to about 10 mg/ml of
carrier solution.
Commercially available insulin is particularly useful which is about 4 mg/ml.
A useful
dose is from about 501 to 1000 ~1 and preferably 100 ~l to 500 ~l from the
stock solution.
The antigen may be administered alone or by formulation in or with an
adjuvant. The
adjuvant is selected from a range of adjuvants which enhance an
immunoregulatory
response including cholera toxin B, heat labile toxin of E. coli, saponin,
Quill A extracts
and other derivatives of saponin, DEAF-dextran, dextran sulphate, aluminium
salts, and
non-ionic block co-polymers. The adjuvant may include other immunomodulators,
such as
cytokines (for example, IL-4 or IL-13), muramyl-dipeptide and derivatives, and
cell wall
components, for example, cell wall lipoprotein from Gram-ve bacteria such as
E.coli, from
species of Mycobacteria or Corynebacteria. The adjuvant formulation may
include a
combination of two or more of the adjuvants listed. These lists are not to be
taken as
exhaustive. The selection of adjuvant is in part dependent on the species
being targeted and
is based on the level and duration of the immune response required and on the
lack of
reactogenicity (i.e. tissue compatibility). The level of active component and
adjuvant are
chosen to achieve the desired level and duration of immune response.
The antigen is administered in a therapeutically effective amount. A
therapeutically
effective amount means that amount necessary at least partly to attain the
desired effect, or
to delay the onset of, inhibit the progression of, or halt altogether, the
onset or progression
of the particular condition being treated. Such amounts will depend, of
course, on the
particular conditions being treated, the severity of the condition and
individual patient
parameters including age, physical conditions, size, weight and concurrent
treatment.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-18-
These factors are well known to those of ordinary skill in the art and can be
addressed with
no more than routine experimentation. It is preferred generally that a maximum
dose be
used, that is, the highest safe dose according to sound medical judgement. It
will be
understood by those of ordinary skill in the art, however, that a lower dose
or tolerable
dose may be administered for medical reasons, psychological reasons or for
virtually any
other reasons.
Generally, daily oral doses of antigen will be from about 0.01 mg/ per dose
per subject per
day to 1000 mg/per dose per subject per day. Small doses (0.01-1 mg) may be
administered initially, followed by increasing doses up to about 1000 mg/kg
per day. In the
event that the response in a subject is insufficient at such doses, even
higher doses (or
effective higher doses by a different, more localized delivery route) may be
employed to
the extent patient tolerance permits. A single dose may be administered or
multiple doses
may be required on an hourly, daily, weekly or monthly basis. Effective
amounts of
antigen vary depending on the individual but may range from about 0.1 ~g to
about 100
mg, preferably from about 1 pg to about 10 mg and more preferably from about 5
pg to 20
mg per dose per subject. In particular, lower doses may be contemplated for
aerosol or
intranasal administration, for example, ng-~ g doses may be optimal.
In a related aspect of the present invention the subject undergoing treatment
may be any
human or animal in need of therapeutic or prophylactic treatment.
The immune status generally, and specifically levels of regulatory T cells and
cytokine
profiles, may be readily determined throughout any treatment regime using
conventional
methods known to those skilled in the art. For example, regulatory T cell
levels may be
monitored by cytometric analysis following labelling with commercially
available
antibodies specific to T-cell subsets. Other examples of methods suitable for
determining
the status of the subject include purification of peripheral blood mononuclear
cells by
density centrifugation followed by stimulation by incubation with well known
antigens
such GAD, IA-2 family members, preproinsulin, proinsulin or insulin or peptide
sequences
from these antigens. Resulting proliferation may be quantified by assaying for
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-19-
incorporation of H3 thymidine. The cytokine profile can be determined
approximately 24-
72 hours after stimulation by antigen. Said cytokines can be detected using, -
for example,
specific cytokine antibodies. Within about 24 hours after stimulation with
antigen,
stimulated cells can be phenotypically characterized by, for example, flow
cytometric
analysis of activation marker expression (for example, CD69, CD44, CTLA4,
CD25).
Following cell surface labelling of activated cells, said cells may be further
fixed and
incubated with fluorochrome labelled antibodies to specific cytokines to
determine
intracellular cytokine levels. In particular, for example, cells may be
further assessed by
double labelling assays. The double labelled cells may be analysed utilizing
flow
cytometric analysis or fluorescence microscopy.
Another aspect of the present invention provides a composition comprising an
antigen
associated with an autoimmune disease in an aerosol formulation including one
or more
pharmaceutically acceptable carriers and/or diluents.
Preferably, the autoimmune disease is IDDM.
Preferably, the antigen is an islet antigen such as modified forms of insulin,
or a precursor
thereof such as preproinsulin, proinsulin or their derivatives (e.g.
proinsulin peptide 24-36)
or GAD or tyrosine phosphatases IA-2 or derivatives thereof wherein said
antigens are
modified to prevent an MHC class I epitope from functioning.
Preferably, the antigen and route of administration induce regulatory T cells,
such as in
relation to whole molecules such as insulin CD8 T cells and most preferably
CD8y8 T cells
or, in relation to smaller molecules such as proinsulin peptide 24-36, CD4 T
cells and most
preferably CD4a~3 T cells.
In an alternative embodiment, a nucleic acid molecule encoding an IDDM-
associated
autoantigen is administered. Generally, according to this embodiment,
intranasal or other
suitable administration of a nucleic acid molecule such as DNA encoding
proinsulin or a
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-20-
modified form thereof induces a population of CD4 T cells which suppresses
development
of diabetes.
Preferably, the nucleic acid molecule encodes a peptide lacking a functional
MHC class I
interacting molecule.
The nucleic acid molecule is preferably DNA such as cDNA or genomic DNA or is
a
DNA:RNA hybrid. It is particularly preferred to have the nucleic acid molecule
in the form
of a plasmid or vector. The nucleic acid molecule may also contain additional
or
substitution analogues of nucleotide bases in order to enhance stability.
Accordingly, another aspect of the present invention contemplates a method of
inducing
immune tolerance, including CTL tolerance, while substantially avoiding CTL
immunity
in response to a mucosal antigen, said method comprising administering to a
subject a
nucleic acid molecule or analogue thereof encoding said mucosal antigen but
wherein said
antigen substantially lacks a functional MHC class I-restricted epitope for a
time and under
conditions sufficient to prevent or reduce induction of CTL immunity.
More particularly, the present invention contemplates a method of preventing
or
suppressing 1DDM while substantially avoiding CTL immunity, said method
comprising
administering to a subject a nucleic acid molecule or analogue thereof
encoding an IDDM-
associated autoantigen which substantially lacks a functional MHC class I-
restricted
epitope for a time and under conditions sufficient to prevent or reduce the
effects of
IDDM.
Still yet another aspect of the present invention contemplates other methods
for
dissociating CTL immunity from CTL tolerance. In particular, the maturation of
CTL to
effector "killer" cells requires priming by antigen-presenting cells such as
dendritic cells.
The dendritic cells present the antigenic peptide (i.e. epitope) as a complex
with MHC
class I molecules to the T-cell receptor of CD8 CTL. The dendritic cell itself
is primed to
perform this function by prior interaction with a "helper" CD4 T cell through
the
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-21 -
interaction between CD40 ligand (CD40L) on the T cell and CD40 on the
dendritic cell
(see Figure 11). CD8 T cells themselves have also been shown to express CD40L.
It is
proposed, therefore, in another embodiment, in conjunction with the
administration of a
mucosal antigen to administer an antagonist of CD40L-CD40 interaction. An
example of
such an antagonist is a CD40L antibody, such as a monoclonal antibody. The
antagonist of
CD40L-CD40 interaction may be administered before, simultaneously with or
sequentially
with the administration of the mucosal antigen. Sequential administration
includes within
seconds, minutes, hours, days or weeks. Simultaneous includes substantially
simultaneously. This extra treatment may be in conjunction with the
administration of a
mucosal antigen or a nucleic acid molecule encoding a mucosal antigen.
Another aspect of the present invention, therefore, contemplates a method of
inducing
tolerance to a mucosal antigen while substantially avoiding CTL immunity to
said antigen,
said method comprising administering said mucosal antigen or a nucleic acid
encoding
1 S same for a time and under conditions sufficient to prevent or reduce CTL
immunity,
before, simultaneously or sequentially with the administration of an
antagonist of CTL
induction and/or maturation.
More particularly, the present invention contemplates a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
comprising administering said mucosal antigen or a nucleic acid encoding same
for a time
and under conditions sufficient to prevent or reduce CTL immunity, before,
simultaneously
or sequentially with the administration of an antagonist of CD40L-CD4
interaction.
Even another aspect of the present invention provides a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
comprising administering an agent for a time and under conditions sufficient
to block or
otherwise delay CTL induction and/or maturation.
More particularly, the present invention provides a method of inducing
tolerance to a
mucosal antigen while substantially avoiding CTL immunity to said antigen,
said method
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-22-
comprising administering an agent for a time and under conditions sufficient
to block or
otherwise delay CTL induction and/or maturation wherein said agent blocks or
otherwise
disrupts CD40-CD40L interaction.
Still another aspect of the present invention contemplates the use of an agent
in the
manufacture of a medicament for the treatment or prophylaxis of a disease
condition, said
agent capable of blocking CTL induction and/or maturation.
The present invention is further described by the following non-limiting
Examples.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
- 23 -
EXAMPLE 1
Aerosol treatment and diabetes assessment
Semi-sealed boxes of eight female NOD mice were each aerosolized by connection
to a
standard, patient electric pump (Maymed Aerosol MKV, Anaesthetic Supplies,
Sydney,
Australia) and Aeroflo nebulizer (Waite & Co., Sydney). Recombinant human
insulin
(Humulin R, Eli Lilly) or control ovalbumin protein, at 4 mg/ml, was delivered
over 10
min at an air flow rate of 12 litres/min. in a rated droplet size of <5.8 ~.m,
to groups of 24-
32 mice. All treatments were given between 0900 and 1100 hours. Protocols and
mouse
care were approved and supervised by the institutional Animal Ethic Committee.
Retro-
orbital venous blood was sampled at least every 28 days from 100 days of age
and mice
considered to be diabetic if their blood glucose, confirmed by a repeat test,
was >11 mM.
Glucose was measured with BM-Test Glycemie (registered trademark) strips and a
Reflolux (registered trademark) II meter (Boehringer-Mannheim), on a drop of
blood
aspirated via a glass capillary tube from the retro-orbital venous plexus of
unanesthetized
mice.
EXAMPLE 2
Histology
Mice were killed by COZ inhalation and the pancreas and salivary glands
immediately
removed into Bouin's fixative and embedded in paraffin. The insulitis score, a
measure of
the severity of islet infiltration, was determined blindly by two independent
investigators
by grading and then averaging a minimum of 15 separate islets in serial 6 pm
pancreas
sections stained with haematoxylin and eosin. The grading scale was: 0, no
filtration, islet
intact; 1, <10 peri-islet lymphoid cells, islet intact; 2, 10-20 peri-and
intra-islet lymphoid
cells, islet intact; 3, >20 peri- and intra-islet lymphoid cells, <5O% of
islet replaced or
destroyed; 4, massive lymphoid infiltrate with >50% of islet replaced or
destroyed.
Infiltration of the salivary glands was graded by the number of lymphoid cells
in clusters:
0, no cells; 1, <10 cells; 2, 10-50 cells; 3, >50 cells.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-24-
EXAMPLE 3
Immune responses
Spleen cells from individual normoglycemic mice were treated with a red cell
lysis buffer,
resuspended and incubated in quadruplicate at 2 x 105/200 p1 of serum-free HL-
1 medium
(Hycor, Irvine, CA) containing 50 pm 2-mercaptoethanol, in round-bottom wells
with the
indicated concentrations of antigen. After 3 days at 37°C in 5% v/v
COZ/air, 100 p1
aliquots from each replicate supernatant were collected and stored at -
70°C for cytokine
assays; the cells were then pulsed with 3H-thymidine, harvested 16 hours later
and counted
on a Topcount (trademark) micro-scintillation counter (Packard, Meriden, CT).
Insulin was
recombinant human (Humulin R, Eli Lilly), as used for aerosol treatments.
Insulin B-chain
peptide corresponding to amino acids 9-23 of mouse insulin II (Peptide
Express, Fort
Collins, CO) was more than 90% pure by HPLC analysis. GAD65 was the
recombinant
human form expressed with a C-terminal hexahistidine in a baculovirus system
and
1 S purified by Ni2+ chelation affinity chromatography. It was resolved as a
single band in
SDS-PAGE and was endotoxin-free by the quantitative Limulus lysate assay
(BioWhittaker, Walkersville, MD).
IL-2, -4, -10 and IFN-y were measured by ELISAs with monoclonal antibody pairs
(Pharmingen); the lower limits of detection were 62, 16, 16 and SS pg/ml,
respectively.
TGF-X31 was measured with an ELISA kit (Promega) with a lower limit of
detection of 16
pg/ml.
To detect insulin antibodies, l2sI-labelled human insulin (approximately
100,000 cpm:
specific activity 120 ~Ci/pg) was incubated with or without excess unlabelled
insulin (10
pg/ml) in phosphate-buffered saline containing a mixture of protease
inhibitors and serial
log dilutions of mouse serum, for 5 days at 4°C. Complexes were then
precipitated with
rabbit anti-mouse globulin anti-serum, washed and counted in a gamma counter.
Positive
control sera (guinea pig anti-porcine insulin serum, human LDDM sera)
maximally
precipitated 37-54% of the total radioactivity. Non-specific binding, in the
presence of
excess unlabelled insulin, was ~.3%.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
- 25 -
EXAMPLE 4
Adoptive transfer of diabetes
Male NOD mice aged 6-9 weeks (16/group) were irradiated (800R) from a Cobalt
source
and 3-6 hours later received 2 x 10' pooled splenocytes from recently-diabetic
14-19 week-
old female NOD mice, together with 2 x 10' splenocytes (or cells fractionated
from this
number) from either aerosol insulin- or ovalbumin-treated mice, in 200 ~1 via
the tail vein.
The onset of diabetes was then monitored by measuring blood glucose starting
two weeks
after transfer.
EXAMPLE 5
Fractionation of spleen cell populations
Spleen cells were treated with red cell lysis buffer and resuspended in mouse
tonicity
phosphate buffered saline. Total T cells were purified by non-adherence to
nylon wool.
CD4 and CD8 cells were positively selected/depleted magnetically with
monoclonal
antibodies directly bound to MACS MicroBeads (Milteny Biotec, GmbH, Germany)
according to the manufacturer's protocols, and counted as viable cells (trypan
blue stain
negative). Flow cytometry revealed 95% depletion of CD4 or CD8 cells, with
recoveries
~80% and ~50% respectively.
yb T cells were positively selected/depleted by incubating T cells from
aerosol-treated
mice first with biotinylated GL3-lA antibody (Pharmingen, San Diego, CA) and
then with
streptavidin-MACS MicroBeads, followed by magnetic separation. By flow
cytometry, y8
cells comprised 1-2% of NOD splenocytes and were totally depleted with GL3-lA
antibody. To purify CD8 y8 T cells, CD8 T cells were first magnetically
selected from total
T cells with anti-CD8-FITC conjugate and anti-FITC MicroBeads. The MicroBeads
were
then released according to the Miltenyi Biotec protocol, and the CD8 cells
magnetically
separated into y8 positive and depleted fractions. Double staining and FACS
analysis
demonstrated total depletion of y8 cells and their recovery as a GL3-lA high
and low
expressing CD8 population.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-26-
EXAMPLE 6
Diabetes and insulitis
Aerosol human insulin or ovalbumin were administered in different schedules to
female
NOD mice from 28 days of age, the earliest time at which insulitis is
detectable in the
colony of mice, and their incidence of diabetes and severity of insulitis
subsequently
measured.
The incidence of diabetes was only marginally affected by a single aerosol
insulin
treatment at 28 days of age, being 75% by 240 days of age compared to 88%
after aerosol
ovalbumin. However, treatment for 3 or 10 consecutive days and then weekly
significantly
delayed the onset and reduced the incidence of diabetes. In five separate
experiments,
diabetes incidence at 156 days of age was reduced from a median of 47% in
ovalbumin-
treated mice to 23% in insulin-treated mice; at 240 days of age, when the
cumulative
incidence of diabetes approaches a maximum, the values were 79% and 49%,
respectively
(p=0.005, Kaplan-Meier survival statistic). There was no difference if the
initial treatment
was for 3 or 10 days. In another experiment, in which treatment was given for
10
consecutive days and then weekly, but not started until 49 days of age when
insulitis was
well-established, aerosol insulin still significantly reduced diabetes
incidence at 156 days
from 58% to 25% (p=0.001). Insulin treatment was associated with a significant
reduction
in the severity of the islet lesion, as judged by the "insulitis score", which
parallelled the
decrease in diabetes incidence (Table 1). Infiltration of the salivary glands
by lymphoid
cells (sialitis), which also occurs in NOD mice, was unaffected by aerosol
insulin.
In the absence of absorption-promoting agents, systemic uptake of insulin from
the naso-
pharyngeal mucosa in humans is insignificant (22). In NOD mice, blood glucose
was not
altered in the short-term by aerosol insulin. Insulin solutions labelled with
10% Evan's
Blue dye were observed to be deposited in the naso-pharynx, trachea and main
bronchial
divisions, as well as the oesophagus. While it may be difficult, if not
impossible, to avoid
some gastrointestinal exposure after aerosol or intranasal delivery of soluble
protein,
delivery into the naso-pharynx alone is sufficient to induce tolerance
(7,23,24).
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-27-
TABLE 1 Severity of insulitis and frequency of diabetes in NOD mice treated
with
aerosol protein
Protein Insulitis score Diabetes frequency
Insulin 1.2 t 0.98' 2/32 (6.3%)'
Ovalbumin 2.6 ~ 0.92 8/32 (25%)
Mice (32/group) were given either aerosol insulin or ovalbumin for 10
consecutive days
and then weekly from 28 days of age. At 105 days of age, five non-diabetic
mice from
each group were killed for pancreas histology. The insulitis score is
expressed as mean ~
SD.
1 The insulitis score in insulin-treated mice was significantly reduced
(p<0.01,
Mann-Whitney U test).
The diabetes frequency in insulin-treated mice significantly reduced (p=0.04,
Fisher's exact test).
EXAMPLE 7
Immune responses
The inventors investigated if aerosol insulin treatment had altered immune
responses to
insulin. Unprimed T-cell proliferative responses to islet antigens, including
insulin, have
been reported in NOD mice (25) but have not always been reproducible (26).
Proliferative
responses of spleen cells (0.5-2.5 x 106/m1) from either insulin or ovalbumin-
treated mice,
aged 56-105 days, to human insulin or ovalbumin (0.2, 2.0, 20 and 40 ~g/ml),
in different
serum-supplemented or serum-free media varied by less than two-fold above
basal and
were usually depressed below basal at the highest concentration of insulin.
Insulin at high
concentrations has been reported to inhibit T-cell responses (27). In
contrast, in the
ovalbumin-treated control mice but not the insulin-treated mice, responses to
insulin B-
chain peptide a 9-23, a dominant epitope for NOD mouse islet-derived T-cell
clones (19),
were significant (Table 2). Furthermore, ovalbumin mice had significantly
higher
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-28-
responses than insulin mice to human glutamic acid decarboxylase 65 (GAD65),
previously reported to stimulate splenic T cells in NOD mice (25). In mice
from both
treatment groups, proliferative responses to non-antigen-specific stimulation
by
concanavalin A or T-cell receptor CD3 monoclonal antibody, 145-2C11, were
similar
(Table 2) and no different to untreated mice, indicating that aerosol
treatment did not cause
general immunosuppression. IL-2, IFN-y and TGF-(31 secretion in response to
insulin B
chain 9-23 were not significantly different between insulin- and ovalbumin-
treated mice;
however, the levels of IL-4 and particularly IL-10 were higher from cells of
insulin-treated
mice (Table 3).
TABLE 2 Proliferative responses of splenocytes from aerosol-treated mice
[ H]-thymidine
uptake (cpm,
mean ~ SD)
Additive Insulin aerosol Ovalbumin aerosol
None 157 ~ 28a 208 ~ 29
Human insulin (40 pg/ml) 134 ~ 27 197 ~ 82
Mouse insulin II B-chain (a 9-23)169 t 80 435 ~ 240e
(40
ml
Human GAD65 (20 ~g/ml) 424 ~ 165 1381 ~ 650I
Concanavalin A (5 pg/ml) 3357 ~ 812 2960 ~ 494
Anti-CD3 antibody (10 pg/ml) 2221 ~ 533 2643 ~ 1126
c v a (p=0.001), a v d (p=0.016),
f v d (p=0.001), a v b (p=0.002),
f v c (p<0.0001)
Splenocytes from three mice per group were assayed in quadruplicate in HL-1
serum-free
medium. Statistical comparisons (Mann-Whitney U tests) were between the 12
results for
each group.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-29-
TABLE 3 Cytokine secretion by splenocytes from aerosol-treated mice to 40
pg/ml of
mouse insulin II B chain peptide (a 9-23)
Cytokine Insulin aerosol Ovalbumin aerosol
IL-2 (pg/ml) 200 ~ 15 186 t 28
IFN-'y (pg/ml) 368 t 71 423 t 31
IL-4 (pg/ml) 36 t 6 not detectable
IL-10 (pg/ml) 222 ~ 149 not detectable
TGF-(3 293 ~ 131 162 ~ 41
S Supernatants from replicate culture wells (Table 2) were sampled after three
days
incubation and assayed for cytokines.
Insulin antibodies were measured by a standard immunoprecipitation assay with
sera
(n=12/group) from insulin- and ovalbumin-treated mice aged 70-105 days.
Precipitation of
l2sl-insulin radioactivity by antibodies in sera from insulin-treated mice
(12.7 ~ 3.6%;
mean precipitated cpm ~ SD) was significantly higher (p<0.01, Mann Whitney U
test) than
in ovalbumin-treated mice (6.9 ~ 2.5%). This increase in the "level" of
insulin antibodies
after aerosol insulin, together with the suppression of T cell proliferation
and the increase
in IL-4 and IL-10 responses to insulin B-chain peptide, is consistent with the
phenomenon
of immune deviation, as described after oral MBP in Lewis rats (1) and
intranasal GAD
peptides in NOD mice (28). ~3-cell destruction within the DTH lesion of IDDM
is an
example of Thl-mediated process (10,11), whose inhibition by aerosol insulin
might be
expected to shift the Thl/Th2 balance towards Th2 in response to key islet
antigens.
Defective suppressor T-cell function has been postulated to shift the balance
towards Thl
in IDDM (11). It seems unlikely that the reduced T cell proliferative response
to GAD
could reflect "bystander" suppression due to the secretion of the Th2
cytokines IL4 and IL-
10 (1) by insulin aerosol-induced regulatory cells because, apart from an
absence of added
insulin in the cultures with GAD, responses to conA and anti-CD3 were not
impaired. A
direct explanation is that the reduced response to GAD reflects the protective
effect of
aerosol insulin on insulitis and [3-cell destruction. This implies that at
least some GAD
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-30-
immunity is secondary and that immunity to (pro)insulin may have a more
proximal role in
~3-cell destruction. Although NOD mouse T-cell responses to human GAD65 have
been
reported to be stronger and to appear earlier than those to native human
insulin (25), it was
found that transgenic expression of mouse proinsulin II in NOD mouse antigen
presenting
cells completely prevents insulitis and diabetes (29).
EXAMPLE 8
Regulatory CD8 y8 T cells
The inventors investigated whether aerosol insulin induced regulatory cells
that could
inhibit the adoptive transfer of diabetes by pathogenic, effector T cells. In
the classic
adoptive transfer model (30) (see Figure 6), spleen cells from diabetic NOD
female mice
transferred intravenously to young, irradiated non-diabetic syngeneic male or
female
recipients cause clinical diabetes in the majority within 4 weeks. When 2 x
107 spleen cells
were co-injected from older, diabetic mice with an equal number of spleen
cells from
aerosol ovalbumin mice, the majority of young recipients developed diabetes
within 4-5
weeks; in contrast, after co-injection with spleen cells from aerosol insulin
mice, only a
minority developed diabetes (Figure 4A). Diabetes incidence was suppressed by
>_75% in
six separate experiments with either splenocytes or nylon wool-non-adherent
splenocytes
(enriched for T cells) from aerosol insulin mice.
Spleen cells were then fractionated to identify the regulatory cells
responsible for the
suppression of diabetes transfer. Depletion and positive selection of CD4 and
CD8 cells
clearly showed that CD8 cells were wholly responsible for the suppression of
transfer
(Figure 4B). Depletion of CD4 cells did not alter the ability of residual
spleen cells from
aerosol insulin mice to suppress transfer (Figure 4B), and positively selected
CD4 cells did
not suppress transfer (Figure 4C). On the other hand, there was no suppression
by CD8-
depleted spleen cells from aerosol insulin mice (Figure 4D), whereas
positively-selected
CD8 cells suppressed transfer (Figure 4E). The partial suppression by
positively-selected
CD8 cells, in contrast to the rapid development of diabetes after their
depletion, is
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-31-
probably due to the inefficient recovery of CD8 cells; in this experiment, 7 x
105 purified
CD8 cells were co-injected into each recipient with 2 x 107 spleen cells from
diabetic mice.
T cells bearing y8 receptors have been shown to have an immunoregulatory role
(31-36).
Interestingly, it has been reported that total peripheral blood y8 cells
decrease
concomitantly with loss of (3-cell function in humans with sub-clinical IDDM
(37). To
determine if the suppression of diabetes transfer that was observed was due to
y8 T cells,
the inventors fractionated spleen cells with the anti-y8 T-cell monoclonal
antibody, GL3-
lA (38). Depletion of y8 T cells, like that of CD8 cells, completely abrogated
the ability of
nylon wool non-adherent spleen cells from insulin aerosol-treated mice to
suppress
adoptive transfer of diabetes (Figure SA). Conversely, relatively small
numbers of y8 T
cells from insulin aerosol-treated mice could suppress transfer. Diabetes
incidence after
transfer was decreased by 50% for at least 70 days when 1.4 x 105 y8 T cells
were co-
injected with 2 x 107 spleen cells from diabetic mice (Figure SA). The splenic
CD8 and y8
T cells that suppressed diabetes transfer were one and the same, and not two
interdependent populations. Thus, the ability of CD8 cells from insulin
aerosol-treated
mice to suppress transfer was abolished if they were first depleted of yb T
cells, whereas
small numbers of y8 cells purified from the CD8 cells prevented transfer
(Figure SB). A
summary of the results from 11 different co-transfer experiments is presented
in Figure 6.
FACS analysis revealed that y8 cells reactive with GL3 antibody constitute 1.6-
2.4% of
total and ~ 1% of CD8+ cells in the spleens of 12-16 week-old female NOD mice.
These
values were no different between groups of mice treated with insulin or
ovalbumin aerosol.
However, because of their low abundance distinct sub-populations of antigen-
specific CD8
y8 T cells would be difficult to distinguish this way. The higher protection
with
fractionated cells, for example, sequentially-purified CD8 y8 cells (Figure
6), is
quantitative and .reflects their higher absolute number relative to that in
unfractionated
cells.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-32-
EXAMPLE 9
Aerosolinization of insulin
Aerosol inhalation as a mode of insulin delivery to the mucosa was as
effective as oral
insulin (22,23) in reducing diabetes incidence in the NOD mouse. The fact that
it was
therapeutic after the onset of insulitis is especially relevant to the
prevention of IDDM in
at-risk humans with sub-clinical disease in whom the presence of circulating
islet-antigen
reactive antibodies and T cells is taken to reflect underlying insulitis.
Indeed, compared to
humans with recently-diagnosed IDDM, NOD mice have more intense insulitis and
the
majority of females to progress to diabetes (10,11,24). Aerosol insulin had no
obvious
metabolic effect but induced a population of regulatory CD8 y8 T cells, small
numbers of
which suppressed the ability of pathogenic effector T cells to adoptively
transfer diabetes.
These antigen-induced "suppressor" T cells protective against cell-mediated
autoimmune
pathology have not been previously described.
Oral tolerance has been associated with a decrease in cellular and sometimes
an increase in
humoral antigen-specific immunity, and with either CD8 or CD4 T cells that
secrete,
respectively, TGF-(3 or IL-4, IL-10 and TGF-(31 (8). However, these regulatory
cells have
not been identified as bearing y8 receptors. In NOD mice, oral tolerance to
insulin was
attributed to regulatory CD4 T cells (21). In accordance with the present
invention CD8 y8
T cells account for the regulatory cells induced by aerosol insulin.
EXAMPLE 10
Intranasal insulin (Figure 1), proinsulin (Figure 2) or proinsulin
peptide 24-36 (Figure 3)
Commercially available insulin at 4 mg/ml, or proinsulin or proinsulin peptide
24-36 at 1-4
mg/ml in either insulin carrier solution or mouse tonicity-phosphate buffered
saline, was
applied in a volume of 10-20 p1 to the nostrils of unanaesthetized, restrained
NOD female
mice at either 28 or 56 days of age. Note that by 56 days of age all mice
exhibit underlying
islet inflammation (insulitis).
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-33-
-*Single doses of insulin, proinsulin or proinsulin peptide 24-36 at either 28
or 56 days of
age each significantly delayed the onset of diabetes in NOD female mice,
compared to
control proteins ovalbumin or hen egg lysozyme. On a comparative dose basis,
proinsulin
and proinsulin peptide 24-36 were more effective than insulin. These effects
are greater
S with repeated doses of these proteins or peptide. In female mice pretreated
with a single
intranasal dose (40 pg) of proinsulin 24-36, whole splenocytes, and whole
splenocytes
depleted of CD8 but not CD4 T cells, significantly suppressed the adoptive
transfer of
diabetes by splenocytes from diabetic mice (Figure 7). Female mice were
treated at 28
days of age, and then killed and their splenocytes taken for adoptive co-
transfers at 56 days
of age.
EXAMPLE 11
Clinical trial of intranasal ihsulin in at-risk individuals
The intranasal insulin trial (INIT) involves administration of intranasal
insulin to at-risk
but otherwise healthy first-degree relatives with immune markers of IDDM,
including
circulating antibodies and T cells reactive with islet autoantigens. Our
subjects have at
least two antibodies, to insulin, GAD or tyrosine phosphatase IA-2, and
peripheral blood T
cell responses to insulin or proinsulin peptide 24-36, and sometimes to GAD
and IA-2
peptides. The rationale is to induce mucosa-mediated immune tolerance to
insulin, based
on the success of this approach in the NOD mouse, and to demonstrate safety.
Commercially-available human recombinant insulin is used, which is normally
given
routinely by subcutaneous or intravenous injection to people with IDDM. No
significant
side effects have been observed in 38 high-risk subjects aged 4-30 (median
11.4 years)
entered into the Trial. The possibility of mucosal irritation exists, but this
has only been
rare and then minor and transient. NOD mice treated with aerosol or intranasal
insulin have
exhibited no clinical complications, or abnormalities at autopsy.
The INIT trial examines the effect of intranasal insulin on the surrogate
immune markers
of IDDM. The design is randomized, double-blind and placebo-controlled, with a
crossover at six months. The placebo is the Garner solution normally used for
insulin. The
aim is to demonstrate significant effects on the levels of antibodies and T
cells to insulin
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-34-
and other beta cell antigens. In addition, first phase insulin release (FPIR)
in response to an
intravenous injection of glucose, a measure of beta cell function, is
monitored at the start,
six months and 12 months. The crossover design gives all subjects the
opportunity of
treatment (an important issue for at-risk relatives), measures if any
treatment effects are
sustained and allows within- and between-group analyses. Treatment is
administered
initially daily for 10 consecutive days, then for two consecutive days weekly.
After six
months, treatment is crossed over (from insulin to placebo, or vice versa).
The administration dose of insulin per nostril is approximately 200 ~l (800
pg) of the
commercial 4 mg/ml solution. The placebo is the Garner solution in which the
insulin is
normally dissolved.
Results
Intranasal insulin was associated with a significant increase (p = 0.01) in
insulin antibodies
overall and with a concomitant decrease in peripheral blood mononuclear (T-
cell)
proliferation to denatured human insulin; insulin antibody levels and T-cell
proliferative
responses to denatured insulin were inversely related in the first (p = 0.05)
and second (p =
0.01) periods. These results are consistent with those found in the NOD mouse.
There was
no change in FPIR in any subject in whom this was initially measurable above
the first
percentile. Changes in immune parameters were not associated with changes in
FPIR, e.g.
an increase in insulin antibodies on insulin was not associated with
deterioration of beta
cell function in subjects with FPIR > 1st percentile. There were no evident
side-effects of
intranasal insulin and the results encourage further trials to determine the
effect of
intranasal insulin on FPIR long-term and diabetes incidence.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-35-
EXAMPLE 12
Modification of proinsulin peptide 24-36 to remove functional MHC
MHC class I interacting eptitope
Following administration of the proinsulin peptide 24-36, although CD4
regulatory T cells
were induced which almost completely blocked the adoptive transfer of diabetes
(when
isolated and transferred with effector "diabetogenic" T cell into young
irradiated NOD
mice), and the onset of spontaneous diabetes was delayed but not prevented.
The inventors
observed that the proinsulin peptide contained predicted epitopes for MHC
class I (H2-
Kd)-restricted CTLs, namely aa26-34 and aa25-34. Accordingly, administration
of
proinsulin peptide 24-36 could result in concomitant induction of CTL immunity
and
tolerance. First, they showed that 26-34 and especially 25-34 could bind to Ka
(Figure 8)
and elicit CTL in NOD mice (shown for as 26-34 in Figure 9). They then
demonstrated the
effect of a series of C-terminal truncations of the proinsulin aa24-36 peptide
(Figure 10).
Inactivation of the MHC class I-binding peptides by deletion of the position 9
anchor
residue (aa34) significantly enhanced the ability of the core MHC class II (I-
Ag7) binding
sequence to prevent diabetes after intranasal administration. The C-terminal
amino acid in
position (p) 9 is a key "anchor" residue required for binding to the MHC class
I molecule.
EXAMPLE 13
Dissociation of cytotoxic T lymphocyte (CTL) immunity from oral tolerance by
targeting CD40L-CD40 signalling
MATERIALS AND METHODS
Mice
Mice were bred and maintained in The Walter and Eliza Hall Institute of
Medical
Research. Oral tolerance and CTL activity were determined in female C57B1/6
mice aged
6 to 8 weeks. Transgenic OT-1 / Rag -/- mice bearing a transgenic CD8 T-cell
receptor
(TCR) for the MHC class I-restricted OVAZS7-26a peptide and OT-II mice bearing
a
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-36-
transgenic CD4 TCR for the MHC class II-restricted OVA3z3-339 peptide were
used
between 6 and 12 weeks as donors of OVA-reactive T cells for adoptive transfer
into
Ly5.1/CD45.2 congenic C57B1/6 mice (OT-I cells) and into RIP-OVA transgenic
mice
(OT-I and OTII cells).
S
Induction of oral tolerance
Oral tolerance was induced with two protocols corresponding to reported high
and low
dose OVA.
OVA (Grade V, Sigma, St. Louis, MO) was administered to female C57B1/6 mice
either at
mg on three alternate days (high dose) or at 0.5 mg on five alternate days
(low dose) via
intragastric intubation under light methoxyflurane (Penthrane (trademark))
anaesthesia.
The endotoxin concentration of an OVA solution (lOmg/ml) measured in the
Limulus
15 lysate assay (BioWhittaker, Walkersville, MD) was <_ 0.5 ng/ml. CD40L
signaling was
blocked by administration of the hamster IgGl anti-mouse CD40L mAb MR-1 (ATCC,
Rockville, MD); the control was the hamster mAb 6C8 specific for human Bcl-2.
Both
mAbs were purified from hybridoma cell culture medium by affinity
chromatography on
protein G-Sepharose (Pharmacia, Uppsala, Sweden) and injected
intraperitoneally (i.p.). in
20 a dose of 250 p.g as indicated.
Cytotoxic T lymphocyte (CTL) assay
CTL were assayed as follows.
Mice were primed i.v. with 20 x 106 OVA-coated H-2Kbm-i spleen cells
(dependant on
CD4 T-cell help) or subcutaneously (s.c.) in the base of tail with 200pg of
OVA peptide
257-264 in CFA in 100 p1 (independent of CD4 T-cell help). Depending on the
experiment, mice were primed 2 or 3 weeks after receiving mAb and oral OVA.
Mice were
killed 7 days after priming, and their spleen cells stimulated in vitro for
another 6 days
before being used as effectors in a S~Cr release assay. Lytic units were
calculated by
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-37-
dividing the total number of effectors generated from each spleen by the total
number of
effectors required for 30% OVA-specific lysis.
Effect of oral OVA ~ anti-CD40L treatment of CTL precursors
S
To determine the response of OVA-specific CTL precursors to oral OVA and the
effect of
CD40L blockade, 3 x 106 OT-I cells were transferred into Ly5.1 congenic female
mice,
which were then given either MR-1 or control 6C8 mAb on days 0 and 3. Oral OVA
20 mg
was given daily on days 1-3. Mice were killed on day 14 and the numbers of
splenic OT-I
cells and their expression of CD44 and CD62L (L-selectin) activation markers)
analyzed
by FACScan using Lysys 2 (trademark) software (Becton Dickinson, San Jose,
CA). Cells
were incubated with FITC-conjugated anti-CD44 and anti-L-selectin mAbs
(PharMingen,
San Jose, CA) together with biotinylated anti-Ly5.2 mAb (PharMingen) and PE-
conjugated anti-CD8 mAb (Sigma), followed by a second-step incubation with
streptavidin- conjugated PerCp (PharMingen) to detect Ly5.2.
Effect of oral OVA f anti-CD40L treatment on diabetes induction in RIP-
OVA~° mice
To examine the effect of CD40L blockade on activation of CTL in vivo be oral
OVA, RIP-
OVA mice which express OVA on their pancreatic (3 cells were adoptively
transferred with
0.3 x 106 OT-1 cells and 0.2 x 106 OT-1 cells and given MR-1 or control 6C8
mAb on the
day of transfer (day 0). Mice were then treated with oral OVA, 0.5 mg on five
alternate
days, starting from day 1. Blood glucose was measured on a drop of retro-
orbital venous
blood with a glucometer, on days 14 and 21 and values above 14 mmol/1 were
considered
to be diagnostic for diabetes.
Evaluation of oral tolerance
To evaluate CTL tolerance to systemic priming, 14 or 21 days after the last
dose of oral
OVA mice were injected i.v. with 20 x 106 OVA-coated H-2Kbm-1 spleen cells or
s.c. with
0.1 mg OVA protein in CFA and their splenic CTL activity subsequently measured
as
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-38-
described above. To evaluate conventional indices of mucosal tolerance, 7 days
after the
last dose of oral OVA mice were immunized by s.c. injection with OVA (0.1 mg)
in CFA
in the base of tail. Ten days later, they were anesthetized, bled with a glass
capillary tube
from the retro-orbital venous plexus, killed by COZ asphyxiation, and their
spleens and
inguinal lymph nodes removed. Serum was harvested and stored at -20~C for
assay of
OVA antibodies. Cell suspensions were prepared from spleens and nodes by
mechanical
disruption through a stainless steel mesh, washed, counted and resuspended in
RPMI-1640
medium containing 2 mM glutamine, 5 x 10-5 2-mercaptoethanol and 5% v/v fetal
calf
serum for assay of proliferative and cytokine responses to OVA.
IgG subclass antibodies to OVA were measured by ELISA using peroxidase-
conjugated
anti-mouse IgG 1, 2a, 2b or 3 antibodies (Southern Biotechnology Associates)
as
previously described .
Proliferative responses to OVA of splenocytes (1 x 106) or inguinal lymph node
cells (5 x
105) in 200 p1 medium were measured in replicates of eight in round-bottom
wells of 96-
well Linbro plates (Flow Labs, McLean, VA), after incubation with or without
0.1 mg/ml
OVA at 37°C in 5% COZ/ air for 96 hours. 3H-thymidine (1 pCi) was added
to each well
for the last 10-16 hours, the cells harvested and washed, and counted on a
TopCount
scintillation counter. Splenocyte or inguinal lymph node cell IFN-y and IL-4
responses to
OVA were measured by ELISPOT assay. Cells (5 x 105/200 p1) were added to wells
of
Multiscreen Immobilon-P membrane 96-well plates (MAIPS4510; Millipore, North
Ryde,
Australia) that had been precoated with monoclonal rat anti-mouse IFNy (clone
R4-6A2)
or IL-4 (clone 11 B11) antibody at S pg/ml PBS overnight Cells were incubated
with or
without 0.1 mg OVA at 37°C in 5% COZ/air for 24 hours, then removed by
washing.
Membrane-bound cytokine was reacted with 4 pg/ml biotin-conjugated monoclonal
rat
anti-mouse IFN-y (clone XMG1.2) or IL-4 (clone BVD6-2462) overnight at
4°C. After
washing, colour was developed with streptavidin-peroxidase followed by 3-amino-
9-
ethylcarbazole (AEC; Dako, Carpinteria, CA). All monoclonal antibodies were
from
Pharmingen, San Diego, CA.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-39-
Statistics
Differences between treatment groups were analyzed by the Fisher's exact test
or the
Mann-Whitney test
S
RESULTS
CD40L blockade impairs CTL induction by oral OVA
After determining the dose (250 pg i.p.) of MR-1 mAb that blocked CTL priming
by the
CD4 T cell help-dependent pathway (Figure 12A) a similar dose of either
control mAb
6C8 or MR-1 mAb was given to C57B1/6 mice that were then fed 20mg OVA on three
alternate days. Oral OVA induced a CTL response in 75% (9/12) of mice treated
with 6C8,
but in only 25% (3/12) of mice treated with MR-1 (Figure 12B).
Activation and expansion of CTL by oral OVA requires CD40L
To demonstrate that CTL induction by oral OVA was associated with activation
and
expansion of CTL precursors, the adoptively transferred OVA-specific
transgenic CTL
(OT-I cells) into naive Ly5.1 congenic recipients and fed them OVA. The role
of CD40L
in the response of OT-I cells to oral OVA was examined by pre-treating
recipient mice
with either control mAb 6C8 or anti-CD40L mAb MR1. In order to allow
activation,
proliferation, recirculation and possible cell death to occur before analyzing
the final
outcome, the inventors examined OT-I cells from the spleen 14 days after the
last dose of
oral OVA. This site and time corresponded to other protocols used, e.g. to
measure OVA
induced CTL. In response to oral OVA and 6C8, OT-I cells in the spleen
expanded greatly
(Figure 13B) and increased CD44 (Figures 13A, C) and decreased CD62L (Figures
13A,
D) expression, indicating that many had acquired an activated/memory
phenotype.
However, in the presence of MR1, the ability of oral OVA to induce expansion
(Figure
13B) and activation (Figures 13A, C, D) of OT-I cells was markedly impaired.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-40-
Anti-CD40L treatment prevents induction of diabetes by oral OVA in RIP-
OVA~° mice
In preliminary experiments, the inventors found that transfer of a minimum of
0.3 x 106
OT-II cells and 0.2 x 106 OT-I cells was necessary for diabetes development
after systemic
priming of recipient RIP-OVA~° mice with OVA-coated splenocytes. These
numbers of
OT-II and OT-I cells were, therefore, transferred into RIP-OVA~° mice
and the following
day the mice were given control mAb 6C8 or anti-CD40L mAb MR1 and then fed 0.5
mg
OVA on five alternate days. Following oral OVA, 60% (9/15) of mice given 6C8
developed diabetes, compared to only 14% (2/14) of mice given MRl (p=0.02)
(Figure
14).
Anti-CD40L treatment does not prevent induction of oral tolerance
Experiments in CD40L gene-targeted mice have indicated that CD40L signalling
is
required for induction of oral tolerance (39). However, this mutation affects
the
development of Peyer's patches (39) and germinal centres (40). Therefore, it
was
important to determine if oral tolerance could be induced in genetically
unmanipulated
mice treated short-term with anti-CD40L mAb. Previously, it was shown that
oral OVA,
while inducing CTL immunity, paradoxically suppressed the further priming of
strong
CTL immunity by systemic OVA (41). To determine if anti-CD40L treatment
influenced
this tolerogenic effect of oral OVA on CTL, C57B1/6 mice were given control
mAb 6C8
or anti-CD40L mAb MRl 250 ~g i.p. and then fed PBS or OVA in PBS. After 14 or
21
days they were primed in a CD4 T cell-dependent manner with i.v. OVA-coated
splenocytes (Figure 15A) or s.c. with 100 ~g OVA in Complete Freund's Adjuvant
(CFA)
(Figure 15B). The latter method directly primes CTLs, independent of CD4 T-
cell help.
These experiments demonstrated that anti-CD40L treatment with MR1 did not
modify the
tolerogenic effect of oral OVA on systemic priming of CTL by either method.
This was
consistent in all experiments, whether mice were challenged 14 or 21 days
after high dose
(20 mg on three alternate days) or low dose (0.5 mg on five alternate days)
oral OVA
(Figures 15C, 15D).
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-41 -
Experiments were then performed to determine whether anti-CD40L treatment
affected
conventional parameters of oral tolerance. A single injection of MR1 before
the first dose
of oral OVA (20 mg three alternate days) did not limit suppression of systemic
OVA-
primed T-cell proliferation or IFN-y production (shown for splenocytes in
Figures 16A and
16B, respectively) or serum anti-OVA antibodies (Figure 16C). Mean stimulation
indices
for proliferation decreased from 3.50 (oral PBS) to 1.96 (oral OVA) (p<0.05)
after 6C8
and from 3.21 to 2.04 (p<0.05) after MR1 (Figure 16A). Suppression of primed
IFN-y
ELISPOT responses following oral OVA was more dramatic and was not affected by
anti
CD40L treatment (Figure 16B). Very few IL-4 ELISPOTS ( /well) were detected
under
any condition.
Mucosal administration of antigen can tolerize subsequent immune responses to
the
antigen and, in the case of autoantigens, suppress development of autoimmune
disease.
Mucosal administration, however, of the model protein antigen ovalbumin (OVA)
also
induces cytotoxic T-cell (CTL) immunity and this may cause disease. The
inventors show
that oral OVA-induced tolerance and CTL immunity can be dissociated by
targeting the
interaction between CD40L and CD40. Monoclonal antibody blockade of CD40L
strengthened tolerance by preventing the simultaneous induction of CTL. This
was
reflected by inhibition of the activation and expansion of adoptively-
transferred OVA-
specific CTL (OT-1-CD8 cells) in response to oral OVA. Furthermore, in mice
with
transgenic expression of OVA on pancreatic (3 cells, CD40L blockade
significantly
inhibited the development of CTL (OT-1 cell)-mediated autoimmune diabetes that
followed oral administration of OVA. These results show that mucosal tolerance
towards
CTL is induced independently of the requirements of CD40L signalling for CTL
priming.
Blockade of CD40L signalling could, therefore, improve the efficacy (and
safety) of
mucosal antigens for preventing CTL-mediated autoimmune disease. To
demonstrate this,
female NOD mice aged 8 weeks were treated with anti-CD40L monoclonal antibody
MR-
1 or control antibody 6C8 (300 pg i.p.) just before administration of aerosol
insulin (4
mg/ml, 10 minutes) or diluent control on days 1, 3, 10, 24 and 38. Anti-CD40L
antibody
treatment with aerosol insulin markedly reduced diabetes incidence compared to
anti-
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-42-
CD40L antibody treatment with diluent or control antibody and either aerosol
insulin or
diluent (Figure 17).
EXAMPLE 14
Intranasal proinsulin DNA induces CD4 T cells which prevent diabetes
MATERIALS AND METHODS
DNA
Mouse proinsulin II cDNA or ovalbumin genomic DNA was subcloned into a plasmid
vector derived from the mammalian expression vector, pCI, under the control of
the CMV
early promoter. The vector was modified and is designated as CIGH. Plasmids
were
prepared from E.coli and purified by PEG precipitation and Triton X114 phase
partition,
diluted to 2 mg DNA per ml in PBS and frozen at -20°C.
Mice and treatment
NOD mice were bred and maintained in The Walter and Eliza Hall Institute of
Medical
Research. At 3 and S weeks of age, 25 ~1 of PBS containing 50 ~g DNA was given
intranasally in repeat 5 ~l portions to non-anaesthetized female mice. In
other experiments
mice were given 25 ~g DNA intranasally for four consecutive weeks beginning at
3 weeks
of age.
Determination of diabetes
Blood glucose was measured using the Advantage monitor (Boehringer Mannheim)
on a
drop of blood obtained via a fine glass capillary tube from the retro-orbital
venous plexus.
Mice were considered to be diabetic if their blood glucose was >11 mM on
consecutive
days. Diabetic donor mice used in adoptive transfer studies has an elevated
blood glucose
for <1 week.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-43-
RESULTS
In initial experiments, spleen cells from intranasal DNA-treated mice at 10
weeks of age
were enriched for T cells (hereafter referred to as splenic T cells) by
passage through nylon
wool, and than either co-transferred i.v. with spleen cells from recently-
diabetic NOD mice
into irradiated 6 week-old male NOD mice or transferred i.v. into
cyclophosphamide-
treated NOD females Cyclophosphamide treatment accelerates the onset of
diabetes on
NOD mice. In both experimental models, a significant reduction in diabetes
incidence was
observed in recipient mice that received cells from proinsulin DNA-treated
donors. In three
experiments in which 5 x 106 splenic T cells were co-transferred with 2 x 107
"diabetic"
spleen cells, the combined incidence of diabetes in recipients 4 weeks after
transfer was
14% in recipients of cells from proinsulin DNA-treated donors compared to 64%
in
recipients of cells from ovalbumin DNA-treated donors (p=0.003) (Table 4). In
parallel, S
x 106 splenic T cells were transferred into 8-10 week-old female NOD mice 2
days after
they had received 300 mg/kg cyclophosphamide i.p. The peak incidence of
diabetes
observed 17 days after cyclophosphamide was 56% in recipients of cells from
ovalbumin
in DNA-treated donors compared to 12.5% in recipients of cells from proinsulin
DNA-
treated donors (p=0.02) (Table 5). No protection was observed when 5 x 106
splenic T cells
from mice that had received two intramuscular injections of proinsulin DNA
were injected
into cyclophosphamide-treated recipients (Table 5).
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-44-
TABLE 4 Splenic CD4 T cells from NOD mice given intranasal proinsulin II DNA
suppress adoptive transfer of diabetes
Intranasal treatment Donor spleen cells Diabetes incidence
of co- after
donor transferred (with 28 days (%)
2 x 107
"diabetic" s teen
cells
Proinsulin DNA S x 10 T cells 4/28 (14)*
Ovalbumin DNA S x 10 T cells 18/28 (64)
Proinsulin DNA 4 x 10 T cells CD4 10/28 (36)**
T cells
Ovalbumin DNA 4 x 10 T cells CD4 20/28 (71)
T cells
Proinsulin DNA 1 x 10 T cells CD8 17/18 (94)
T cells
Ovalbumin DNA 1 x 10 T cells CD8 15/18 (83)
T cells
*p=0.003, **p=0.02 compared to ovalbumin DNA control; Fisher's exact test
TABLE 5 Splenic CD4 T cells from NOD mice given intranasal proinsulin II DNA
suppress cyclophosphamide-induced diabetes
Treatment of donor Donor spleen cells injectedDiabetes incidence
(2 after
days after 17 da s
c clo hos hamide
Proinsulin DNA intranasal5 x 10 T cells 2/16 (12.5)*
Ovalbumin DNA intranasal5 x 10 T cells 9/16 (56)
Proinsulin DNA intramuscular5 x 10 T cells 4/8 (50)
Proinsulin DNA intranasal4 x 10 T cells CD4 T 3/18 (17)**
cells
Ovalbumin DNA intranasal4 x 10 T cells CD4 T 10/18 (56)
cells
Proinsulin DNA intranasal2.5 x 10 T cells CD8 5/6 (83)
T cells
Ovalbumin DNA intranasal2.5 x 10 T cells CD8 4/6 (67)
T cells
*p=0.002, **p=0.04 compared to ovalbumin DNA control; Fisher's exact test
The inventors next sought to identify the phenotype of the T cell responsible
for protection
by transfernng fractional splenic T-cell population. Splenic T cells were
incubated with
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
- 45 -
either anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies conjugated to
magnetic
MACS MicroBeads and purified on positive selection columns (Miltenyi). The
purity of
CD4 and CD8 T cells by FACS analyses was >95% and >85%, respectively. Either 4
x
106 CD4 T cells or 1 x 106 CD8 T cells were then co-transferred with 2 x 107
diabetic
spleen cells into 6 week-old irradiated males. Diabetes incidence 4 weeks
after transfer
was 36% in recipients of CD4 T cells from proinsulin DNA-treated donors
compared to
71 % in recipients of CD4 T cells from ovalbumin DNA-treated controls (p=0.02)
(Table
4). In contrast, there was no difference in diabetes incidence (94 v 83%) in
recipients of
co-transferred CD8 cells from either proinsulin DNA-or ovalbumin DNA-treated
mice
(Table 4). Similar results were obtained when either 4 x 106 CD4 T cells or
2.5 x 106 CD4-
depleted (CD8 T cell-enriched) splenic T cells were injected into
cyclophosphamide-
treated 10 week-old female mice. Thus, diabetes incidence in mice that
received CD4 T
cells from proinsulin DNA-treated donors was significantly reduced (17%)
compared to
mice that received CD4 T cells from ovalbumin DNA-treated donors (56%)
(p=0.04),
whereas there was no difference in diabetes incidence in mice that received
CD4-depleted
splenic T cells from proinsulin DNA- or ovalbumin DNA-treated donors (Tables 5
and 6).
TABLE 6 CD451tBh' and CD45RB~° CD4 T cells from NOD mice given
intranasal
proinsulin II DNA suppress cyclophosphamide-induced diabetes
Intranasal Donor spleen cells injected (2 daysDiabetes
treatment of after incidence
donor cyclophosphamide) after
17 da s
Proinsulin DNA 3 x 10 CD45RB' T cells (43% of splenic3/16 (19)*
T cells)
Ovalbumin DNA 3 x 10 CD45RB' T cells (43% of splenic9/16 (56)
T cells)
Proinsulin DNA 1 x lOb CD4512B' T cells (14% of 4/16 (25)**
splenic T cells)
Ovalbumin DNA 1 x 10 CD45RB' T cells (14% of splenic6/14 (42)
T cells)
None None 5/10 (50)
*p=0.07, **p=0.4 compared to ovalbumin DNA control; Fisher's exact test
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-46-
In both humans and NOD mice the development of hyperglycaemia is preceded by
"insulitis", which ranges form a peri-islet accumulation of lymphocytes at the
vascular
pole or boundary of the islet to massive infiltration into the islet
associated with ~3-cell
destruction. The inventors then examined islets of NOD mice and found that the
degree of
insulitis was significantly less in proinsulin DNA- compared to ovalbumin DNA-
treated
mice at both 70 and 100 days of age (0.89 t 0.08 versus 1.6 ~ 0.12, p < 0.01;
Mann-
Whitney test).
Those skilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all. such variations and modifications. The
invention also
includes all of the steps, features, compositions and compounds referred to or
indicated in
this specification, individually or collectively, and any and all combinations
of any two or
more of said steps or features.
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-47-
BIBLIOGRAPHY
1. Weiner, H.L., et al., . Annu. Rev. Immunol. 12: 809-837, 1994
2. Kikutani et al., Adv. Immunol. 5l: 285-322, 1992
3. Adorini et al., Springer Semin. Immunopathol. 14: 187-199, 1992
4. Muir et al., Diabet.lMetab. Rev. 9: 279-287, 1993
5. Tisch et al., Proc. Natl. Acad. Sci. USA 91: 437-438, 1994
6. Harnson, L.C., Mol. Med. 1: 722-727, 1995
7. Holt et al., Immunol. Today 8: 14-15, 1987
8. Wells, H.G., J. Infect. Dis. 9: 147-151, 1911
9. Chen et al., Science 265: 1237-1239. 1994
10. Honeyman et al., Springer Semin. Immunopethol. 14: 253-274, 1993
11. Bach, J.F. 1994. Endocrine Rev. I5: 516-542, 1994
12. Mordes et al., Diab. Metab. Rev. 3: 725-750, 1987
13. Harrison, L.C., Immunol. Today. 13: 348-352, 1992
14. Dean et al., Diabetologia 29: 339-342, 1986
15. Ziegler et al., Diabetologia 36: 402-408, 1993
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
- 48 -
16. Harrison et al., J. Clin. Invest. 89: 1161-1165, 1992
17. Rudy et al., Mol. Med. l: 625-633, 1995
18. Ziegler et al., Diabetes 38: 358-363, 1989
19. Daniel et al., Proc. Natl. Acad. Sci. USA 93: 956-958, 1986
20. Zhang et al., Proc. Natl. Acad. Sci. USA 88: 10252-10256, 1991
21. Bergerot et al., J. Autoimmunity 7: 655-663, 1994
22. Moses et al., Diabetes 32: 1040-1047, 1983
23. Metzler et al., Inter. Immunol. S: 1159-1165, 1993
24. Waldo et al., Clin. Immunol. Immunopathol. 73: 30-34, 1994
25. Kaufinan, D.L., Nature 366: 69-72, 1993
26. Hurtenback et al., J. Autoimmunity 2: 151-161, 1989
27. Hunt et al., J. Immunol. 136: 3994-3996, 1986
28. Tian et al., J. Exp. Med. 183: 1561-1567, 1996
29. French et al., Diabetes 46: 34-39, 1997
30. Wicker et al., Diabetes 35:855-60, 1986
CA 02387652 2002-04-16
WO 01/30378 PCT/AU00/01299
-49-
31. Kabelitz, D., Crit. Rev. Immunol. 1l: 281-303, 1992
32. Kaufman et al., Proc. Natl. Acad. Sci. USA 90: 9620-9624, 1993
33. Cardillo et al.,. Eur Jlmmunol 23: 2597-2605, 1993
34. Gorczynski, R.M., Immunology 81: 27-35, 1994
35. Seo et al., Cancer Immunol. Immunother. 40: 358-366, 1995
36. Nakata et al., Immunol Immunopathol 3: 217-222, 1995
37. Lang et al., J. Autoimmunity 6: 107-119, 1993
38. Goodman et al., J. Exp. Med. 170. 1569-1581, 1989.
39. Kweon et al., J. Immunol. 162: 1904-1909, 1999
40. Xu et al., Immunity 1: 423-431, 1994
41. Blanas et al., Science 274: 1707-1709, 1996
