U.S. patent application number 12/434346 was filed with the patent office on 2009-09-10 for treating eye disorders using intestinal trefoil proteins.
Invention is credited to Daniel K. Podolsky.
Application Number | 20090227513 12/434346 |
Document ID | / |
Family ID | 23215677 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090227513 |
Kind Code |
A1 |
Podolsky; Daniel K. |
September 10, 2009 |
TREATING EYE DISORDERS USING INTESTINAL TREFOIL PROTEINS
Abstract
Intestinal trefoil factors and nucleic acids encoding intestinal
trefoil factors are disclosed. The intestinal trefoil factors
disclosed are resistant to destruction in the digestive tract and
can be used for the treatment of peptic ulcer diseases,
inflammatory bowel diseases, eye disorders and other insults.
Inventors: |
Podolsky; Daniel K.;
(Wellesley, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
23215677 |
Appl. No.: |
12/434346 |
Filed: |
May 1, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10449456 |
May 30, 2003 |
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12434346 |
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10313642 |
Dec 6, 2002 |
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10449456 |
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09313434 |
May 17, 1999 |
6525018 |
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10313642 |
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10362310 |
Feb 19, 2003 |
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PCT/US97/06004 |
Apr 11, 1997 |
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09313434 |
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08631469 |
Apr 12, 1996 |
6221840 |
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10362310 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/575 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Claims
1. A method for enhancing corneal epithelial wound healing in a
patient, said method comprising administering to the eye of said
patient a polypeptide selected from the group consisting of human
ITF (intestinal trefoil factor) and biologically active fragments
thereof, wherein said biologically active fragments are wound
healing fragments, and wherein said polypeptide is administered in
an amount sufficient to enhance corneal epithelial wound
healing.
2. The method of claim 1, wherein said polypeptide is human
ITF.
3. The method of claim 1, wherein said corneal wound is caused by a
bacterial infection.
4. The method of claim 1, wherein said polypeptide is administered
in a dimeric form.
5. A method for treating keratoconjunctivitis sicca (dry eyes) in a
patient, said method comprising administering to the eye of said
patient a polypeptide selected from the group consisting of the
intestinal trefoil factor (ITF) set forth in SEQ ID NO.: 2 or SEQ
ID NO.: 4 and biologically active fragments thereof, wherein said
biologically active fragments are wound healing fragments.
6. The method of claim 5, wherein said polypeptide is the ITF set
forth in SEQ ID NO.: 4 or a biologically active fragment
thereof.
7. The method of claim 5, wherein said polypeptide is administered
in a dimeric form.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/449,456, filed May 30, 2003, which is a continuation of U.S.
application Ser. No. 10/313,642, filed Dec. 6, 2002, which is a
continuation of U.S. application Ser. No. 09/313,434, filed May 17,
1999, issued as U.S. Pat. No. 6,525,018.
[0002] This application is a continuation-in-part of U.S.
application Ser. No. 10/362,310, filed Feb. 19, 2003, which is the
National Stage of International Application No. PCT/US97/06004,
filed Apr. 11, 1997, which was published in English under PCT
Article 21 (2), and which is a continuation-in-part of U.S.
application Ser. No. 08/631,469, filed Apr. 12, 1996, issued as
U.S. Pat. No. 6,221,840, each of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The field of the invention is peptides useful for treatment
of disorders of the digestive system, disorders of the eye and
disorders associated with unwanted apoptosis.
BACKGROUND
[0004] Jorgensen et al. (1982, Regulatory Peptides 3:231) describe
a porcine pancreatic peptide, pancreatic spasmolytic peptide (PSP).
PSP was found to inhibit "gastrointestinal motility and gastric
acid secretion in laboratory animal after parenteral as well as
oral administration." It was suggested that "if the results in
animal experiments can be confirmed in man, PSP may possess a
potential utility in treatment of gastroduodenal ulcer
diseases."
[0005] pS2 is a small cysteine-rich protein which is expressed and
secreted from human breast tumours. In addition, pS2 protein is
expressed in normal stomach mucosa and in regenerative tissues in
ulcerative diseases of the gastrointestinal tract (Rio et al.,
Cancer Cells, 1990, 2:269-74).
SUMMARY OF THE INVENTION
[0006] In a first aspect, the invention features a purified nucleic
acid encoding an intestinal trefoil factor (ITF).
[0007] In preferred embodiments, the ITF is a mammalian ITF,
preferably human, rat, bovine, mouse, monkey or porcine ITF. In
another preferred embodiment, the purified nucleic acid encoding an
ITF is present within a vector. In one embodiment, the ITF is in a
monomer form. In another embodiment, two monomer ITF can be linked
by a disulfide bond to form a dimer.
[0008] In a related aspect, the invention features a cell that
includes a vector encoding an ITF.
[0009] In another related aspect, the invention features a
substantially pure ITF. In a preferred embodiment, the polypeptide
is detectably labelled. In a related aspect, the invention features
a therapeutic composition that includes an ITF and a
pharmacologically acceptable carrier.
[0010] In another aspect the invention features ITF variants.
[0011] In another aspect, the invention features a monoclonal
antibody which preferentially binds (i.e., forms an immune complex
with) an ITF. In a preferred embodiment, the monoclonal antibody is
detectably labelled.
[0012] In a related aspect, the invention features a method for
detecting human ITF in a human patient. The method includes the
steps of contacting a biological sample obtained from the patient
with a monoclonal antibody which preferentially binds ITF, and
detecting immune complexes formed with the monoclonal antibody. In
preferred embodiments the biological sample is an intestinal
mucosal scraping, or serum.
[0013] In a related aspect, the invention features a method for
treating digestive disorders in a human patient, which method
involves administering to the patient a therapeutic composition
that includes an ITF and a pharmacologically acceptable carrier. In
one embodiment, a wild-type ITF protein, e.g., a human ITF protein
(FIG. 6, SEQ ID NO: 4), is used to treat a digestive disorder. A
wild-type ITF protein is resistant to destruction in the digestive
tract, and can be used for treatment of a digestive disorder such
as a peptic ulcer disease, an inflammatory bowel disease, and can
be used to protect the intestinal tract from injury caused by
insults such as radiation injury or bacterial infection.
[0014] In another related aspect, the invention features a method
for treating an eye disorder in a human patient, which method
involves administering to the patient a therapeutic composition
that includes an ITF protein and a pharmacologically acceptable
carrier. An ITF protein and biologically active fragments or
variants thereof can be used for the treatment of eye disorders
such as a corneal ulcer, or an ocular inflammatory disease. An ITF
and biologically active fragments or variants thereof can be used
to treat corneal injury or lesion associated with corneal
transplantation, lens implantation and other types of eye surgery.
ITF can also be used to treat traumatic physical injury to the eye.
The methods of the invention also include treating eye disorders
with SP or pS2 protein, e.g., a human Sp or pS2 protein (FIG. 9,
SEQ ID NO:14 and FIG. 10, SEQ ID NO:16), respectively. In addition,
biologically active fragments or variants of SP or pS2 can be used
to treat eye disorders. Any or all of the trefoil proteins can be
administered to treat an eye disorder (see Sands, Annual Rev.
Physiol 58:253-73). ITF or pS2 can be administered in monomer form
or can be administered in a dimer form.
[0015] In yet another related aspect, the invention features a
method for modulating apoptosis in a human patient, which method
involves administering to the patient a therapeutic composition
that modulates expression or activity of ITF and a
pharmacologically acceptable carrier. The methods of the invention
also include a method of modulating apoptosis by administering a
therapeutic composition that modulates expression or activity of SP
or pS2. In addition, biologically active fragments or variants of
SP or pS2 can be used to modulate apoptotic disorders. Any or all
of the trefoil proteins can be administered. ITF or pS2 can be
administered in monomer form or can be administered in a dimer
form.
[0016] In another aspect, the invention features a method for
detecting binding sites for ITT in a patient. The method involves
contacting a biological sample obtained from the patient with the
factor, and detecting the factor bound to the biological sample as
an indication of the presence of the binding sites in the sample.
By "binding sites," as used herein, is meant any antibody or
receptor that binds to an ITF protein, factor, or analog. The
detection or quantitation of binding sites may be a useful
reflection of abnormalities of the digestive tract.
[0017] In another aspect, the invention features substantially pure
ITF. In preferred embodiments, the ITF is human, porcine, mouse,
rat, guinea pig, monkey, or bovine trefoil factor.
[0018] By "intestinal trefoil factor" ("ITF") is meant any protein
that is substantially homologous to rat ITF (FIG. 2, SEQ ID NO:2)
or human ITF (FIG. 6, SEQ ID NO:4) and which is expressed in the
large intestine, small intestine, or colon to a greater extent than
it is expressed in tissues other than the small intestine, large
intestine, or colon. Also included are: allelic variations; natural
mutants; induced mutants; proteins encoded by DNA that hybridizes
under high or low stringency conditions to ITF encoding nucleic
acids retrieved from naturally occurring material; and polypeptides
or proteins retrieved by antisera to ITF, especially by antisera to
the active site or binding domain of ITF. The term also includes
other chimeric polypeptides that include an ITF.
[0019] The term ITF also includes analogs of naturally occurring
ITF polypeptides. Analogs can differ from the naturally occurring
ITF by amino acid sequence differences or by modifications that do
not affect sequence, or by both. Analogs of the invention will
generally exhibit at least 70%, more preferably 80%, more
preferably 90%, and most preferably 95% or even 99%, homology with
all or part of a naturally occurring ITF sequence. The length of
comparison sequences will generally be at least 8 amino acid
residues, usually at least 20 amino acid residues, more usually at
least 24 amino acid residues, typically at least 28 amino acid
residues, and preferably more than 35 amino acid residues.
Modifications include in vivo, or in vitro chemical derivatization
of polypeptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying
the glycosylation patterns of a polypeptide during its synthesis
and processing or in further processing steps, e.g., by exposing
the polypeptide to enzymes that affect glycosylation derived from
cells that normally provide such processing, e.g., mammalian
glycosylation enzymes. Also embraced are versions of the same
primary amino acid sequence that have phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
phosphothreonine. Analogs can differ from naturally occurring ITT
by alterations of their primary sequence. These include genetic
variants, both natural and induced. Induced mutants may be derived
by various techniques, including random mutagenesis of the encoding
nucleic acids using irradiation or exposure to ethanemethylsulfate
(EMS), or may incorporate changes produced by site-specific
mutagenesis or other techniques of molecular biology. See,
Sambrook, Fritsch and Maniatis (1989), Molecular Cloning: A
Laboratory Manual (2d ed.), CSH Press, hereby incorporated by
reference. Also included are analogs that include residues other
than naturally occurring L-amino acids, e.g., D-amino acids or
non-naturally occurring or synthetic amino acids, e.g., .beta. or
.gamma. amino acids.
[0020] In addition to substantially full-length polypeptides, the
term ITF, as used herein, includes biologically active fragments of
the polypeptides. As used herein, the term "fragment," as applied
to a polypeptide, will ordinarily be at least 10 contiguous amino
acids, typically at least 20 contiguous amino acids, more typically
at least 30 contiguous amino acids, usually at least 40 contiguous
amino acids, preferably at least 50 contiguous amino acids, and
most preferably at least 60 to 80 or more contiguous amino acids in
length. Fragments of ITF can be generated by methods known to those
skilled in the art and described herein. The ability of a candidate
fragment to exhibit a biological activity of ITF can be assessed by
methods known to those skilled in the art and are described herein.
Also included in the term "fragment" are biologically active ITF
polypeptides containing amino acids that are normally removed
during protein processing, including additional amino acids that
are not required for the biological activity of the polypeptide, or
including additional amino acids that result from alternative mRNA
splicing or alternative protein processing events.
[0021] An ITF polypeptide, fragment, or analog is biologically
active if it exhibits a biological activity of a naturally
occurring ITF, e.g., the ability to alter gastrointestinal motility
in a mammal or the ability to enhance corneal epithelial wound
healing.
[0022] The invention also includes nucleic acid sequences, and
purified preparations thereof, that encode the ITF polypeptides
described herein. The invention also includes antibodies,
preferably monoclonal antibodies, that bind specifically to ITF
polypeptides.
[0023] ITF, SP and pS2 are referred to as trefoil proteins and are
members of the trefoil family. These proteins are designated
trefoil proteins because they have a trefoil shaped secondary
structure which is stabilized by intrachain disulfide bonds.
Members of the trefoil family typically will have at least one of
the following properties in common: (i) a common structural domain,
e.g., the trefoil shaped secondary structure, (ii) a degree of
amino acid or nucleotide sequence homology, or (iii) a common
functional characteristic.
[0024] Members of the trefoil family, e.g., ITF, are used in the
treatments discussed above. Skilled artisans may review these
proteins in Sands et al. (1996, Ann. Rev. Physiol. 58:253-273). As
stated above, the invention encompasses biologically active
fragments of the trefoil proteins. Fragments that retain the
trefoil structure (i.e., the three loop structure) or that lie
within regions of the protein that are highly conserved may prove
particularly useful. Thus, portions of ITF, pS2, or SP from about
the first cystine involved in a disulfide bond of the three loop
structure to about the last cysteine involved in a disulfide bond
of the three loop structure are useful. Biologically active
fragments of ITF, pS2, SP can include 5, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, or 129 amino acids.
[0025] Variants of a selected trefoil protein are least 60%,
preferably at least 75%, more preferably at least 90%, and most
preferably at least 95% identical to the selected trefoil protein,
preferably a human trefoil protein, more preferably human ITF.
[0026] The term "identical," as used herein in reference to
polypeptide or DNA sequences, refers to the subunit sequence
identity between two molecules. To determine the percent sequence
identity of two amino acid sequences or of two nucleic acids, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of a first amino acid or nucleic
acid sequence for optimal alignment with a second amino or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions (e.g., overlapping positions).times.100). In one
embodiment the two sequences are the same length.
[0027] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
[0028] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0029] An isolated nucleic acid molecule encoding a "variant
protein" can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of ITF, pS2 or SP such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Alternatively, mutations can
be introduced randomly along all or part of the coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that retain
activity. Following mutagenesis, the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined.
[0030] In the case of amino acid sequences that are less than 100%
identical to a reference sequence, the non-identical positions are
preferably, but not necessarily, conservative substitutions for the
reference sequence. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. Sequence identity is typically measured
using sequence analysis software such as the Sequence Analysis
Software Package of the Genetics Computer Group at the University
of Wisconsin (Biotechnology Center, 1710 University Avenue,
Madison, Wis. 53705), and the default parameters specified
therein.
[0031] A variant of a selected trefoil protein preferably has the
amino acids present in the naturally-occurring form of the selected
trefoil protein at the more highly conserved amino acid positions
of the protein. Thus, a variant of human ITF preferably is
identical to naturally-occurring human ITF at all or nearly all of
the more highly conserved positions. Sequence conservation among
trefoil proteins is evident in Table 1 of Sands et al. (supra)
which can be used by those skilled in the art to identify more
conserved residues. In one embodiment, a human SP variant
polypeptide is identical to wild-type SP polypeptide at 129 (127,
125, 123, 121, 119, 117, or 115) of the 130 amino acid residues of
the wild-type SP. Preferably the cysteine residues and the trefoil
structure is preserved. The remaining amino acids can be replaced
by conservative substitutions. In another embodiment, a human pS2
variant polypeptide is identical to wild type pS2 at 83 (81, 79,
77, 75, 73, 72, or 71) of the 84 amino acid residues of the
wild-type pS2. Preferably the cysteine residues and the trefoil
structure is preserved. The remaining pS2 amino acid residues can
be replaced by conservative substitutions. In yet another
embodiment, a human ITF variant polypeptide is identical to
wild-type human ITF at 72 (71, 69, 67, 65, 63, 61 or 59) of the 73
amino acid residues of the wild-type human ITF. Preferably the
cysteine residues and the trefoil structure is preserved. The
remaining amino acids can be replaced by conservative
substitutions.
[0032] As used herein, the term "substantially pure" describes a
compound, e.g., a nucleic acid, a protein, or a polypeptide, e.g.,
an ITF protein or polypeptide, that is substantially free from the
components that naturally accompany it. Typically, a compound is
substantially pure when at least 60%, more preferably at least 75%,
more preferably at least 90%, and most preferably at least 99%, of
the total material (by volume, by wet or dry weight, or by mole
percent or mole fraction) in a sample is the compound of interest.
Purity can be measured by any appropriate method, e.g., in the case
of polypeptides, by column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
[0033] By "isolated DNA" is meant DNA that is free of the genes
which, in the naturally-occurring genome of the organism from which
the given DNA of the invention is derived, flank the DNA. The term
"isolated DNA" thus encompasses, for example, cDNA, cloned genomic
DNA, and synthetic DNA. A "purified nucleic acid", as used herein,
refers to a nucleic acid sequence that is substantially free of
other macromolecule (e.g., other nucleic acids and proteins) with
which it naturally occurs within a cell. In preferred embodiments,
less than 40% (and more preferably less than 25%) of the purified
nucleic acid preparation consists of such other macromolecule.
[0034] "Homologous", as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules, or two polypeptide
molecules. When a subunit position in both of the molecules is
occupied by the same monomeric subunit, e.g., if a position in each
of two DNA molecules is occupied by adenine, then they are
homologous at that position. The homology between two sequences is
a direct function of the number of matching or homologous
positions, e.g., if half, e.g., 5 of 10, of the positions in two
compound sequences are homologous then the two sequences are 50%
homologous; if 90% of the positions, e.g., 9 of 10, are matched or
homologous the two sequences share 90% homology. By way of example,
the DNA sequences 3`ATTGCC`5 and 3`TATGGC`5 share 50% homology. By
"substantially homologous" is meant largely but not wholly
homologous.
[0035] "Treatment of lesions" encompasses both the inhibition of
the formation of lesion and the healing of lesions already formed.
Biologically active fragments and variants of a trefoil protein,
particularly ITF, which promote healing of lesions, e.g., eye
lesion, intestinal lesion, or inhibit the formation of lesions,
e.g., eye lesions or intestinal lesions, are useful in the
treatments of the invention.
[0036] "Disorders of the eye" refers to any disturbance, defect, or
abnormality in eye function or structure (e.g., the eye disorder is
a disorder resulting from a disruption of the corneal epithelium).
An eye disorder can be congenital, hereditary, or can be the result
of a trauma such as a physical injury, an illness, inflammation, an
autoimmune disease, a virus (e.g., adenoviruses, herpes simplex
virus), a blepharitis, a keratitis sicca, a trachoma, a corneal
foreign body, ultraviolet light exposure (e.g., welding arcs,
sunlamps), contact lens overwear, a systemic drug (e.g., adenine
arabinoside), a tropical drug, or invasion by a microbe.
[0037] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a depiction of the nucleotide sequence of rat
trefoil factor (SEQ ID NO:1).
[0039] FIG. 2 is a depiction of the deduced amino acid sequence of
rat trefoil factor (SEQ ID NO:2).
[0040] FIG. 3 is a depiction of the amino acid sequences of rat
trefoil factor, pS2 protein, and pancreatic spasmolytic polypeptide
(SP). The sequences are aligned to illustrate the amino acid
sequence homology between the proteins. Dashes (-) indicate the
insertion of spaces which optimize alignment. Bars indicate
sequence identity.
[0041] FIG. 4 depicts the disulfide bond structure proposed for pS2
(panel A) and PSP (panel B).
[0042] FIG. 5 is a depiction of the proposed disulfide bond
structure of rat ITF.
[0043] FIG. 6 is a depiction of the nucleotide sequence of the
human ITF cDNA and the corresponding deduced amino acid sequence
(SEQ ID NO:3).
[0044] FIG. 7 is a diagram depicting the strategy used to mutate
the ITF gene in embryonic stem cells.
[0045] FIG. 8 is a graph depicting survival following
administration of Dextran Sulfate Sodium (DSS; 2.5% w/v in drinking
water for 9 consecutive days), shown as Kaplan-Meier transform of
probability versus days of DSS treatment.
[0046] FIG. 9 is a depiction of the nucleotide sequence of the
mature human spasmolytic cDNA and the corresponding deduced amino
acid sequence (SEQ ID NO:4).
[0047] FIG. 10 is a depiction of the nucleotide sequence of the
human pS2 cDNA and the corresponding deduced amino acid sequence
(SEQ ID NO:5).
[0048] FIG. 11 is a graph depicting dose-dependent effects of the
trefoil peptides ITF and hSP on corneal epithelial migration.
Values are mean.+-.SEM.
[0049] FIG. 12 is a graph depicting the effects of neutralizing
anti-TGF-.beta..sub.1-antibody on ITF and hSP stimulated corneal
epithelial restitution. Values are mean.+-.SEM.
[0050] FIG. 13 is a graph depicting the effects of trefoil peptides
on proliferation of primary rabbit corneal epithelial cells. Values
are mean.+-.SEM.
DETAILED DESCRIPTION
Treatment of Digestive Tract Disorders
[0051] Trefoil proteins, and in particular ITF, can be used to
treat a number of different conditions. ITF is useful for treatment
of disorders associated with digestive tract because of its
generally protective effect on the digestive tract. ITF promotes
the maintenance of mucosal integrity. ITF can also be used to
inhibit adhesion to or colonization of the mucosa by Helicobacter
pylori (H. pylori). H. pylori is one of the most common bacterial
infections leading to active, chronic gastritis and is frequently
associated with syndromes such as duodenal ulcer, gastric ulcer,
gastric cancer, MALT lymphoma, or Menetrier's syndrome. Eradication
of H. pylori has been shown to reduce the recurrence of duodenal
and gastric ulcers. Furthermore, it has been postulated that
widespread treatment of H. pylori will reduce the incidence of
gastric carcinoma, which is the second leading cause of cancer
related death world-wide.
[0052] Long-standing gastritis associated with H. pylori infection
is often associated with the expression of intestinal-like features
in the gastric mucosa. This condition, referred to as intestinal
metaplasia (IM), may signal an increased risk of gastric cancer.
The etiology of IM is unclear; it could represent a mutational
adaptation or defense against H. pylori infection. For example, the
metaplastic mucosa may produce mucus or other substances that
create an environment that is hostile to H. pylori. ITF can be used
in the treatment of H. pylori infection and conditions associated
with H. pylori infection (e.g., ulcers, gastric carcinoma,
non-ulcer dyspepsia, gastritis, and esophageal lesions associated
with gastro-esophageal reflux disease). In this application ITF or
fragments or variants thereof which inhibit adhesion or
colonization of the mucosa by H. pylori are useful. Such molecules
can be identified using assays known to those skilled in the art,
including the H. pylori binding assay described below.
[0053] ITF may also be used to promote healing of tissues damaged
by conditions associated with H. pylori infection. In this regard,
it is important that addition of trefoil proteins to wounded
monolayers of confluent intestinal epithelial cells increases the
rate of epithelial cell migration into the wound. This effect is
enhanced by concomitant addition of mucin glycoproteins, the other
dominant product of goblet cells.
[0054] Just as ITF can be used to protect other parts of the
digestive tract, it can be used to protect the mouth and esophagus
from damage caused by radiation therapy or chemotherapy. ITF can
also be used to protect against and/or to treat damage caused by
alcohols or drugs generally.
[0055] The invention features a method for the treatment of lesions
in the alimentary canal of a patient by administering to the
patient a trefoil peptide, or a biologically active fragment
thereof. The lesions typically occur in the mucosa of the
alimentary canal, and may be present in the mouth, esophagus,
stomach, or intestine of the patient. The lesions can be caused in
several ways. For example, the patient may be receiving radiation
therapy or chemotherapy for the treatment of cancer. These
treatments typically cause lesions in the mouth and esophagus of
the patient. Skilled artisans will recognize that it may be useful
to administer the proteins of the invention to the patient before
such treatment is begun. Alternatively, the lesions can be caused
by: (1) any other drug, including alcohol, that damages the
alimentary canal, (2) accidental exposure to radiation or to a
caustic substance, (3) an infection, or (4) a digestive disorder
including but not limited to non-ulcer dyspepsia, gastritis, peptic
or duodenal ulcer, gastric cancer, MALT lymphoma, Menetrier's
syndrome, gastro-esophageal reflux disease, and Crohn's disease.
For the treatment of human patients it is expected that the peptide
will be expressed by a human gene. However, eukaryotic ITF
proteins, such as those cloned from the rat and mouse genomes may
also prove effective. These peptides may be isolated from a
naturally occurring source or synthesized by recombinant
techniques. It is expected that the typical route of administration
will be oral. Determining other routes of administration, and the
effective dosage are well within the skills of ordinary artisans
and will depend on many factors known to these artisans.
Treatment of Eye Disorders
[0056] Injury to the corneal epithelium results in the rapid
formation of a layer of cells that covers the denuded corneal
surface, preventing infection and loss of vision. After wounding,
resealing of the surface epithelium occurs over a period of several
hours, resulting in the formation of a migratory leading edge.
Proliferation through mitotic burst is observed in cells
surrounding the original wound margin after 36 hours.
[0057] The invention features a method for treating an eye disorder
(trauma or lesion) in a patient. The method involves administering
to the patient a trefoil peptide, or a biologically active fragment
thereof. In one embodiment, trefoil can be administered to a
patient having a corneal transplant. A trefoil protein (e.g., ITF,
pS2, SP and biologically active fragments or variants thereof) can
be used to treat an eye condition. A trefoil protein, and
biologically active fragments or a variants thereof can also be
used to maintain eye structural integrity and protect the eye.
Biologically active fragments or variants of a trefoil protein can
be identified using assays known to those skilled in the art,
including the in vitro or in vivo eye assay's described below. ITF
or pS2 can be administered as a monomer or as a dimer.
[0058] An eye disorder may affect any part of the eye, e.g., the
cornea, the sclera, the retina, the conjunctiva, the ciliary body,
the posterior chamber, or the anterior chamber. In a preferred
embodiment the eye disorder affects the cornea, e.g., the corneal
epithelium, or the conjunctiva. Eye disorders include but are not
limited to superficial puntate keratitis, corneal ulcer, herpes
simplex keratoconjunctivitis, ophthalmic herpes zoster,
phlyctenular keratoconjunctivitis, keratoconus, conjunctiva,
keratoconjunctivitis sicca (dry eyes), ocular inflammation, corneal
ulcers and cicatricial penhigoid. Eye disorders can be caused by
viruses (e.g., adenoviruses, herpes simplex virus), blepharitis,
keratitis sicca, trachoma, corneal foreign bodies, ultraviolet
light exposure (e.g., welding arcs, sunlamps), contact lens
overwear, systemic drugs (e.g., adenine arabinoside), tropical
drugs, bacteria, fungi, or by a hypersensitive reaction to an
unknown antigen. Physical eye trauma can also result in an eye
disorder. Physical trauma to the eye includes an abrasion to the
cornea (e.g., caused by a foreign body), perforation of the cornea
(e.g., caused by a blunt injury that disrupts the continuity of the
cornea), or chemical burns to the cornea (e.g., exposure to NaOH).
The eye disorder generally results in damage and disruption of eye
function or structure. For example, the disorder may cause the
corneal epithelium to tear, cause necrosis of the cornea, cause
corneal ulcers or damage the conjunctiva. Any of the eye disorders
listed above can be treated by administering trefoil. For the
treatment of human patients with eye disorders it is expected that
the trefoil protein will be expressed by a human gene. However,
eukaryotic trefoil proteins, such as those cloned from the rat and
mouse genomes may also prove effective. These proteins may be
isolated from a naturally occurring source or synthesized by
recombinant techniques. It is expected that the typical route of
administration will be topical. Determining other routes of
administration, and the effective dosage are well within the skill
of an ordinary artisan and will depend on many factors known to the
artisan and can be determined based on studies in animal models and
humans. The trefoil proteins may be administered singly, or in
combination with another protein/preparation.
Modulating Apoptosis
[0059] In another related aspect, the invention features a method
for modulating apoptosis (e.g., the method includes inhibiting
unwanted apoptosis) in a patient, which method involves
administering to the patient a therapeutic composition that
modulates expression or activity of a trefoil protein. The method
also includes administering a trefoil or a biologically active
fragment thereof in order to inhibit aberrant apoptosis, activate
apoptosis or enhance apoptosis in a patient. Unwanted apoptosis
includes any condition where a patient has abnormal apoptosis.
These conditions include but are not limited to the following:
retinitis pigmentosa, Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS), spinal muscular atrophy,
various forms of cerebellar degeneration, decreased production of
blood cells, chronic neutropenia, myelodysplastic syndromes,
myocardial infarctions and stroke.
Example 1
Purification and Cloning of rITF
[0060] This example details how rat ITF was cloned. An inhibitor of
soft agar colony formation by human breast carcinoma-derived BT-20
cells (ATTC HTB79) was isolated from cytology-positive human
malignant effusions (Podolsky et al., 1988, Cancer Res. 48:418;
hereby incorporated by reference). The factor also inhibited soft
agar colony formation by human colon carcinoma-derived HCT15 cells
(ATTC-CCL225). Inhibition was not observed for polyoma and murine
sarcoma virus transformed rodent fibroblast lines. The isolated
factor (transformed cell-growth inhibiting factor or TGIF) had an
apparent molecular weight of 110,000 kD and appeared to consist of
two 55,000 kD subunits linked by sulfhydryl bonds.
[0061] The purified protein was partially sequenced. The sequence
from the amino terminal 14 amino acids was used to produce a set of
degenerate oligonucleotide probes for screening of a rat intestinal
epithelial cell cDNA library.
[0062] A rat intestinal cDNA library (Lambda ZAP.degree. II,
Stratagene, La Jolla, Calif.) was produced by standard techniques
(Ausubel et al., Eds., Current Protocols in Molecular Biology, John
Wiley & Sons, New York, 1989) using cells purified by the
method of Weisner (1973, J. Biol. Chem. 248:2536). Screening of the
cDNA library with the fully degenerate oligonucleotide probe
described above resulted in the selection of 21 clones. One of the
clones (T3411) included a core sequence which encoded a single open
reading frame. The nucleotide sequence of the open reading frame
and flanking DNA is presented in FIG. 1 (SEQ ID NO: 1). The insert
present in T3411 was nick translated (Ausubel et al., supra) to
produce a radioactively labelled probe for Northern blot analysis
of rat poly(A).sup.+ RNA. Northern analysis demonstrated that RNA
corresponding to the cloned cDNA fragment was expressed in small
intestine, large intestine, and kidney; no expression was detected
in the lung, spleen, heart, testes, muscle, stomach, pancreas, or
liver. In the tissues in which the RNA was expressed, the level was
comparable to that of actin.
[0063] The open reading frame of clone T3411 encoded an 81 amino
acid peptide (FIG. 2; SEQ ID NO:2). Comparison of the sequence of
the encoded peptide, referred to as rat intestinal trefoil factor
(rITF), to the sequence of proteins in the Genebank database
revealed significant homology to human breast cancer associated
peptide (pS2; Jakowlev et al., 1984, Nucleic Acids Res. 12:2861)
and porcine pancreatic spasmolytic peptide (PSP; Thim et al., 1985
Biochem. Biophys. Acta. 827:410). FIG. 3 illustrates the homology
between rITF, PSP and pS2. Porcine pancreatic spasmolytic factor
(PSP) and pS2 are both thought to fold into a characteristic
structure referred to as a trefoil. A trefoil structure consists of
three loops formed by three disulfide bonds. pS2 is thought to
include one trefoil (FIG. 4A), and PSP is thought to include two
trefoils (FIG. 4B). The region of rITF (nucleotide 114 to
nucleotide 230 which encodes cys to phe), which is most similar to
PSP and pS2, includes six cysteines all of which are in the same
position as the cysteines which make up the trefoil in pS2 (FIG.
3). Five of these six cysteines are in the same position as the
cysteines which form the amino terminal trefoil of PSP (FIG. 3).
FIG. 5 depicts the proposed disulfide bond configuration of
rITF.
[0064] Based on homology to PSP and pS2 (Mori et al., 1988,
Biochem. Biophys. Res. Comm. 155:366; Jakowlew et al., 1984 Nucleic
Acids Res. 12:2861), rITF includes a presumptive pro-sequence (met1
to ala22) in which 12 of 22 amino acids have hydrophobic side
chains.
Example 2
Production of Anti-rITF Antibodies
[0065] This example details how rat anti-ITF antibodies were
generated.
[0066] A peptide corresponding to the carboxy-terminal 21 amino
acids of rITF was synthesized and coupled to bovine serum albumin
(BSA). This conjugate (and the unconjugated peptide) was used to
raise polyclonal antibodies in rabbits. All procedures were
standard protocols such as those described in Ausubel et al.
(supra). The anti-rITF antibodies were used in an indirect
immunoflouresce assay for visualization of rITF in rat tissues.
Cryosections of rat tissues were prepared using standard
techniques, and fluorescein labelled goat anti-rabbit monoclonal
antibody (labelled antibodies are available from such suppliers
Kirkegaard and Perry Laboratories, Gaithersberg, Md.; and
Bioproducts for Science, Inc., Indianapolis, Ind.) was used to
detect binding of rabbit anti-rITF antibodies. By this analysis
rITF appears to be present in the globlet cells of the small
intestine but not in the stomach or the pancreas.
Example 3
Cloning of Human ITF
[0067] This example details how human ITF was cloned.
[0068] DNA encoding the rat ITF can be used to identify a cDNA
clone encoding the human ITF (hITF). This can be accomplished by
screening a human colon cDNA library with a probe derived from rITF
or with a probe derived from part of the hITF gene. The latter
probe can be obtained from a human colon or intestinal cDNA using
the polymerase chain reaction to isolate a part of the hITF gene.
This probe can then serve as a specific probe for the
identification of clones encoding all of the hITF gene.
[0069] Construction of a cDNA Library
[0070] A human colon or intestinal cDNA library in .lamda.gtlO or
.lamda.gtll, or some other suitable vector is useful for isolation
of hITF. Such libraries may be purchased (Clontech Laboratories,
Palo Alto, Calif.: HLI034a, HLI0346b). Alternatively, a library can
be produced using mucosal scrapings from human colon or intestine.
Briefly, total RNA is isolated from the tissue essentially as
described by Chirgwin et al. (1979, Biochemistry 18:5294; see also
Ausubel et al., supra). An oligo (dT) column is then used to
isolate poly(A).sup.+ RNA by the method of Aviv et al. (1972, J.
Mol. Biol. 134:743; see also Ausubel et al., supra).
Double-stranded cDNA is then produced by reverse transcription
using oligo (dT).sub.12-18 or random hexamer primers (or both).
RNAse H and E. coli DNA poll are then used to replace the RNA
strand with a second DNA strand. In a subsequent step E. coli DNA
ligase and T4 DNA polymerase are used to close gaps in the second
DNA strand and create blunt ends. Generally, the cDNA created is
next methylated with EcoRI methylase and EcoRI linkers are added
(other linkers can be used depending on the vector to be used). In
subsequent steps the excess linkers are removed by restriction
digestion and the cDNA fragments are inserted into the desired
vector. See Ausubel et al., supra and Sambrook et al. (1990,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.) for detailed protocols.
Useful vectors include: .lamda.gtll, .lamda.gtlO, Lambda ZAP.RTM.
II vector, Lambda Uni-ZAP.TM. XR vector, all available from
Stratagene (La Jolla, Calif.).
[0071] The cDNA library must be packaged into phage; this is most
readily accomplished by use of a commercial in vitro packaging kit,
e.g., Gigapack.RTM. II Gold or Gigapack.RTM. II Plus (Stratagene,
La Jolla, Calif.). See Ausubel et al. (supra) for packaging
protocols and suitable host strains. The library is preferably
amplified soon after packaging; this step generates sufficient
clones for multiple screening of the library. See Ausubel et al.
supra or Sambrook et al. supra for details of amplification
protocols and procedures for storing the amplified library.
[0072] Screening of the cDNA Library
[0073] To screen the library it must be placed on an appropriate
host strain (e.g., Y1090 or Y1088 for .lamda.gtlO libraries,
C600hflA for .lamda.gtlO libraries). After plating the phage,
plaques are transferred to nitrocellulose or nylon filters (See
Ausubel et al., supra and Sambrook et al. supra). The filters are
then probed with .alpha..sup.32P-labelled nick translated probe
derived from rITF. The probe is preferentially generated using a
portion of the region of rITF DNA coding for the trefoil structure
(nucleotides 114 to 230 of SEQ ID NO:1, which encode cys32 to phe71
of SEQ ID NO:2). This region is conserved between rITF, pS2 and
PSP, and it is likely that this region is conserved between rITF
and hITF. Once a plaque is identified, several cycles of plaque
purification are required to isolate a pure clone encoding hITF. A
phage DNA isolation is performed and the cDNA insert can be
subcloned into an appropriate vector for restriction mapping and
sequencing. If the phage vector is Lambda ZAP.RTM. II, coinfection
with helper phage allows rescue and recircularization of
pBluescript SK.sup.- phagemid vector (Stratagene, La Jolla, Calif.)
harboring the cDNA; alternatively the phage clone is purified and
the cDNA insert is subcloned into a vector suitable for restriction
mapping and sequencing. If the clone does not contain the entire
hITF gene (as assessed by homology to rITF and the presence of
start and stop codons), the library can be rescreened with the
original rITF probe or, preferably, with a probe generated from the
hITF clone obtained. If none of the clones contain the intact gene,
it can be reconstructed from clones which bear overlapping
fragments of hITF.
[0074] Direct Isolation of an hITF Probe by PCR
[0075] It is possible to isolate part of the hITF gene directly
from the packaged library or cDNA. To isolate a portion of hITF
directly from the packaged library, a pair of oligonucleotide
primers and Taq polymerase are used to amplify the DNA
corresponding to the hITF gene. The primers used would be
approximately 15-20 nucleotides long and correspond in sequence to
the 5'-most and 3'-most portions of the rITF coding sequence.
Friedman et al. (in PCR Protocols: A Guide to Methods and
Applications, Innis et al., Eds., Academic Press, San Diego)
describe a procedure for such amplification. Briefly, phage
particles are disrupted by heating; Taq polymerase, primers (300
pmol of each), dNTPs, and Taq polymerase buffer are added; and the
mixture is thermally cycled to amplify DNA. The amplified DNA is
isolated by agarose gel electrophoresis. The ends of the fragment
are prepared for ligation into an appropriate vector by making them
flush with T4 polymerase and, if desired, adding linkers.
Alternatively, a restriction site may be engineered into the
fragment by using primers which have sequence added to their 5'
ends which sequence will generate an appropriate sticky end when
digested. For example the sequence: 5'-GGGCGGC CGC-3' (SEQ ID NO:5)
can be added to the 5' end of each primer. This sequence includes
the NotI restriction site flanked at the 5' end by the sequence:
GG. The additional nucleotides prevent the 5' ends from denaturing
and interfering with subsequent restriction digestion with NotI.
The gel purified DNA of the appropriate size is next cloned into a
cloning vector for sequencing and restriction mapping. This clone
will not have the entire hITF sequence, rather it will be a
combination of hITF (the region between the sequences corresponding
to the primers) and rITF (the 5' and 3' ends which correspond to
the primer sequences). However, this DNA can be used to generate a
labelled probe (produced by nick translation or random primer
labelling) which, since it is the correct hITF sequence, can be
used in a high stringency screening of the library from which the
cDNA was originally isolated. In an alternative approach, cDNA can
be used in the above procedure instead of a packaged library. This
eliminates the steps of modifying the cDNA for insertion into a
vector as well as cDNA packaging and library amplification. Ausubel
et al. supra provides a protocol for amplification of a particular
DNA fragment directly from cDNA and a protocol for amplification
from poly(A)+ RNA.
[0076] Identification of a Presumptive Human ITF clone
[0077] A nick translated probe derived from rITF cDNA
(corresponding to nucleotides 1 to 431 of SEQ ID NO:1) was used for
Northern blot analysis of poly(A).sup.+ RNA derived from human
intestinal mucosal scrapings. Probe hybridization and blot washing
were carried out according to standard procedures. Probe
(5.times.10.sup.5 cpm/ml hybridization buffer) was hybridized to
the filter at 45.degree. C. in 5.times.SSC with 30% formamide. The
filter was then washed at 60.degree. C. in 5.times.SSC with 40%
formamide. Using this protocol a band was clearly visible after an
overnight exposure of the filter with an intensifying screen. This
result indicated that there is sufficient homology between rITF and
hITF to allow the use of probes derived from the sequence of the
rITF gene for identification of the hITF gene.
[0078] A human intestinal cDNA library was obtained from Clontech
(Palo Alto, Calif.). Alternatively, a human intestinal cDNA library
may be produced from mucosal scrapings as described above. Four
oligonucleotide probes were selected for screening the library
cDNA. Two of the probes correspond to sequences within the region
of rITF encoding the trefoil and are referred to as internal probes
(5'GTACATTCTGTCTCTTGCAGA-3'; SEQ ID NO:6) and
5'-TAACCCTGCTGCTGCTGGTCCTGG-3'; SEQ ID NO:7). The other two probes
recognize sequences within rITF but outside of the trefoil encoding
region and are referred to as external probes
(5'-GTTTGCGTGCTGCCATGGAGA-3'; SEQ ID NO:8) and
5'-CCGCAATTAGAACAGCCTTGT-3'; SEQ ID NO:9). These probes were tested
for their utility by using them to screen the rat intestinal cDNA
library described above. Each of the four probes could be used to
identify a clone harboring all or part of the rITF gene. This
result indicates that these probes may be used to screen the human
intestinal library for the presence of hITF.
[0079] The internal probes were used as described above to amplify
a DNA fragment from human colon library cDNA (Clontech, Palo Alto,
Calif.). Linkers were added to the isolated DNA fragment which was
then inserted into pBluescript phagemid vector (Stratagene, La
Jolla, Calif.). The region of this clone corresponding to the
sequence of human cDNA (i.e., not including the sequence
corresponding to the internal probes) was used to make a
radioactively labelled probe by random oligonucleotide-primed
synthesis (Ausbel et al., supra). This probe was then used to
screen the human colon cDNA library. This screening led to the
identification of 29 clones. One of these clones (HuPCR-ITF) was
nick-translated to generate a probe for Northern analysis of
poly(A).sup.+ RNA isolated from human intestinal mucosal scrapings.
A single band of roughly the same size as the rat transcript
(approximately 0.45 kD) was observed.
[0080] Northern analysis of poly(A).sup.+ isolated from human
tissues indicated that RNA corresponding to this probe was
expressed in the small intestine and the large intestine but not in
the stomach or the liver. These results indicate that the clone
does not encode the human homolog of porcine PSP. Porcine PSP is
expressed in porcine pancreas and is not significantly expressed in
the small or large intestine. These results also distinguish the
cloned gene from pS2 which is expressed in the stomach.
[0081] FIG. 6 shows the nucleic acid sequence information (SEQ ID
NO:3) from the human ITF cDNA clone that was deposited with the
ATCC on ______, 1998, along with the deduced amino acid sequence in
one-letter code (SEQ ID NO:4). This clone was obtained in the
experiment described above.
Example 4
Human ITF
[0082] This example details recombinant production of human ITF,
purification procedures for ITF, and procedures for generating
antibodies against human ITF.
[0083] Production of hITF
[0084] The isolated hITF gene can be cloned into a mammalian
expression vector for protein expression. Appropriate vectors
include pMAMneo (Clontech, Palo Alto, Calif.) which provides a
RSV-LTR enhancer linked to a dexamethasone-inducible MMTV-LTR
promoter, an SV40 origin of replication (allows replication in COS
cells), a neomycin gene, and SV40 splicing and polyadenylation
sites. This vector can be used to express the protein in COS cells,
CHO cells, or mouse fibroblasts. The gene may also be cloned into a
vector for expression in drosophila cells using the bacoluvirus
expression system.
[0085] Purification of ITF
[0086] ITF can be purified from intestinal mucosal scrapings of
human, rats or any other species which expresses ITF (pigs and cows
may provide a source of ITF). The purification procedure used for
PSP will be useful for the purification of ITF since the proteins
are likely to be homologous. Jorgensen et al. describes a method
for purification of PSP (1982, Regulatory Peptides 3:207). The
preferred method is the second approach described by Jorgensen et
al. (supra). This method involves chromatography of SP-Sephadex
C-25 and QAE Sephadex A-25 columns (Sigma, St. Louis, Mo.) in
acidic buffer.
[0087] Anti-ITF Monoclonal Antibodies
[0088] Anti-ITF monoclonal antibodies can be raised against
synthetic peptides whose sequences are based on the deduced amino
acid sequence of cloned hITF (SEQ ID NO:4). Most commonly the
peptide is based on the amino- or carboxy-terminal 10-20 amino
acids of the protein of interest (here, hITF). The peptide is
usually chemically cross-linked to a carrier molecule such as
bovine serum albumin or keyhole limpet hemocyanin. The peptide is
selected with the goal of generating antibodies which will
cross-react with the native hITF. Accordingly, the peptide should
correspond to an antigenic region of the peptide of interest. This
is accomplished by choosing a region of the protein which is (1)
surface exposed, e.g., a hydrophobic region or (2) relatively
flexible, e.g., a loop region or a .beta.-turn region. In any case,
if the peptide is to be coupled to a carrier, it must have an amino
acid with a side chain capable of participating in the coupling
reaction. See Hope et al. (1983, Mol. Immunol. 20:483; 1982, J.
Mol. Biol. 157:105) for a discussion of the issues involved in the
selection of antigenic peptides. A second consideration is the
presence of a protein homologous to hITF in the animal to be
immunized. If such a protein exists, it is important to select a
region of hITF which is not highly homologous to that homolog.
[0089] For hITF, peptides that correspond to the amino-terminal or
carboxy-terminal 15 amino acids are likely to be less homologous
across species and exposed to the surface (and thus antigenic).
Thus they are preferred for the production of monoclonal
antibodies. Purified hITF can also be used for the generation of
antibodies.
Example 5
Genetic Disruption of a Trefoil Protein Impairs the Defense of
Intestinal Mucosa
[0090] This example details disruption of the mouse endogenous ITF
gene and the phenotype of the ITF deficient mouse.
[0091] Isolation of the Murine ITF Gene and Generation of
ITF-Deficient Mice
[0092] The murine ITF gene was isolated from a phage genomic
library using the rat ITF cDNA sequence as a probe, and its
identity was confirmed by nucleotide sequencing using standard
techniques (Mashimo et al., 1995, Biochem. Biophys. Res. Comm.
210:31).
[0093] A targeting vector for disrupting the gene by homologous
recombination in embryonic stem (ES) cells was designed and
constructed, as shown in FIG. 7. The entire second exon (Ex2) of
the murine ITF gene, which is contained within the XbaI-EcoRI
fragment shown, was replaced with the neomycin resistance (neo)
gene cassette. As the deleted sequence encodes most of the "trefoil
domain", the ability of any resultant peptides to produce the
looping structure characteristic of trefoil proteins is abolished.
A positive-negative selection strategy (Mansour et al., 1988,
Nature 336:348) was used to enrich for homologous recombination
events in the embryonic stem (ES) cells by selecting for neo within
the homologous DNA and against a herpes simplex virus thymidine
kinase gene (hsv-tk) placed at the 3' end of the targeting vector.
The pPNT plasmid (Tybulewicz et al., 1991, Cell 65:1153) was used
to construct the targeting vector. The targeting vector was
linearized with the restriction enzyme NotI and electroporated into
pluripotent J1 ES cells (Li et al., 1992, Cell 69:915) under
conditions previously described (Strittmatter et al., 1995, Cell
80:445). Disruption of the ITF gene in ES cells following
homologous recombination was distinguished from random integration
of the targeting vector by Southern blot analysis of genomic DNA
from individual clones of cells digested with the restriction
enzyme XhoI. The pITF2 probe identified a 19 kb "wild type"
fragment and a 23 kb "knock out" fragment created by introduction
of an XhoI site upon homologous insertion of the targeting vector.
Approximately 10% of neomycin-resistant ES clones were found to
have undergone homologous ITF recombination using this method.
[0094] The polymerase chain reaction (PCR) was used to confirm the
targeted mutation as follows. A 200 bp region of DNA was amplified
using primers spanning exon 2 of ITF
(5'-GCAGTGTAACAACCGTGGTTGCTGC-3' (SEQ ID NO: 10) and
5'-TGACCCTGTGTC ATCACCCTGGC-3' (SEQ ID NO:17)); and a 400 bp region
of the neo gene was amplified with a second set of primers
(5'-CGGCTGCTCTGATGGCCGCC-3' (SEQ ID NO:18) and
5'-GCCGGCCACAGTCGATGAATC-3' (SEQ ID NO:19)) The DNA template for
the PCR reaction was obtained from tail tissue. Approximately 0.5
cm of the tail was cut off each animal, and the samples were
digested with proteinase-K (200 .mu.l at 0.5 mg/ml in 50 mM
Tris-HCl pH 8.0 and 0.5% Triton X-100; Sigma, St. Louis, Mo.) at
55.degree. C. overnight. One .mu.l of this mixture was added
directly to a 25 .mu.l PCR reaction (per Stratagene, Menosha,
Wis.). The reaction was begun with a "hot start" (incubation at
96.degree. C. for 10 minutes), and the following cycle was repeated
30 times: 72.degree. C. for 120 seconds (hybridization and
elongation) and 96.degree. C. for 30 seconds (denaturation). Ten
.mu.l of each reaction mixture was electrophoresed on a 2% agarose
gel. Wild type animals were identified by the presence of a 200 bp
fragment, corresponding to an intact ITF gene, heterozygous animals
were identified by the presence of this band and, in addition, a
400 bp fragment produced by amplification of the neo gene, and
ITF-deficient (knock out) animals were identified by the presence
of only the fragment corresponding to the neo gene.
[0095] Two ES clones, which arose independently, were used to
derive two lines of mice lacking ITF. These mice were screened by
Southern genomic blot analysis as described for ES clones, or by
PCR.
[0096] Analysis of Trefoil Peptide Expression in Wild Type and
Mutant Mice
[0097] Although expression of ITF is abolished in the mutant mice,
expression of other trefoil genes is preserved. Northern blot
analysis was performed using cDNA probes for ITF (Suemori et al.,
1991, Proc. Natl. Acad. Sci. USA 88:11017), SP (Jeffrey et al.,
1994 Gastroenterology 106:336), and, as a positive control,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The nucleic acid
probe for murine pS2 was made by reverse transcription-polymerase
chain reaction (RT-PCR) using the oligonucleotide pairs:
5'-GAGAGGTTGCTGTTTTGATGACA-3' (SEQ ID NO:20) and 5'-GCCAAGTCTTGATGT
AGCCAGTT-3' (SEQ ID NO:21), which were synthesized based on the
published mouse pS2 cDNA sequence (GenBank Accession Number:
Z21858). The GeneAmp RNA PCR Kit (Perkin Elmer) was used according
to the manufacturer's instructions, as was the pCR.TM.II
(Invitrogen) cloning vector. RNA was extracted from the following
tissues from both wild type and ITF-deficient (knock out) mice:
stomach, duodenum, terminal ileum, right colon, appendix,
transverse colon, left colon, and rectum. Fifteen .mu.g of total
RNA from each sample were electrophoresed on a 1% agarose gel, and
transferred to nitrocellulose paper. Following hybridization,
washing, and autoradiography, wild type mice exhibited a pattern of
tissue expression considered normal: ITF was expressed in the small
intestine and colon, which is the same expression pattern seen for
ITF in the rat and human. The analysis of mutant mice confirmed the
lack of ITF expression in the gastrointestinal tract. In contrast,
the expression of the other trefoil proteins, SP and pS2, are
unaltered in the gastrointestinal tract of mutant mice. SP was
expressed in the stomach and, at lower levels, in the duodenum of
both wild type and mutant mice. Similarly, pS2 was expressed in the
stomach of both wild type and ITF-deficient mice.
[0098] Immunocytochemistry Reveals that ITF is not
[0099] Expressed in the Colon of ITF-Deficient Mice
[0100] In order to confirm that ITF protein was not expressed by
ITF knock out mice, immunocytochemistry was performed as follows.
Tissue from the colon and small intestine was fixed in the course
of perfusion, immersed in 4% paraformaldehyde (McLean et al., 1974,
J. Histochem. Cytochem. 22:1077), and embedded in paraffin.
Sections were collected and stained either with a polyclonal
antibody raised against a synthetic peptide from the predicted 18
carboxy-terminal amino acids of murine ITF or a monoclonal antibody
against colonic mucin (Podolsky et al., 1986, J. Clin. Invest.
77:1263). Primary antibody binding was visualized with a
biotinylated secondary antibody, Avidin DH, biotinylated
horseradish peroxidase H, and diaminobenzidine tetrahydrochloride
reagents according to the manufacturer's instructions (VectaStain
ABC, Vector Laboratories, Bulingame, Calif.). Following
immunocytochemistry, the sections were counterstained with
hematoxylin and viewed. Goblet cells in the colon of wild type mice
were immunoreactive with both antibodies, staining positively for
ITF and mucin. In contrast, the goblet cells in the colon of
ITF-deficient mice lacked detectable ITF but continued to express
colonic mucin.
[0101] Induction of Mild Colonic Epithelial Injury with Dextran
Sulfate Sodium
[0102] ITF-deficient mice derived from each ES clone appear to
develop normally and are grossly indistinguishable from
heterozygous and wild type litter mates. Their growth is not
retarded and they reach maturity without evident diarrhea or occult
fecal blood loss. However, the colon of ITF-deficient mice may be
more prone to injury than the colon of wild type mice. To
investigate this hypothesis, dextran sulfate sodium (DSS), which
reproducibly creates mild colonic epithelial injury with ulceration
in mice (Kim et al., 1992, Scand. J. Gastroent. 27:529; Wells et
al., 1990, J. Acquired Immune Deficiency Syndrome 3:361; Okayasu et
al., 1990, Gastroenterology 98:694) was administered in the
animals' drinking water. After standardization of DSS effects in
comparable wild type mice, a group of 20 wild type and 20
ITF-deficient mice (litter mates from heterozygous crosses,
weighing >20 grams each) were treated with 2.5% DSS in their
drinking water for nine days.
[0103] Although 85% of wild type mice and 100% of ITF-deficient
mice treated with DSS demonstrate occult blood (using Hemoccult,
Smith Kline Diagnostics, San Jose, Calif.) in their stool during
the period of treatment, ITF-deficient mice were markedly more
sensitive to the injurious effects of DSS. Fifty percent of
ITF-deficient mice developed frankly bloody diarrhea and died (FIG.
8). In contrast, only 10% of wild type mice treated similarly
exhibited bloody diarrhea, and only 5% died. Weight loss was also
significantly more pronounced in ITF-deficient mice than wild type
mice receiving DSS.
[0104] ITF-Deficient Mice Treated with Dextran Sulfate
[0105] Sodium (DSS) Develop Severe Colonic Erosions
[0106] After seven days of treatment with DSS (2.5% w/v), the
colons of wild type and ITF-deficient mice were examined
histologically. Left colon transections were fixed in 4%
paraformaldehyde, mounted in paraffin, and stained with hematoxylin
and eosin. Multiple sites of obvious ulceration and hemorrhage were
present in the colon of ITF-deficient mice, while the colons of
most wild type mice were grossly indistinguishable from those of
untreated mice. Histological examination of the DSS-treated
ITF-deficient colon confirmed the presence of multiple erosions and
intense inflammatory changes including crypt abscesses. Damage was
more pronounced in the distal colon, i.e., the descending colon,
sigmoid colon, and rectum, which contained large, broad areas of
mucosal ulceration. When similarly inspected, mucosal erosions
could be seen in the tissue of 80% of the DSS-treated wild type
mice, but most were small lesions that also appeared to be healing,
with complete re-epithelialization of most lesions. There was no
evidence of re-epithelialization in the colons of ITF-deficient
mice exposed to DSS.
[0107] During the normal course of growth and development,
intestinal epithelial cells originate from stem cells in the
intestinal crypts and rapidly progress up the crypt and villus to
be extruded from the villus tip within five days. After intestinal
injury, the epithelial covering is repopulated by cells which
appear to generate signals to heal the lesion by modulation of
epithelial and mesenchymal cell growth and matrix formation
(Poulsom et al., 1993, J. Clin. Gastroenterol. 17:S78). In vitro
evidence suggests that trefoil proteins play a key role in
re-establishing mucosal integrity after injury. Despite the normal
restriction of SP and pS2 expression to the proximal
gastrointestinal tract, these trefoil proteins and ITF are
abundantly expressed at sites of colonic injury and repair. The DSS
model described above provides a system for testing the protective
effects of ITF, other trefoil peptides, or active polypeptide
fragments or variants thereof. One can administer a molecule to be
tested to DSS-treated mice, either wild type or ITF-deficient mice,
and determine whether the molecule has therapeutic effects by
performing the assays described above.
[0108] In addition to the use of DSS, any chemical compound that is
known to damage the mucosa lining the digestive tract can be used
to assay the proteins of the invention. These compounds include,
but are not limited to, alcohol, indomethacin, and methotrexate.
For example, methotrexate (MTX) can be administered
intraperiotoneally to mice at a dose of 40 mg/kg. One group of
MTX-treated animals could be given, in addition, the protein in
question. Various parameters, such as body weight, the presence of
lesions in the digestive tract, and mortality of these animals
could then be compared to equivalent measurements taken from
animals that were not treated with the protein.
[0109] Anti-Apoptotic Effect of ITF
[0110] ITF knock-out mice were generated as described above.
Analysis of colonic crypts for apoptotic colonic epithelial cells
revealed that more apoptotic colonic epithelial cells were observed
in the colonic crypts of the ITF knockout mice than in the wild
type mice.
Example 6
In Situ H. pylori Binding Assay
[0111] This example describes a method for determining whether ITF
(or protein fragment or variant thereof) prevents or can be used to
treat diseases associated with H. pylori infection.
[0112] In order to determine if ITF is useful as a protein that can
prevent or treat diseases associated with H. pylori infection, an
established animal model of H. pylori infection can be used. One
such model was recently developed by Falk et al. (1995, Proc. Natl.
Acad. Sci. USA 92:1515-1519). This model involves the use of
transgenic mice that express the enzyme
.alpha.-1,3/4-fucosyltransferase and, as a consequence, express
Le.sup.b on the surface of mucosal cells that bound clinical
isolates of H. pylori. If the addition of a protein, such as ITF,
to this system reduces the level of H. pylori binding to the
mucosal cell, the protein would be considered an inhibitor of H.
pylori. More specifically, the assay could be carried out as
follows. H. pylori are obtained, for example, from patients with
gastric ulcers or chronic active gastritis, grown to stationary
phase, and labeled, for example with digoxigenin or fluorescein
isothiocyanate (FITC). The labeled bacteria are then exposed,
together with the protein of interest, to frozen sections prepared
from the stomach, duodenum, ileum, or liver of adult transgenic
mice (as described above). As a control, the experiment could be
performed in parallel using tissue from a wild type littermate. The
sections are fixed with ice-cold methanol for 5 minutes, rinsed
three times with wash buffer (TBS; 0.1 mM CaCl.sub.2, 1 mM
MnCl.sub.2, 1 mM MgCl.sub.2; 10 minutes/cycle), and treated with
blocking buffer (Boehringer Mannheim; see also Falk supra).
Bacteria are diluted to an OD.sub.600 of 0.05 with dilution buffer
[TBS; 0.1 mM CaCl.sub.2, 1 mM MnCl.sub.2, 1 mM MgCl.sub.2
containing leupeptin (1 .mu.g/ml), aprotinin (1 .mu.g/ml),
[-1-p-tosylamido-2-phenylethyl chloromethyl ketone (100 .mu.g/ml),
phenylmethylsulfonyl fluoride (100 .mu.g/ml), and pepstatin A (1
.mu.g/ml)] and overlaid on the sections for 2 hours at room
temperature in a humidified chamber. Slides are then washed six
times in wash buffer on a rotating platform (5 minutes/cycle at
room temperature). Digoxigenin-labeled bacteria are visualized on
washed slides with FITC-conjugated sheep anti-digoxigenin
immunoglobulin (Boehringer Mannheim) diluted 1:100 in histoblocking
buffer. Nuclei were stained with bisbenzimide (Sigma). For blocking
controls, digoxigenin-conjugated stationary-phase bacteria can be
suspended in dilution buffer to an OD.sub.600 of 0.05 and shaken
with or without Le.sup.b-HSA or Le.sup.a-HSA (final concentration,
50 .mu.g/ml; reaction mixture, 200 .mu.l) for 1 hour at room
temperature. The suspension is then overlaid on methanol-fixed
frozen sections.
Example 7
Mutants of ITF
[0113] This example describes the generation of ITF mutants and the
determination of which residues are critical for the biological
function of ITF. Biological functions of ITF include cell
migration, EGF receptor phosphorylation and anti-apoptotic
effects.
[0114] ITF has a unique three loop structure which is formed by
intrachain disulfide bonds between six cysteine residues. These six
cysteine residues were mutated in turn to serine residues thereby
generating six mutants which have incomplete tertiary loop
structure. The mutants were generated using site-directed
mutagenesis of the ITF cDNA sequence.
[0115] A "non-dimerized" mutant of ITF was generated by converting
the seventh cysteine residue to a serine. The seventh cysteine
residue is located near the carboxy terminus of the protein which
is thought to permit homodimer formation.
[0116] A C-terminal truncated ITF was generated by removing the 10
C-terminal amino acids (truncated after the 6th cysteine).
Example 8
Protease Resistance of ITF
[0117] This example details the effect of different proteases on
wild-type ITF and mutant ITF.
[0118] 10 microgram of mutant ITF proteins were incubated with 0.0,
0.01, 0.1, 1.0 and 10.0 microgram of trypsin or 0.0, 0.01, 0.1, 1.0
and 10.0 microgram of pepsin for 4 hours and electrophoresed on the
PAGE gels. The amount of digested and undigested protein was
measured. Results showed that mutated and C-terminal truncated
ITF-fusion proteins were significantly more easily digested by the
proteases than wild type ITF protein.
Example 9
Promotion of Cell Migration by Mutant ITF
[0119] This example details an experiment for determining if the
mutant ITF can cause migration of cells.
[0120] The mutant ITF proteins were added to cultured colonic
epithelial cells (IEC-6) and subjected to a cell migration assay.
Briefly, a confluent monolayer of cultured IEC-6 cells was wounded
by a razor blade and then incubated with wild-type and mutant ITF
for 24 hours. The number of migrating cells across the wounded edge
was then counted. The seven mutated ITF proteins showed a decrease
in migration activity of 40 to 60% as compared to the wild type ITF
protein. The C-terminal truncated ITF protein showed no migration
activity. Thus, for epithelial cell migration, the C-terminal
portion as well as the trefoil motif of ITF is important.
Example 10
Phosphorylation of EGF Receptor by ITF Mutants
[0121] The ability of the fusion proteins to phosphorylate the
epidermal growth factor receptor was determined. HT29 cells were
stimulated by the addition of 100 microgram/ml of an ITF proteins
for 5 minutes and lysed. Each cell lysate was immunoprecipitated by
the anti-human EGFR antibody (4G10) and immunoblotted.
Phosphorylation of the EGF receptor was only detected when
wild-type ITF protein was added to the cells. This result suggests
that tyrosine phosphorylation of EGF receptor requires the complete
structure of the ITF peptide.
Example 11
Anti-apoptotic Effect of ITF Mutants
[0122] The anti-apoptotic effect of the ITF mutant proteins was
investigated by measuring apoptosis following etoposide-induced
apoptosis in cultured cells.
[0123] HCT116 cells were preincubated with 3 mg of ITF protein or
BSA overnight and then further incubated for 24 hours with 1 mM of
etoposide. Cells lysates were electrophoresed on the SDS-PAGE and
subjected to western blotting using anti-Poly (ADP-ribose)
polymerase (PARP) antibody. Apoptosis-related cleavage fragments
were detected in cells which were incubated with mutated ITF
protein. In contrast, no apoptosis cleavage fragments were detected
for HCT116 which were treated with wild-type ITF. This result
suggests that the anti-apoptotic effect of ITF requires the
complete structure of ITF, including the trefoil motif.
Example 12
ITF Promotes Eye Wound Healing
[0124] This example describes experiments that were undertaken to
determine if ITF has a role in eye wound healing. The experiments
involved isolating and culturing primary rabbit corneal epithelial
cells, wounding the cells, and then performing a migration or
restitution assay. In addition further experiments were performed
to compare the effect of trefoil proteins and other known growth
factors such as TGF on wound healing.
[0125] Isolation and Culture of Primary Rabbit Corneal Epithelial
Cells
[0126] Primary corneal epithelial cells were isolated from New
Zealand rabbits (2.5 kg). In brief, 9 mm discs were removed by
trypsinization, stroma was separated from the overlying epithelium
and underlying endothelium and cells were passaged. Fibroblasts
were removed by scalpel and remaining corneal epithelial cells
seeded onto 12-well (restitution assay) or 24-well plates
(proliferation assay). Cells were cultured in 4.5 g/l glucose
containing Dulbecco's modified Eagle medium, (DMEM, Cellgro,
Mediatech Inc., Herndon, Va.) containing 4 mM L-glutamine, 100
I.U/ml penicillin/100 .mu.g/ml streptomycin, and 10%
heat-inactivated FBS (fetal bovine serum, Sigma Chemical Co., St.
Louis, Mo.). Mitomycin C (Sigma Chemical Co.) was added to the
serum-starved monolayers for 2 hours before starting the
restitution assay described above to inhibit cell proliferation and
was used at concentrations of 1 .mu.g/ml.
[0127] In Vitro Corneal Epithelial Cell Migration/Restitution
Assay
[0128] Primary rabbit corneal epithelial wound assays were
performed by modification of techniques described for intestinal
epithelial cells. Cells were grown to confluency, then washed and
cultured in serum-deprived (0.1% FBS containing) medium for 12
hours. Monolayers were then wounded with a sterile blade (3-5
wounds of 5-7 mm length per dish/wound area) and subsequently
stimulated with increasing doses of the purified recombinant
trefoil peptide SP which is normally predominantly expressed in the
proximal gastrointestinal tract, or recombinant human ITF (hITF)
which is expressed in the small and large intestine. Effects of
trefoil peptides were evaluated over a range of concentrations from
0.1 to 1 .mu.g/.mu.l.
[0129] Trefoil peptides, hITF and hSP, were observed to cause a
substantially enhanced number of cells to migrate over the original
wound edge 12 hours after wounding of the corneal epithelial
monolayers. Since proliferation of confluent serum-starved
monolayers of primary corneal epithelial cells was very low, the
number of cells counted over the original wound scratch mark after
12 hours represents cell migration but does not reflect cell
proliferation. This result is consistent with previous observations
in an in vitro restitution model using intestinal epithelial
cells.
[0130] Assessment of the Functional Role of Trefoil Peptides on
Corneal Epithelial Restitution
[0131] Wound areas were viewed under the microscope and wound
margins labelled with a marker to avoid later observer bias. After
washing, wounded corneal epithelial monolayers were cultured in
serum-deprived medium in the presence or absence of hSP (0.1-1
.mu.g/.mu.l), hITF (0.1-1 .mu.g/.mu.l), TGF-.alpha. (100 ng/ml),
TGF-.beta..sub.1 (5 ng/ml). BSA (1 .mu.g/.mu.l) was used as
non-specific control protein. Anti-TGF-{acute over (.alpha.)} and
anti-TGF-.beta. antibodies and normal rabbit IgG control were used
at a concentration of 10 .mu.g/ml. After 12 hours, cell culture
medium was aspirated and cells were fixed with a 2%
glutaraldehyde/PBS.sup.+ solution. Cell migration was assessed in a
blinded fashion by counting cells found across the former wound
margin. The previously labelled standardized wound areas were
photographed at 100-fold magnification using an inverted Nikon
Diaphor TMS microscope (Nikon Inc., Melville, N.Y.). Migration
studies were performed in triplicate, and at least 3 wound areas
per plate were analyzed for cell migration.
[0132] The restitution enhancing effects of both, hITF and hSP on
wounded corneal epithelial cells were dose-dependent as shown in
FIG. 11. For both trefoil peptides, restitution promoting effects
compared to medium containing control protein BSA were most
pronounced at a final concentration of 0.3 .mu.g/.mu.l.
[0133] Since it is known that peptide growth factors promote
migration of corneal as well as intestinal epithelial cells, the
effects of trefoil factors hITF and hSP were compared to effects of
TGF-.alpha. and TGF-.beta..sub.1. As demonstrated in FIG. 12,
addition of 100 ng/ml TGF-.alpha. to wounded corneal epithelial
monolayers resulted in a five-fold increase in restitution compared
to control medium (p=0.011), whereas addition of TGF-B.sub.1 at a
concentration of 5 ng/ml effected a four-fold increased migration
rate (p=0.015). The restitution enhancing effects of both
TGF-B.sub.1 and of TGF-.alpha. were substantially inhibited by
addition of neutralizing anti-TGF-.beta.-antibody suggesting that
TGF.beta. is necessary or part of a final common pathway through
which diverse peptide growth factors and cytokines promote
restitution. The magnitude of the observed restitution promoting
effects of trefoil peptides was comparable to the effects of
TGF-.beta..sub.1. However, the effects of hITF and hSP on corneal
epithelial restitution were not significantly affected by addition
of neutralizing anti-TGF-.beta.-antibody.
[0134] Mitogenic Assays
[0135] Primary rabbit corneal epithelial cells were seeded into
24-well plates (1-5.times.10.sup.4/well) in DMEM containing 10%
FBS. When cells were approximately 50% confluent, cells were washed
and cultured for 18 hours in medium containing 0.1% FBS. Cells were
then cultured in 0.1% FBS containing medium in the presence or
absence of trefoil peptides, growth factors, neutralizing
antibodies, control protein or IgG. After 20 hours,
[.sup.3H]thymidine was added (1.8 .mu.Ci well of 24 well plate).
After 4 hours, the incorporation of radiolabeled thymidine was
determined (Clacci, Gastro93). Briefly, cells were washed with PBS
and fixed with methanol-acetic acid (3:1, v/v). Acid-insoluble
material was then lysed with NaOH, and radioactivity was counted
using a liquid scintillation counter. Results as shown in FIG. 13
showed that the stimulation of subconfluent primary corneal
epithelial cells with hITF resulted in a somewhat lower
[.sup.3H]-thymidine incorporation (291.+-.12 cpm) compared to the
effects of control medium (373.+-.4 cpm), a difference which was
not significant. In addition, hSP also did not significantly affect
corneal epithelial proliferation (344.+-.17 cpm). In contrast
addition of TGF-.alpha. (589.+-.45 cpm; p=0.0074) significantly
enhanced and TGF-.beta. (95.3.+-.23 cpm; p=0.0002) significantly
decreased proliferation of corneal epithelial cells.
Example 13
In Vivo Eye Wound Treatment Assay
[0136] Animal models can be used to evaluate the in vivo efficacy
of treatment of ITF or other trefoil proteins on corneal epithelial
wound healing. Corneal wounding can be inflicted using mechanical
force (Stiebel-Kalish et al., Eye, 1998, 12:829-333; Reidy et al.
British Journal of Opthalmolgy, 1994, 78:377-380; and Xie et al.,
Australian and New Zealand Journal of opthalmolgy, 1998, 26:47-49),
exposure to an alkali (Perry et al., Cornea, 12:379-382, 1993), or
exposure to iodine vapours (Rieck et al., Exp. Eye Res. 54,
987-998, 1992).
[0137] In addition, the role of trefoil as a protector against
virus or bacterial invasion can be determined in vivo, e.g., using
a murine model of herpes simplex virus type 1 (Brandt et al.
Antimicrobial agents and chemotherapy, 1078-1084, 1996).
[0138] Rabbit Mechanical Trauma Model
[0139] Corneal wounding can be inflicted using mechanical trauma
(Crosson et al., Opthalmol Vis Sci, 1986; 27:464-73). Briefly, an
artificial, controlled wound of identical size and depth is
inflicted to the corneas of a group of rabbit eyes in order to
measure their healing rates. The controlled wound is inflicted
using a 7.12 mm rotating disc which causes an identical epithelial
defect (surface area 40 mm.sup.2, depth 40 um). The depth of the
wound is controlled by an adjustment knob on the rotating disc.
After wound infliction, a trefoil protein can be administered
topically. For topical administration, the right rabbit eye is
treated with a trefoil protein (e.g., ITF or PS) and the left eye
is untreated and serves as a control. Timing of the treatment is
planned so that during the acute phase of healing (first 48 hours)
medication is administered as often as six times daily.
[0140] The rabbits eyes are examined at 8 hour intervals by
slitlamp microscopy and color photographs taken. Before each
examination the rabbits are anaesthetized and their corneas stained
with 2% fluorescein solution. The photographs are projected from a
fixed distance onto a millimetric grid scale and the area of
erosion that has closed is measured in square magnification. The
photographs were taken at the same magnification (X100) throughout
the study.
[0141] After complete re-epithelization the rabbits are killed,
eyes fixed and stained with haematoxylin-eosin for microscopic
examination of the corneal epithelium.
[0142] Rabbit Alkali-Burn Model
[0143] Alternatively, corneal wounding can be caused by chemicals,
e.g., Sodium hydroxide. Briefly, a circular plastic well with an
11-mm inner diameter is sequentially placed on the eyes of all
rabbits and filed with 0.5 ml of 1N sodium hydroxide (Levinson et
al., Invest Opthalmol Vis Sci, 1976:15:986-93). After 30 seconds
the alkali was removed by hypodermic syringe, and the eye was
rinsed for 5 seconds with normal saline solution. Trefoil can be
administered topically. For topical administration, the right eye
of the rabbit serves as a control while the left eye receives
treatment with trefoil. As described above, eyes are examined for
epithelial healing.
[0144] Rabbit Iodine-Vapor Model
[0145] Both eyes of each rabbit are wounded following exposure to
iodine (Moses et al. Invest. Ophthal mol. Vis. Sci 1979,
18:103-106). Briefly, iodine vapours from a glass cylinder
(internal diameter of 7.4 mm) containing iodine crystals are
applied to the corneal central epithelium. Following exposure to
iodine the right eye of each animal receives topical application of
trefoil and the left eye is the control eye and receives no
treatment. Treatment of trefoil is applied once or more times
daily. Wound healing was determined as above.
[0146] Murine Model of Herpes Simplex virus type 1 (HSV-1)
[0147] To determine the effect of trefoil on Herpes Simplex virus
type 1 ocular disease the right and left cornea of a mouse is
scratched three times vertically or three times horizontally with a
sterile 30-gauge needle. A drop of DMEM containing approximately
10.sup.6 PFU of HSV-1 is placed on the left damaged cornea and left
for 30 seconds. Excess inoculum is removed by adsorption with a
sterile swab.
[0148] The mice are treated topically once or more times a day with
trefoil protein. Briefly, mice are anesthetized by inhalation
halothane and trefoil is applied with sterile micropipette tip to
cover of cornea.
[0149] On days 1, 2, 3 6 or 10 the amount of infectious virus is
determined by plague assay on vero cell monolayers.
[0150] Use
[0151] In the practice of the present invention, ITF may be
administered orally, intravenously, or intraperitoneally for
treatment of peptic ulcer diseases, inflammatory bowel diseases,
eye disorders, for protection of the intestinal tract from injury
caused by bacterial infection, radiation injury or other insults,
for promoting healing of corneal tissue or preventing wounding of
corneal tissue. The mode of administration, dosage, and formulation
of ITF will depend upon the condition being treated.
[0152] Skilled pharmacologists are able to readily determine
appropriate dosage regimens. As trefoil peptides are not degraded
within the digestive tract, it is expected that the route of
administration will be oral for treating disorders of the digestive
tract. However, trefoil proteins can be administered topically for
treatment of eye disorders. The dosage will range from 1 to 500 mg,
taken once to three times per day. The peptide could be
administered, for example, in the form of a tablet, capsule, cream,
or pill, or could be suspended in a solution, such as a syrup, that
the patient swallows. Alternatively, the solution containing the
peptide may be administered as a gastric lavage. The peptide may
also be included in a solution that is administered as an enema, or
it may be administered as a suppository. ITF or pS2 can be
administered as a monomer or can be administered as a dimer.
Other Embodiments
[0153] ITF may be used to produce monoclonal antibodies for the
detection of ITF in intestinal tissue or blood serum by means of an
indirect immunoassay. ITF may be detectably labelled and used in an
in situ hybridization assay for the detection of ITF binding sites.
Labels may include, but are not limited to, fluorescein or a
radioactive ligand.
[0154] ITF may be used to protect and stabilize other proteins.
This protection is accomplished by forming a hybrid molecule in
which all or part of ITF is fused to either the carboxy-terminus or
the amino-terminus (or both) of the protein of interest. Because
ITF is resistant to degradation in the digestive system, it will
protect the protein of interest from such degradation. As a
consequence, the protein of interest is likely to remain active in
the digestive system and/or will be more readily absorbed in an
intact form.
[0155] Stably dimerized trefoil protein can be used in the methods
of the invention. Such molecules can be prepared by stably
crosslinking monomers of trefoil or by expressing a gene encoding a
tandem repeat of a trefoil protein (e.g., ITF) or a portion thereof
(e.g., a portion capable of forming the three loop structure
characteristic of trefoil proteins).
[0156] Also useful in the method of the invention are trefoil
proteins produced by chemical synthesis.
[0157] Trefoil proteins can be used to treat other disorders, e.g.,
Crohn's disease.
[0158] Trefoil proteins can be used to treat patients undergoing
corneal transplants.
[0159] Other embodiments are within the following claims.
Sequence CWU 1
1
261166PRTHomo sapiens 1Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg
Ser Ser Asn Phe Gln1 5 10 15Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly
Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro
Glu Glu Ile Lys Gln Leu Gln 35 40 45Gln Phe Gln Lys Glu Asp Ala Ala
Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe Arg
Gln Asp Ser Ser Ser Thr Gly Trp Asn65 70 75 80Glu Thr Ile Val Glu
Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95His Leu Lys Thr
Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly
Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120
125Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr
130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn
Arg Leu145 150 155 160Thr Gly Tyr Leu Arg Asn 1652166PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa = Leu, Ser, Tyr, or Thr 2Met Ser Tyr
Asn Xaa Leu Gly Xaa Leu Gln Arg Ser Ser Asn Xaa Gln1 5 10 15Cys Gln
Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys
Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Xaa Gln 35 40
45Gln Xaa Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln
50 55 60Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp
Asn65 70 75 80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His
Gln Ile Asn 85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Xaa Glu Lys
Glu Asp Xaa Thr 100 105 110Arg Gly Lys Xaa Met Ser Ser Xaa His Leu
Lys Arg Tyr Tyr Gly Arg 115 120 125Ile Leu His Tyr Leu Lys Ala Lys
Glu Tyr Ser His Cys Ala Trp Thr 130 135 140Ile Val Arg Val Glu Ile
Xaa Arg Asn Phe Tyr Phe Ile Asn Arg Leu145 150 155 160Thr Gly Tyr
Leu Arg Asn 1653166PRTHomo sapiens 3Met Ser Tyr Asn Ser Leu Gly Ser
Leu Gln Arg Ser Ser Asn Ser Gln1 5 10 15Cys Gln Lys Leu Leu Trp Gln
Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe
Asp Ile Pro Glu Glu Ile Lys Gln Ser Gln 35 40 45Gln Ser Gln Lys Glu
Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala
Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn65 70 75 80Glu Thr
Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95His
Leu Lys Thr Val Leu Glu Glu Lys Ser Glu Lys Glu Asp Ser Thr 100 105
110Arg Gly Lys Ser Met Ser Ser Ser His Leu Lys Arg Tyr Tyr Gly Arg
115 120 125Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala
Trp Thr 130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Ser
Ile Asn Arg Leu145 150 155 160Thr Gly Tyr Leu Arg Asn
165425DNAArtificial sequenceDescription of Artificial Sequence
Primer for Homo sapiens beta-interferon, wherein nucleotides are
altered to substitute a serine for another amino acid 4ctcccttgga
ttcctacaaa gaagc 25525DNAartificial sequenceDescription of
Artificial Sequence Primer for Homo sapiens beta-interferon,
wherein nucleotides are altered to substitute a serine for another
amino acid 5cttgcttgga tccctacaaa gaagc 25628DNAartificial
sequenceDescription of Artificial Sequence Primer for Homo sapiens
beta-interferon, wherein nucleotides are altered to substitute a
serine for another amino acid 6agcagcaatt ctcagtccca gaagctcc
28728DNAArtificial sequenceDescription of Artificial Sequence
Primer for Homo sapiens beta-interferon, wherein nucleotides are
altered to substitute a serine for another amino acid 7agcagcaatt
ttcagtccca gaagctcc 28827DNAArtificial sequenceDescription of
Artificial Sequence Primer for Homo sapiens beta-interferon,
wherein nucleotides are altered to substitute a serine for another
amino acid 8ttaagcagtc ccagcagttc cagaagg 27927DNAArtificial
sequenceDescription of Artificial Sequence Primer for Homo sapiens
beta-interferon, wherein nucleotides are altered to substitute a
serine for another amino acid 9ttaagcagct gcagcagtcc cagaagg
271030DNAArtificial sequenceDescription of Artificial Sequence
Primer for Homo sapiens beta-interferon, wherein nucleotides are
altered to substitute a serine for another amino acid 10gaagaaaaat
ccgagaaaga agatttcacc 301130DNAArtificial sequenceDescription of
Artificial Sequence Primer for Homo sapiens beta-interferon,
wherein nucleotides are altered to substitute a serine for another
amino acid 11gaagaaaaac tggagaaaga agattccacc 301225DNAArtificial
sequenceDescription of Artificial Sequence Primer for Homo sapiens
beta-interferon, wherein nucleotides are altered to substitute a
serine for another amino acid 12aaaatccatg agcagtctgc acctg
251325DNAArtificial sequenceDescription of Artificial Sequence
Primer for Homo sapiens beta-interferon, wherein nucleotides are
altered to substitute a serine for another amino acid 13aaaactcatg
agcagttccc acctg 251428DNAArtificial sequenceDescription of
Artificial Sequence Primer for Homo sapiens beta-interferon,
wherein nucleotides are altered to substitute a serine for another
amino acid 14acttttactc cattaacaga cctacagg 281525DNAArtificial
sequenceDescription of Artificial Sequence Reverse primer for Homo
sapiens beta-interferon, wherein nucleotides in the forward primer
are altered to substitute a serine for another amino acid
15ttgtagctca tatgtaagta tttcc 251629DNAArtificial
sequenceDescription of Artificial Sequence Reverse primer for Homo
sapiens beta-interferon, wherein nucleotides in the forward primer
are altered to substitute a serine for another amino acid
16tctttgtagg aatccaagca agttgtagc 291727DNAArtificial
sequenceDescription of Artificial Sequence Reverse primer for Homo
sapiens beta-interferon, wherein nucleotides in the forward primer
are altered to substitute a serine for another amino acid
17tctcctcagg gatgtcaaag ttcatcc 271827DNAArtificial
sequenceDescription of Artificial Sequence Reverse primer for Homo
sapiens beta-interferon, wherein nucleotides in the forward primer
are altered to substitute a serine for another amino acid
18caggactgtc ttcagatggt ttatctg 271923DNAArtificial
sequenceDescription of Artificial Sequence Reverse primer for Homo
sapiens beta-interferon, wherein nucleotides in the forward primer
are altered to substitute a serine for another amino acid
19cccctggtga aatcttcttt ctc 232027DNAArtificial sequenceDescription
of Artificial Sequence Reverse primer for Homo sapiens
beta-interferon, wherein nucleotides in the forward primer are
altered to substitute a serine for another amino acid 20ttcttaggat
ttccactctg actatgg 272129DNAArtificial sequenceDescription of
Artificial Sequence Reverse primer for Homo sapiens
beta-interferon, wherein nucleotides are altered to substitute a
serine for another amino acid 21tctttgtagg gatccaaggg agttgtagc
292226DNAArtificial sequenceDescription of Artificial Sequence
Reverse primer for Homo sapiens beta-interferon, wherein
nucleotides are altered to substitute a serine for another amino
acid 22cccctggtgg aatcttcttt ctcgga 2623166PRTHomo sapiens 23Met
Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln1 5 10
15Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu
20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Ser
Gln 35 40 45Gln Ser Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met
Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr
Gly Trp Asn65 70 75 80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val
Tyr His Gln Ile Asn 85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Ser
Glu Lys Glu Asp Ser Thr 100 105 110Arg Gly Lys Ser Met Ser Ser Ser
His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125Ile Leu His Tyr Leu Lys
Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140Ile Val Arg Val
Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu145 150 155 160Thr
Gly Tyr Leu Arg Asn 16524166PRTHomo sapiens 24Met Ser Tyr Asn Leu
Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln1 5 10 15Ser Gln Lys Leu
Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg
Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Ser Gln 35 40 45Gln Ser
Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn
Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn65 70 75
80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn
85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Ser Glu Lys Glu Asp Ser
Thr 100 105 110Arg Gly Lys Ser Met Ser Ser Ser His Leu Lys Arg Tyr
Tyr Gly Arg 115 120 125Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser
His Cys Ala Trp Thr 130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn
Phe Tyr Phe Ile Asn Arg Leu145 150 155 160Thr Gly Tyr Leu Arg Asn
16525166PRTHomo sapiens 25Met Ser Tyr Asn Ser Leu Gly Ser Leu Gln
Arg Ser Ser Asn Phe Gln1 5 10 15Ser Gln Lys Leu Leu Trp Gln Leu Asn
Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile
Pro Glu Glu Ile Lys Gln Ser Gln 35 40 45Gln Ser Gln Lys Glu Asp Ala
Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe
Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn65 70 75 80Glu Thr Ile Val
Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95His Leu Lys
Thr Val Leu Glu Glu Lys Ser Glu Lys Glu Asp Ser Thr 100 105 110Arg
Gly Lys Ser Met Ser Ser Ser His Leu Lys Arg Tyr Tyr Gly Arg 115 120
125Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr
130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn
Arg Leu145 150 155 160Thr Gly Tyr Leu Arg Asn 16526166PRTHomo
sapiens 26Met Ser Tyr Asn Ser Leu Gly Ser Leu Gln Arg Ser Ser Asn
Phe Gln1 5 10 15Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu
Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile
Lys Gln Ser Gln 35 40 45Gln Ser Gln Lys Glu Asp Ala Ala Leu Thr Ile
Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser
Ser Ser Thr Gly Trp Asn65 70 75 80Glu Thr Ile Val Glu Asn Leu Leu
Ala Asn Val Tyr His Gln Ile Asn 85 90 95His Leu Lys Thr Val Leu Glu
Glu Lys Ser Glu Lys Glu Asp Ser Thr 100 105 110Arg Gly Lys Ser Met
Ser Ser Ser His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125Ile Leu His
Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140Ile
Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu145 150
155 160Thr Gly Tyr Leu Arg Asn 165
* * * * *
References