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TREATMENT OF CELIAC DISEASE
WITH INTERLEUKIN-1S ANTAGONISTS
The invention relates to celiac disease, and in particular to floe treatment
of celiac disease.
Celiac disease (CD) is caused by the ingestion of glia~din in genetically
predisposed
individuals 1, 2 generally leading to a wide spectrum of clinical symptoms.
This
pathology is characterised by specific changes at the level of the small
intestine with
characteristic villus atrophy 1, intraepithelial lymphocytes. migration 1 and
production of
anti-endomysium antibodies 3, 4. The latter have been shown to be a specific
marker for
disease. also representing a useful tool to study the incidence of the disease
4-6. Since
gluten drives this disease, a simple gluten free-diet controls all the signs
of this pathology.
The pathogenic mechanisms leading to foil-blown CD are; however not yet
clarified. it is
generally accepted that gluten is recognised by muccrsal T cells, thus
initiating an
immunological cascade that finally leads to the injury of the mucosa and other
disease
specific signs l, 2. Consequentially, several studies on C:D have focused
their attention on
the role of T cells 7, in particular in defying the conditions that induce T
cell activation
and possibly tissue damage. Despite numerous efforts, no study has provided a
definitive
explanation of how T cells might cause this pathology, although
intraepithelial
lymphocytes (IEL) have been considered as the "key players" in this disease 1.
The
inventors have investigated which factors) was driving the massive
intraepithelial
migration observed upon gliadin challenge. Interleukin 15 (IL-15) has recently
gained a
pivotal rule in inducing T cell migration 8 as well as in altering the
functional
characteristics of the targeted T cells 9. The inventors assessed the effect
of IL-15 on
mucosal T cells in an in vitro organ culture of small intestine 10, 11. The
inventors
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observed that IL-15. can induce mucosal T lymphocyte migration in celiac as
well as
normal individuals, although some discriminatory differences were also
observed. since
IL-I S has been reported to modulate the function of intestinal epithelial
cells 12, the
inventors studied whether IL-15 could cause, on mucosal epitlhelial cells of
CD patients as
well as on long term established human intestinal epithelial cell lines,
modifications
compatible with the ones driven by gliadin challenge in celiac patients. The
obtained
results indicated that IL-15 might have been the cardinal factor involved in
the
pathogenesis of CD. ,:The key role of IL-15 was further supported by the
selective
over-representation of IL-15+ cells in the small intestine of untreated CD.
Furthermore by
using neutralising anti-IL-15 monoclonal antibody the inventors have proved
that IL-15
was essentially mediating all the effects induced by gliadin challenge in an
organ culture
model of CD. The inventors have also provided evidence that IL-15 plays a key
role in
modulating intraepithelial migration. These findings indicate that IL-15 is
directly
involved in the initiation and maintenance of CD, providing a novel pathogenic
interpretation of this disease.
Accordingly, a first aspect of the invention provides a method of treating an
inflammatory
bowel disease. such as celiac disease. by administration of an antagonist of
IL-15 to a
patient.
A further aspect of the invention provides the use of an antagonist of IL-IS
to treat an
inflammatory bowel disease, such as celiac disease.
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A still further aspect of the invention provides an antagonist of IL- I 5 for
use in the
manufacture of a medicament to treat an inflammatory bowel disease, such as
celiac
disease.
Preferably, the antagonist of IL-15 activity interferes W ith the signal
transduction of IL-15
through its receptor complex. In particular. the IL-15 antagonists used in the
invention are
preferably selected from the group consisting of (a) a mutein of mature. or
native, IL-15
capable of binding to the a-subunit of the IL-15 receptor and incapable of
transducing a
signal through the ~i and/or r-subunits of the IL-15 receptor complex; (b) a
monoclonal
antibody against IL-15 that prevents IL-15 from effecting signal transduction
through. the ~3
and/or ;~-subunits of the IL-15 receptor complex; and {c) an IL-15 molecule
that is
covalently bonded with a chemical group that interferes with the ability of IL-
15 to effect a
signal tranduction through either the ~i or y-subunits of the; IL-I5 receptor
complex, but
does not interfere with IL-15 binding to IL-lSRa. Antagonists for use in the
invention also
include monoclonal antibodies against IL-i 5.
Preferably. the antagonist used is selected from mature, or native, simian IL-
15 molecules
having the sequence of amino acids 49-I62 of SEQ ID NO:l or human IL-I5
molecules
having the sequence of amino acids 49-162 of SEQ ID NCI:2, that have been
mutated in
order to produce an antagonist of IL-15. Such IL-15 muteins are capable of
binding to the
IL-lSRa subunit, and are incapable of transducing a signal through the ~3 or ~-
subunits of
the IL-I5 receptor complex. These are the subject of patent application number
WO
96/26274.
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Preferably the antagonist is supplied in a pharmaceutically effective amount.
That is, an
amount sufficient to reduce or remove the clinical symptoms ~pf the
inflammatory disease.
L-15 Muteins
There are many possible mutations of IL-15 that can produce antagonists. Such
mutations
can be made at specific amino acid sites believed to be responsible for (3 or
y-subunit
signalling; or mutations can be made over entire regions of IL-1 S that are
considered
necessary for ~3- or ~r-subunit signalling. Typically; mutations may be made
as additions,
substitutions or deletions of amino acid residues. Preferably, substitution
and deletion
muteins are preferred with substitution muteins being most preferred.
It is believed that the Asp56 affects binding with the ~i-subur~it and that
the Glnl56 affects
binding with the y-subunit of the iL-15 receptor complex. Adding or
substituting other
naturally-occurnng amino acid residues near or at sites Asp56 and G1n156 can
affect the
binding of IL-15 to either or both of the ~3 or ~~-subunits of the IL-15
receptor complex.
Indeed. removing the negatively-charged aspartic acid residue and replacing it
with
another negatively-charged residue may not be as effective at blocking
receptor binding as
if the aspartic acid were replaced with a positively-charged amino acid such
as arginine, or
uncharged residues such as serine or cysteine.
Recombinant production of an IL-15 mutein first requires isolation of a DNA
clone (i.e.,
cDNA) that encodes an IL-15 mutein. cDNA clones are derived from primary cells
or cell
lines that express mammalian IL-15 polypeptides. First total cell mRNA is
isolated, then a
cDNA library is made from the mRNA by reverse transcription. A cDNA clone may
be
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isolated and identified using the DNA sequence information ;provided herein to
design a
cross-species hybridization probe or PCR primer as described above. Such cDNA
clones
may have the sequence of nucleic acids 1-489 of SEQ ID N0:3 and SEQ ID NO:4.
The isolated cDNA is preferably in the form of an open reading frame
uninterrupted by
internal nontranslated sequences. or introns. Genomic DNA containing the
relevant
nucleotide sequences that encode mammalian IL-15 polypeptides can also be used
as a
source of genetic information useful in constructing coding sequences. The
isolated
cDNA c'an be mutated utilising techniques known in the art fo provide IL-15
antagonist
activity.
Equivalent DNA constructs that encode various additions or substitutions of
amino .acid
residues or sequences, or deletions of terminal or internal resid~,ues or
sequences not needed
for activin~ are encompassed by the invention. For example. N-glycosylation
sites in IL-15
can be modified to preclude glycosylation, allowing expression of a reduced
carbohydrate
analog in mammalian and yeast expression systems. N-glycosylation sites in
eukaryotic
polypeptides are characterised by an amino acid triplet Asn-~;-Y, wherein X is
any amino
acid except Pro and Y is Ser or Thr. The simian IL-15 protein. comprises two
such triplets,
at amino acids 127-129 and 160-162 of SEQ ID NO:1. The human IL-1~ protein
comprises three such triplets. at amino acids 119-121, 127-1.29 and 160-162 of
SEQ ID
0:2. Appropriate substitutions. additions or deletions to the nucleotide
sequence
encoding these triplets will result in prevention of attachment of
carbohydrate residues at
the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is
replaced by a
different amino acid, for example. is sufficient to inactivate an N-
glycosylation site.
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Known procedures for inactivating N-glycosylation sites in proteins include
those
described in U.S. Patent 5.071,972 and EP 276,846, hereby incorporated by
reference..
Recombinant expression vectors include synthetic or cDI~IA-derived DNA
fragments
encoding an IL-15 mutein. The DNA encoding an IL-15 mutein is operably linked
to a
suitable transcriptional or translational regulatory or structwral nucleotide
sequence, uch
as one derived from mammalian, microbial, viral or insect genes. Examples of
regulatory
sequences include, for example, a genetic sequence having a regulatory role in
gene
expression {e.g., transcriptional promoters or enhancers), an optional
operator sequence to
control transcription. a sequence encoding suitable mRNA ribosomal binding
sites, and
appropriate sequences that control transcription and translation initiation
and termination.
Nucleotide sequences are operably linked when the regulatory sequence
functionally
relates to the structural gene. For example, a DNA sequence for a signal
peptide (secxetory
leader) may be operably linked to a structural gene DNA sequence for an IL-15
mutein if
the signal peptide is expressed as part of a precursor amino acid sequence and
participates
in the secretion of an IL-IS mutein. Further, a promoter nucleotide sequence
is operably
linked to a coding sequence (e.g., structural gene DNA) if the promoter
nucleotide
sequence controls the transcription of the structural gene: nucleotide
sequence. Still
further. a ribosome binding site may be operably linked to a structural gene
nucleotide
coding sequence (e.g. IL-15 mutein) if the ribosome binding site is positioned
within the
vector to encourage translation.
Suitable host cells for expression of an IL-I S mutein include prokaryotes,
yeast or higher
eukaryotic cells under the control of appropriate promoters. Prokaryotes
include gram
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negative or gram positive organisms, for example E. coli or bacilli. Suitable
prokaryotic
hosts cells for transformation include, for example, E. coli, Bacillus
subtilis, Salmonella
typhimuriurn. and various other species within the genera Pseudomonas,
Streptomyces,
and Staphylococcus. As discussed in greater detail below, examples of suitable
host cells
also include yeast such as S. cerevisiae, a mammalian cell I:ine such as
Chinese Hamster
Ovary (CHO) cells. or insect cells. CeII-free translation systems could also
be employed
to produce an IL-I ~ mutein using RNAs derived from the DNA constructs
disclosed
herein. Appropriate cloning and expression vectors for use with bacterial,
insect. yeast,
and mammalian cellular hosts are described, for example. in Pouwels et al.
Cloning
Vectors: A Laboratory Manual. Elsevier, New York, 1985.
When an IL-15 mutein is expressed in a yeast host cell, the nucleotide
sequence (e.g.,
structural gene) that encodes an IL-15 mutein may include a leader sequence.
The leader
sequence may enable improved extracellular secretion of translated polvpeptide
by a yeast
host cell.
IL-IS muteins may be expressed in yeast host cells, preferably from the
Saccharomyces
genus (e.g., S. cerevisiae). Other genera of yeast. such as Pichia or
Kluyveromyces, may
also be employed. Yeast vectors will often contain an origin of replication
sequence from
a 2m yeast plasmid, an autonomously replicating sequence (ARS), a promoter
region;
sequences for polyadenylation, and sequences for transcription termination.
Preferably,
yeast vectors include an origin of replication sequence and selectable marker.
Suitable
promoter sequences for yeast vectors include promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Che~m. 255:2073. 1980) or
other
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glycolvtic enzymes (Hess et al.. J. Adv. Enzyme Reg. 7: /, 49, 1968; and
Holland et al.,
Biochem. 17:4900, /978), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase,
hexokinase. pyruvate decarboxyiase, phosphofructokinase, glucose-6-phosphate
isomerase. 3-phosphoglycerate mutase, pyruvate kinase:, triosephosphate
isomerase,
phosphoglucose isomerase, and glucokinase. Other suitable; vectors and
promoters for use
in yeast expression are further described in Hitzeman, EP-A-73,657
Yeast vectors can be assembled, for example, using DNA sequences from pBR322
for
selection and replication in E. coli (Ampr gene and origin of replication).
Other yeast
DNA sequences that can be included in a yeast expression construct include a
glucose-repressible ADH2 promoter and a-factor secretion leader. The ADH2
promoter
has been described by Russell et al. (J. Biol. Chem. 25f.:2674, 1982) and
Beier et al.
(Nature 300:724, 1982). The yeast a-factor leader sequence directs secretion
of
heterologous polypeptides. The a-factor leader sequence is often inserted
between the
promoter sequence and the structural gene sequence. See, e;.g., Kurjan et al.,
Cell 30:933,
1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330., 1984. A leader
sequence rnay
he modified near its 3' end to contain one or more restriction sites. This
will facilitate
fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those skilled in the art. One such
protocol is
described by Hinnen et al:, Proc. Natl. Acad. Sci. USA 75:1929, 1978. The
Hinnen et al.
protocol selects for Trp+ transformants in a selective medium, wherein the
selective
medium consists of O.b7% yeast nitrogen base, 0.5% casamino acids, 2% glucase,
IO
mg/ml adenine and 20 mglml uracil.
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Yeast host cells transformed by vectors containing ADH2 promoter sequence may
be
grown for inducing expression in a "rich" medium. An example of a rich medium
is one
consisting of 1 % yeast extract. 2% peptone, and 1 % glucose supplemented with
80 mg/ml
adenine and 80 mg/ml uracil. Repression of the ADH2 promoter is lost when
glucose is
exhausted from the medium.
Alternatively, in a prokaryotic host cell, such as E. coli, the IL-15 mutein
may include an
~I-terminal rnethionine residue to facilitate expression of the recombinant
polypeptide in a
prokaryotic host cell. The N-terminal Met may be c;ieaved from the expressed
recombinant IL-15 mutein.
The recombinant expression vectors carrying the recombinant IL-1S rnutein
structural gene
nucleotide sequence are transfected or transformed into a suitable host
microorganism or
mammalian cell line.
Expression vectors transfected into prokaryotic host cells ge:neraIly comprise
one or more
phenoypic selectable markers. A phenotypic selectable marker is, for example:
a gene
encoding proteins that confer antibiotic resistance or that supply an
autotrophic
requirement. and an origin of replication recognised by the host to ensure
amplification
«.-ithin the host. Other useful expression vectors for prolkaryotic host cells
include a
selectable marker of bacterial origin derived from commercially available
plasmids. This
selectable marker can comprise genetic elements of the cloning vector pBR322
(ATCC
.: ~ O l 7). pBR322 contains genes for ampiciliin and tetracycline resistance
and thus
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provides simple means for identifying transformed cell:>. The pBR322
"backbone"
sections are combined with an appropriate promoter and a. IL-15 mutein
structural gene
sequence. Other commercially available vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMf 1 (Promega Biotec,
Madison,
WI, USA).
Promoter sequences are commonly used for recombinant prokaryotic host cell
expression
vectors. Common promoter sequences include ~i-lact~unase (penicillinase),
lactose
promoter system (Chang et al., Nature 275:6I5, 1978; and (Joeddel et ai.,
Nature 281:544,
1979), tryptophan (trp) promoter system (Goeddel et aL, N'ucl. Acids Res.
8:4057, 1980;
and EPA 36:776) and tac promoter (Saunbrook et al., Molecular Cloning: A
Laboratory
Manual. Cold Spring Harbor Laboratory, ( I989)). A particularly useful
prokaryotic host
cell expression system employs a phage I PL promoter and a cI857ts
thermolabile
repressor sequence. Plasmid vectors available from the American Type Culture
Collection
that incorporate derivatives of the 1 PL promoter include plasmid pHUB2
(resident in E.
coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RRl {ATCC
53082)).
Mammalian or insect host cell culture systems also could be employed to
express
recombinant IL- I 5 muteins. Examples of suitable mammalian host cell lines
include the
COS-7 lines of monkey kidney cells (Gluzman et aL, Cel.l 23:175, { 1981 );
ATCC CRL
165 I ). L cells, C 127 cells, 3T3 cells (ATCC CCL 163), CHO cells, HeLa cells
(ATCC
CCL 2), and BHK (ATCC CRL 10) cell lines. Suitable mammalian expression
vectors
include nontranscribed elements such as an origin of replication, a promoter
sequence, an
enhancer linked to the structural gene, other 5' or 3' flanking nontramscribed
sequences,
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such as ribosome binding sites. a polyadenylation site, splice donor and
acceptor sites, and
transcriptional termination sequences.
Transcriptional and translational control sequences in m~unmalian host cell
expression
vectors may be provided by viral sources. For example, commonly used mammalian
cell
promoter sequences and enhancer sequences are derived from Polyoma, Adenovirus
2,
Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from
the
SV40 viral genome, for example. SV40 origin, early and late promoter,
enhancer, splice,
and polyadenylation sites may be used to provide the other genetic elements
required for
expression of a structural gene sequence in a mammalian host cell. Viral early
and late
promoters are particularly useful because both are easily obtained from a
viral genome as a
fragment that may also contain a viral origin of replication (Hers et ai.,
Nature ? 73:113,
1978). Smaller or larger SV40 fragments may also be used, provided the
approximately
2~0 by sequence extending from the Hind III site toward the Bgl I site located
in the SV40
~-iral origin of replication site is included.
E~cemplan~ mammalian expression vectors can be constructed as disclosed by
Okayama
and Berg (Mol. Cell. Biol. 3:280. 1983). Additional useful mammalian
expression vectors
are described in U.S. Patent Application Serial No. 07/480,694 filed February
14, 1990
and U.S. Patent x,350,683.
Purification of Recombinant IL-15 Muteins
In general. IL- I 5 mutein polypeptides may be prepared by culturing
transformed host cells
under culture conditions necessary to express IL-I ~ mutein polypeptides. The
resulting
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expressed mutein may then be purified from culture media or cell extracts. An
1L-15
mutein may be concentrated using a commercially available; protein
concentration filter,
for example. an Amicon or Millipore Pellicon ultrafiltratio:n unit. With or
without the
concentration step, the culture media can be applied to a purification matrix
such as a
hydrophobic chromatography medium. Phenyl Sepharose" CL-4B (Phatmacia) is the
preferred medium. Alternatively, an anion exchange resin caar be employed, for
example,
a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can
be, acrylamide, agarose, dextran, cellulose ar other types commonly employed
in protein
purification. Alternatively, gel filtration medium can be used.
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC)
steps employing hydrophobic RP-HPLC media. e.g., silica gel having pendant
butyl or
other aliphatic groups, can be employed to further purify IL-15 muteins. An S
Sepharose
(Pharmacia) cation exchange column may also be employed as a final buffer
exchange
step. Some or all of the foregoing conventional purification steps, in various
combinations. can also be employed to provide a substantially homogeneous
recombinant
protein.
Recombinant protein produced in bacterial culture is usually isolated by
initial disruption
of the host cells, centrifugation. extraction from cell pellets if an
insoluble polypeptide, or
from the supernatant if a soluble polypeptide, followed by one or more
concentration,
salting-out. ion exchange or size exclusion chromatography ;steps. Finally, RP-
HPLC can
be employed for final purification steps. Microbial cells can be disnzpted by
any
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convenient method. including freeze-thaw cycling, sonication, mechanical
disruption, or
use of cell lysing agents.
Transformed yeast host cells are preferably employed to express an IL-15
mutein as a
secreted polvpeptide. Secreted recombinant polypeptide from a yeast host cell
fermentation can be purified by methods analogous to those disclosed by Urdal
et al. (J.
Chromatog. 296:171. 1984). Urdal et al. describe two sequential. reversed-
phase HPLC
steps for purification of recombinant human IL-2 on a preparative FIPLC
column.
Preferably. a mutein of IL-15 is used wherein at least on.e of the amino acid
residues
:~sp56 or GIn156 of IL-IS (simian IL-I5 having the seduence of amino acid
residues
.I9-I62 shown in SEQ ID NO:1 or human IL-15 having the sequence of amino acid
residues 49-162 shown in SEQ ID N0:2) is deleted or substituted with a
different
naturally-occurring amino acid residue. Any combination of substitutions
and/or deletions
can be made. For example, Asp56 can be deleted while Ci1n156 is substituted
with any
other amino acid, or both Asp56 and Glnl56 are each substituted with the same
or
different amino acid moiety. Further, Asp56 can be substituted with any amino
acid while
Glnl56 is deleted. Generally, substitution muteins are preferred, and more
preferred are
those that do not severely affect the natural folding of the IL-15 molecule.
Substitution
muteins preferably include those wherein Asp56 is substituted by serine or
cysteine; or
wherein G1nI56 is substituted by serine or cysteine, or wherein both Asp56 and
Glni56
are each substituted with a serine or cysteine. Examples of deletion muteins
include those
wherein Asp56 and Glnl56 of mature IL-15 are both deleted; wherein only Asp56
is
,ieleted: or wherein only G1nI56 is deleted. It is possible that other amino
acid residues in
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the region of either Asp~6 and G1nI56 can be substituted or deleted and still
have an effect
of preventing signal tranduction through either or both of the ,l3 or y-
subunits of the IL~-1 S
receptor complex. Therefore. the invention further encompasses muteins wherein
amino
acid residues within the region of Asp~6 and G1n156 are either substituted or
deleted, and
that possess IL-15 antagonist activity. Such muteins can be made using the
methods
described herein and can be assayed for IL-15 antagonist activity using
conventional
methods.
Conjugated IL-15 Molecules and IL-15 Muteins
The mature IL-15 polypeptides disclosed herein (mature sinnian IL-15
comprising the
sequence of amino acids 49-162 of SEQ ID NO:I and mature human IL-15 having
the
sequence of amino acid residues 49-162 shown in SEQ ID 1\f0:2), as well as the
IL-15
muteins, may be modified by forming covalent or aggregative conjugates with
other
chemical moieties. Such moieties can include PEG, mPEG, dextran, PVP, PVA,
polyamino acids such as poly-L-lysine or polyhistidine, albtunin and gelatin
at specific
sites on the IL-15 molecule that can interfere with binding of IlL-15 to the
~i or y-chains of
the IL-15 receptor complex, while maintaining the high affinity of IL-15 for
the IL-ISRa.
Additionally, IL-15 can be specifically glycosylated at sites that can
interfere with binding
of IL-15 to the ~i or ;~-chains of the IL-15 receptor complex, while
maintaining the high
affinity of IL-l~ for the IL-lSRa. Preferred groups for conjugation are PEG,
dextran and
PVP. Most preferred for use in the invention is PEG, wherein the molecular
weight of the
PEG is preferably between about 1,000 to about 20,000. A molecular weight of
about
X000 is preferred for use in conjugating IL-15, although PEG molecules of
other weights
would be suitable as well. A variety of forms of PEG are suitable for use in
the invention.
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For example. PEG can be used in the form of succinimidyl succinate PEG (SS-
PEG)
which provides an ester linkage that is susceptible to h.ydrolvtic cleavage in
vivo,
succinimidvl carbonate PEG (SC-PEG) which provides a wethane linkage and is
stable
against hydrolytic cleavage in vivo. succinimidyl propionate 1PEG (SPA-PEG)
provides an
ether bond that is stable in vivo, vinyl sulfone PEG (VS~-PEG) and maleimide
PEG
(Mal-PEG) all of which are commercially available from Shearwater Polymers,
Inc.
(Huntsville. AL). In general, SS-PEG. SC-PEG and SPA-PEG react specifically
with
lysine residues in the polypeptide. whereas VS-PEG and Mfal-PEG each react
with free
cysteine residues. However. Mal-PEG is prone to react with lysine residues at
alkaline pH.
Preferably. SC-PEG and VS-PEG are preferred, and SC-PE(J is most preferred due
to its
in viv o stability and specificity for lysine residues.
The PEG moieties can be bonded to IL-15 in strategic sites to take advantage
of PEGOs
large molecular size. As described above, PEG moieties can be bonded to IL-15
by
utilising lysine or cysteine residues naturally occurring in the protein or by
site-specific
PEGylation. One method of site specif c PEGylation is through methods of
protein
engineering wherein cysteine or lysine residues are introduced into IL-15 at
specific amino
acid locations. The large molecular size of the PEG chains) conjugated to IL-
15 is
believed to block the region of IL- I S that binds to the ~3 and/or ~-subunits
but not the
a-subunit of the iL-15 receptor complex. Conjugations can be made by a simple
addition
:eaction wherein PEG is added to a basic solution containing IL-I5. Typically,
PEGylation is carried out at either (I) about pH 9.0 and apt molar ratios of
SC-PEG to
lysine residue of approximately 1:1 to 100:1, or greater: or (2) at about pH
7.0 and at
anolar ratios of VS-PEG to cysteine residue of approximately 1:1 to 100:1, or
greater.
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Characterization of the conjugated PEGylated IL-15 molecules can be performed
by
SDS-PAGE on a 4-20 % gradient polyacrylamide gel, available from Novex Corp.,
San
Diego, California. Conventional silver staining may be emplayed, or
conventional
Western blotting techniques can be utilised for highly PECiylated proteins
that are not
visualised easily by silver staining. Purification of the PEGy:lated IL-i S
molecules can be
performed using size exclusion chromatography, dialysis, ultrafiltration or
aff nity
purification. '
The extent of modification and heterogeneity of PEGyIated Ii:-15 can be
determined using
conventional matrix assisted laser desarption ionization mass spectrometry
(MALDI).
Since human IL-1 S has a molecular weight of about 13,000 and by using PEG
having a
molecular weight of 5000, MALDI indicates that preparations weighing 13,000
are
unPEGylated, those weighing / 8.000 indicate that 1 molecule of IL- I S is
bonded to one
PEG molecule; those weighing 23,000 signify that one IL-1S molecule is bound
witri two
PEG molecules, etc.
Monoclonal Antibodies Against IL-15
Alternatively, an antagonist according to the invention can take the form of a
monoclonal
antibody against IL-IS that interferes with the binding of IL-IS to any of the
a, ~3 or
y-subunits of the IL-1 S receptor complex. Within one aspect of the invention,
IL-1 S,
including derivatives thereof, as well as portions or fragments of these
proteins such as
IL-15 peptides, can be used to prepare antibodies that specii~cally bind to IL-
1S. Within
the context of the invention. the term "antibodies" shoulld be understood to
include
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polyclonal antibodies, monoclonal antibodies. fragments therf:of such as
F(ab')2 and Fab
fragments. as well as recombinantly produced binding partners. The affinity of
a
monoclonal antibody or binding partner may be readily determined by one of
ordinary skill
in the art (see Scatchard. Anti. N.Y. Acad. Sci., 51: 660-672 (1949)).
In general, monoclonal antibodies against IL-15 can be generated using the
following
procedure. Purified IL-15, a fragment thereof, synthetic peptides or cells
that express
IL-15 can be used to generate monoclonal antibodies against IL-15 using
techniques
known per se. for example, those techniques described in U.S~. Patent
4,411,993. Briefly,
mice are immunised with IL-L~ as an immunogen emulsified in complete Freund's
adjuvant or RIBI adjuvant (RIBI Corp., Hamilton, Montan<~), and injected in
amounts
ranging from 10-100 ,ug subcutaneousiy or intraperitoneally. Ten to twelve
days later, the
immunised animals are boosted with additional IL-i ~ emulsified in incomplete
Freund's
adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly
immunisation
schedule. Serum samples are periodically taken by retro-orbital bleeding or
tail-tip
excision to test for IL-IS antibodies by dot blot assay, ELISA {Enzyme-Linked
Immunosorbent Assay) or inhibition of IL-15 activity on CTLL-2 cells.
Following detection of an appropriate antibody titer. positive animals are
provided an
additional intravenous injection of IL-15 in saline. Three to four days Later,
the animals
are sacrificed. spleen cells harvested. and spleen cells are fizsed to a
murine myeloma cell
line. e.g., NSl or preferably P3x63Ag8.653 (ATCC CRI:. 1580). Fusions generate
acbridoma cells, which are plated in multiple microtiter plates in a HAT
(hypoxanthine,
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aminopterin and thvmidine) selective medium to inhibit proliferation of non-
fused
myelama cells and myeloma hybrids.
The hybridoma cells are screened by ELISA for reactivity against purified IL-1
S by
adaptations of the techniques disclosed in Engvall et al., Immunochem. 8:871,
1971 and in
U.S. Patent 4,703,004. A preferred screening technique is the antibody capture
technique
described in Beckmann et al., (J. Immunol. 144:4212, 1990). Positive hybridoma
cells can
be injected intraperitoneally into syngeneic Balb/c mice to produce ascites
containing high
concentrations of anti-IL-15 monoclonal antibodies. Alternatively, hybridoma
cells can be
grown in vitro in flasks or roller bottles by various techniques. Monoclonal
antibodies
produced in mouse ascites can be purified by ammonium sulfate precipitation,
followed by
gel exclusion chromatography. Alternatively, affinity chromatography based
upon binding
of antibody to protein A or protein G can also be used. as can affinity
chromatography
based upon binding to IL-15.
Other "antibodies" can be prepared utilising the disclosure provided herein,
and thus fall
within the scope of the invention. Procedures used to generate humanized
antibodies can
be found in U.S. Patent No. 4.816,567 and WO 94/10:332; procedures to generate
microbodies can be found in WO 94/09817; and procedures to generate transgenic
antibodies can be found in GB 2 272 440, all of which are incorporated herein
by
reference.
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To determine which monoclonal antibodies are antagonists, use of a screening
assay is
preferred. A CTLL-2 proliferation assay is preferred for this purpose. See,
Giliis and
Smith. Nature 268:15 (1977), which is incorporated herein by reference.
Preferably the IL-15 antagonists are formulated according to known methods
used to
prepare pharmaceutically useful compositions. An IL-15 antagonist can be
combined in
admixture. either as the sole active material or with other known active
materials, with
pharmaceutically suitable diluents (e.g., Tris-HCI, acetate, phosphate),
preservatives (e.g.,
Thimerosal. benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants
andlor carriers.
Suitable carriers and their formulations are described in l~emington's
Pharmaceutical
Sciences. 16th ed. 1980. Mack Publishing Co. In addition. such compositions
can contain
an IL-I~ antagonist complexed with polyethylene glycol (PEG), metal ions, or
incorporated into polymeric compounds such as polyacetic acid, polyglycolic
acid,
hydrogels. etc., or incorporated into liposomes, microemulsions, micelles,
unilamellar or
multilamellar vesicles, etythrocwe ghosts or spheroblasts. Such compositions
will
influence the physical state, solubility, stability, rate of in vivo release.
and rate of in vivo
clearance of an IL-15 antagonist. An IL-15 antagonist can also be conjugated
to antibodies
against tissue-specific receptors, ligands or antigens, or coupled to ligands
of
tissue-specific receptors.
The IL-I~ antagonist of the invention can be administered topically, orally,
parenterally,
rectail~- or by inhalation. The term "parenteral" includes subcutaneous
injections,
intravenous, intramuscular, intracisternal injection. or infusion techniques.
These
compositions will typically contain an effective amount of an IL-IS
antagonist, alone or in
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combination with an effective amount of any other active ;material. Such
dosages and
desired drug concentrations contained in the compositions may vary depending
upon many
factors, including the intended use, patient's body weight and age, and route
of
administration. Preliminary doses can be determined according to animal tests,
and the
scaling of dosages for human administration can be performed according to art-
accepted
practices.
Figure 1: Effect on T lymphocyte migration of IL-15 in in vitro cultured
treated CD (A)
and control (B) intestine.
A: Treated CD intestine:
Migration to the subepithelial {SE) compartment of the lamin;~ propria. number
of positive
cells per mm-' of Lamina propria, mean + SD and migration into the IEC; number
of
positive cells per mm epithelium, mean + SD.
~p < 0.01 and ~ ~ p<0.05 vs cultures with medium alone.
B: Control intestine:
Migration to the subepithelial (SE) compartment of the lamina propria and to
the
intraepithelial (IE) compartment.
Migration to SE, number of positive cells per. mm2 of lamina propria, and
migration to the
IE, number of positive cells per mm epithelium, mean + SD.
~~<0.05 vs cultures with medium alone.
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Figure 2: Migration of CD3+ cells to the upper mucosal layers in treated CD
intestine
after incubation with medium alone (A) or with IL-15 (B).
Note the massive migration of CD3+ cells to the subepithelia.l compartment of
the lamina
propria and the infiltration of the surface epithelium after IL-15 treatment
(B). Only a few
intraepithelial lymphocytes are found within the surface epithelium after
incubation with
medium alone (A).
(Original rnagnifrcation x 160 (A), I80(B)~
Figure 3: Effect on T cell activation of IL-15 in in vitro cultured treated CD
and control
mtesune
A: Treated CD intestine
of CD3+ cells per mm' of lamina propria, mean + SD.
~ p< 0.01 and ~ ~ p<0.05 vs cultures with medium alone
B: Conwol intestine
of CD3+ cells per mm'- of lamina propria, mean + SD.
~ ~ p<0.05 vs cultures with medium alone.
Figure -l: Expression of FAS by enterocytes in treated CD intestine after
challenge with
medium alone (A) or with IL-15 (B).
Ven- low but negligible staining is observed after culture wiith medium
alone(A). Intense
FAS expression by enterocytes is detected after culture with IL-15. The
staining is mainly
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PCT/GB99/022U1
detected on the basolateral membranes and in the basal cytoplasm (B). Original
magnification, x 160f
Figure ~: Effect of anti-1L-1 ~ MoAb on treated CD intestine: modulation of
the
expression of FAS-L by enterocytes after 24 h of organ culri;~re in the
presence of gliadin
digest.
Intense expression of FAS-L is detected on cellular membranes and the whole
cytoplasm
after incubation with giiadin. The staining is also detected iin some lamina
propria cells.
The same pattern is observed in this case after incubation with the sole
medium or with
IL-15 (A). A marked reduction of FAS-L expression is observed after treatment
of
biopsies challenged with gliadi.n added with anti-IL-15 MoAb MI10 (B)
~Ornginal
magnification, x180).
Figure 6: Effect of anti-IL-15 MoAb on untreated CD 'intestine with villus
atrophy:
inhibition of enterocy~te DNA fragmentation induced by 24 lz organ culture in
the presence
of gliadin digest.
Many enterocvtes are TLTNEL+ after challenge with gliadin (A) ; a dramatic
decrease in
the number of TLTNEL+ enterocvtes is observed after incubation with gliadin
added with
M1 IO anti-IL-15 MoAb (B) (Original magnification, x180)
Figure 7: Effect of IL-15 and IL-2 on the expression of irtvnunological
markers after 24 h
of incubation in CACO-2 cell line.
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A-B: Expression of Kib7 antigen: intense expression of Ki67 is detected in
almost all cells
after incubation with IL-15 (A) and IL-2 (B).
C-D: Expression of FAS: intense expression of FAS is detected on the cell
surface of
almost all cells after IL-1~ treatment (C), whereas weak but negligible
staining is detected
after incubation with IL-2 (D), as well as with medium alone.
E-F: Expression of FAS-Ligand: intense staining is detected on the cell
surface of almost
all cells after IL-S 1 treatment {E}, whereas a weak staining is detected only
in a few cells
after IL-2 treatment (F), as well as with medium alone.
Original magnification, x180 (A. B),. x240 (C, D, E, F); .
Figure 8: Effect of IL-i5 on cell death in CACO-2 cell line simultaneously
cultured with
IL-l~ vs medium alone. or vs IL-7, IL-2, gIiadin, IL-IS+M3M:oAb: Trvpan-Blue+
cells in
culture supernatants.
umber of Trvpan-Blue+ cells x2x 10~/mi culture medium
Callures with IL-15 vs cultures with: medium alone(n=-7); IL-7(n=7); IL-
2(n=7);
aliadin(n=7}; IL-15+M3 MoAb{n=5)
~~.IL-l~ vs medium alone, IL-7;IL-2,gliadin,IL-15+M3 MoAb, respectively,p<0.05
Fiwre 9: Effect of IL-15 on CACO-2 cell line: induction of apoptosis after 24
h of
incubation with IL-15.
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After 24 h of IL-I ~ treatment. incubation with FITC Annexin-V (green colour),
and
propidium iodide (orange colour) leads some cells to show green colour and
others to
co-express nuclear orange colour (A); note green staining on c;eli surface
together orange
colour in the nucleus and in the cytoplasm in some cells (B).
(Original magnification, x, 180 (A), .r280 (B)
METHODS
1. Patients and organ tissue cultures of duodenal expiants
Patients. 14 untreated CD patients with villas atrophy and crypt hyperplasia
(mean age
44.5, range 18-60), 14 treated CD patients on gluten-free diet for at least 12
months and 8
non-CD controls (mean age 38.x, range 19-53) underwent duodenal endoscopy for
diagnostic purposes. Informed consent was obtained from all patients. AlI
treated C:D
patients showed normal mucasal histology, with VIC ratio >~ 3 and absence of
serum
EMA, although EMA were detected in one of these treated :patients in small
intestine
organ culture challenged for 24 hours with medium alone. Non-~CD controls were
affected
by oesophagitis (3/8), gastritis (2/8) and chronic non-specific diarrhoea
(3/8). They were
all EMA negative and showed normal villas length. Intestinal specimens were
obtained at
the duodenal jejunal flexure by peroral biopsy from all patients. All
specimens were
washed in p.15 M sodium chloride and examined with a dissecting microscope.
One
specimen from each patient was oriented and embedded in optimal cutting
temperature,
OCT compound (Tissue Tek, Miles Laboratories, Elkhart, T1V, USA), snap frozen
in
isopenthane cooled in liquid nitrogen and then stored at -70°C until
cryosectioning. 5 ,um
sections were stained with hematoxylin and used for diagnosis.
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Preparation of the culture medium and mucosal tissue culture. Duodenal
explants
from treated CD patients, from 4 untreated CD with villus atrophy and 5
controls were
cultured in vitro for 24 hours as previously reported ". IL-1 ~ and IL-7 were
obtained from
Immunex (Seattle, USA), IL-2 from Roche (Basel, Switzerland), and IL-4 from
Sandoz,
Basel, Switzerland. Anti-IL-15 MoAbs M110 and M111 were obtained from immunex.
IL-15. IL-7, IL-2 and IL-4 were added to the medium at the final concentration
of 10
ng/ml. in 4 treated CD samples. just before the in vitro culttue, anti-IL-15
MoAbs (M110
or Vi 111 ) were added at the f nal concentration of 5 ,ug/nnl to the medium
containing
iL-15. In 3 samples taken from treated CD patients and in 4 samples taken from
untreated
CD with villus atrophy, just before the in vitro culture. M110 MoAb was added
to the
medium containing PT digest. and .carefully mixed to reach a final
concentration of 5
,ugiml. Anti-human lactase MoAb mlacl!6 .was. used as isotype control
antibody.
Duodenal explants from non-CD controls were cultured 'in the presence of the
sole
medium or of medium added with PT gliadin digest ( 1 m~;/ml) or medium added
with
IL-1~ t 10 mgiml).
Immunohistochemistry. Cryostat sections (5 Vim) were individually tested with
MoAbs
to different immunological markers {ICAM-1 {Dako, Copenhagen. Denmark. 1:400),
CD2~ (Dako. 1:30), CD3 (Dako, 1:200), CD8 {Dako, 1:200), ~Id (T Cell
Diagnostics, Inc.
Cambridge. MA, 1:25), Ki67 (Dako 1:25)} and immunostai:ning was done following
the
alkaline phosphatase/anti-alkaline phosphatase or perxoidase staining
techniques".
VioAhs M3 and M38 and M38 FAS specifc'2 (Immunex, mouse MoAbs, 1:30) and FAS-L
specific (Alexis Bingham, UK. rat biotinyiated MoAb 804-009B-Ti00. 1:0)33 were
also
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used and detected by peroxidase staining technique. Intraepithelial
lymphocytes stained by
anti-CD3 or CD8 or yld MoAbs were numbered per mm epithelium; the number of
stained cells in the lamina propria was calculated per mm2 of lamina propria
as previously
reported and referred as percentage of CD3+ cells within the same mucosal
area". The
number of dividing cells expressing Ki67 in crypts was calculated as
previously described
as percentage of crypt enterocytes';. Staining of epithelial cells by anti-FAS
or anti-FAS-L
was arbitrarily graded from 0 to +2. This evaluation was based on the
intensity of staining
of the cells [undetectable (0), low (1+), intense (2+)}. Guidelines for this
scoring system
were established at the start of the study and the samples were independently
analysed by
two observers: the results were compared afterwards. At least 5 slides for
each sample
were blindly evaluated for all tested markers. Two colours
immunohistochemistry for the
characterisation of mononuclear cell populations was performed as previously
reported'4.
For control of specificity of the immunohistochemical data we have performed:
i)
incubation with mouse IgG or IgM directed against inappropriate blood group
antigens and
detected by peroxidase-conjugated streptavidin, as well as by peroxidase anti-
peroxidase
(PAP) staining technique; ii) incubation with Rat Ig MoAb against IL-2; iii)
omission of
primary antibody; iv) repeated experiments (three times each) in the presence
of already
tested positive and negative samples as internal experimental controls; v)
parallel analysis
of samples cultured in the presence of medium or gliadin or tested cytokines
in the same
experimental conditions (each subject provides an internal control).
Antiendomysial antibodies (EMA) detection in culture supernatants. EMA
detection
was sought for in undiluted culture supernatants by immunofluorescence
(Eurospitai
Pharma. Trieste, Italy) according to experimental procedure previously
reported'S. The
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results were blindly evaluated by two distinct observers. In cultured samples
positivity
was weak but clear and only detectable in undiluted supernatants and always
blindly
compared to the other samples belonging to the same patient.
2. Cell culture.
Cell culture chemicals were obtained from GIBCO-Life 'Technologies (Milan,
Italy).
Human intestinal cells CACO-2 were purchased from the Istituto Zooprofilattico
dells
Lombardia a deil'Emilia (Brescia, Italy} and used at passage ?S-40. Cells were
grown in
Duibecco's modified Eagle's medium (DMEM) containing ?5 mmallL glucose and
supplemented with 10% fetal bovine serum, 1 % non-essential amino acids,
2mmol/L
L-glutamine. 1% penicillin-streptomycin, 1% sodium pyruvate. Cells were
maintained in
a humidified atmosphere of 5% CO~ in air at 37°C. Single cell
suspensions were obtained
from 70-80% confluent cultures by incubation with 0.05% trypsin and then 10'
cells were
seeded in 60x 1 S mm Petri dishes containing 20x20 mm
glass coverslips.
Detection of cell deati~ in culture supernatants
One ml culture supernatant was centrifuged ( 10.000 rpm for ~ min) in a
centrifuge
Eppendorf ~=ll5c (Hamburg, FRG). The pellet was re-suspended in 20 ,ul medium
to which
?0 ul of Trepan Blue (Sigma Chemical Co, St Louis. MO) were added. After short
worteting cells were loaded in a Neubaumer chamber (C:arlo Erba, Milan, Italy)
and
counted.
W resin-V test and TI1NEL.
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Detection of DNA fagmentation on cryostat tissue samples was performed by
deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labellin
g
(TUNEL) method and visualised by peroxidase staining as previously
described34.
ApoAlertTM Annexin V Apoptosis Kit (Clonotech Laboratories, Inc., Palo Alto,
CA) was
used for detection of apoptosis in cultured CACO-2 cells. The experiments were
carried
out according to the manufacturer's recommendations. Briefly, coverslips were
rapidly
rinsed in phosphate buffer (PBS) pH 7.4, and incubated for 15 min with' 200,u1
lx binding
buffer. Then, ,I O ;ul enhanced Annexin V-FITC (final concentration O.S
,ug/rnl) and 10 1
propidium iodiae were added and incubation took place for I0 min in the dark.
Coverslips
were mounted and analysed under fluorescence microscope using a dual filter
set for FITC
& rhodamine:
Immunocytochemistry
Coverslips with cells were fixed in acetone for S min and air dried. They were
then rinsed
in Tris buffered saline pH 7.36 and incubated with non-immune goat serum
(/:200,
DAKO) for 15 min. This followed by 1 h incubation with primary MoAb (anti-FAS,
FAS-L, Ki67, as described for immunohistochemistry on tissue sections) and
then by Link
antibody goat anti-mouse biotynilated Ig ( / :300, Dako) only after anti-FAS
and anti-Ki67
Ab incubation and finally by FITC streptavidin { I :50, DAKO) after alI
antibody
incubation. Each step was followed by careful washing with 'TRIS saline
buffer. The
coverslips were then mounted and observed at optical microscope equipped with
fluorescence.
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3. Statistical analysis.
The samples belonging to each category were compared to each other. Studen's
two-tailed
t test was used to compare intestinal samples for the expression of
immunological markers
in the lamina propria, for calculation of stained intraepithelial lymphocytes"
and. for Ki67
antigen expression3~. Non parametric tests (Wilcoxon two-tailed) were also
applied and
the results were concordant with those obtained using paramt;tric tests. For
FAS, FAS-L
and TFR expression samples with undetectable or low staiining have been pooled
for
analysis and Fisher's test was applied to compare tissues with undetectable or
low (0 to
1-) staining with those showing intense (2+) staining. For thE: analysis of
EMA in culture
supernatants Fisher's test was applied as well. For the analysis of Trypan-
Blue+ cells in
culture supernatants, paired t test was used to compare sample, simultaneously
cultured in
the presence of IL-I S vs. cultures in the presence of IL-2, IL-~7, gliadin,
medium alone or
IL-I S+M3MoAb. Student's t test for independent samples was used to compare
the total
number of experiments.
RESULTS
IL-15 induces migration and activation of T cells in l;reated celiac and
control
intestine
The inventors tested in an organ culture model'°~" to investigate
whether challenging
biopsies of treated CD with IL-15 induced the migration o~f CD3+ cells both
into the
subepithelial (SE) and intraepithelial (IE) mucosal compartments". The T cell
distribution
«-as studied in 8 treated CD patients after IL-15 or gliadin challenge, or
after challenge
~tith IL-7. IL-2 and IL-4. These cytokines were used as control for IL-IS
since thew all
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share the same j~ chain receptor and are reported to act in a similar fashion
on T cells'3 (Fig
IA). IL-I5, but not the other tested cytokines, was as effective as gliadin in
modulating
SE T cell migration as well as IE T cell i~ltration (Fig IA, Fig 2A-B). IL-I5,
but not
gliadin, was also competent in inducing migration of yb+ ce:lls to the IEC
(Fig lA). IL-7,
but not IL-4 or IL-2, induced a significant although less vi~;orous, IE
infiltration of y8+ T
cells (Fig I A).
The effects of IL-I S on T cell migration were not limited to CD patients,
since this
cytokine induced T cell migration (with exception of yb-~- T cells) in non-CD
control
biopsies (Fig. 1 B). In non-CD control intestines gliadin was incompetent to
induce any T
cell migration, as previously reported (Fig I B) ".
Because of the well known activities of IL-15 on T cell activation and surface
antigen
modulation9, the inventors determined whether IL-1 ~ challenge of biopsies
from treated
CD patients induced expression of intercellular adhesion molecule-1 (ICAM-I)
and IL-2
receptor on lamina propria CD3+ cells. A significant proportion of CD3+ cells
was
influenced by IL-I S (Fig 3A), and similar results were observed after
incubation. with
gliadin alone, as previously reported" (Fig 3A). Even in this case no effect
was observed
after incubation of treated CD biopsies with IL-7, IL-2 or I1G-4 (Fig 3A). IL-
15, but not
gliadin, induced ICAM-l and IL-2 receptor expression by lamina propria CD3+
cells also
in the intestine of non-CD controls (Fig 3B).
Further studies by the inventors (results not shown) have indicated that IL-
I5, but not IL-7,
IL-4 or IL-2 induced an intraepithelial increase in CD8-~- cells in celiac and
control
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intestine. IL-15 also increases the number of intraepithelial CD94+ cells in
celiacs only.
IL-7 was not found to increase CD94+ cells in celiac biopsies.
Gliadin has been demonstrated {results not shown) to induce epithelial
migration of CD3+,
CD8+ (p<0.001 ) and CD94+ (p<p.05) cells in celiacs but not in controls.
IL-IS challenge induces epithelial changes in treated celiac but not in
control
intestine
To analyse the mitogenic activity of IL-15 on epithelial cells, the inventors
studied the
expression of Ki67, one of the earliest markers of cell proliferation'. In
treated CD
biopsies (n=10) IL-15 challenge induced the expression of Ki67 by crypt
epithelial cells
was induced by IL-I5 challenge [percentage of stained cells/100 crypt
enterocytes, mean
(SD), 8.5{5.7), p=0.007 vs cultures with medium alone (n=10), mean {SD)
1.5(2.1)]. This
effect was not observed in the 5 tested cases of treated CD biopsies after
incubation with
IL-7 [3.2(1.7)], IL-4 [2.5 {2.6)], IL-2 [5.2(4.9)], nor after IL-I5 challenge
of all 8 tested
non-CD control biopsies (data not shown). In 4 out of 10 treated CD biopsies a
few Ki67+
enterocytes were also detected in villus after IL-15 treatment. No Ki67
expression by
crypt enterocytes is observed after a short challenge (24h) with gliadin (n=6)
[2 (2.6)], as
previously reported ".
Expression of Ki67 in crypt enterocytes is a characteristic feature of
untreated CD intestine
and correlates with mucosal damage (Maiuri L. et al., submiated).
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The inventors have recently observed that FAS is over-a;xpressed by small
intestine
epithelial cells of untreated celiac patients (Maiuri L. et al., submitted).
In view of the
epithelial modifications induced by IL-15, the inventors determined whether
FAS was also
up-regulated on epithelial cells by IL-15. After IL-15 challenge, the
expression of FAS
was intense (2+) in 7 out 11 samples and low in 4/11; whil:ct FAS expression
was low to
undetectable in 12 out of 14 samples cultured with sole medium (Table l, Fig
4A-B).
Similarly. gliadin challenge was effective ~in enhancing epithelial expression
of FAS in 8
out of I 4 tested samples (Table I ), in agreement with our previous report
(Maiur~i L et al,
submitted}, whilst no up-regulation of FAS expression was detected after
incubation with
IL-7, IL-:1 or IL-2 (Table 1). IL-15 challenge did not induce expression of
FAS in the
enterocytes of non-CD controls (low or undetectable FAS expression in all 8
tested cases
after culture with IL-15).
Untreated CD patients also over-express FAS-L on their small intestine
epithelial cells
(Maiuri L et al, unpublished data). The inventors consequently verified
whether IL-I5
could also induce surface expression of FAS-L on intestinal epithelial cells
(Table I ).
Biopsies obtained from 5 treated CD patients were studied, in 3 of these cases
FAS-L was
not observed after 24h culture with medium alone, whilst in. two cases FAS-L
expression
was elevated. In two of the 3 biopsies that remained FAS-L negative after 24
hours
incubation with medium, IL-15 induced an increase of the expression of FAS-L.
C)n the
contrary, in all these three negative biopsies 24 h of in vitro challenge
gliadin could not
induce an increase in FAS-L expression (Table 1 ). In none c>f the 5 tested
non-CD control
biopsies the expression of FAS-L was increased after culture in presence of IL-
i 5 or
medium alone.
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IL-15 induces production of antiendomysium antibodies iin treated celiac but
not in
control intestine
Since the inventors have recently described that antiendomysium antibodies
(EMA} are
induced upon in vztro gliadin challenge's, the inventors also tested the
effects of IL-15 on
the production of these autoantibodies. They observed that IL-15 was effective
in
inducing production of EMA in 4 out of 7 tested treated CDR biopsies (Table
2}. On the
contrary, no other cytokine tested. IL-7, IL-2 or IL-4, induced any variation
in the EMA
levels. Similarly to IL-15. only a challenge with gliadin was able to increase
the
production of EMA in two out of 4 tested samples, as we previously reported'S.
IL-15, as
gliadin. did not induce EMA in any of the 5 tested non-CD control cases.
Anti-IL-15 neutralising antibodies control the epithelial changes and the
production
of EMA induced by IL-15 as well as by gliadin challenge in treated celiac
intestine
The results described indicated that IL-15 might play a pivotal role in CD.
The key test
would be. however, to induce an abrogation of the alternations observed in CD
by using
anti-IL-15 neutralising MoAbS (monoclonal antibodies}. Two MoAbs (M 110 and M
I I 1 }
that share the same neutralising characteristics were used for this
experiment. In 3 out of 4
treated CD intestine biopsies anti-IL-I5 MoAbs were effective in controlling
the
modifications caused by IL-15, such as expression of FAS, Ki67, as well as EMA
production. in 2 out of 3 tested samples anti-IL-15 MoAbs prevented the
epithelial
expression of FAS and the production of EMA induced by gliadin challenge.
Finally in
the two samples where FAS-L expression was already intense after incubation
with the
medium alone. M I 10 MoAb was effective in down regulating enterocytes
expression (Fig
SUBSTTTUTE SHEET (RULE 26)
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SA-B). On the contrary, neutralising monoclonal antibodies were not effective
in
controlling the gliadin-induced migration of CD3+ cells into the IEC, as well
as the
gliadin-induced T cell activation. Anti-human lactase MoAb miacl, used as
isotype
control, was devoid of any in vitro effect.
Anti-IL-15 Neutralising antibodies control gliadin-induced apoptosis of
enterocytes
in untreated celiac intestine.
The inventors have found that a 24 h of in vitro with gliadiin challenge in
untreated CD
intestine biopsies is effective in producing enterocytes's DNA fragmentation
(Maiuri L. et
aI, submitted). In 3 out of 4 tested cases M110 MoAb was effective in
preventing the
increase in the number of TUNEL+ enterocytes induced by gliadi:n [mean (SD)
46(2.6)
after gliadin challenge, vs 27.2(7.5) after IL-IS treatment, p < 0.05 (Fig 6 A-
B)].
Expression of IL-15 by lamina propria mononuclear cells (LPMNC) of celiac and
control intestines
To further support the role of IL-15 in the pathogenesis of C;D, The inventors
determined
by immunoistochemistry, the number of LPMNC expressing IL-15, in patients and
controls. In untreated celiac patients ( 14 cases) a significant: increase of
IL-I S+ cells was
detected compared to the intestine from controls (8 cases) (p <0.005) (Table
3). Upon
gluten free diet (10 cases) the number of IL-15+ cells returned to the normal
values (p
>0.05 vs controls) (Table 3). Most of the IL-IS+ cells were. CD68+, and not
surprisingly
none was CD3+.
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Effect of IL-15 on CACO-2 cell lines
Although alI the results previously described strongly indicated that IL-I S
was causing all
the pathognomonic features of CD the inventors could not exclude that some of
the
modifications, particularly the ones observed on epithelial cells, were not
directly induced
by IL-I5. The inventors therefore challenged with IL-15 the CACO-2 epithelial
cell Line,
which is considered to be a faithful prototype of human intestinal epithelial
cells 12, 16,
17. The C ACO-2 cells were
tested at different stages of in vitro maturation.
IL-15 and IL-2, but not IL-7 nor gliadin induces Ki67 antigen expression.
Induction of Ki67 is observed in small intestine epithelial cells of untreated
CD patients
(Maiuri L et aI, submitted), as well as previously shown upon in vitro IL-15
challenge of
small intestine biopsies. The inventors thus analysed the expression of this
marker of
proliferation on CACO-2 cells challenged with different cyto~kines. An intense
expression
of Ki67 was detected in all three experiments in the large majority of the
cells (more than
70% of cellsimmz) after incubation with IL-15 (Fig 7A) or IL-2 (Fig 7B),
whilst in the
same experiments Ki67, expression was restricted to a lower number of cells
(less than
30% of cells/mm2) after incubation with medium alone, IL-7 or gliadin.
IL-15 but not IL-2, IL-7 nor gliadin induces FAS and FAS-L expression
As previously indicated, expression of FAS and FAS-L b~y epithelial cells is a
newly
defined (Maiuri et al submitted) feature of CD. In 4/6 experiments FAS
expression was
induced on cell surface after IL-15 challenge (Fig 7C), while it was low after
incubation
with medium alone, IL-2 (Fig 7D), IL-7 or gliadin (Table 4). Similarly, IL-1~
induced
SUBSTITUTE SHEET (RULE 26)
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36
expression of FAS-L in all 4 experiments (Fig 7E), compared to the lower
expression
observed after incubation with medium alone, IL-2 (Fig 7F), Il:; 7 or gliadin
(Table 4).
IL-15 but not IL-2, IL-7 nor gliadin induces cell death
Since IL-15 was able to induce the co-expression of FAS and FAS-L on CACO-2
cells the
inventors monitored if this event could initiate a suicides/fratricide
outcome. After
incubation with IL-IS the number of Trypan-Blue positive cells in culture
supernatants
was higher than mean+3SD of the values observed after inculbation with medium
alone in
15/16 experiments. The values were significantly higher after IL-15 {(n=16}
mean of
Trypan-Blue+ cells x2 x1041m1, 50.2, SD 41.7; median 36.75, range 7.5-173.5}
than after
incubation with medium alone (n=7, p=0.0008), IL-2 (n=7, p== 0.007), IL-7
(n=7, p=0.001)
or gliadin (n=7, p=0.02). In the experiments in which CACCi-2 cells were
simultaneously
tested with IL-I S vs IL-2 (n=7), or IL-7 (n=7), or gliadin (n=7) or medium
alone (n=7), the
number of Trypan-Blue+ cells in culture supernatants was significantly higher
after IL-15
incubation then after incubation with IL-2 (p = 0.05), IL-7 (~p=0.05), gliadin
(p=0.02) or
medium alone (p=0.01 ) (Fig 8}.
in order to identify whether or not IL-1 S-induced cell death was mediated by
FAS
engagement the inventors performed experiments where IL-:15 was mixed to
neutralising
anti-FAS M3 MoAb. In 5 tested cultures the number of T'rypan-Blue+ cells in
culture
supernatants was significantly lower after incubation with i:L-15 supplemented
with M3
MoAb than after incubation with IL-I 5 alone (p = 0.01 ) (Fig 8). After
incubation with
IL-15 detection of apoptosis by annexin-V and propidi~um iodide revealed in
4/6
experiments that more than 20% of cells showed signs of apoptosis: positive
nuclei
SUBSTITUTE SHEET (RULE 26}
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37
(orange colour) together with surface staining (green colour) (Fig 9A, 9B) in
some of
them. and only green colour in others. On the contrary, in 4lti experiments
less than 5% of
cells became stained after incubation with medium alone or other tested
cytokines.
SUBST TTUTE SHEET (RULE 26a
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DISCUSSION
CD has always been considered as the prototype of an immuno-mediated disease
in which
a single antigen, gliadin, induces T cell activation leading to disease'' z.
Several aspects of
this pathology have pointed in this direction. The first is the strong HLA
associatian, in
particular with the heterodimer DQA1*0501, DQBl*0201'g, which has been
considered to
be the element of genetic restriction for T cells recognising the triggering
antigen (gliadin)
The second is being the presence of a massive T lymphocyte inf ltration of the
small
intestine with a dramatic increase of intraepithelial lyrnphacytes '. Thus,
much of the
studies performed in this pathology have been directed tow~~rds the dissection
of the role
and function of T cells '~'9 (Maiuri et al submitted) and related cytolunes
2°''~ (Maiuri et al
submitted). There were, however, signs that the simple T cell immunological
recognition
of gliadin could not explain all the pathogenic steps of this disease. The
first is the
unusually high incidence of self autoantibodies: the EMA in CD3'~ with the
evidence of
their synthesis, after gliadin challenge, at the mucosal site 'S~ z3. The
autoantigen
recognised by EMA has been recently defined as tissue transglutaminase
2°. It was also
difficult to explain how gliadin could induce the migration of T lymphocytes.
Finally, it
has been so far impossible to define how T cells could induce the mucosal
damage. The
inventors have reported that small intestine enterocytes co-express FAS and
FAS-L on
their surface (Maiuri, et al., submitted), and that the epithelial cells show
clear signs of
apoptosis '5 (Maiuri, et aI submitted). They also observed that gliadin
rapidly induces FAS
on these epithelial cells, in a way that apparently did not require T cell
activation (Maiuri;
et al. submitted).
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39
The inventors have demonstrated that a single cytokine, IL-15, can reproduce
almost all
the features of celiac disease, and more significantly that tlZe modifications
induced by
gliadin could be, by enlarge, controlled with neutralising anti-IL-15
monoclonal
antibodies. These results indicate that IL-15, likely induced at the mucosal
level by
gliadin, has a central role in the pathogenesis of CD in ?~ different ways.
Firstly by
engaging T cells, secondly, by inducing EMA and thirdly by directly affecting
epithelial
cell function. Why IL-15 should have such a dominant role? It has already been
reported
that this cvtokine might have a fundamental part in other imW uno-mediated
diseases such
as Rheumatoid Arthritis, by favouring T cell migration g and ;in 'activating'
T cells 9. Even
in this study, only IL-I5 and not the other cytokines (with the partial
exception of IL-7 on
ya, induced T cell migration although the effects of IL-l:> were not selective
for CD
patients. This indicates that the activity of IL-15 on T sell migration is not
disease
restricted. However IL-IS has another unique characteristic, which ftu~ther
suggests a
potential role in CD: the ability to directly modulate small intestine
epithelial cells'2.
Using the inventors' organ culture model they could demonstrate that small
intestine
epithelial cells over-expressed transferrin receptor after IL-1S challenge
(data not shown),
though other two cytokines, IL-4 and IL-2, were also able ta~ modulate this
receptor. This
is not surprising since it has been reported that intestinc; epithelial cells
express the
receptor and respond to IL-2'6~ ' 7, although very high doses of IL-2 are
needed to induce
epithelial cell responses'Z. Only IL-15 however was able to up-regulate Ki67
suggesting a
trophic action of IL-15, and unexpectedly to consistently induce FAS
expression and,
although less dramatically, FAS-L upregulation. This is important since in the
small
intestine of untreated CD patients these markers are over-expressed and the
engagement of
FAS by its ligand initiate the apoptosis process (Maiuri et a submitted).
Moreover, a 24
SUBSTTTUTE SHEET (RULE 26~
CA 02333923 2001-O1-10
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hour challenge with IL-1 S of. 4 biopsies from untreated celiacs induced a
statistically
significant increase of apoptotic epithelial cells compared to cultures
incubated with
medium alone (data not shown). These findings, induction of FAS, FAS-L and
initiation
of apoptosis by IL-I S on epithelial cells was completely supported by the
studies
performed with the CACO-2 cell line, demonstrating that IL-i 5 directly
influenced
intestinal epithelial cell. In this context it has to be mentioned that the
inventors' results
differ from a recent report in which IL-15 was shown to protect T, B cells and
hepatocytes
from FAS induced apoptosisz6. The reasons for such an app~~rent discrepancy
might lie in
the different experimental procedures, the fact that different; species were
studied, in our
case human and not mouse cells, different target cells and snore important, we
relate our
findings to a well-defined pathology. That IL-15 has a central role in CD is
fiurther
supported by the compelling evidence that EMA, the most specific marker of CD,
are
directly induced by this cytokine, and that the gliadin induced production of
EMA is
blocked by neutralising anti-IL-15 monoclonal antibody. In this context it is
of interest,
the finding that the spontaneous release of EMA in culture, observed in the
supernatant of
one treated CD biopsy, was controlled by neutralising anti-IL,-15 monoclonal
antibodies.
IL-15 could control the induction of EMA by different, and not conflicting,
ways for
instance by directly influencing B cellsz' as well as acting on T cellsz8~ z9
or by unmasking
the EMA antigen (tissue transglutaminase). Indeed, tissue transglutaminase the
autoantigen recognised by EMAz4, is normally up-regulated in epithelial cells
undergoing
apoptosis3°. Thus a scenario might be envisaged in which IL-15 could
control the
induction of EMA by unmasking the autoantigen, via the autoantigen, via the
induction of
small intestine epithelial apoptosis, thus promoting the expression of tissue
SUBSTITUTE SHEET (MULE 26)~
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4I
transglutanninase. In this scheme IL-15 fulfils the role of an agent unmasking
a 'hidden'
autoantigen (translutaminase). IL-15 may further influence the production of
EMA acting
as a locally available growth and differentiation factor for T and B cellsz'~
is, 29.
From our analysis it is apparent that the cells producing IL-15 are, not
surprisingly,
mononuclear cells in the subepithelial compartment, since T' cell are not able
to produce
II,-153'. Epithelial cells seem to be not involved, although small intestine
epithelial cells
have been shown to produce IL-15'2. It remains to be clarified how gliadin
induces IL-15
and why only epithelial cells of CD patients, as well as CACO-2 cells, are
sensitive to the
action of this cytokine. These results therefore suggest that two bottle-necks
control the
induction of CD. The first is the restricted ability of monocyrtic cells of
cellars to produce
IL-15 after gliadin challenge. The second being the specific effects of IL-15
on epithelial
cells of cellars thus, by inducing proliferation, as defined bw the expression
of Ki67, and
ultimately death by allowing the induction of FAS and likely FAS-L. In
conclusion we
have provided powerful evidence that IL-15 has a central role in CD by
directly
influencing T cells, inducing EMA production and controlliing epithelial
damage. In the
final analysis the inventors' study provides a totally novel interpretation of
the pathogenic
mechanisms governing the evolution of one of the most common diseases,
provides novel
therapeutic targets, and sheds light to define the possible genes involved in
this pathology.
SUBSTITUTE SHEET (RULE 2ti)
CA 02333923 2001-O1-10
WO 00/02582 PCT/GB99/02201
42
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CA 02333923 2001-O1-10
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CA 02333923 2001-O1-10
WO 00102582 PCT/GB99/02201
44
a
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beta chain of the interleukin-2 receptor. Science 264, 965-8 (1994).
30. Cummings, M. et aL Apaptosis of epithelial cells in vivo involves tissue
tt~ansgiutaminase upregnlation
IL-15: a pleiotropic cytokine with diverse receptorisignaling pathways whose
expression
is controlled at mnitiple levels
A lymphokine, provisionally designated fnterieukin T and produced by a human
adult
T-c~eil leukemih lice, stimulates T-cell proliferation and th;e iaductian of
lymphokine-
activated killer cells
Cloning of a T cell growth factor that interacts with the beta chain of the
interieukin-2
receptor. J Patlsod 179: 288-93 (1996).
31. Bamford, R. N., Battiata. A. P.; Burton. J. D., Sharma. H. & Waldmann. T.
A.
Iatetleukin (11,) ISIIL-T production by the adult T-cell leukemia cell line
HuT-102 is
associated with a human T-cell Iymphatrophic virus type i ,region /IL-15
fusion message
that lacks many upstream AUGs that normally attenuates IL-IS mRNA translation.
Proc Natl Acad Sci U S A 93, 2897-902 (1996]. ,
32. Alderson, M. R. et aL Regulation of apoptosis anal T cell activation by
Fas-
specific mAb. Irzt Immunol 6, I?99-806 (1994).
33. Hahne. M. et aL Melanoma cell expression of Fas(Apo-I/CD95) Iigand:
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This
lauraal is curreatiy taken by both CY & CW Libraries ISSN: 403b-8075 (I99b).
SUBSTITUTE SHEET (RULE 2G)
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34. Maiuri, L. et al. DNA frab nentation is a feature oi; cystic fibrosis
epithelial cells:
a disease with inappropriate apoptosis. FEB,S Letters 408, ZZ~-231 (I997).
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SEQUENCE LISTING
<110> The Mathilda and Terence 'Kennedy Institute o f Rheu
<120> Treatment Of Coeliac Disease With Interleuk.in-15
antagonists
<130> DGB/DE/PCT128
<140>
<142>
<150> GB 9814892.7 ..
<151> 1999-07-10-
<I60> 4
<170> PatentIn Ver. 2.1
<210> 1
- <2I1> 162
<212> PRT
<213> SIMIAN
<400> 1
Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser I:le Gln Cys Tyr
1 S 10 15
Leu Cys Leu Leu Leu Lys Ser His Phe Leu Thr Glu Ala Gly Ile His
20 25 30
Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro lays Thr Glu Ala
35 40 45
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
50 55 60
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu ~Ser Asp Val His
65 70 75 80
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
85 90 95
Val Ile Ser His Glu Ser Gly Asp Thr Asp Ile His Asp Thr Val Glu
100 205 110
Asn Leu Ile Ile Leu Ala Asn Asn Ile Leu Ser Ser Asn Gly Asn Ile
215 120 125
SUBSTITUTE SHEET (RULE 26)
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Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Nfet Phe Ile Asn
145 150 155 160
Thr Ser
<210> 2
<211> 162
<212> PRT
<213> Homo Sapiens
<400> 2
Met Arg ile Ser Lys Pro His Leu Arg Ser Ile Ser :Lle Gln Cys Tyr
1 5 10 15
Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu ~Ala Gly Ile His
20 25 30
Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala
35 40 45
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile G1u Asp Leu Ile
50 55 60
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
65 70 75 80
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
85 90 95
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
100 I05 110
Asn Leu Ile Ile Leu Aia Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
115 120 125
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
245 150 155 160
Thr Ser
SUBSTITUTE SHEET (RULE 26)
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<210>3
<21I>489
<212>DNA
<2I3>SIMIAN
<400> 3
atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctacct gtgtttactt 60
ctaaagagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120
gcagggctcc ctaaaacaga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180
gaagatctta ttcaatctat gcatattgat gctactttat atac;agaaag tgatgttcac 240
cccagttgca aggtaacagc aatgaagtgc tttctcttgg agtt:gcaagt tatttcacat 300
gagtccggag atacagatat tcatgataca gtagaaaatc ttat:catcct agcaaacaac 360
atcttgtctt ctaatgggaa tataacagaa tctggatgca aagaatgtga ggaactagag 420
gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgi:ccaaat gttcatcaac 480
489
acttcttga
<210>4
<211>489
<212>DNA
<213>Homo sapiens
<400> 4
atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60
ctaaacagtc attttctaac tgaagctggc attcatgtct tca,ttttggg ctgtttcagt 120
gcagggcttc ctaaaacaga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180
gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 240
ccca:gttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatatcactt 300
gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 360
agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 420
gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480
489'
acttcttga
SUBSTTTUTE SHEET (RULE 26)
