Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOREACTIVE AND IMMUNOTHERAPEUTIC MOLECULES WHICH
INTERACT IN SUBJECTS WITH INSULIN-DEPENDENT DIABETES
MELLITUS (IDDM)
This is a divisional of Canadian Patent Application
2,213,301 filed February 20, 1996.
The present invention relates generally to molecules such as peptides,
polypeptides
and proteins which interact immunologically with antibodies or T-cells in
subjects
having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDIvi).
These
molecules are preferentially immunoreactive to T-cells in subjects having pre-
clinical
or clinical IDDM and are useful in the development of diagnostic, therapeutic
and
prophylactic agents for IDDM.
Amino acid sequences are referred to herein by sequence identity numbers (SEA
ID
NOs) which are defined at the end of the specification.
1 S Throughout this specification, unless the context requires otherwise, the
word
"COmprtSe", Or VSriatlOItS Sll~li 8S "COmprlSeS" Or "Comprising", will be
undefStOOd t0
imply the inclusion of a stated element or integer or group of elements or
integers,
but not to the exclusion of any oø~er element or integer or group of elements
~r
integers. .
Insulin - Dependent Diabetes Mellitus is a serious disease resulting from the
destruction of insulin-secreting ~3 - cells, probably mediated by T cells that
recognise
~-cell autoantigens. A major antigen implicated in T-cell mediated ~-cell
destruction
characteristic of IDDM is glutamic acid decarboxylase (GAD), which o~"ccurs in
two
major isoforms, GAD 65 and GAD 67. These two isoforms have appro~:imately 6~%
similarity at the amino acid sequence level. Subjects with IDDM or at high-
risk of
the disease show autoantibody and autoreactive T-cell responses to GAD insulin
Qr
both autoantigens. In N4D mice, an animal model for spontaneous IDDM, GA,I3 is
a
dominant and early target antigen (Tisch et al Nature 366:72-75, 1993).
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Identification of the immunodominant epitope(s) of pathogenic autoantigens
involved
in ,B-cell autoimmunity could lead to improved methods of diagnosis as well as
therapeutic strategies to prevent IDDM.
In work leading up to the present invention, the inventors sought to identify
immunodominant epitopes in GAD and proinsulin molecules in order to improve
upon current diagnostic procedures and to further develop therapeutic and
prophylactic compositions and treatment approaches for IDDM.
In accordance with the present invention, peptides were synthesised based on a
thirteen amino acid region of high similarity between the sequences of human
GAD
65 (amino acid residue numbers 506-518) and human proiinsulin (amino acid
residue
numbers 24-36), whicr~ region of similarity also extends to human GAD 67 and
mouse proinsulins and mouse GADS (Figure 1 ). The immunoreactivity of these
peptides is identified in accordance with the present invention on the basis
of
interactivity of peripheral blood cells or T-cells obtained from the
peripheral blood of
subjects with pre-clinical or clinical IDDM, thereby forming the basis for a
new
range of diagnostic, therapeutic and prophylactic procedures for IDDM.
Accordingly, one aspect of the present invention provides a recombinant or
synthetic
peptide or chemical equivalents thereof of the formula:
XiXzX3
wherein:
X, and X3 may be the same or different and each is an amino acid sequence
comprising from 0 to 40 naturally or non-naturally occurring amino acid
residues;
X2 is any amino acid sequence of from 10 to 100 residues derived from,
homologous
to or contiguous within amino acids 506 to 518 inclusive or derivatives
thereof of
human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of
human
proinsulin; and wherein said peptide molecule is capable of reacting with T
cells and
modifying T-cell function when incubated with cells from subjects having pre-
clinical
or clinical Insulin-Dependent Diabetes Mellitus (IDDM). Preferred cells
include but
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are not limited to peripheral blood mononuclear cells (PBMCs), andcoagulated
whole
blood and tissue biopsy cells.
Reference to a "peptide" includes reference to a polypeptide or protein or
parts
thereof.
In a preferned embodiment XZ comprises not less than. about 10 and not greater
than
about 50, amino acid residues, more preferably not less than about 10 and not
greater
than about 30 amino acid residues and even more preferably not less than about
10
and not greater than about 15.
In a particularly preferred embodiment XZ has either of the following amino
acid
sequences:
FFYTPKTRREAED [SEQIDNO:1];or
FWYIPPSLRTLED [SEQIDN0:2].
According to this preferred embodiment, there is provided a recombinant or
synthetic
peptide or chemical equivalent thereof comprising the sequence:
X,XiX3
wherein
X, and x3 may be the same or different and each is an amino acid sequence
comprising from 0 to 15 naturally or non-nahwaUy occurring amino acid
residues;
X2 is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or
chemical equivalent thereof and wherein said peptide is capable of reacting
with T
cells and modifying T-cell function when incubated with cells from subjects
with pre.
clinical or clinical IDDM and determining reactivity by an appmpziate assay.
Preferred cells include but are not limited PBMCs, anti-coagulated .whole
blood or
tissue biopsy cells and de~teimining reactivity by an appropriate assay,
The peptides of the present invention may be prepared by recombinant or
chemically
synthetic mesas. According to a preferred aspect of the present invention,
there is
provided a recombinant peptide which is preferentially immunologically
reactive with
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T-cells from individuals with clinical or pre-clinical IDDM, which is prepared
by the
expression of a host cell transformed with a cassette coding for the peptide
sequences
of the present invention. The peptide may be fused to another peptide,
polypeptide
or protein. Alternatively, the peptide may be prepared by chemical synthetic
techniques, such as by the Merrifield solid-phase synthesis procedure. The
synthetic
or recombinant peptide may or may not retain GAD activity or proinsulin
activity.
Furthermore, although synthetic peptides of the formula given above represent
a
preferred embodiment, the present invention also extends to biologically pure
preparations of the naturally occurring peptides or fragments thereof. By
I0 "biologically pure" is meant a preparation comprising at least about 60%,
preferably
at least about 70%, more preferably at least about 80% and still more
preferably at
least about 90% or greater as determined by weight, activity or other suitable
means.
By "pre-clinical IDDM" as used herein means those subjects who may or may not
be
first degree relatives of someone with IDDM who have genetic and/or immune
markers of pancreatic islet (~i) cell autoimmunity. By "immune markers" is
meant
amongst other parameters known to those in the art to include circulating
antibodies
and/or T-cells reactive with islet (~i) cell autoantigeas.
By "derivatives" as used herein is taken to include any single or multiple
amino acid
substitution, deletion and/or addition relative to the naturally occurring
amino acid
sequence in the native molecule from which the peptide is derived including
any
single or multiple substitution, deletion and/or addition of other molecules
associated
with the peptide, including carbohydrate, lipid and/or other proteinacious
moieties.
Such derivatives, therefore, include glycosylated or non-glycosylated forms or
molecules with altered glycosylation patterns.
By the term "reacting with T cells and modify'mg T-cell ftmction" as used
herein is
takcn to include T-cell activation, T-cell inactivation andlor T-cell dcath.
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The present invention also covers chemical analogues of the subject peptides
which
include, but is not limited to, modifications to side chains, incorporation of
unnatural
amino acids and/or their derivatives, during peptide synthesis and the use of
cross-
Linkers and other methods which impose conformational constraints on the
peptides
or their analogues.
Examples of side chain modifications contemplated by the present invention
include
modifications of amino groups such as by reductive alkylation by reaction with
an
aldehyde followed by reduction with NaBH4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups with cyanate;
trinitrobenzyLation of amino groups with 2, 4, 6-trinitrobenzene sulphonic
acid
(TNBS); acylation of amino groups with succinic anhydride and
tetrahydrophthalic ~,, .
anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed
by
reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of
heterocyclic condensation products with reagents such as 2,3-butanedione,
phenylglyoxal and glyoxal.
zo
The carboxyl group may be modified by carbodiimide activation via O-
acylisourea
formation followed by subsequent derivitisation, for example, to a
con~esponding
amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with
iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid;
formation of mixed disulphides with other thiol compounds; reaction with
maleimide,
malefic anhydride or other substituted maleimide; formation of mercurial
derivatives
using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,
phenylmercury
chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with
cyaaate at alkaline pH.
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Tryptophan residues may be modified by, for example, oxidation with N-
bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl
bromide or sulphenyl halides. Tyrosine residues on the other hand, may be
altered
by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished
by
alkylation with iodoacetic acid derivatives or N-carbethoxylation with
diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide
synthesis include, but are not limited to, use of norleucine, 4-amino butyric
acid, 4-
amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine,
norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-
methylheptanoic
acid, 2-thienyl alanine and,~or D-isomers of amino acids.
IS
Crosslinkers can be used, for example, to stabilise 3D conformations, using
homo-
bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n
spacer
groups with n=1 to n=6, glutaraldehyde, N-hydmxysuccinimide esters and hetero-
bifunctional reagents which usually contain an amino-reactive moiety such as N-
hydroxysuccinimide and another group specific-reactive moiety such as
maleimido or
dithio moiety (SH) or carbodiimide (COOI-n. In addition, peptides can be
conformationally constrained by, for example, incorporation of Ca and Na
methylamino acids, introduction of double bonds between CQ and C~ atoms of
amino
acids and the formation of cyclic peptides or analogues by introducing
covalent
bonds such as forming an amide bond between the N and C termini, between two
side chains or between a side chain and the N or C terminus.
The invention also extends to use of the peptides, or derivatives thereof of
the present
invention in the tr~a~ent of patients. In this latter aspect, such methods of
treatment
include their use as an adsorbent to remove autoantibodies or autoreactive
cells from
a patient, their use in direct administration to a patient as a means of
dGSensitising or
inducing immunological tolerance or other mechanisnns to eliminate or diminish
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reactivity of autoreactive T-cells or autoantibodies to IDDM autoantigens or
to
generate T-cell lines or clones to be used for or as therapeutic agents.
According to this aspect of the present invention, there is provided a method
of
treatment comprising administering to a subject an effective amount of a
peptide or
chemical equivalent thereof for a time and under conditions sufficient to
remove or
substantially reduce the presence or function in said subject of autoreactive
T-cells
and/or autoantibodies to IDDM autoantigens wherein the peptide comprises the
formula:
X,X~X3
wherein:
X, and X3 may be the same or different and each is an amino acid sequence
comprising from 0 to 40 naturally or non-naturally occurring amino acid
residues;
Xz is any amino acid sequence of from 10 to 100 residues derived from,
homologous
to or contiguous within amino acids 506 to 518 inclusive or derivatives
thereof of
human GAD65 andlor amino acids 24 to 36 inclusive or derivatives thereof of
human
proinsulin; and wherein said peptide molecule is capable of reacting with T
cells and
modifying T-cell function when incubated with cells from subjects having
clinical or
pre-clinical Insulin-Dependent Diabetes Mellitus (IDDM). Preferred cells
include but
are not limited to peripheral blood mononuclear cells (PBMCs), anticoagulated
whole
blood and tissue biopsy cells.
The method of treatment contemplated herein includes, but is not limited to,
the
following examples. A first example of treatment is desensitisation or
tolerance
induction using an effective amount of synthetic peptide or derivative thereof
to alter
T-cell recognition of or response to GAD and/or pro-insulin and/or other IDDM
antigens and/or induce T-cell suppression or regulation. This may be achieved
by
using the known effect of certain ultraviolet wavelengths, especially LTV-B,
to modify
antigen presentation through the skin or transmucosal or systemic
administration.
Effective amounts of the peptides or derivatives thereof would be applied
epicutaneously to the skin of subjects exhibiting peripheral blood T-cell
reactivity to
GAD or proinsulin peptides or polypeptides. After exposure of skin to IJV-B
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_g_
radiation, treatment would be repeated until such time that T-cell reactivity
to GAD
or proinsulin was suppressed.
A second example of treatment is to induce mucosal-mediated tolerance using an
effective amount of the subject peptides or derivatives thereof to alter T-
cell
recognition of or response to GAD and/or pro-insulin and/or other IDDM
antigens
and/or induce T-cell suppression using an effective amount of peptide or
derivative
thereof to alter T-cell recognition of or response to GAD and/or pro-insulin
and/or
other IDDM antigens and/or induce T-cell suppression by the administration of
the
peptide or derivatives thereof by oral, aerosol or intranasal means amongst
other
routes of mucosal administration.
Another treatment involves application of the subject peptides to the skin
together
with one or more cytokines such as but not limited to TNFa or ,8. A further
treatment involves systemic administration of soluble peptide via subcutaneous
or
intravenous routes to induce immunological tolerance. Yet another treatment
involves T-cell immunisation whereby T-cell lines are generated to GAD or
proinsulin peptide or polypeptide or fragments thereof by standard procedures,
cells
attenuated by fixation with agents such as glutaraldehyde or
parafortnaldehyde,
washed under sterile conditions and re-injected into patients for a time and
under
conditions to cause suppression of the endogenous T-cell response to
autoantigens.
These approaches are applicable to the prevention of IDDM progression in
asymptomatic subjects with pre-clinical IDDM or subjects with recent - onset
clinical IDDM, as well as to the recurrence of IDDM in subjects who have
received
pancreas, islet cell or insulin-producing cell transplants. These approaches
are also
applicable to Stiff Man Syndrome (SMS) and other diseases where GAD and/or
proinsulin is an autoandgen.
In accordance with the present invention, the effective amount of peptide is
0.1 erg to
10 mg per dose and preferably 1.0 frg to 1 mg per dose. A dose may comprise a
single administration or protocol comprising single or multiple administration
hourly,
daily, weekly ,or monthly_ or at other suitable times. Administration magic be
.by any- _ _. _ _.. _ .
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convenient means such as, but not limited to, intravenous, subcutaneous,
epicutaneous, infusion, oral, topical, intranansal, aerosol suppository or
intraperitoneal
administration. The peptide may be administered alone or in combination with
one
or more other active molecules such as molecules which facilitate the activity
or
action of the peptide for example lipopolysaccharide (LPS), choleratoxin ~3-
chain,
Lymphocyte Functional Associated Antigen-3 (LFA-3), other adjuvants and in
particular, tumour necrosis factor a (TNF-a), tumour necrosis factor ~3 (TNF-
~) or
leukaemia inhibitory factor (LIF).
In yet a further embodiment, the present invention contemplates the use of the
peptides described herein to measure reactivity of a subject's cells to the
IDDM
autoantigen. The peptides or derivatives thereof may be added in solution or
bound
to a solid support together with cells derived from peripheral blood or from
tissue r
biopsies either unfractionated, fractionated or derived as continuous cell
lines.
Reactivity to the autoantigen may then be measured by standard proliferation
assays
such as incorporation of tritiated thymidine, standard cytotoxic assays such
as release
of marker radioactivity from target cells, measurements of expressed or
secreted
molecules such as surface markers, cytokines or other standard assays of
cellular
reactivity which are well known in the art.
According to this aspect of the present invention, there is provided a method
of
assaying the reactivity of a subject to IDDM autoantigen, said method
comprising
contacting a peptide or chemical equivalent thereof comprising the formula:
X~XzX3
wherein:
X, and X3 may be the same or different and each is an amino acid sequence
comprising from 0 to 40 :naturally or non-naturally occurring amino acid
residues;
XZ is any amino acid sequence of from 10 to 100 residues derived from,
homologous
to or contiguous within amino acids 506 to 518 inclusive or derivatives
thereof of
human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of
human
pminsulin; and wherein said peptide molecule is capable of reacting with T
cells and
modifying T-cell function when incubated-with cells from subjects having pre-
clinical
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or clinical Insulin-Dependent Diabetes Mellitus (IDDM) and determining
reactivity
by appropriate assay. In accordance with this assay, any cell type may be used
but is
preferably selected from PBMC's, anti-coagulated whole blood cells or tissue
biopsy
cells.
S
Preferably, the present invention contemplates a method of assaying the
reactivity of
a subject to IDDM autoantigen said method comprising contacting a peptide or
chemical equivalent thereof comprising the formula:
X,X~X3
wherein:
X, and x3 may be the same or different and each is an amino acid sequence
comprising from 0 to 1 S naturally or non-naturally occurring amino acid
residues;
XZ is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or
chemical equivalent thereof and wherein said peptide is capable of reacting
with T
cells and modifying T-cell function when incubated with ~ cells from subjects
with pre-
clinical or clinical IDDM aad determining reactivity by an appropriate assay.
Preferably, cells include but are not limited to peripheral blood mononuclear
cells
(PBMCs), aaticoagulated whole blood and tissue biopsy cells.
In another embodiment of the present invention, there is provided a diagnostic
kit for
assaying T cells. Standard 96 - well plates, as used in ELISA, are pre-coated
with a
monoclonal antibody (MAb) to a T-cell cytokine such as Y-interferon (7-IFI~
with or
without antigen. Alternatively, antigen is added in soluble form together with
aliquots of peripheral blood, peripheral blood mononuclear cells or T-cells.
Incubation is allowed to pmceed for one or more days, the supernatant
(comprising
medium and plasma) and the cells are washed off, wells washed again and plates
developed with a labelled second MAb to the cytokine such as anti-~-IFN
conjugated
with alkaline phosphatase or horsc,~radish pemxidase. Colorimetric reaction
and read-
out systems can then be utilised. Alternatively, soluble eytokines (eg: Y-
IF'N) are
min the supernatant by standard assays such as ELISA; finther it is possible
to visualise mipically by the ELISPOT technique individual spots on bottoms
of wells representing cytokine produced at the single cell level thereby
enabling the
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frequency of peptide- epitope-reactive T-cells to be determined.
The present invention will now be further described with reference to the
following
non-limiting Figures and Examples.
In the Figures:
Figure 1 shows a comparison of the regions of similarity among mouse and human
proinsulins and GADS. Similarities are boxed; identities within boxes are
shaded.
The C-terminus of the mature insulin B-chain and the pro-insulin cleavage site
are
indicated by the vertical line and arrow respectively.
Figure 2 is a graphical representation showing the level of cellular
proliferation
expressed as the delta score following the stimulation of peripheral blood
mononuclear cells taken from IDDM at-risk (as described in Example 1) or
control
subjects with the following peptides: human GAD65 (residues 506-518); human
proinsulin (residues 24-36); irrelevant control peptide; or tetanus toxoid
(CSL .Ltd.,
Melbourne, Australia).
Figure 3 is a graphical representation showing proliferation (mean + sem) of
pbmc to
proinsulin (aa 24-36) and insulin (aa 1-15) in pre-clinical and control
subjects.
Figure 4 is a graphical representation showing IFN-gamma response (mean + sem)
to
proinsulin (aa 24-36) and insulin beta chain (aa 1-15) in pre-clinical and
control
subjects.
Figure 5 is a graphical representation showing IL 10 response (mean + sem) to
proinsulin (aa 24-36) and insulin beta-chain (aa 1-15) in pre-clinical and
control
subjects.
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The following single and three letter abbreviations are used for amino acid
residues:
Amino Acid Three-letter One-letter
Abbreviation Symbol
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine _ Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
Any residue Xaa X
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EXAMPLE 1
Subjects
Subjects at-risk for IDDM were from the Melbourne Prediabetes Family Study,
Victoria, Australia. Each was entered on the basis of having at least one
first degree
relative with IDDM and islet cell antibodies (ICA)~0 JDF units and/or insulin
autoantibodies (IAA)_>IOOnU/ml. All had normal fasting blood glucose and
glycated
hemoglobin and had had repeat antibody and metabolic tests at six monthly
intervals.
Control subjects were HLA-DR matched, asymptomatic, and without history of
IDDM.
All subjects gave informed, signed consent and the study was approved by the
Ethics
Committees of the Royal Melbourne Hospital and the Walter and Eliza Hall
Institute
of Medical Research. Details of Subjects are described in Table 1.
F;XANIPLE 2
IILA typing and assays of ICA, LA,A, GAD Ab, FPIR:
HLA Typing:
HLA class I (A, B, C) and HLA class II (DR,DQ) typing was performed using
populations of T and B lymphocytes respectively. The cells were isolated from
anticoagulated blood using magnetic beads (Dynal) coated with monoclonal
antibodies to CD8 (class I) or a monomorphic determinant on the class II beta
chain
(class II). The enriched cell populations were typed in a standard
microlymphocytotoxicity assay using a battery of 240 allosera for class I and
120
allosera for class II.
Antibody assays:
ICA were assayed using indirect immunoffuorescence on blood group O donor
pancreas. Titres, in JDF units, were determined by doubling dilution of
positive sera
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and comparison with standard sera run in each assay. The assay has been
included in
all International Diabetes Workshops and proficiency programs.
IAA were assayed by a radiobinding assay which has been internationally
standardised. The upper limit for normal control sera is 40 nU insulin
bound/ml
serum.
GAD antibodies were assayed by immunoprecipitation of GAD enzymatic activity
from piglet brain extract . The mean plus (three) 3 SD of 72 healthy subjects,
460nU/ml, was used to define the normal range.
First phase insulin release (FPIR):
FPIR was calculated as the sum of serum insulin concentrations at 1 and 3
minutes
following the completion of intravenous glucose (O.Sg/kg body weight) injected
over
3 minutes.
EXAMPLE 3
T-cell proliferation assay
Blood was drawn from paired IDDM at-risk and HLA-DR matched controls at the
same time (within 30 minutes) and processed similarly to reduce the effects of
diurnal variation and handling artefacts. Peripheral blood mononuclear cells
were
isolated from heparinised whole blood by Ficoll-Paque (Pharmacia Biotech)
density
centrifugation, washed and resuspended in RPMI 1640 medium (Biosciences Pty
Ltd)
containing 20mM Hepes (CSL Ltd), 10 5 M 2-mercaptoethanol (BDH), penicillin
( 1 OOU/ml), streptomycin ( 1 OO~cg/ml) and 10% v/v autologous plasma.
Aliquots of
2001 (2x105 cells) were transferred into wells of a 96-well, round-bottomed
plate
(Falcon) and incubated in replicates of six with the following peptides at
final
concentrations of 10, 2, and 0.4~cg/ml: human GAD65 (506-518), human
proinsulin
(24-36) (synthesised using an Applied Biosystems Model 431A synthesiser (ABI,
Foster city, CA), and an irrelevant control peptide (CRFDPQFALTNIAVRK)
(Macmmolecular Resources, Fort Collins, CO). Tetanus toxoid (CSL Ltd,
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Melbourne, Australia) at final concentrations of 1.8, 0.18 and 0.018 LfU/ml
was used
as a positive control. Twelve "autologous only" wells containing cells but
without
antigen were included as the background control. Plates were incubated at
37°C in a
5% v/v C02 humidified incubator for 6 days; 0.25~Ci of [3H]thymidine (ICN) was
added to each well for the last 6 hours. The cells were than harvested onto
glass
fibre filters and incorporated radioactivity measured by beta-particle
counting
(Packard Model 2000 Liquid Scintillation Counter). The level of cellular
proliferation was expressed as the delta score (DS=mean counts per minute
(cpm)
incorporated in the presence of antigen, minus the mean cpm of the "autologous
only" wells).
EXAMPLE 4
T-cell Proliferative Responses
1 S T-cell proliferative responses to the similar 13-mer peptides from
proinsulin and
GAD were compared for ten pairs of HLA-DR matched at-risk and control
subjects.
HLA-DR matching was thought to be important not only because of the
specificity of
peptide binding to MHC class II alleles but also because of the known
association
between MHC class II and IDDM. Therefore, T-cell responses would reflect IDDM
rather than MHC specificity . Responses to the highest concentration of either
peptide were significantly (proinsulin, p<0.008; GAD, p<0.018 - Wilcoxon one-
tailed
paired analysis) greater among IDDM at-risk than control subjects. The results
are
summarised in Table 2.
Reactivity to the proinsulin sequence was confined almost entirely to IDDM at-
risk
subjects, whereas some controls also responded to the GAD peptide (Table 2,
Fig. 2).
Both groups responded similarly to tetanus, and no subject reacted to the
unrelated
control peptide.
For six of these pairs (#1, 2, 3, 5, 6, 7) the assay was performed on a
separate
occasion, but using twice as many cells (4x105 per well). Exhaustion of the
media
resulted in unreliable results in three cases. In two of the other three (#5
and 6), the
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results were consistent with those tabulated here, while in the third (#3) the
at-risk
subject displayed greater reactivity to both antigens at the higher cell
number.
EXAMPLE S
T-cell cytokine secretion assays
In a second cohort of 18 paired IDDM at-risk and HLA-DR-matched controls,
PBMCs indicated as per Example 3 were incubated with human proinsulin 24-36
and
human insulin B chain 1-15 each at 0.5, 5 and SO ~g/ml under the conditions as
per
Example 3. In addition to harvesting cells for the measurement of
proliferation by
[3HJ thymidine uptake after 6 days, as per Example 3, incubation media above
the
cells was sampled after 2 days for the measurement of IFN-Y and interleukin-
(IL-) 10
by standard ELISA methods.
1 S EXAMPLE 6
T-CeU Responses
T-cell proliferative and IFN-Y and IL-10 secretory responses to human
proinsulin 24-
36 and human insulin B 1-1S were compared for 18 pairs of HLA-DR matched
IDDM at-risk and control subjects. As per Example 4, there was a significantly
greater (p~.003) proliferative response of IDDM at-risk subjects to the
proinsulin
peptide (Figure 3). In addition, both IFN Y and IL-10 secretion in response to
the
proinsulin peptide were significantly increased (p=O.OOS and p~.001,
respectively)
compared to matched control subjects (Figures 4, S).
2S
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.
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CA 02566828 2006-11-16
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-21 -
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT: (Other than US): AMRAD OPERATIONS PTY LTD
(US only): HARR.ISON, L; HONE~'MAN, M;
RUDY, G; and LEW, A.
(ii) TITLE OF INVENTION: Immunoreactive and Immunotherapeutic Molecules"
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DAVIES COLLISON CAVE
(B) S TREET: 1 LITTLE COLLINS STREET
(C) CITY: MELBOURNE
(D) STATE: VICTORIA
(E) COUNTRY: AUSTRALIA
(F) ZIP: 3000
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT INTERNATIONAL
(B) FILING DATE: 20-FEB-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PN1239/95
(B) FILING DATE: 20-FEB-1995
(A) APPLICATION I~ltIMBER: PN5172/95
(B) FILING DATE: 04-SEP-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HUGHES DR, E JOHN L
(C) REFERENCE/DOCKET NUMBER: EJH/EK
(ix) TELECOhwiUNICATION INFORMATION:
(A) TELEPHONE: +61 3 9254 2777
(B) TELEFAX: +61 3 9254 2770
CA 02566828 2006-11-16
WO 96126218 PCTIAU96muU85
-22-
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp
10
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Phe Trp Tyr Ile Pro Pro Ser Leu Arg Thr Leu Glu Asp
5 10
