Note: Descriptions are shown in the official language in which they were submitted.
CA 0222~14~ 1997-12-18
11957P W0/WWjumy
In vivo diabetes test
Description
The present invention concerns the use of autoreactive
substances to produce an agent for the diagnosis of
cell-mediated diseases or a predisposition to cell-
mediated diseases which influence the immune system.
The elucidation of the molecular relationships in the
development of auto-immune diseases such as rheumatoid
arthritis and juvenile diabetes (IDDM) has advanced
rapidly during recent years and has now revealed
concrete applications for the early diagnosis and a
causal therapy of these diseases.
It has now been established that, in addition to a
genetic disposition, environmental factors also play a
role in the development of these diseases. At the level
of genetic risk factors only a few alleles of the MHC
class II antigens are, for example in the case of IDDM,
closely associated with this disease. Hence it is
possible to define a risk group for IDDM by analysing
these alleles (cf. e.g. Thomson et al., Am. J. Hum.
Genet. 43 (1988), 799-816 or Todd et al., Nature 329
(1987), 599-604).
The environmental factors involved in the development of
IDDM are probably exogenous immunogenically active
peptide sequences. ~_ral antigens among others have been
discussed in this connection which have partial
homologies to endogenous structures. Under special
CA 0222~14~ 1997-12-18
circumstances, in particular in the postnatal phase,
antigens that are taken up through the food such as
bovine serum albumin can induce an immune response
which, due to homologies to endogenous structures, can
initiate an autoaggressive process.
The progressive destruction of the pancreatic ,B cells by
cytotoxic lymphocytes is typical for the course of the
disease in IDDM. This process begins a long time before
an impairment of glucose metabolism becomes apparent.
When manifestations of diabetes can be detected already
over 80 % of the ,~ cells are destroyed. It would
therefore be extremely important to be able to detect
these autoaggressive T cells at an early stage in
persons at risk ln order to be able to provide the
affected individuals with a causal therapy.
It has nowadays been established that the destruction of
endogenous tissue in auto-immune diseases initially
progresses very slowly. In the initial stage of this
process the autoaggressive T cells probably only
recognize one or a few autoantigens. Publications by
Kaufman et al. (Nature 368 (1993), 69-72) and Tisch et
al. (Nature 368 (1993), 72-78) on an animal model (NOD
mouse) of type I diabetes have shown that in the case of
spontaneously occurring diabetes in this mouse strain
the initial T cell-mediated auto-immune reaction is
directed towards glutamic acid decarboxylase. Initially
only 1 to 2 epitopes at the C-terminus of glutamic acid
decarboxylase (GAD) are recognized in the NOD mouse in
this process. As described above changes in glucose
metabolism cannot yet be detected at this stage whereas
in contrast a perinsulitis is already detectable. It is
not until the disease progresses further that the
spectrum of the peptides of GAD that are recognized by
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-- 3
the autoaggressive T cells becomes broader. Once the
diabetes is manifest it is also possible to detect pre-
activated T cells against other islet cell antigens e.g.
peripherin, heat shock protein HSP 65 and
carboxypeptidase H.
There is evidence that also in humans the immune
response towards GAD is causally related to the
development of type I diabetes. Hence for example auto-
antibodies to GAD can be detected in about 80 % of pre-
diabetics although the aetiological role of these auto-
antibodies is estimated to be low. Rather it is assumed
that in type I diabetes there is a progressive
destruction of the pancreatic ~-cells by T lymphocytes.
These T lymphocytes directed towards GAD have already
been detected by several research groups (Harrison et
al., J. Clin. Invest. 89 (1992), 1161; Honeyman et al.,
J. Exp. Med. 177 (1993), 535). T lymphocytes found by
these groups reacted with a peptide fragment of the GAD
67 kd molecule comprising amino acids 208 to 404.
EP-A-0 519 469 discloses auto-immunely reacting
polypeptides from the human GAD 65 kd molecule. These
polypeptides have the amino acid sequence:
X-P-E-V-K-(T or E)-K-Z
in which X is an optional sequence selected from 1 to 10
amino acids and Z is an optional sequence selected from
1 to 8 amino acids.
The European Patent Application No. 95 100 764.0 also
proposes autoreactive peptide sequences from human GAD
65 as well as a pharmaceutical composition containing
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these peptides. It also describes the use of this
pharmaceutical composition to produce an agent for the
diagnosis of diseases or a predisposition to diseases
which influence the immune system or of tumour diseases
or a predisposition to tumour diseases.
One of the objects of the present invention was to
provide a diagnostic method for cell-mediated diseases
of the immune system, in particular auto-immune
diseases, which on the one hand causes as little
discomfort as possible for the patient and on the other
hand enables a rapid and reliable diagnosis.
This object is achieved by the use of autoreactive
substances for the production of an agent for the
diagnosis of cell-mediated diseases or a predisposition
to cell-mediated diseases wherein the agent is
administered intradermally or intracutaneously and the
diagnosis is made on the basis of the occurrence or
absence of a positive reaction at the site of
application.
The diagnostic method according to the invention differs
from the presently available methods on the market that
are used for a general test for cell-mediated immunity
in that these tests use foreign antigens to which the
patient has already been exposed e.g. purified protein
derivative (PPD), tetanus toxoid, Candida etc.. These
tests are used to determine the general state of the
immune system of the test person. Cell-mediated diseases
of the immune system are not diagnosed.
In another widely used test such as the prick test used
to determine allergic reactions, the antigens are not
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administered intradermally. A positive reaction which is
not T cell-mediated can be detected within four hours
and is characterized by the appearance of a superficial
swelling but not by the appearance of a nodule.
The present invention provides a method for the in vivo
detection of the occurrence of a cell-mediated immune
reaction towards autoreactive substances. This cell-
mediated immune reaction indicates the presence of
specific diseases of the immune system, in particular
auto-immune diseases, or a predisposition to such
diseases. Furthermore the cell-mediated immune reaction
can also indicate the presence of tumour diseases or a
predisposition thereto.
The autoreactive substances or auto-antigens that are
suitable for the method according to the invention are
natural, synthetic or recombinant substances such as
nucleic acids, lipids, saccharides, polypeptides,
proteins, peptides and complexes thereof e.g. complexes
of peptides with polypeptides e.g. MHC molecules or
peptide-binding derivatives. Further suitable
autoreactive substances are specific immunogenic
epitopes, fragments, analogues, mimetics or derivatives
which are for example produced by chemical modification
as well as mixtures of the aforementioned substances.
The autoreactive substances can be administered as such
or in a carrier-bound form e.g. in the form of
lipopeptides. The autoreactive substances can be
administered alone or together with additional
substances such as accessory stimulating components or
adjuvants. The auto-antigens can be produced from
natural sources, by recombinant DNA technology or by
synthetic methods e.g. by peptide solid phase synthesis.
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In addition to autoreactive substances in the sense of
endogenous antigens, it is also possible to use those
autoreactive substances which, although being foreign
antigens, are immunologically related to auto-antigens.
Examples of such foreign antigens are peptides which
have a homology to autoreactive peptide sequences from
endogenous proteins. Specific examples are a peptide
from bovine ~-casein A with the sequence
L-V-Y-P-G-P-I-P-N (aa 60-68) which has a homology region
to the endogenous glucose transporter GLUT-2 in the
region of the sequence Q-I-G-P-G-P-I-P-W (aa 412-420)
and a peptide from the Coxsackie virus protein P2-C with
the sequence K-V-K-I-L-P-E-V-K-E-K-H-E (aa 33-45) which
has a homology to glutamate decarboxylase in the region
of the sequence R-F-K-M-F-P-E-V-K-E-K-G-M (aa 155-167).
The method according to the invention comprises the
intradermal administration of the respective auto-
antigens and the determination of the occurrence or
absence of a local positive cellular reaction at the
site of administration. This positive reaction is
determined at a time which is characteristic for a T
cell-mediated immune response preferably at a time which
is at least 24 hours after the administration
particularly preferably during a period of 24 h to 1
week after the administration and most preferably during
a period of 40 to 80 h after the administration.
The positive reaction preferably comprises the
appearance of a nodule at the site of administration and
can be determined qualitatively by visual observation
or/and by palpation and also quantitatively e.g. by
ultrasound or photographic methods.
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On the other hand it is also possible to determine the
products of a positive reaction e.g. cytokines such as y
interferon which are released at the site of
administration by infiltrating cells e.g. leucocytes.
The method according to the invention is particularly
suitable for the diagnosis of auto-immune diseases or a
predisposition to auto-immune diseases.
Examples of auto-immune diseases which can be diagnosed
by the method according to the invention are diabetes
mellitus in particular type I diabetes (IDDM), multiple
sclerosis, rheumatoid arthritis, Basedow disease,
ankylosing spondylitis, acute anterior uveitis, Good-
pasture syndrome, myasthenia gravis, systemic lupus
erythematodes, pemphigus vulgaris, immune thyreoiditis,
sclerodermia, Crohn's disease, Sjogren syndrome,
Reiter's syndrome, inflammatory bowel disease or Graves'
disease. The method according to the invention is
particularly preferably used to diagnose T cell-mediated
diseases such as IDDM, rheumatoid arthritis or multiple
sclerosis and the method according to the invention is
most preferably used to diagnose IDDM.
Autoreactive substances that are for example used to
diagnose IDDM are glutamic acid decarboxylase (GAD)
65 kd or 67 kd, tyrosine phosphatase (38 kd antigen),
carboxypeptidase H, insuline, heat shock protein, 38 kd
insulin secretory granule protein or parts thereof.
Human GAD 65 kd or peptide partial sequences thereof are
particularly preferably used as the autoreactive
substance.
Analogous diagnostic applications are, however, also
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possible for other auto-immune diseases. Examples of
such auto-immune diseases are multiple sclerosis in
which reactive T cells e.g. against myelin basic protein
or the proteolipid protein can be determined, rheumatoid
arthritis in which reactive T cells e.g. against
collagen type II, cytokeratins, dnaJ protein from E.
coli and Hsp 65 can be determined and Basedow disease in
which reactive T cells e.g. against thyroid peroxidase
can be determined. In the case of myasthenia gravis it
is possible to determine reactive T cells against the
acetylcholine receptor or other relevant autoreactive
substances. In the case of lupus erythematodes there are
reactive T cells against HSP 90 and in the case of
Graves disease against the TSH receptor.
In general a diagnostic application is possible for all
diseases which influence the immune system such as e.g.
also in the case of arteriosclerosis. In this case the
disease has been proven to be associated with an immune
response against the heat shock protein Hsp 65 (Xu et
al., _ancet 341, 8840 tl993), 255-259).
A further application is the diagnostic detection of T
cells which react with tumour antigens. Examples of this
are T cells against a melanoma-associated antigen MAGE 1
which were isolated from melanoma patients (van der
Bruggen et al., Science 254 (1991), 1643-1647). These T
cells can be already detected at a stage in which the
tumour is not yet detectable by conventional methods
because the cell mass is still too small. Furthermore
specifically reacting T cells could also be used to
monitor an anti-tumour vaccination.
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In addition it is also possible to use peptides, peptide
derivatives or molecules which bind analogously as
autoreactive substances. For the diagnosis of IDDM it is
preferable to use one or several peptides from GAD in
particular from human GAD 65 kd or peptide derivatives
or peptide mimetics derived therefrom.
For the diagnosis of IDDM peptides or peptide
derivatives are particularly preferably used which
comprise:
(a) the amino acid sequence (I)
D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R,
(b) the amino acid sequence (II)
S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K,
(c) the amino acid sequence (III)
N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T,
(d) the amino acid sequence (IV)
T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G,
(e) the amino acid sequence (V)
P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W,
(f) the amino acid sequence (VI)
T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R,
(g) the amino acid sequence (VII)
F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I,
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(h) the amino acid sequence (VIII)
G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A,
(i) the amino acid sequence (IX)
E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K,
(j) one of the amino acid sequences shown in fig. 1
or 2,
(k) partial regions of the amino acid sequences shown in
(a) to (i) with a length of at least 6 amino acids
or/and
(1) amino acid sequences which have an essentially
equivalent specificity or/and affinity of binding to
MHC molecules as that of the amino acid sequences
shown in (a) to (j).
The amino acid sequences (I) to (VII) correspond to the
amino acid residues 86-105 (I), 246-265 (II), 146-165
(III), 166-186 (IV), 176-195 (V), 206-225 (VI) and
556-575 (VII) of human GAD 65 kd. The amino acid
sequences I-VII are stated in the sequence protocols
SEQ ID NO. 1-7.
The amino acid sequence (VIII) corresponds to the amino
acid residues 266-290 of human GAD 65 kd and the amino
acid sequence (IX) corresponds to the amino acid
residues 306-325 of human GAD 65 kd. The amino acid
sequences shown in figs. 1 and 2 are also partial
sequences of human GAD 65 kd. The amino acid sequences
of figs. 1 and 2 are given in the sequence protocols
SEQ ID NO. 8-28. The amino acid sequences (VIII) and
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(IX) are given in the sequence protocols SEQ ID NO. 29
and 30.
It was surprisingly found that peptides which contain
the aforementioned regions of human GAD 65 react
specifically with T cell subpopulations that have been
isolated from recently discovered type I diabetics.
Hence the peptides according to the invention are early
autoepitopes the use of which enables a very early
diagnosis to type I diabetes.
The amino acid sequences (I) to (IX) are partial regions
from the 65 kd isoform of human glutamic acid
decarboxylase (GAD) the complete amino acid sequence of
which was described by Bu et al. (Proc. Nat. Acad. Sci.
USA 89 (1992), 2115 ff.). The amino acid sequences (I)
to (IX) were found by setting up T cell lines from the
peripheral blood of type I diabetics and subsequently
stimulating them in vitro with GAD from porcine brain or
with recombinant human GAD and testing these T cell
lines in a proliferation assay with synthetic peptide
sequences which were derived from the human GAD
sequence.
The peptides can be produced by known synthetic
processes using chemical methods or they can be produced
by genetic engineering by cloning and expressing a DNA
sequence coding for these peptides in a suitable host
cell in particular E. coli.
Peptides are also preferred which have partial regions
of the specified amino acid sequences (I) to (IX) or of
the amino acid sequences shown in figs. 1 and 2 which
have a length of at least 6 amino acids, preferably of
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-- 12 --
at least 8 amino acids, particularly preferably of at
least 10 amino acids and most preferably of at least 15
amino acids. The minimum length of a peptide according
to the invention is determined by its ability to
recognize a MHC molecule, to bind specifically to it and
to react with the corresponding T cell receptor.
The maximum length of the sections of a peptide
according to the invention which are derived from GAD
and bind to MHC is preferably 100 amino acids,
particularly preferably 50 amino acids and most
preferably 25 amino acids.
In addition to the aforementioned peptides it is also
possible to use peptides which have an essentially
equivalent specificity or/and affinity of binding to MHC
molecules as the aforementioned sequences and which are
preferably derived from the aforementioned amino acid
sequences by substitution, deletion or insertion of
individual amino acid residues or short sections of
amino acid residues or modified substances which bind in
an analogous manner.
In particular the present invention also concerns
peptide variants whose sequence does not fully
correspond to that of the aforementioned amino acid
sequences but instead only have the same or closely
related "anchor positions". In this connection the term
"anchor position" means an amino acid residue that is
essential for binding to an MHC molecule in particular
to a MHC molecule of the classes DR3, DR4 or DQ. The
anchor position for the DRB10401 binding motif is stated
for example in Hammer et al., Cell 74 (1993), 197-203.
Such anchor positions are conserved in peptides
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according to the invention or optionally replaced by
amino acid residues with chemically very closely related
side chains (e.g. alanine by valine, leucine by
isoleucine and vice versa). The anchor positions in the
peptides according to the invention can be determined in
a simple manner by testing variants of the
aforementioned specific peptides for their ability to
bind to MHC molecules. A characteristic of peptides
according to the invention is that they have an
essentially equivalent specificity or/and affinity of
binding to MHC molecules as the aforementioned peptides.
The peptides derived from these peptides preferably have
a sequence homology of at least 30 %, particularly
preferably of at least 50 % and most preferably of at
least 60 % to the initial peptides or partial sequences
thereof.
Examples of variants of the specified peptides are the
corresponding homologous peptide sections from human GAD
67, the complete amino acid sequence of which has also
been described by Bu et al., supra.
The term "essentially equivalent specificity or/and
affinity of binding to MHC molecules" also includes a
binding specificity or/and affinity that is improved in
comparison to the amino acid sequences (I) to (VIII) or
to the amino acid sequences shown in figures 1 and 2
which is found especially in the case of shortened
peptides which have a length of preferably 8 to 15 amino
acids.
The present invention also concerns peptide derivatives.
This term includes peptides in which one or several
amino acids have been derivatized by a chemical
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reaction. Examples of peptide derivatives according to
the invention are in particular those molecules in which
the backbone or/and reactive amino acid side groups e.g.
free amino groups, free carboxyl groups ortand free
hydroxyl groups have been derivatized. Specific examples
of derivatives of amino groups are sulfonamides or
carboxamides, thiourethane derivatives and ammonium
salts e.g. hydrochlorides. Examples of carboxyl group
derivatives are salts, esters and amides. Examples of
hydroxyl group derivatives are O-acyl or 0-alkyl
derivatives. The term peptide derivative according to
the present invention also includes those peptides in
which one or several amino acids are substituted by
naturally occurring or non-naturally occurring amino
acid homologues of the 20 "standard" amino acids.
Examples of such homologues are 4-hydroxyproline, 5-
hydroxylysine, 3-methylhistidine, homoserine, ornithine,
~-alanine and 4-amino butyric acid.
Additional suitable peptide derivatives are complexes or
covalent bonds between peptides and adjuvant components
which are administered together with the autoantigen
e.g. lipids. In this case the peptides can also be used
as lipopeptides.
The present invention also encompasses polypeptides in
which the MHC-binding peptide section is a component of
a larger polypeptide unit wherein the linkage between
the MHC-binding peptide and the remainder of the
polypeptide unit preferably has a pre-determined
breaking point e.g. a protease cleavage site.
The invention also concerns peptide-mimetic substances
which have an essentially equivalent specificity or/and
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affinity of binding to MHC molecules as the
aforementioned peptide or peptide derivatives. Peptide-
mimetic substances or peptide-mimetics are compounds
which can replace peptides with regard to their
interaction with the MHC molecules and, compared to the
native peptides, have a higher metabolic stability,
improved bioavailability and longer duration of action.
Methods for the preparation of peptide-mimetics are
described in Giannis and Kolter, Angew. Chem. 105
(1993), 1303-1326, Lee et al., Bull. Chem. Soc. Jpn. 66
(1993), 2006-2010 and Dorsch et al., Kontakte
(Darmstadt) (1993) (2), 48-56. Reference is made to the
disclosure of these literature references with regard to
the preparation of peptide-mimetic substances according
to the invention.
A further suitable autoreactive substance is a complex
which contains at least one peptide, peptide derivative
or peptide-mimetic and at least one MHC molecule or a
peptide-binding derivative of an MHC molecule. In this
complex a peptide, peptide derivative or peptide mimetic
with a binding constant of preferably at least
10-7 l/mol particularly preferably in the range of 10-8
- 10-9 l/mol is bound to a MHC molecule or a peptide-
binding derivative of a MHC molecule. Alternatively the
peptide, peptide derivative or peptide mimetic can also
be covalently coupled to the MHC molecule e.g. via a
photolinker or a covalent genetic peptide-MHC fusion.
Such a peptide-MHC fusion protein preferably contains a
HLA-DR beta chain and an autoreactive peptide which is
genetically fused thereto. The complex particularly
preferably contains a MHC class II molecule or a
peptide-binding derivative thereof.
The nucleotide sequences of the genes coding for MHC
.. . .. ..
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class II molecules are published in Corell et al., (Mol.
Immunol. 28 (1991), 533-543). Reference is herewith made
to the contents of this publication.
The term "peptide-binding derivative of a MHC molecule"
includes fragments of MHC molecules which are prepared
by proteolytic cleavage of native MHC molecules or by
recombinant DNA techniques and which have essentially
retained their peptide-binding properties. This term is
also understood to include fusion proteins which contain
further polypeptide components in addition to a MHC part
that is responsible for the peptide binding.
The peptide-MHC complexes are preferably prepared by
associating peptide-free MHC molecules or MHC molecule
derivatives with the peptides, peptide derivatives or
peptide mimetics according to the invention. Peptide-
free MHC molecules can for example be prepared by
unfolding native MHC molecules in order to dissociate
bound peptides and refolding the empty MHC molecules
(see Dornmair and McConnell, Proc. Natl. Acad. Sci. USA
87 (1990), 4134-4138 and WO91/14701). Other methods for
the preparation of complexes of peptides and MHC
molecules are described in the European Patent
application No. 95 100 764Ø Reference is herewith made
to this disclosure.
The invention also concerns the use of a composition
which contains an autoreactive substance e.g. a protein,
polypeptide, peptide, peptide derivative, peptide-
mimetic or/and a peptide-MHC complex as the active
component optionally in combination with adjuvants or
other common pharmaceutical additives in a diagnostic
method which comprises the intradermal administration of
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the composition. The composition can in addition contain
an accessory stimulating component e.g. cytokines such
as IL-2 and IL-4 or/and the surface antigen B7 (Wyss-
Coray et al., Eur. J. Immunol. 23 (1993), 2175-2180;
Freeman et al., Science 262 (1993), 909-911) which can
bind to the surface molecule CD-28 on a T cell. The
presence of the accessory stimulating component can
improve or/and modify the diagnostic effect of the
compositlon.
Furthermore it is also preferable to carry out
additional determinations with positive or/and negative
controls in the method according to the invention. One
can for example use a non-reactive polypeptide such as
human serum albumin or a preparation that only contains
additives, e.g. the buffer, as the negative control.
Purified protein derivative (PPD) from the culture
medium in which tubercle bacteria have been cultured
(commercially available for example from Statens Serum
Institute, Copenhagen Denmark) or tetanus-toxoid which
is known to cause a strong T cell stimulation in many
people can for example be used as a positive control
antigen.
In certain embodiments of the invention it is also
preferable to administer the autoreactive substances
together with an adjuvant. Examples of suitable
adjuvants are aluminium hydroxide, incomplete Freund's
adjuvant, aluminium phosphate and lipids which are
suitable for coupling to peptides (lipopeptides).
The autoreactive substances can be administered by known
methods in which for example a normal hypodermic
syringe, e.g. with a size 26G needle, is used. The
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.
- 18 -
composition containing the autoreactive substance is
preferably administered intracutaneously in a volume of
20 ~l to 500 ~1, preferably 50 ~l to 200 ~l at a
suitable site of the body which is most suitable for the
autoantigen presented to the cells of the immune system.
For example to diagnose IDDM it can be injected into the
forearm (front side).
The concentration of the autoantigen in the composition
can be varied over wide ranges depending on the
autoantigens used in each case. Concentrations of 1 to
100 ~g/ml in particular of 5 to 20 ~g/ml are preferably
used.
Furthermore it is also possible to carry out the
administration in a device which comprises one chamber
for holding a preparation containing autoreactive
substances, one chamber for holding a preparation for
the positive control and one chamber for holding a
preparation for the negative control. Such a device can
for example contain 2 to 10 chambers for holding
preparations containing autoreactive substances. This
device can be used in a simple manner to test several
autoreactive substances simultaneously.
A further subject matter of the present invention is the
determination of a specific T cell subpopulation in
which the sample containing T cells which is preferably
derived from a tissue sample from the area of the site
of administration is contacted with an autoreactive
substance and the reaction of T cells with the substance
is determined. A specific reaction of T cells with the
autoantigen can for example be detected by an increased
T cell proliferation which can be measured by the
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-- 19 --
incorporation of radioactivity. On the other hand the
reaction of T cells can also be determined directly by
using a labelled autoantigen e.g. in a complex form with
a soluble MHC molecule. In this embodiment the
autoreactive substance is preferably used with a
fluorescent labelling group that is coupled thereto. The
evaluation can for example be carried out by FACS
analysis in which the T cells are contacted with a first
fluorescent marker that is coupled to a T cell specific
antibody and then contacted with the autoantigen which
is coupled to a second fluorescent marker and the
presence of double-labelled cells is determined by
fluorographic analysis. In this manner a T cell
subpopulation is determined which is characterized by
its reactivity with an autoantigen. The method can
optionally also include a selection for pre-activated T
cells e.g. a selective enrichment of IL-2 receptor-
positive T cells by incubation with IL-2 or/and by
incubation with IL-2 receptor antibodies and subsequent
separation of the antibody-binding cells for example
using immuno-magnetic methods. On the other hand the
preactivated cells can be first selected after contact
of the T cells with the autoantigen.
In a modification of this method it is also possible to
determine the ratio of preactivated autoreactive T
cells, i.e. T cells with the IL-2 receptor as a surface
marker, to non-activated autoreactive T cells i.e. T
cells without the IL-2 receptor.
A further subject matter of the present invention is the
isolation of T cell subpopulations which react with an
autoantigen. In such a method a tissue sample containing
T cells which is derived from the area of the site of
administration is contacted with an autoantigen, the T
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cells reacting with the autoantigen are identified and
they are optionally separated from other T cells. In
this case it is also possible to select for preactivated
T cells, i.e. T cells containing the IL-2 receptor,
before or/and after contact of the T cells with the
autoantigen. In such a method one can use the
autoantigen for example in the form of a complex with an
MHC molecule, in an immobilized form on a carrier which
simplifies the separation of the positively reacting T
cell population from other T cells. T cell lines can be
established from the T cell subpopulations isolated in
this manner by restimulation. These autoreactive T cell
lines can then be used to immunize patients.
In the diagnostic method for identifying specific T cell
subpopulations it is also possible to use an anti-
idiotypic antibody instead of the autoantigens which
imitates the action of the MHC peptide complex. Such
antibodies can be readily obtained by using a T cell
subpopulation which is specific for a particular antigen
as an immunogen for producing an antibody te.g. in a
mouse) or by firstly producing a first antibody to the
autoantigen and then an anti-idiotypic antibody to the
first antibody.
Specific T cell subpopulations can also be identified by
identifying the products of these in vivo activated T
cells instead of isolating the T cells. This can be
achieved by collecting a tissue sample (e.g. fine needle
aspirate) and determining the cytokines that are
contained therein. Alternatively the cytokines can also
be determined in situ by applying a plaster for a long
time in which antibodies to various cytokines are
immobilized. The cytokines are detected by a
conventional solid phase immunoassay method using enzyme
CA 0222~14~ 1997-12-18
-- 21 --
substrates which yield an insoluble coloured product
which is either read off directly or using a scanner.
Finally the invention concerns a reagent kit for the in
vivo diagnosis of cell-mediated diseases or a
predisposition thereto comprising:
(a) at least one autoreactive substance in the form
of a pharmaceutically acceptable composition,
(b) optionally at least one control substance in the
form of a pharmaceutically acceptable
composltlon
(c) device for the intradermal administration of the
autoreactive substance and the control substance
and
(d) optionally device for the diagnostic evaluation
of the test result.
The compositions for the autoreactive substance and the
control substance preferably contain adjuvants and
optionally other common pharmaceutical carrier
substances, diluents and additives.
It is intended to further elucidate the invention by the
following examples in conjunction with figures 1 and 2
and the sequence protocols SEQ ID NO. 1-30.
Fig. l shows autoreactive amino acid sequences from
human GAD 65 kd,
... ..
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-- 22 --
Fig. 2 shows further autoreactive amino acid sequences
from human GAD 65 kd,
SEQ ID NO. 1-7 show autoreactive amino acid sequences
from human GAD (I) - (VII),
SEQ ID NO. 8-11 show autoreactive amino acid sequences
from human GAD according to fig. 1,
SEQ ID NO. 12 -2 8 show autoreactive amino acid sequences
from human GAD according to fig. 2,
SEQ ID NO. 29 and 30 show autoreactive amino acid
sequences from human GAD (VIII) and (IX).
EXAMPLE 1 (comparative example)
In vitro investi~ation
Peripheral venous blood was collected from eight
patients that had recently contracted IDDM and from
twelve healthy persons. The peripheral blood lymphocytes
were isolated by Ficoll-Hypaque density gradient
centrifugation and were used for an in vitro stimulation
with:
a) recombinant human GAD 65 kd as the test antigen;
b) purified protein derivative (PPD) as a positive
control antigen;
c) human serum albumin (HSA) as a negative control
antigen.
1 x 105 peripheral blood lymphocytes (PBL) were
incubated for five days at 37~C and 5 ~ C02 in a well of
CA 0222~14~ 1997-12-18
- - 23 -
a 96-well round bottom plate in 100 ~l of a culture
medium (RPMI 1640 medium, 5 % human serum, 2 mmol/l
glutamine, 10 U/ml penicillin, 10 ~g/ml streptomycin and
50 nmol/l 2-mercaptoethanol) in the presence of the
aforementioned antigens or in medium alone.
After 5 days 20 ~l RPMI 1640 medium containing 0.5 ~Ci
3H thymidine was added per well. The incubation was
continued for 16 hours and then the cells were
harvested. The incorporation of thymidine was measured
by liquid scintillation counting. The results are stated
as a stimulation index (SI, i.e. cpm in the presence of
the antigen divided by cpm in the absence of the antigen
i.e. medium alone).
The proliferation reactions of lymphocytes from IDDM
patients and control persons towards the individual
antigens are summarized in tables 1 and 2.
TABLE 1
S.I.=cpm PBL stimulated/
cpm PBL non-stimulated
IDDM base value PPD rhGAD 65 HSA
patients cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml
1 730 17.9 2.6 1.1
2 833 16.7 8.8 1.2
3 639 23 6.6 0.9
4 328 28.9 0.8 2.9
1211 10.6 1.9 0.5
6 1041 11.9 1.8 2.2
7 520 19.8 7.2 1.0
8 412 2.7 8.3 0.8
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- 24 -
TABLE 2
S.I.=cpm PBL stimulated/
cpm PBL non-stimulated
healthy base value PPD rhGAD 65 HSA
persons cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml
1 1108 25 1.8 0.7
2 1035 2.1 3.0 0.5
3 390 6.5 2.7 1.1
4 578 6.2 0.9 1.2
243 1.3 0.9 1.9
6 375 6.3 2.5 2.6
7 648 12.5 4.2 0.8
8 730 20.6 4.2 0.5
9 542 0.8 0.9 2.0
gl5 0.5 2.8 2.8
11 567 90.6 3.5 2.3
12 1247 12.5 1.0 0.6
A statistical analysis of the investigation showed that
the proliferation of PBL from recently diagnosed IDDM
patients with rhGAD 65 kd could be regarded as positive
with an SI of more than 4.7 (mean of the control persons
+ 2 SD).
Hence an in vitro T cell reaction to rhGAD 65 kd was
observed in 50 % of the IDDM patients (patients 2, 3, 7
and 8). Antibodies to GAD were found in patients 1, 2, 4
and 8 but not in patients 3 and 5. Patients 6 and 7 were
not tested for antibodies to GAD. The control persons
did not react to GAD 65 kd.
CA 0222~14~ 1997-12-18
-- 25 --
The diabetics and control persons exhibited no
statistically significant difference at all in their
reaction to the controls HSA and PPD.
EXAMPLE 2
In vivo investigation
100 ~l rhGAD 65 kd at a concentration of 10 ~g/ml was
injected intradermally into the right forearm of the
eight IDDM patients from example 1.
The antigen was diluted in physiological saline pH 7.3
containing 1 % HSA. The same volume (100 ~l) HSA at a
concentration of 10 ~g/ml was injected into the right
upper arm as a negative control.
PPD was used as a positive control antigen in 5 of the
18 IDDM patients. HSA was used as a negative control in
all patients.
For the in vivo diagnosis the formation of a nodule
(specific increase of the derma thickness) at the site
of administration was regarded as a positive reaction.
The diagnosis was made 48 h after the administration.
The results were confirmed and quantified by ultrasound
measurements in the area of the administration site. No
side reactions were observed.
The results of this investigation are shown in table 3.
CA 02225145 1997-12-18
-- 26
TABLE 3
IDDM PPD rhGAD 65 HSA time of
patients 10 ~/ml 10 ~g/ml 10 ~g/ml evaluation
1 + ++ - 48h
2 n.t. ++ - 48h
3 n.t. + - 48h
4 - - - 48h
_ 48h
6 - - - 48h
7 n.t. +++ - 48h
8 + + - 48h
Five of the eight tested IDDM patients reacted
positively to rhGAD 65.
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- 27 -
SEQUENCE PROTOCOL
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Boehringer Mannheim GmbH
(B) ROAD: Sandhofer Str. 112-132
(C) CITY: Mannheim
(E) COUNTRY: DE
(F) POSTAL CODE (ZIP): 68305
(ii) TITLE OF THE INVENTION: In vivo diabetes test
(iii) NUMBER OF SEQUENCES: 30
(iv) COMPUTER-READABLE FORM:
(A) DATA CARRIER: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, version #1.30 (EPA)
(vi) DATA OF ORIGINAL APPLICATION:
(A) APPLICATION NUMBER: DE 19526561.0
(B) DATE OF APPLICATION: 20-July-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asp Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala Cys Asp
1 5 10 15
Gly Glu Arg
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
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- 28 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro Glu Val
1 5 10 15
Lys Glu Lys
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
- (C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Asn Trp Glu Leu Ala Asp Gln Pro Gln Asn Leu Glu Glu Ile Leu Met His
1 5 10 15
Cys Gln Thr
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Thr Leu Lys Tyr Ala Ile Lys Thr Gly His Pro Arg Tyr Phe Asn Gln Leu
1 5 10 15
Ser Thr Gly
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- 29 -
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Pro Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly Leu Ala
1 5 10 15
Ala Asp Trp
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
~C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val Thr Leu Lys
1 5 10 15
Lys Met Arg
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(i.) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
.... .
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- 30 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp
1 5 10 15
Phe Leu Ile
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 14 amino acids
(8) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCECE DESCRIPTION: SEQ ID NO: 8:
Ile Leu Ile Lys Cys Asp Glu Arg Gly Lys Met Ile Pro Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Leu Gly Ile Gly Thr Asp Ser Val Ile Leu Ile Lys Cys Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Leu Ala Phe Leu Gln Asp Val Met Asn Ile Leu Leu Gln Tyr
1 5 lO
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Ala Leu Gln Cys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Val Ser Tyr Gln Pro Leu Gly Asp Lys Val Asn Phe Phe Arg
1 5 lO
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Leu Ala Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr Asn Met
1 5 lO
CA 0222~14~ 1997-12-18
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Leu Leu Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gly Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Leu Leu Gln Tyr Val Val Lys Ser Phe Asp Arg Ser Thr Lys
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr
l 5 lO
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
~B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Leu Glu Tyr Val Thr Leu Lys Lys Met Arg Glu Ile Ile Gly
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro
1 5 10
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- - 34 -
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Lys Ile Trp Met His Val Asp Ala Ala Trp Gly Gly Gly Leu
l 5 10
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Trp Gly Gly Gly Leu Leu Met Ser Arg Lys His Lys Trp Lys
l 5 10
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Glu Gly Tyr Glu Met Val Phe Asp Gly Lys Pro Gln His Thr
l 5 10
....
CA 0222~14~ 1997-12-18
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
~B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Trp Leu Thr Ser Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu
1 5 lO
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val
1 5 lO
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(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Leu Val Ser Ala Thr Ala Gly Thr Thr Val Tyr Gly Ala Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Tyr Ile Pro Pro Ser Leu Arg Thr Leu Glu Asp Asn Glu Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp Phe
l 5 10
. . .
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(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Gly Met Ala Ala Leu Pro Arg Leu Ile Ala Phe Thr Ser Glu His Ser His
1 5 10 15
Phe Ser Leu Lys Lys Gly Ala Ala
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRAND FORM:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Glu Arg Gly Lys Met Ile Pro Ser Asp Leu Glu Arg Arg Ile Leu Glu Ala
1 5 10 15
Lys Gln Ly~
