Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NON-INFLAMMATORY GLUTEN PEPTIDE ANALOGUES AS BIOMARKERS
FOR CELIAC SPRUE
GOVERNMENT RIGHTS
[01] This invention was made with Government support under contract DK063158
awarded by the National Institutes of Health. The Government has certain
rights in this
invention.
[02] In 1953, it was first recognized that ingestion of gluten, a common
dietary protein
present in wheat, barley and rye causes disease in sensitive individuals.
Gluten is a
complex mixture of glutamine- and proline-rich glutenin and gliadin molecules,
which is
thought to be responsible for disease induction. Ingestion of such proteins by
sensitive
individuals produces flattening of the normally luxurious, rug-like,
epithelial lining of the
small intestine known to be responsible for efficient and extensive terminal
digestion of
peptides and other nutrients.
[03] Clinical symptoms of celiac sprue include fatigue, chronic diarrhea,
malabsorption of
nutrients, weight loss, abdominal distension, anemia, as well as a
substantially enhanced
risk for the development of osteoporosis and intestinal malignancies (lymphoma
and
carcinoma). The disease has an incidence of approximately 1 in 200 in most
populations.
Although no non-dietary therapy has been approved thus far for the treatment
of celiac
sprue, several efforts are under way to develop oral enzyme therapies
(hereafter referred to
as "glutenases") that accelerate the digestion, detoxification and
assimilation of
proteolytically resistant, immunotoxic gluten peptides in the celiac patient's
gastrointestinal
tract. Other types of drugs are also being considered for treatment of celiac
sprue.
[04] A related disease is dermatitis herpetiformis, which is a chronic
eruption
characterized by clusters of intensely pruritic vesicles, papules, and
urticaria-like lesions.
IgA deposits occur in almost all normal-appearing and perilesional skin.
Asymptomatic
gluten-sensitive enteropathy is found in 75 to 90% of patients and in some of
their relatives.
Onset is usually gradual. Itching and burning are severe, and scratching often
obscures the
primary lesions with eczematization of nearby skin, leading to an erroneous
diagnosis of.
eczema. Strict adherence to a gluten-free diet for prolonged periods may
control the
disease in some patients, obviating or reducing the requirement for drug
therapy. Dapsone,
sulfapyridine and colchicines are sometimes prescribed for relief of itching,
although the
underlying disease is unaffected by these drugs. Given the close relationship
between
celiac sprue and dermatitis herpetiformis pathogenesis, the above-mentioned
therapies are
also expected to be useful for the treatment of dermatitis herpetiformis.
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[05] The environmental trigger of celiac sprue is dietary gluten from common
food grains
such as wheat, rye, and barley. Duodenal digestion of ingested gluten releases
proteolytically resistant, immunotoxic peptide fragments, such as the
immunodominant 33-
mer from a-gliadin (Shan et al. (2002) Science 297: 2275-2279). These peptides
traverse
the mucosal epithelium by unknown mechanisms and are deamidated at specific
glutamine
residues by an endogenous enzyme, tissue transglutaminase (tTG) (Molberg et
al. (1998)
Nat Med 4: 713-717; Arentz-Hansen et al. (2000) J Exp Med 191: 603-612).
Deamidated
peptides bind with high affinity to the primary genetic determinant of celiac
sprue, human
leukocyte antigen (HLA) DQ2 (Quarsten et al. (1999) Eur J Immunol 29: 2506-
2514; Kim et
al. (2004) Proc Natl Acad Sci USA 101: 4175-4179), a class II major
histocompatibility
complex (MHC) molecule found in >90% of diagnosed celiac sprue cases
(Spurkland et al.
(1997) Tissue Antigens 49: 29-3), and the remaining cases are associated with
HLA DQ8
(Karell et al. (2003) Hum Immunol 64: 469-477). Upon encountering DQ2-gluten
complexes
on the surface of antigen presenting cells (APC), gluten-specific, DQ2-
restricted CD4+ T
cells are activated to induce a Th1 response comprising the secretion of pro-
inflammatory
cytokines, such as IFN-y, and the recruitment of CD8+ intraepithelial
lymphocytes, ultimately
causing mucosal damage (Jabri et al. (2005) Immunol Rev 206: 219-231).
Additionally,
CD4+ T cells give help to a humoral immune response comprising production of
both gluten-
specific antibodies and TG2-specific autoantibodies (Sollid LM (2002) Nat Rev
Immunol 2:
647-655).
[06] In many affected individuals, this molecular pathogenesis is manifested
symptomatically as nutrient malabsorption, wasting, and/or chronic diarrhea,
and chronic
inflammation caused by recurrent exposure to gluten is associated with the
increased
incidence of T cell lymphoma of the small intestine (Green and Jabri (2006)
Annu Rev Med
57: 207-221). Inflammation, antibody production, and clinical symptoms are
gluten-
dependent, such that strict adherence to a gluten-free diet causes remission,
while
reintroduction of dietary gluten causes relapse. However, a gluten-free diet
is extremely
difficult to maintain due to the ubiquity of gluten in human foods (Pietzak
(2005)
Gastroenterology 128: S135-141). Consequently, non-dietary therapies could
substantially
improve the health and quality of life of celiac sprue patients.
[07] Several recent studies have highlighted the potential of orally
administered gluten-
specific proteases (i.e., glutenases) for treating celiac sprue (reviewed in
Stepniak and
Koning (2006) Trends Biotechnol 24: 433-434; Cerf-Bensussan et al. (2007) Gut
56: 157-
160). Since the proteolytic resistance of gluten peptides stems from their
high proline
(-15%) and glutamine (-35%) content, efforts are primarily focused on enzymes
capable of
cleaving proximal to these residues.
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[08] Bacterial prolyl endopeptidases (PEP) detoxify immunodominant gluten in
vitro
(Stepniak et al. (2006) Am J Physiol Gastrointest Liver Physiol 291: G621-
629), and are
especially effective when complemented with a barley endoprotease, EP-B2, that
preferentially cleaves gluten C-terminal to glutamine residues (Siegel et al.
(2006) Chem
Biol 13: 649-658; Gass et al. (2007) Gastroenterology 133: 472-480).
Importantly, the
cleavage sites for EP-B2 in the immunodominant peptide, 33-mer, are coincident
with those
glutamine residues that are selectively deamidated by tTG (Bethune et al
(2006) Chem Biol
13: 637-647), suggesting EP-B2-catalyzed cleavage of gluten may interfere
specifically with
this critical step in disease progression. The therapeutic promise of EP-B2 is
further
underscored by its digestion of gluten in vivo (see Gass et al. (2006) J
Pharmacol Exp Ther
318: 1178-1186) and by its ability to protect a gluten-sensitive rhesus
macaque against
gluten-induced clinical relapse when dosed orally (Bethune et al. (2008) PLoS
ONE 3:
e1857).
[09] To bring therapeutic glutenases to bear on the human condition of celiac
sprue,
however, safe and effective means of assessing enzyme efficacy in human celiac
patients
are needed. A similar need also exists for therapies that have alternative
modes of action.
For example, AT-1001 (Alba Therapeutics Corp.) is an investigational drug for
celiac
disease that is thought to reverse tight junction dysfunction in celiac
patients, thereby
preventing gluten transport across the epithelial layer.
[10] There is an urgent need for the development of sensitive, specific and
non-invasive
biomarkers for assessing drug efficacy in the treatment of patients with
enteropathic
diseases such as celiac sprue. The ideal biomarker would not only facilitate
clinical trials of
drug candidates, but would also find utility in disease management of patients
who are
prescribed such medications. Current diagnostic methods for celiac sprue, such
as ELISA-
based methods in which either anti-gliadin or anti-tTG antibodies in the
patient's serum are
detected or T cell methods in which cell proliferation or y-IFN secretion is
measured upon
stimulation with gliadin, are unsuitable for this purpose. Antibody tests are
unsuitable
because patients must be exposed to relatively high doses of gluten over
extended
durations before they seroconvert. T cell proliferation assays are more
sensitive, but they
require invasive procedures (e.g. withdrawal of a small intestinal biopsy or
relatively large
quantities of blood to harvest adequate numbers of peripheral blood
mononuclear cells) and
are deemed to be too expensive for routine use. The present invention
addresses this
emerging but unmet medical need.
SUMMARY OF THE INVENTION
[11] Methods are provided for diagnosis and clinical monitoring of
enteropathic disease,
which diseases include, without limitation, celiac sprue, Crohn's disease and
irritable bowel
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syndrome, and inflammation associated with intestinal infection, e.g.
infection with rotavirus,
giardia, enteroaggregative E. coli, Cryptosporidium, and the like. In some
embodiments the
methods are utilized for monitoring ongoing therapeutic regimens for such
enteropathic
diseases. In other embodiments, the methods of the invention are used in
determining the
efficacy of a therapy for treatment of an enteropathic disease, either at an
individual level, or
in the analysis of a group of patients, e.g. in a clinical trial format. Such
embodiments
typically involve the comparison of two or more time points for a patient or
group of patients.
The patient status is expected to differ between the two time points as the
result of
administration of a therapeutic agent, therapeutic regimen, or challenge with
a disease-
inducing agent to a patient undergoing treatment.
[12] The efficacy of therapy in a patient with an enteropathic disease is
assessed by
detecting the ability of the patient to metabolize an orally administered
gluten peptide
analog, where the term "metabolize" refers to proteolytic cleavage of gluten
peptides and
peptide analogs; or refers to the ability of the gut to maintain normal
permeability. Such
gluten analog peptides are substantially similar to native gluten peptides in
susceptibility to
proteolytic cleavage; but are substantially decreased in pathogenic properties
of the native
gluten peptide, i.e. are not presented by the DQ MHC protein; and are not a
substrate for
deamidation by tissue transglutaminase. As a result, these peptides are not
presented to
gluten-specific T cells and are consequently non-inflammatory. Such analog
peptides can
be safely administered at a higher dose than native gluten proteins or
peptides. Gluten
analog peptide compositions are also a feature of the invention.
[13] The patient metabolism of the peptide may be monitored in a variety of
ways. The
peptide analog may be labeled, e.g. with an isotopic, fluorescent, etc. label,
and the
appearance of labeled amino acids that result from metabolism of the peptide
is detected in
a patient sample over a period of time following oral administration, e.g. in
urine, plasma,
breath, saliva, etc. Alternatively, the level of labeled or unlabeled peptide
itself (or a partially
proteolyzed metabolite) in a bodily fluid may be determined by various
methods, including
immunoassays. The determination of the presence of the peptide is used in
determining
peptide cleavage, or for assessing the extent of leakiness of the epithelial
barrier of the
small intestine in the context of celiac sprue, or other inflammatory bowel
diseases where
mucosal leakiness is elevated.
[14] In other embodiments, the methods of the invention are used to diagnose
celiac
sprue. The enhanced permeability of the epithelial barrier in patients with
active disease is
correlated to more established measures of diagnosis such as tTG auto-
antibodies and
upper intestinal villus atrophy.
[15] In one embodiment, the sequence of a 33-residue immunotoxic gluten
peptide
derived from a2-gliadin (see Genbank accession number CAB76964) is modified so
that
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three key glutamine (Q) residues that are selectively deamidated by TG2 are
substituted
with non-substrate amino acids, e.g. by asparagine (N) or histidine (H).
Labeled or
unlabeled biomarker peptides can safely be administered orally to celiac sprue
patients in
conjunction with a drug or placebo.
[161 Various formats may be used in the pharmacokinetic analysis. In some
embodiments, a patient sample is obtained prior to treatment, as a control,
and compared to
samples from the same patient following treatment. In other embodiments, the
CYP3A
function is assessed over long periods of time to monitor patient status.
BRIEF DESCRIPTION OF THE DRAWINGS
[17] Figure 1. Synthetic gluten and biomarker peptide sequences. Sequences are
shown
for the native 33-mer (designated QQQ-33-mer) derived from (2-gliadin, the
synthetically
deamidated 33-mer (EEE-33-mer), the biomarkers NNN-33-mer and HHH-33-mer, and
a
non-inflammatory control peptide of unrelated sequence derived from myoglobin.
Bonds
that are scissile to EP-B2-mediated cleavage are designated by arrowheads.
Glutamine
residues that are selectively deamidated by TG2 or synthetically replaced in
the biomarker
peptides are in bold. Leucine residues that are isotope-labeled for in vivo
experiments are
underlined.
[18] Figure 2. The 33-mer gluten peptide and gluten peptide-based biomarkers
are
similarly resistant to cleavage by pepsin and susceptible to cleavage by the
glutenase EP-
B2. (a-f) Reverse-phase HPLC traces for (a,d) QQQ-33-mer, (b,e) NNN-33-mer,
and (c,f)
HHH-33-mer (300 M) after simulated gastric digestion for specified durations
with either (a-
c) pepsin alone or (d-f) pepsin and 120 g/ml EP-B2. The TAME internal
standard (T), intact
peptide (peak 5), minimally processed peptide lacking only the N-terminal LQ
(peak 4), and
other major digestion products are indicated for each peptide trace overlay.
(g-j) Integrated
area-under-the-curve analysis for intact peptides (g) QQQ-33-mer, (h) NNN-33-
mer, (i)
HHH-33-mer, and (j) myoglobin peptide showing dose- and time-dependency of EP-
B2-
mediated digestion. Each peptide (300 M) was digested in vitro with pepsin
supplemented
with specified concentrations of EP-B2, and reaction products were analyzed by
HPLC. The
area-under-the-curve for each intact peptide peak (and, where applicable, the
minimally-
processed -LQ peptide peak) was calculated and normalized to that for the
internal
standard. Values are expressed as the % of intact peptide remaining after a
given digestion
duration relative to the initial peak area. (k) LC-MS identification of major
digestion products
from simulated gastric digests with pepsin EP-B2. HPLC peak number
corresponds to the
peak numbers in panels (a-f). Data shown are representative of 3 independent
experiments.
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[19] Figure 3. The 33-mer gluten peptide and gluten peptide-based biomarkers
are
similarly resistant to cleavage by pancreatic enzymes and susceptible to
cleavage by prolyl
endopeptidase. (a) Integrated area-under-the-curve analysis for intact QQQ-33-
mer, NNN-
33-mer, and HHH-33-mer after simulated intestinal digestion supplementation
with prolyl
endopeptidase from Flavobacterium meningosepticum (FM PEP). Following
treatment with
pepsin, each peptide (300 M) was digested in vitro with pancreatic proteases
(trypsin,
chymotrypsin, elastase, carboxypeptidase A (TCEC)) and rat intestinal brush
border
membrane (BBM) 1.2 U/ml FM PEP. Reaction products were analyzed by reverse-
phase
HPLC. The area-under-the-curve for each intact peptide peak was calculated and
normalized to that for the internal standard. Values are expressed as the % of
intact peptide
remaining after a given duration relative to the initial peak area. (b)
Cleavage map derived
from LC-MS/MS analysis of major digestion products following simulated
gastrointestinal
digests. Blue arrowheads designate major cleavage sites resulting from EP-B2
supplementation. Red arrowheads designate major cleavage sites resulting from
FM PEP
supplementation. Underlined, numbered sequences designate major products of
digestion
with EP-B2. (c-e) HPLC traces for (c) QQQ-33-mer, (d) NNN-33-mer, and (e) HHH-
33-mer
(300 M) after simulated gastric digestion with pepsin + 120 g/ml EP-B2 for
60 min
followed by treatment with TCEC + BBM 1.2 U/ml FM PEP for 10 or 60 min. T,
TAME
internal standard. HPLC peak numbers corresponds to the numbered sequences in
panel
(b). Data shown are representative of 2 independent experiments.
[20] Figure 4. Biomarkers are non-inflammatory in the context of celiac sprue.
(a) Specific
activity of transglutaminase 2 (TG2; 5 M) in the presence of 100 M gluten
peptide QQQ-
33-mer, biomarkers NNN-33-mer or HHH-33-mer, control myoglobin peptide, or no
peptide.
Means s.d. for triplicate assays are shown. Data are representative of 3
independent
experiments. Statistical comparisons were performed with respect to samples
containing
QQQ-33-mer. *** p < 0.001. (b) Ratio of DQ2-bound to unbound fluorescein-
conjugated (f-)
peptides following incubation of thrombin-cleaved DQ2-(I-gliadin peptide
complexes (9.4
M) with 0.185 M f-QQQ-33-mer, f-EEE-33-mer, f-NNN-33-mer, -f-HHH-33-mer, or f-
myoglobin peptide in a citrate-PBS buffer, pH 5.5 for 45 h at 37 C. Means
s.d. for
triplicate assays are shown. Data are representative of 3 independent
experiments.
Statistical comparisons were performed with respect to samples containing f-
EEE-33-mer.
**p<0.01.
[21] Figure 5. Biomarker transepithelial transport parallels gluten peptide
transport in vitro
and in vivo. (a) Experimental design for in vitro studies. Transwell supports
bearing mature
T84 epithelial cell monolayers were preincubated with media alone or with 600
U/ml IFN-y in
the basolateral chamber. After 48 h, 20 M Cy5-labeled QQQ-33-mer, NNN-33-mer,
or
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HHH-33-mer was added to the apical chamber, and samples from the apical and
basolateral chambers were sampled over time to determine the stability and
apical-to-
basolateral flux of each intact peptide. (b) Apical-to-basolateral flux of
each Cy5-labeled
peptide under basal (0 U/ml IFN-y) and simulated inflammatory (600 U/ml IFN-y)
conditions.
Means s.d. for triplicate assays are shown. Data are representative of 2
similar
experiments. Statistical comparisons were performed with respect to QQQ-33-
mer; no
significant differences were observed. (c-e) Reverse-phase HPLC traces from LC-
MS
analysis of samples taken from the apical and basolateral chambers at 0 and 10
h. Elution
of (c) Cy5-QQQ-33-mer, (d) Cy5-NNN-33-mer, and (e) Cy5-HHH-33-mer were
monitored by
their absorbance at 640 nm. Intact Cy5-peptides elute as the major peak
between 9-10 min.
The peak eluting in each trace at 8 min. is Cy5-LQ, signifying limited
processing of the N-
terminus by T84 cells. (f) Area-under-the-curve analysis quantifying the data
from panels (c-
e).
DETAILED DESCRIPTION
[22] Enteropathic inflammatory disease is clinically monitored by measuring
the
metabolism and digestion kinetics of gluten peptide analogs. In preferred
embodiments
such substances are orally administered as a solution, capsule, enteric
formulation, etc.
[23] As used herein, the term "therapeutic drug" or "therapeutic regimen"
refers to an
agent used in the treatment or prevention of a disease or condition,
particularly an
enteropathic condition for the purposes of the present invention. Of interest
are clinical
trials using such therapies, and monitoring of patients undergoing such
therapy.
[24] In some embodiments, the therapy involves treatment of celiac sprue
patients with
glutenase. In other embodiments, the therapy involves treatment of celiac
sprue patients
with a permeabilibity modifying agent. Assessment of treatment may utilize a
gluten
challenge. In some embodiments, 1-14 days of a moderate dose (at least about 1
g/day, at
least about 5 g/day, at least about 10 g/day, or more) of oral gluten is
utilized for this for this
purpose. Patients may be control patients that have not been treated, or
patients subject to
a clinical regimen of interest, e.g. dietary restriction of gluten, treatment
with permeability
modifier, treatment with glutenase, and the like.
[25] A "patient," as used herein, describes an organism, including mammals,
from which
samples are collected in accordance with the present invention. Mammalian
species that
benefit from the disclosed systems and methods for therapeutic drug monitoring
include,
and are not limited to, apes, chimpanzees, orangutans, humans, monkeys; and
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domesticated animals (e.g., pets) such as dogs, cats, mice, rats, guinea pigs,
and
hamsters.
[26] The term "pharmacokinetics," refers to the mathematical characterization
of
interactions between normal physiological processes and a therapeutic drug
over time (i.e.,
body effect on drug). Certain physiological processes (absorption,
distribution, metabolism,
and elimination) will affect the ability of a drug to provide a desired
therapeutic effect in a
patient. Knowledge of a drug's pharmacokinetics aids in interpreting drug
blood stream
concentration and is useful in determining pharmacologically effective drug
dosages
[27] The term "patient sample" or "sample" as used herein refers to a sample
from an
animal, most preferably a human, seeking diagnosis or treatment of a disease,
e.g. an
enteropathic disease. Samples of the present invention include, without
limitation, urine,
saliva, breath, and blood, including derivatives of blood, e.g. plasma, serum,
etc.
[28) Sample analysis. Patient samples are analyzed to determine the metabolism
of a
gluten peptide analog, usually an orally administered gluten peptide analog.
Sample may
be quantitatively analyzed for the presence of the substrate and/or its
metabolites by any
suitable assay, which are well-known in the art. Methods of analysis include
liquid
chromatography-mass spectroscopy (see Kanazawa et al. (2004) J. Chromatography
1031:213-218, Gorski et al., supra.); HPLC; ion-monitoring gas
chromatography/mass
spectroscopy (see Paine et al., supra.); gas chromatography; semiconductive
gas sensors;
immunoassays; mass spectrometers (including proton transfer reaction mass
spectrometry), infrared (IR) or ultraviolet (UV) or visible or fluorescence
spectrophotometers
(i.e., non-dispersive infrared spectrometer); binding assays involving
aptamers or
engineered proteins etc.
[29] In some embodiments, the biological sample is patient urine. The
concentration of
the peptide and its metabolites, i.e. amino acids, dipeptides, etc. can be
monitored in a 6-
hour urine collection.
[30] Conditions of interest for monitoring methods of the present invention
include a
variety of enteropathic conditions, particularly inflammatory chronic
conditions. In some
embodiments of the invention, a patient is diagnosed as having an enteropathic
condition,
for which treatment is contemplated. The patient may be initially tested for
activity prior to
treatment, in order to establish a baseline level of activity. Alternatively,
the patient may be
released from a treatment regimen for a period of time sufficient to induce an
enteropathic
state, in which state the patient is tested for activity in order to establish
a baseline level of
activity. Enteropathic conditions of interest include, without limitation,
celiac sprue,
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herpetiformis dermatitis, irritable bowel syndrome (IBS); Crohn's Disease; and
inflammation
associated with intestinal infection.
[31] Celiac sprue is an immunologically mediated disease in genetically
susceptible
individuals caused by intolerance to gluten, resulting in mucosal
inflammation, which causes
malabsorption. Symptoms usually include diarrhea and abdominal discomfort.
Onset is
generally in childhood but may occur later. No typical presentation exists.
Some patients
are asymptomatic or only have signs of nutritional deficiency. Others have
significant GI
symptoms.
[32] Celiac sprue can present in infancy and childhood after introduction of
cereals into
the diet. The child has failure to thrive, apathy, anorexia, pallor,
generalized hypotonia,
abdominal distention, and muscle wasting. Stools are soft, bulky, clay-
colored, and
offensive. Older children may present with anemia or failure to grow normally.
In adults,
lassitude, weakness, and anorexia are most common. Mild and intermittent
diarrhea is
sometimes the presenting symptom. Steatorrhea ranges from mild to severe (7 to
50 g
fat/day). Some patients have weight loss, rarely enough to become underweight.
Anemia,
glossitis, angular stomatitis, and aphthous ulcers are usually seen in these
patients.
Manifestations of vitamin D and Ca deficiencies (eg, osteomalacia, osteopenia,
osteoporosis) are common. Both men and women may have reduced fertility.
[33] The diagnosis is suspected clinically and by laboratory abnormalities
suggestive of
malabsorption. Family incidence is a valuable clue. Celiac sprue should be
strongly
considered in a patient with iron deficiency without obvious GI bleeding.
Confirmation
usually involves a small-bowel biopsy from the second portion of the duodenum.
Findings
include lack or shortening of villi (villous atrophy), increased
intraepithelial cells, and crypt
hyperplasia. Because biopsy results may be non-specific, serologic markers can
aid
diagnosis. Anti-gliadin antibody (AGA) and anti-endomysial antibody (EMA, an
antibody
against an intestinal connective tissue protein) in combination have a
positive and negative
predictive value of nearly 100%. These markers can also be used to screen
populations
with high prevalence of celiac sprue, including 1st- degree relatives of
affected patients
and patients with diseases that occur at a greater frequency in association
with celiac
sprue. If either test is positive, the patient may have a diagnostic small-
bowel biopsy
performed. If both are negative, celiac sprue is unlikely. Other laboratory
abnormalities
often occur and may be sought. These include anemia (iron-deficiency anemia in
children
and folate-deficiency anemia in adults); low albumin, Ca, K, and Na; and
elevated alkaline
phosphatase and PT. Malabsorption tests are sometimes performed, although they
are not
specific for celiac sprue. If performed, common findings include steatorrhea
of 10 to 40
g/day and abnormal D- xylose and (in severe ileal disease) Schilling tests.
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[34] Conventional treatment is a gluten-free diet (avoiding foods containing
wheat, rye, or
barley). Gluten is so widely used that a patient needs a detailed list of
foods to avoid.
Patients are encouraged to consult a dietitian and join a celiac support
group. The response
to a gluten-free diet is usually rapid, and symptoms resolve in 1 to 2 months.
Ingesting
even small amounts of food containing gluten may prevent remission or induce
disease.
[35] Complications include refractory sprue, collagenous sprue, and the
development of
intestinal lymphomas. Intestinal lymphomas affect 6 to 8% of patients with
celiac sprue,
usually presenting in the patient's 50s. The incidence of other GI
malignancies (eg,
carcinoma of the esophagus or oropharynx, small-bowel adenocarcinoma)
increases.
Adherence to a gluten-free diet can significantly reduce the risk of
malignancy.
[36] Dermatitis herpetiformis is a chronic eruption characterized by clusters
of intensely
pruritic vesicles, papules, and urticaria-like lesions. The cause is
autoimmune. Diagnosis is
by skin biopsy with direct immunofluorescence testing. Treatment is usually
with dapsone or
sulfapyridine.
[37] This disease usually presents in patients 30 to 40 yr old and is rare in
blacks and
East Asians. It is an autoimmune disease. Celiac sprue is present in 75 to 90%
of dermatitis
herpetiformis patients and in some of their relatives, but it is asymptomatic
in most cases.
The incidence of thyroid disease is also increased. Iodides may exacerbate the
disease,
even when symptoms are well controlled. The term "herpetiformis" refers to the
clustered
appearance of the lesions rather than a relationship to herpesvirus.
[38] Patients may have skin biopsy of a lesion and adjacent normal-appearing
skin. IgA
deposition in the dermal papillary tips is usually present and important for
diagnosis.
Patients should be evaluated for celiac sprue.
[39] Strict adherence to a gluten-free diet for prolonged periods (eg, 6 to 12
mo) controls
the disease in some patients, obviating or reducing the need for drug therapy.
When drugs
are needed, dapsone may provide symptomatic improvement. It is started at 50
mg po
once/day, increased to bid or tid (or a once/day dose of 100 mg); this usually
dramatically
relieves symptoms, including itching, within 1 to 3 days; if so, that dose is
continued. If no
improvement occurs, the dose can be increased every week, up to 100 mg qid.
Most
patients can be maintained on 50 to 150 mg/day, and some require as little as
25 mg/wk.
Although less effective, sulfapyridine may be used as an alternative for those
who cannot
tolerate dapsone. Initial oral dosage is 500 mg bid, increasing by 1 g/day q 1
to 2 wk until
disease is controlled. Maintenance dosage varies from 500 mg twice/wk to 1000
mg
once/day. Colchicine is another treatment option. Treatment continues until
lesions resolve.
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[401 Gluten peptide analog. Gluten peptide analogs of the present invention
have
properties of native gluten peptides, non-limiting examples of which are
provided in Table 1,
but are modified to decrease the pathogenic properties of stable binding to
MHC DQ
proteins, and of acting as a substrate for tissue transglutaminase.
[41] Native gluten peptides suitable for modification are at least about 14
amino acids in
length, and not more than about 40 amino acids in length, and may be obtained
by partial
proteolytic digestion of a grain gluten or gliaden polypeptide, e.g. wheat,
barley, oats, etc.
as is known in the art, for example as set forth in Shan et al. (2005) J.
Proteome Res.
4:1732-1741 (herein specifically incorporated by reference). Such
oligopeptides have the
properties of being (i) resistant to digestion with mammalian pancreatic and
gastric
proteases, e.g. as described in detail in U.S. Patent no. 7,303,871, and in
Shan et al.
(2002), supra. (each herein specifically incorporated by reference); (ii) are
substrates for
deamidation by tissue transglutaminase, e.g. as described in Bethune et al.
(2006) supra.
(herein specifically incorporated by reference); and (iii) are presented by
human MHC DQ,
e.g. as described by Quarsten et al. (1999), supra., and Kim et al. (2004),
supra., (each
herein specifically incorporated by reference).
Table 1
Native Gluten Peptides
Sequence Sequence
identifier
SEQ ID NO:1 QPFPQPQLPYPQPQLPYPQPQLPYPQPQP
SEQ ID NO:2 PFPQPQLPYPQPQLPYPQPQLPYPQPQP
SEQ ID NO:3 LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF
SEQ ID NO:4 QPFPQPQLPYPQPQPFRPQ
SEQ ID NO:5 PFPQPQLPYLQPQPFRPQQPYPQPQP
SEQ ID NO:6 QPFPQPQLPYPQPQPFRPQQ
SEQ ID NO:7 PLFQLVQGQGIIQPQQPAQLEVIRSLVLG
SEQ ID NO:8 QVPQPQQPQQPFLQPQQPFPQQPQQPFPQTQQPQQPFPQQP
SEQ ID NO:9 FLQPQQPFPQQPQQPFPQTQQPQQPFPQQP
SEQ ID NO:10 PQPQQPQQPFLQPQQPFPQQPQQP
SEQ ID NO:11 PQQPQQPFLQPQQPFPQQPQQP
SEQ ID NO:12 PFLQPQQPFPQQPQQPFP
SEQ ID NO:13 LQPQQPFPQQPQQPFPQ
SEQ ID NO:14 QQSEQIIPQQLQQPFPLQPQQPFPQQPQQPFP
SEQ ID NO:15 QPFPLQPQQPFPQQPQQPFPQPQQPIPVQ
SEQ ID NO:16 QPFPLQPQQPFPQQPQQPFPQPQQPIP
SEQ ID NO:17 PQQPQQPFPQTQQPQQPFPQQPQQPFPQTQQPQQPFPQQP
SEQ ID NO:18 TQQPQQPFPQQPQQPFPQTQQPQQPFPQQPQQPFPQ
SEQ ID NO:19 TQQPQQPFPQQPQQPFPQTQ
SEQ ID NO:20 FPQTQQPQQPFPQQPQQPFP
SEQ ID NO:21 TQQPQQPFPQQPQQPFPQ
SEQ ID NO:22 TQQPQQPFPQQPQQPFP
SEQ ID NO:23 PQQLFPELQQPIPQQPQQPFPLQPQQPFPQQPQQPFPQQP
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SEQ ID NO:24 FPELQQPIPQQPQQPFPLQPQQPFPQQPQQP
SEQ ID NO:25 PQQPFPQQPQQPVPQQSQQPFPQTQQPQQ
SEQ ID NO:26 QPQQPTPIQPQQPFPQQPQQPQQPFP
SEQ ID NO:27 QPFPQQSQQPFPQQPQQS
SEQ ID NO:28 QQSQQPFPQQPQQS
SEQ ID NO:29 PQQPQQPFPQQPQQP
SEQ ID NO:30 QPQQPFPQQPQ
SEQ ID NO:31 PRQPYPQQPQQP
SEQ ID NO:32 SQQQQPPFSQQQPPFSQQQQPV
SEQ ID NO:33 SQQQPPFSQQQQPV
SEQ ID NO:34 SQQQLPPFSQQQPPFSQQQQPV
SEQ ID NO:35 PPFSQQQQPVLPQQPPFSQQQQQQQQQPPFSQQQQPV
SEQ ID NO:36 VLPQQPPFSQQQQPVLPPQQSPFPQ
SEQ ID NO:37 FSQQQLPPFSQQLPPFSQQQQQVLPQQPPFSQQQQPV
SEQ ID NO:38 LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF
SEQ ID NO:78 FLQPQQPFPQQPQQPYPQQPQQPFPQ
[42] The amino acid sequence of a native gluten peptide is modified from the
native
peptide, e.g. a peptide selected from SEQ ID NO:1-SEQ ID NO:38 to reduce the
pathogenicity without substantially changing the metabolic profile of the
peptide. Analog
peptides of the invention are typically altered from a native peptide in the
replacement of at
least one, at least two, at least three, and not more than about five amino
acid residues.
[43] The analog peptides are similarly resistant to gastrointestinal
proteases, as
compared to native gluten peptides, e.g. SEQ ID NO:38. Under test conditions,
e.g. as set
forth in the Examples, as set forth in U.S. Patent no. 7,303,871, etc., an
analog peptide of
interest has at least about 75% of the resistance of the native gluten
peptide, at least about
85% of the resistance, at least about 95% of the resistance.
[44] Analog peptides of interest have significantly decreased activity as a
tissue
transglutaminase (tTG) substrate. Under test conditions, e.g. as set forth in
the Examples,
as set forth in Bethune et al. (2006) supra., etc., an analog peptides of
interest has less than
about 10% of tTG substrate activity; less than about 5% of tTG substrate
activity; less than
about 1 % of tTG substrate activity.
[45] Analog peptides of interest have low affinity for DQ2, thereby minimizing
presentation to T cells, which also correlates with having poor
immunostimulatory capacity
toward gluten-specific T cells derived from celiac patient intestinal
biopsies. This minimizes
the risk of inducing inflammation upon oral administration of the biomarker.
Under test
conditions, e.g. as set forth in the Examples, as set forth in Quarsten et al.
(1999), supra.,
and Kim et al. (2004), supra. etc., an analog peptides of interest has less
than about 10% of
T cell stimulatory or DQ binding affinity activity; less than about 5% of T
cell stimulatory or
DQ binding affinity activity; less than about 1% of T cell stimulatory or DQ
binding affinity
activity.
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[46) In some embodiments of the invention, the criteria set forth above are
accomplished
by substituting one, two or three glutamine residues of a native gluten
peptide, e.g. a
peptide set forth in SEQ ID NO:1-SEQ ID NO:38, with amino acids other than
glutamine and
other than a negatively charged amino acid. In some embodiments, the
substituting amino
acid is a histidine, an asparagine, or a combination thereof.
[47] Glutamine residues susceptible to deamidation by tTGase can be
experimentally
determined, as described above and as known in the art. In some embodiments,
the
deamidated glutamine is the underlined residue in the motif PQPQLPY. In other
embodiments, the deamidated glutamine is present in the motif QXP, where Q is
a
glutamine targeted by tTG and X is an amino acid intervening between Q and P.
In such
motifs, the deamidated glutamine is replaced by X, where X is an amino acid
other than
glutamine.
[481 Examples of analog peptides, without limitation, are provided in Table 2,
where one
or more X residues in a given peptide are an amino acid other than glutamine,
preferably a
neutral or positively charged amino acid, and which may be histidine or
asparagine. One of
skill in the art will understand that all X residues need not be substituted,
and some may
remain as a glutamine, provided that such glutamine does not result in a
pathogenic peptide
as described above.
Table 2
Analog Gluten Peptides
Sequence Sequence
identifier
SEQ ID NO:39 QPFPQPXLPYPQPXLPYPQPXLPYPQPQP
SEQ ID NO:40 PFPQPXLPYPQPXLPYPQPXLPYPQPQP
SEQ ID NO:41 LQLQPFPQPXLPYPQPXLPYPQPXLPYPQPQPF
SEQ ID NO:42 QPFPQPXLPYPQPQPFRPQ
SEQ ID NO:43 PFPQPXLPYLQPQPFRPXQPYPQPQP
SEQ ID NO:44 QPFPQPXLPYPQPQPFRPQQ
SEQ ID NO:45 PLFXLVQGQGIIQPXQPAXLEVIRSLVLG
SEQ ID NO:46 QVPXPXQPXQPFLQPXQPFPXQPXQPFPQTXQPQQPFPXQP
SEQ ID NO:47 FLQPQXPFPQXPQXPFPXTQQPQXPFPQQP
SEQ ID NO:48 PQPQQPQXPFLXPQQPFPQQPQQP
SEQ ID NO:49 PQQPXQPFLQPXQPFPXQPQQP
SEQ ID NO:50 PFLXPXQPFPXQPXQPFP
SEQ ID NO:51 LQPXQPFPXQPXQPFPQ
SEQ ID NO:52 QQSEQIIPQQLXQPFPLQPXQPFPXQPXQPFP
SEQ ID NO:53 QPFPLQPXQPFPXQPXQPFPQPXQPIPVQ
SEQ ID NO:54 QPFPLXPXQPFPXQPXQPFPQPQQPIP
SEQ ID NO:55 PXQPXQPFPXTXQPXQPFPXQPXQPFPXTXQPXQPFPXQP
SEQ ID NO:56 TXQPXQPFPXQPXQPFPXTXQPXQPFPXQPXQPFPQ
SEQ ID NO:57 TXQPXQPFPXQPXQPFPQTQ
SEQ ID NO:58 FPXTXQPXQPFPXQPXQPFP
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SEQ ID NO:59 TXQPXQPFPXQPXQPFPQ
SEQ ID NO:60 TXQPXQPFPXQPXQPFP
SEQ ID NO:61 PQQLFPELXQPIPXQPXQPFPLQPXQPFPXQPXQPFPXQP
SEQ ID NO:62 FPELXQPIPXQPXQPFPLQPXQPFPXQPXQP
SEQ ID NO:63 PXQPFPXQPXQPVPQQSXQPFPQTXQPQQ
SEQ ID NO:64 QPXQPTPIQPXQPFPXQPXQPXQPFP
SEQ ID NO:65 QPFPQQSXQPFPXQPQQS
SEQ ID NO:66 QQSXQPFPXQPQQS
SEQ ID NO:67 PXQPXQPFPXQPXQP
SEQ ID NO:68 QPXQPFPXQPQ
SEQ ID NO:69 PRQPYPXQPXQP
SEQ ID NO:70 SQQXQPPFSQXQPPFSQQXQPV
SEQ ID NO:71 SQXQPPFSQQXQPV
SEQ ID NO:72 SQQQLPPFSQXQPPFSQQXQPV
SEQ ID NO:73 PPFSQQXQPVLPXQPPFSQQQQQQQXQPPFSQQXQPV
SEQ ID NO:74 VLPXQPPFSQQXQPVLPPQQSPFPQ
SEQ ID NO:75 FSQQQLPPFSQQLPPFSQQQQQVLPXQPPFSQQXQPV
SEQ ID NO:76 LQLQPFPQPXLPYPQPXLPYPQPXLPYPQPQPF
SEQ ID NO:79 FLQPXQPFPXQPXQPYPXQPXQPFPQ
[49] In some embodiments, the analog peptide consists or comprises of the
sequence
set forth in SEQ ID NO:76, where each X is histidine, asparagine, or a
combination thereof,
i.e. one histidine substitution and two asparagine substitutions; three
histidine substitutions;
three asparagine substitutions; and the like. In one embodiment of the
invention, the
peptide is LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID
NO:77)
[50] Analog peptides optionally comprise one, two or more labeled amino acids,
where
the label provides for specific detection of the peptide and its metabolites.
Preferred labels
are non-radioactive, biogenic moieties, e.g. 13C, 2H, etc. Other labels of
interest include
radiography moieties (e.g. heavy metals and radiation emitting moieties),
positron emitting
moieties, magnetic resonance contrast moieties, and optically visible
moieties, e.g.,
fluorescent or visible-spectrum dyes, quantum dots, visible particles, FRET
pairs; etc.
Among the most commonly used positron-emitting nuclides in PET are included
11C, 13 N,
150, 18F, and 1241. Isotopes that decay by electron capture and/or 7 emission
are used in
SPECT, and include 1251, 1281, 1231 and 99mTc. Optically visible moieties
include fluorescent
dyes, or visible-spectrum dyes, visible particles, and other visible labeling
moieties.
Fluorescent dyes such as ALEXA dyes, fluorescein, coumarin, rhodamine, bodipy
Texas
red, and cyanine dyes, are useful when sufficient excitation energy can be
provided to the
site to be inspected visually. Acceptable dyes include FDA-approved food dyes
and colors,
which are non-toxic, although pharmaceutically acceptable dyes which have been
approved
for internal administration are preferred.
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Methods of the Invention
[51] The ability of an individual to metabolize a gluten peptide analog via an
intestinal
route is analyzed by administering an oral dose of a gluten peptide analog to
an individual
suffering from an enteropathic disorder, and quantitating the presence of the
gluten peptide
analog and/or its metabolite(s) in at least one patient sample.
[52] In some embodiments, the method comprises identifying a patient as having
an
enteropathic disorder, e.g. by criteria described above for specific disease
conditions;
administering an oral dose of a gluten peptide analog to an individual
identified as having an
enteropathic disorder, and quantitating the presence of the gluten peptide
analog and/or its
metabolite(s) in at least one patient sample.
[53] Patient samples include a variety of bodily fluids in which the gluten
peptide analog
and/or metabolites will be present, e.g. blood and derivatives thereof, urine,
saliva, breath,
etc. The samples will be taken prior to administration of the peptide, and at
suitable time
points following administration, e.g. at 15 minutes, 30 minutes, 1 hour, 1.5
hours, 2 hours,
2.5 hours, 3 hours, 4 hours, 6 hours, etc., following administration.
[54] In some preferred embodiments, the methods of the invention are used in
determining the efficacy of a therapy for treatment of an enteropathic
disease, either at an
individual level, or in the analysis of a group of patients, e.g. in a
clinical trial format. Such
embodiments typically involve the comparison of two time points for a patient
or group of
patients. The patient status is expected to differ between the two time points
as the result
of a therapeutic agent, therapeutic regimen, or disease challenge to a patient
undergoing
treatment.
[55] Examples of formats for such embodiments may include, without limitation,
testing
peptide metabolism at two or more time points, where a first time point is a
diagnosed but
untreated patient; and a second or additional time point(s) is a patient
treated with a
candidate therapeutic agent or regimen. An additional time point may include a
patient
treated with a candidate therapeutic agent or regimen, and challenged for the
disease,
particularly for celiac sprue and/or dermatitis herpetiformis, which may be
challenged with
administration of gluten.
[56] In another format, a first time point is a diagnosed patient in disease
remission, e.g.
as ascertained by current clinical criteria, as a result of a candidate
therapeutic agent or
regimen. A second or additional time point(s) is a patient treated with a
candidate
therapeutic agent or regimen, and challenged with a disease-inducing agent,
particularly for
celiac sprue and/or dermatitis herpetiformis, which may be challenged with
administration of
gluten.
[57] In such clinical trial formats, each set of time points may correspond to
a single
patient, to a patient group, e.g. a cohort group, or to a mixture of
individual and group data.
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Additional control data may also be included in such clinical trial formats,
e.g. a placebo
group, a disease-free group, and the like, as are known in the art. Formats of
interest
include crossover studies, randomized, double-blind, placebo-controlled,
parallel group trial
is also capable of testing drug efficacy, and the like. See, for example,
Clinical Trials: A
Methodologic Perspective Second Edition, S. Piantadosi, Wiley-Interscience;
2005, ISBN-
13: 978-0471727811; and Design and Analysis of Clinical Trials: Concepts and
Methodologies, S. Chow and J. Liu, Wiley-Interscience; 2003; ISBN-13: 978-
0471249856,
each herein specifically incorporated by reference.
[58] Specific clinical trials of interest include analysis of therapeutic
agents for the
treatment of celiac sprue and/or dermatitis herpetiformis, where a patient is
identified as
having celiac sprue by conventional clinical indicia. For example, in celiac
sprue a daily
dose of 5-10 g gluten (equivalent to 2-3 slices of bread) for two weeks can
induce
malabsorption, as measured by a 72-hour quantitative fecal fat collection or a
D-xylose
urinary test (Pyle, 2005), providing for a means to challenge the efficacy of
a treatment.
[59] In one embodiment, a blinded crossover clinical trial format is utilized.
A patient
alternates for a set period of time, e.g. one week, two weeks, three weeks, or
from around
about 7-14 days, or around about 10 days, between a test drug and placebo,
with a 4-8
week washout period. The patient is challenged with gluten during both
alternating time
periods with around about 1 g gluten, about 5 g. gluten, about 10 g. gluten,
or more, usually
not more than about 25 g gluten daily. Subjects are tested with a gluten
peptide analog, as
described above, at the beginning and end of each alternating time period. The
duration of
gluten challenge may be about 1, about 3, about 5, about 7, about 10 days,
about 14 days,
and the like. By decreasing the duration of the gluten challenge or the
magnitude of the
daily gluten dose, adverse symptoms can be minimized.
[60] In another embodiment a randomized, double-blind, placebo-controlled,
parallel
group trial is used to test drug efficacy. In one embodiment, individuals
identified as having
celiac sprue, who are on a gluten-free diet, undergo three sequential
treatment periods,
each of 1-14 day durations. Subjects will be assessed with the gluten peptide
analog at
entry and at the end of each treatment period. During the entire study,
subjects will
consume regular gluten-free meals plus drug or placebo as indicated. During
the first
treatment period (run-in), all subjects will receive placebo. During the
second treatment
period, the subjects will be randomized into drug or placebo groups. During
the third
treatment period, subjects will remain on the same (drug or placebo) treatment
as in the
second period. In addition, all subjects will receive 1-5 g gluten with each
meal. Drugs that
are effective will show a statistically lower frequency of relapse in the
treatment arm versus
placebo arm of the study.
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[61] In all such methods, the gluten peptide analog is administered at a dose
that is
sufficient to monitor the metabolism over time, which will vary with the
specific peptide that
is selected. The dose may be at least about 1 mg, at least about 5 mg at least
about 10
mg, at least about 25 mg, at least about 100 mg, at least about 500 mg, and
not more than
about 1 g.
[62] The peptide may be administered in any conventional formulation, e.g.
solution,
suspension, tablets, powders, granules or capsules, for example, with
conventional
additives, such as lactose, mannitol, corn starch or potato starch; with
binders, such as
crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins;
with
disintegrators, such as corn starch, potato starch or sodium ca rboxymethylcel
I u lose; with
lubricants, such as talc or magnesium stearate; and if desired, with diluents,
buffering
agents, moistening agents, preservatives and flavoring agents. An alternative
formulation is
an aqueous solution containing 5 g lactulose, 2 g mannitol and 22.3 g glucose
as an
osmotic filler. The lactulose-mannitol cocktail in this solution facilitates
an independent
assessment of intestinal permeability.
[63] In one embodiment of the invention, the oral formulations comprise
enteric coatings,
so that the active agent is delivered to the intestinal tract. Such
formulations are created by
coating a solid dosage form with a film of a polymer that is insoluble in acid
environments,
and soluble in basic environments. Exemplary films are cellulose acetate
phthalate,
polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and
hydroxypropyl
methylcellulose acetate succinate, methacrylate copolymers, and cellulose
acetate
phthalate. Other enteric formulations comprise engineered polymer microspheres
made of
biologically erodable polymers, which display strong adhesive interactions
with
gastrointestinal mucus and cellular linings and can traverse both the mucosal
absorptive
epithelium and the follicle-associated epithelium covering the lymphoid tissue
of Peyer's
patches. The polymers maintain contact with intestinal epithelium for extended
periods of
time and actually penetrate it, through and between cells. See, for example,
Mathiowitz et
al. (1997) Nature 386 (6623): 410-414. Drug delivery systems can also utilize
a core of
superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh
et al.
(2001) J Control Release 71(3):307-18.
[64] The presence of the peptide may be determined by an affinity assay. For
example
an antibody that specifically binds to the peptide may be used in a
quantitative or semi-
quantitative assay. Such antibodies are known in the art, e.g. see Moron et
al. (2008) PLoS
ONE 3(5): e2294. For such assays it is not necessary to label the peptide.
[65] Other assays may utilize a labeled peptide, where the presence of the
label is
determined, e.g. by a chromatographic separation of peptide and free amino
acids, by
detection of cleavage through a peptide labeled with a FRET pair, and the
like.
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DATABASES OF METABOLIC ANALYSES
[661 Also provided are databases of metabolic analyses. Such databases will
typically
comprise analysis profiles of various individuals following a clinical
protocol of interest etc.,
where such profiles are further described below.
[67] The profiles and databases thereof may be provided in a variety of media
to facilitate
their use. "Media" refers to a manufacture that contains the expression
profile information
of the present invention. The databases of the present invention can be
recorded on
computer readable media, e.g. any medium that can be read and accessed
directly by a
computer. Such media include, but are not limited to: magnetic storage media,
such as
floppy discs, hard disc storage medium, and magnetic tape; optical storage
media such as
CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these
categories
such as magnetic/optical storage media. One of skill in the art can readily
appreciate how
any of the presently known computer readable mediums can be used to create a
manufacture comprising a recording of the present database information.
"Recorded" refers
to a process for storing information on computer readable medium, using any
such methods
as known in the art. Any convenient data storage structure may be chosen,
based on the
means used to access the stored information. A variety of data processor
programs and
formats can be used for storage, e.g. word processing text file, database
format, etc.
1681 As used herein, "a computer-based system" refers to the hardware means,
software
means, and data storage means used to analyze the information of the present
invention.
The minimum hardware of the computer-based systems of the present invention
comprises
a central processing unit (CPU), input means, output means, and data storage
means. A
skilled artisan can readily appreciate that any one of the currently available
computer-based
system are suitable for use in the present invention. The data storage means
may
comprise any manufacture comprising a recording of the present information as
described
above, or a memory access means that can access such a manufacture.
[69] A variety of structural formats for the input and output means can be
used to input
and output the information in the computer-based systems of the present
invention. Such
presentation provides a skilled artisan with a ranking of similarities and
identifies the degree
of similarity contained in the test expression profile.
REAGENTS AND KITS
[[701 Also provided are reagents and kits thereof for practicing one or more
of the above-
described methods. The subject reagents and kits thereof may vary greatly.
Reagents of
interest include reagents specifically designed for use in production of the
above described
analysis. Kits may include a gluten peptide analog, reagents for analysis of
the peptide
and/or metabolites, and such containers as are required for sample collection.
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(71] The kits may further include a software package for statistical analysis
of one or
more phenotypes. In addition to the above components, the subject kits will
further include
instructions for practicing the subject methods. These instructions may be
present in the
subject kits in a variety of forms, one or more of which may be present in the
kit. One form
in which these instructions may be present is as printed information on a
suitable medium or
substrate, e.g., a piece or pieces of paper on which the information is
printed, in the
packaging of the kit, in a package insert, etc. Yet another means would be a
computer
readable medium, e.g., diskette, CD, etc., on which the information has been
recorded. Yet
another means that may be present is a website address which may be used via
the
internet to access the information at a removed site. Any convenient means may
be
present in the kits.
[72] This invention also provides rapid commercial test methods and devices
that use at
least one gluten analog peptide of the invention. In such methods, detection
may utilize, for
example, standard immunochromatographic technology with visible colorimetric
readout. A
positive result for the increased presence of at least one gluten analog
peptide in a
biological fluid, e.g. blood, urine, saliva, etc., from the patient being
tested, relative to a
normal control or normal reference sample, indicates active disease, i.e.
increased intestinal
permeability and/or lack of gluten peptide metabolism.
[73] Such assay devices may contain a single test membrane, or two or more
test
membranes. The test membranes may be present within a cassette, each receiving
fluid
from a single application, such as through an aperture in a test cassette
equipped with
means such as tubules for distributing said fluid to each test membrane. This
invention also
provides kits which comprise one or more test membrane strips comprising
binding partners
for the antigens used in the assay method. Assay devices may include puncture
or other
physical means known to the art, e.g. finger prick devices.
[74] Preferred binding partners for gluten analog peptides are antibodies
specific thereto.
They can be polyclonal or monoclonal antibodies. A first antibody may bind to
the gluten
analog peptide of the invention to form a complex, and a second antibody may
bind to the
complex. Either the first or second antibody may be labeled, and either the
first or second
antibody may be immobilized on a substrate such as a test membrane for ease of
detection.
[75] This invention further provides an assay system comprising: (1) a
cassette; (2) a test
membrane housed within the cassette; (3) first antibodies specific to one or
more gluten
analog peptide(s), said first antibodies being capable of binding to the
corresponding
peptides to form first antibody-antigen complexes; (4) binding partners
specific to such
complex; and (5) labels attached to said first antibodies or said binding
partners. The
binding partners capable of binding to the complexes may be second antibodies
specific to
said gluten analog peptide; or they may be second antibodies specific to the
first antibodies.
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The binding partners may also be detection antigens capable of binding
specifically to each
of the first antibodies.
[76] The binding partners for the complex, or the first antibodies, may be
immobilized on
a substrate such as, a test membrane. The first antibodies or the binding
partners for the
complex may be labeled. Preferably, the labels used are detectably different
for detecting
each antigen. Immobilized antibodies or binding partners for the complexes may
be laid
down on the substrate in different patterns.
[77] The assay system may be in the form of a cassette comprising all needed
antibodies
and antigens, or may be in the form of a kit which includes necessary antigens
and/or
antibodies or other reagents as separate reagents. In some embodiments, the
kit includes
a cassette comprising all needed antibodies; and a predetermined dose of
gluten analog
peptide, which may be in a form suitable for ingestion.
[78] A preferred cassette comprises a sample aperture for introducing sample
fluid into
the assay, preferably with a sample pad positioned beneath the sample
aperture. The
cassette also comprises a substrate such as a test membrane for immobilizing
antibodies
and/or antigens. A filter may be positioned downstream from the sample
aperture. The
cassette also preferably comprises a test window positioned above the point on
the test
membrane wherein labeled first antibodies, labeled second antibodies, or
labeled detection
antigens are immobilized. The human eye or a detection device may be used to
view test
results through the test window.
[791 The cassette may also comprise control peptides. These may or may not be
immobilized on the test membrane and may or may not be labeled. The cassette
also
comprise binding partners for the control antigens. The binding partners may
be labeled
and may be immobilized on the test membrane. Preferably, the cassette also
comprises a
control window positioned above the test membrane at the point where the
control antigens
or their binding partners are immobilized, so that results can be viewed
through the control
window by the human eye or a detection device.
[80] While the methods of this invention can be carried out using an
immunological assay
device as described above, other testing methods known in the art for
measuring antigens
and gluten analog peptide of the invention levels, either directly or
indirectly, such as
western blot, sandwich blot, ELISA, dot blot, slot blot, Northern blot, PCR,
and antibody
precipitation, are also useful in the methods of this invention.
[81] The following examples are put forth so as to provide those of ordinary
skill in the art
with a complete disclosure and description of how to make and use the present
invention,
and are not intended to limit the scope of the invention or to represent that
the experiments
below are all or the only experiments performed. Efforts have been made to
ensure
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accuracy with respect to numbers used (e.g., amounts, temperature, and the
like), but some
experimental errors and deviations may be present. Unless indicated otherwise,
parts are
parts by weight, molecular weight is weight average molecular weight,
temperature is in
degrees Centigrade, and pressure is at or near atmospheric.
EXAMPLE 1
[82] Our strategy for designing a biomarker for disease management and
clinical drug
development was inspired by the physical, chemical and biological properties
of
immunotoxic gluten peptides such as the 33-mer from a-gliadin. Specifically,
we sought to
engineer a gluten peptide analogue that mimics the 33-mer with respect to some
criteria but
can be differentiated from the natural product with respect to others. Like
the 33-mer, the
biomarker must be resistant to gastrointestinal proteases, so that it is not
rapidly cleared
from the stomach or intestinal lumen. Also like the 33-mer, it must be
efficiently proteolyzed
by therapeutic glutenases, so that its amino acid metabolites are rapidly
assimilated into the
bloodstream in a glutenase dose dependent manner. And finally, like the 33-
mer, it must be
able to penetrate across the intestinal epithelium. However, unlike 33-mer,
the biomarker
must neither be recognized by human TG2 nor HLA-DQ2. Consequently, it must be
unable
to stimulate disease-specific T cells so as to elicit an inflammatory
response. We present
two examples of biomarkers that meet these criteria, and demonstrate their
utility in animal
models.
[83] Design of gluten peptide-based biomarkers. In order to engineer a
biomarker with
the desired properties, we replaced the reactive GIn residues in the 33-mer
(QQQ-33-mer)
with either Asn (NNN-33-mer) or His (HHH-33-mer) residues (Figure 1). The
rationale for
the Q--).N analogue was that, within the scope of naturally occurring (i.e.
dietary) amino
acids, this conservative change would minimally perturb recognition by the EP-
B2
glutenase34 (Figure 1, arrowheads). The rationale for the Q----).H analogue
was that, in the
acidic environment of the post-prandial stomach and the upper small intestine,
these
substitutions were expected to position positive charges at sites where TG2-
mediated
introduction of negative charge increases peptide affinity for HLA-DQ25,6.
Importantly,
neither Asn nor His is a preferred residue for cleavage by gastrointestinal
proteases, so
these biomarkers were expected to be as proteolytically resistant in the
gastrointestinal
lumen as the natural 33-mer peptide.
[84) As controls for our study, we also synthesized a peptide derived from
myoglobin with
moderate proteolytic resistance but unrelated sequence, as well as EEE-33-mer,
the
synthetically deamidated analogue of the 33-mer (Figure 1). The structures and
purity of all
peptides were confirmed by liquid chromatography-assisted mass spectrometry
(LC-MS).
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[85] Biomarkers are resistant to gastrointestinal digestion but susceptible to
therapeutic
glutenases. To evaluate the resistance of the 33-mer and the derivative
biomarkers to
gastrointestinal proteolysis, we first performed simulated gastric digests at
37 C, pH 4.5,
using commercially available pepsin, the major protease present in the
stomach. The extent
of digestion at multiple time-points was determined by analytical high
performance liquid
chromatography (HPLC) and LC-MS. As previously observed, the 33-mer was not
cleaved
by pepsin under simulated gastric conditions (Figure 2A). Similarly, both
biomarkers were
completely resistant to pepsin digestion over the course of 60 min (Figure
2B,C). To test
whether the experimental glutenase EP-B2 could accelerate digestion of these
biomarkers,
we performed identical digests with the addition of proEP-B2, the acid-
activated proenzyme
form of EP-B2. The addition of proEP-B2 rapidly degraded the 33-mer (Figure
2D), as well
as the derivative biomarkers (Figure 2E,F) in a time- and dose-dependent
manner (Figure
2G,H,I). By contrast, pepsin alone catalyzed nearly complete cleavage of the
myoglobin
control peptide within 10 min (Figure 2J). The major products of biomarker
digestion
identified by LC-MS were similar to those produced by 33-mer cleavage (Figure
2K),
indicating that the substitutions in these 33-mer analogues did not
substantially alter the
sites of susceptibility to EP-B2-mediated digestion.
[861 We next performed simulated duodenal digests to determine whether the
biomarkers are resistant to pancreatic proteases (trypsin, chymotrypsin,
elastase, and
carboxypeptidase A (collectively, TCEC)), as well as to the exopeptidases
contained in the
intestinal brush border membrane (BBM). Following a 60 minute gastric digest
containing
either pepsin alone or pepsin and EP-B2, reactions were adjusted to pH 6.0 and
commercial TCEC was added with BBM purified from rat intestine. Intestinal
digests were
carried out at 37 C for 60 minutes, over which period several time points
were taken and
analyzed as above. Both biomarkers were highly resistant to high
concentrations of
intestinal proteases, with -80% of intact NNN-33-mer remaining after 60
minutes, similar to
33-mer, and -60% of intact HHH-33-mer remaining (Figure 3A). Supplementation
of these
digests with the proline-specific glutenase Flavobacterium meningosepticum
(FM) PEP
resulted in complete digestion of the 33-mer, as previously reported, and also
of both
biomarkers within 10 min (Figure 3A). Tandem mass spectrometry of biomarker
digests
revealed complementary EP-B2 and FM PEP cleavage patterns (Figure 3B),
demonstrating
these biomarkers can be used to assess efficacy of combination enzyme
therapies. Indeed,
addition of FM PEP enabled further digestion of fragments remaining after
treatment of the
33-mer and biomarkers with EP-B2 (Figure 3C,D,E).
[87] Biomarkers are non-inflammatory in the context of celiac sprue. For
biomarkers to
be administered safely to celiac sprue patients, they must not be deamidated
by TG2, bind
HLA DQ2, or stimulate a strong immune response by pre-existing gluten-specific
T cells. To
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determine the capacity for gluten peptide-based biomarkers to elicit an
inflammatory T cell
response in celiac sprue patients, these characteristics were tested in vitro
and compared to
the immunodominant 33-mer gluten peptide. The extent to which each biomarker
is
deamidated by TG2 was determined by a spectrophotometric assay in which the
ammonium ion released by TG2-catalyzed substrate deamidation is coupled to
glutamate
dehydrogenase-catalyzed oxidation of NADH36. Consistent with previous results,
the 33-
mer was readily deamidated by TG2 (Figure 4A).
[88] By contrast, TG2 activity in the presence of NNN-33-mer and HHH-33-mer
was
significantly reduced, 33.1- fold and 25.0-fold, respectively, relative to
that in the presence
of native 33-mer. Activity in the presence of the biomarkers was slightly
higher than that
detected in the absence of a peptide substrate, though this difference was not
significant for
HHH-33-mer (p = 0.04 for NNN-33- mer). The control myoglobin peptide, which
lacks
glutamine residues entirely (Figure 1), elicited no activity from TG2,
suggesting the residual
activity elicited by the biomarkers might be attributable to deamidation of
glutamines at
positions other than the preferred sites that were synthetically altered. The
affinity of each
biomarker for HLA-DQ2 was determined using a peptide exchange assay in which
fluorescein-labeled peptides were incubated with soluble HLA-DQ2 molecules at
pH 5.5, 37
C to simulate the endocytic environment. After 45 h, DQ2-bound and free
fluorescein-
labeled peptides were separated by high-performance size exclusion
chromatography
(HPSEC) and the ratio of their peak heights determined by fluorometry.
Consistent with
previous results, >80% of synthetically deamidated 33-mer (EEE-33-mer) bound
HLA-DQ2,
a 9.9-fold increase over the native peptide and a 15.6-fold increase over the
NNN-33-mer
biomarker (Figure 4B). Neither HHH-33-mer nor the myoglobin control peptide
exhibited any
detectable binding to HLA-DQ2. The immunostimulatory capacity of each
biomarker was
measured via T cell proliferation assays employing gluten-specific T cells
derived from
celiac patient intestinal biopsies. The low immunostimulatory capacity of the
biomarkers
toward any of the cell lines and clones precluded response saturation and EC50
determination. However, it is apparent that the immunostimulatory capacity of
NNN-33-mer
was reduced -1000-fold relative to TG2-treated 33-mer. The HHH-33-mer
biomarker was
even less immunogenic, eliciting minimal or no response at micromolar
concentrations.
Treatment of the biomarkers with TG2 did not increase their immunostimulatory
capacity.
[89] Biomarker transepithelial transport parallels gluten peptide transport
under basal
and inflammatory conditions. In addition to reporting on glutenase activity in
vivo,
noninflammatory gluten peptide-based biomarkers are useful tools for
understanding the
factors and mechanisms that modulate intestinal permeability of immunogenic
dietary
peptides, as well as for practical applications related to the diagnosis of
celiac sprue and its
treatment with modulators of epithelial permeability. To be used in such
applications, these
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biomarkers must be similar to inflammatory gluten peptides in terms of their
transport and
transepithelial stability across healthy and inflamed mucosa. The T84
epithelial cell line was
used to model the intestinal epithelium because its responsiveness to IFN-y,
the major
inflammatory cytokine present in celiac lesions, has been extensively studied.
Additionally,
the effect of IFN-y on the intact transport of the 33-mer and other gluten
peptides across
T84 epithelial monolayers has recently been described. To simulate transport
under healthy
and inflammatory states, media alone or media containing IFN-y was incubated
for 48 hours
on the basolateral side of T84 epithelial cells cultured on transwell
supports, and the apical-
to-basolateral flux of Cy5-labeled 33-mer and biomarkers was measured
thereafter. The
flux of Cy5-33-mer and both Cy5-labeled biomarker peptides was -6 pmol/cm2/h
under
basal conditions. Following exposure of T84 monolayers to IFN-y, the flux of
all 3 peptides
was increased -10-fold. No significant difference in flux was observed between
33-mer and
either biomarker under basal or simulated inflammatory conditions. Some
processing of the
33-mer may occur upon its transport across the intestinal epithelium.
Therefore, to evaluate
the stability of apical and translocated Cy5-labeled biomarkers, the apical
and basolateral
media were analyzed by LC-MS immediately after peptide addition to the apical
chamber
and 10 h thereafter. The absorbance at 640 nm was monitored during
chromatographic
separation. Both Cy5-labeled biomarkers, as well as Cy5-labeled 33- mer,
eluted between
9-10 min as a single major peak. A smaller peak, eluting at -8.min in all
samples, was
identified as Cy5-LQ, indicating that some processing of the peptides' N-
terminus occurred
in the presence of epithelial cells. Nonetheless, after 10 h, no other
breakdown products
were identifiable by mass spectrometry, and >95% of each intact peptide
remained in the
apical chamber of control cells. Intact Cy5-peptides were present at somewhat
lower levels
(>80% of initial) after 10 h in the apical chambers of those cells
preincubated with IFN-y.
This was due to increased N-terminal processing, evidenced by the more
apparent Cy5-LQ
peak present in these samples, as well as to IFN-y-induced enhancement of
apical-to-
basolateral flux. All three Cy5-labeled peptides remained intact during
transport. After 10 h,
0.2-0.3% and 2-3% of the initial 20 M apical peptide was observed intact on
the
basolateral side of cells preincubated with media alone or with IFN-y,
respectively. Thus,
both biomarkers were highly stable in the presence of epithelial cells, and
were translocated
intact to a similar extent as the 33-mer under basal and simulated
inflammatory conditions.
To examine biomarker transport under basal and inflammatory conditions in
vivo,
catheterized rats were administered 20 mg [73C3]-HHH-33-mer biomarker by oral
gavage
following 2 days of daily intravenous treatment with vehicle or IFN-y. The
level of intact
peptide in peripheral plasma 60 min after peptide administration was
determined by 3Q
LCMS/ MS. As predicted by the lack of a serological response to ingested
gluten in rodents,
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biomarker was detected in the plasma of only 1 of 8 control rats administered
oral
biomarker. Pretreatment with IFN-y did not elicit a general increase in
biomarker transport in
the test animal group relative to controls, as only 1 of 4 IFN-y-treated
animals exhibited
plasma biomarker. However, the level of plasma biomarker in this IFN-y-treated
animal
(101.7 nM) was -10-fold higher than that in the control animal exhibiting
detectable plasma
biomarker (9.5 nM), a difference similar to that caused by IFN-y in vitro.
Additionally, 33-mer
dosed together with [13C3]-HHH-33-mer was detected in the plasma of both
animals
exhibiting plasma biomarker, but not in other animals, suggesting this
inflammatory gluten
peptide and its non-inflammatory counterpart are transported in parallel in
vivo.
[90] A recent report demonstrated that the 33-mer is absorbed intact across
the gut
epithelium of an enteropathic gluten-sensitive rhesus macaque, but not across
that of a
healthy control. To determine if similar absorption of a non-inflammatory
gluten peptide-
based biomarker occurs, a gluten-sensitive macaque with chronic diarrhea and
elevated
plasma anti-gliadin antibodies was administered 100 mg [13C3]-HHH-33-mer
intragastrically.
Peripheral blood samples were collected at hourly intervals and analyzed for
biomarker
content by 3Q LC-MS/MS. The labeled biomarker was clearly detected (2.3 0.1
nM) in
peripheral blood 60 min after administration, similar to the extent and rate
of absorption
reported for the 33-mer gluten peptide. In a repeat experiment,
intragastrically administered
labeled biomarker was again detected in the peripheral blood of this gluten-
sensitive
macaque (2.2 0.5 nM), but was not detected in samples from two identically
dosed
healthy controls.
[91] Celiac sprue affects up to 1% of many human populations, but despite the
wide
prevalence and serious manifestations of the disease, the only treatment
available remains
a life-long gluten-free diet. Compliance with this burdensome dietary
treatment is poor, and
recurrent exposure to gluten causes chronic inflammation, increased morbidity,
and more
serious health effects over time. Moreover, in asymptomatic celiac sprue
patients, disease
management is especially difficult, as invasive histological evaluations are
the only reliable
way to assess response to a gluten-free diet. Finally, the development of non-
dietary
treatment alternatives to the gluten-free diet requires a long-term gluten
challenge in celiac
patients, which is inherently problematic. Therefore, novel tools for
monitoring compliance
with the gluten-free diet, and safely evaluating non-dietary treatments in
vivo are needed.
[92] Here we used the disease-causing agent in celiac sprue as a template for
designing
noninflammatory analogs as tools for fundamental and translational research.
Celiac sprue
is uniquely suited for this biomarker strategy because we know the
environmental trigger
and have structural information about its binding mode to the primary genetic
determinant
CA 02722996 2010-10-29
WO 2009/139887 PCT/US2009/002997
for the disease. Additionally, gluten peptides are extraordinarily stable in
the relevant
physiological compartment, making them ideal scaffolds for drug and biomarker
design.
[93] Research into the molecular basis for celiac sprue has elucidated many of
the
properties of gluten peptides that allow them to persist through
gastrointestinal proteolysis,
access the gut-associated lymphoid tissue, and interact with the key players
in disease
pathogenesis: TG2, DQ2, and gluten-specific T cells. Guided by these findings,
our goal
was to abrogate those properties that contribute to the inflammatory response
to gluten
while retaining those properties that render biomarker metabolism and
transport relevant to
disease. By altering key residues in a gluten peptide scaffold, we developed
peptide
biomarkers that mimic gluten peptides in their resistance to gastrointestinal
proteases and
susceptibility to therapeutic glutenases, but that are neither substrates for
TG2 nor ligands
for DQ2. As a result, these peptides are not presented to gluten-specific T
cells and are
consequently noninflammatory.
[94] In the present study, the 33-mer from a2-gliadin was used as a scaffold
for
biomarker design because its metabolism in the presence and absence of
glutenases has
been extensively characterized and because its intact transepithelial
translocation has been
demonstrated. Other examples of disease-relevant gluten peptide sequences that
are
suitable for biomarker design include the 26-mer from y5-gliadin and the p31-
49 peptide that
stimulates an innate immune response in mucosal biopsies from celiac sprue
patients.
[95] Oral protease therapy is one of the more promising non-dietary treatments
being
developed for celiac sprue, but few studies have been conducted in vivo.
Celiac patients
administered an undefined enzyme mixture from animal digestive extracts showed
modest
improvement in a clinical trial. More recently, clinical efficacy of oral EP-
B2 was
demonstrated in a gluten sensitive rhesus macaque. These studies relied on
histological,
clinical, and serological readouts, complex parameters which require weeks to
register a
response and which are indirect measures of glutenase-mediated gluten
detoxification. By
contrast, in the present study, the metabolism of [13C3]-HHH-33-mer in rats
dosed with EP-
B2 provided an immediate and direct readout for glutenase activity in vivo.
This effect was
observed by mass spectrometric analysis of gastric contents. Studies will also
focus on
optimizing non-invasive readouts for biomarker metabolism, such as the
measurement of
isotope-labeled (or alternatively labeled) amino acid metabolite
concentrations in bodily
fluids such as serum or urine. Alternatively, a stable isotope breath test
(e.g. for 13 C02) may
be adapted to the detection of biomarker metabolites.
[96] In animal studies, we observed intact biomarker absorption across the
intestinal
epithelium of a subset of tested rats as well as an enteropathic gluten-
sensitive macaque.
Whereas the results of our rodent studies support the hypothesis that
proteolytically stable
gluten oligopeptides can be transported intact across the gut epithelium, they
also highlight
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the need for a considerably larger study to quantify inherent permeability
differences
between individual animals and to explore the role of IFN-y in enhancing
intestinal
permeability. In the glutensensitive macaque, the extent and kinetics of
biomarker transport
during active disease were similar to those reported for the 33-mer itself in
a previous
experiment.
[97] Biomarker transport was not observed in healthy controls, also consistent
with the
33-mer experiment. These data demonstrate that the level of the intact peptide
in blood (or
urine) can be used as a disease-relevant biomarker for intestinal barrier
function. Such a
metric would facilitate the evaluation of drugs intended to modulate
intestinal permeability.
One such drug candidate, AT-1001, has shown evidence of decreasing
paracellular
permeability in a diabetes-prone rat model and in preliminary clinical trials.
By analogy to
the use of neutralizing antibodies against TNF-ato treat Crohn's disease, anti-
IFN-
y antibodies may represent another candidate for reducing gut permeability and
inflammation in celiac sprue.
[98] Lastly, strict adherence to a gluten-free diet reduces intestinal
permeability in a
majority of celiac patients and this reduction precedes measurable
improvements in
histology. Therefore, biomarkers are a useful clinical tool for monitoring
adherence to a
gluten-free diet as well. Notwithstanding the effect of a gluten-free diet on
intestinal
permeability, epithelial uptake of gluten remains altered in treated celiac
patients with
respect to healthy controls. This is likely related to the 7.6-fold higher
levels of IFN-y present
in treated patients relative to healthy controls. In our experiments, both
biomarkers were
translocated intact across epithelial monolayers to a 10-fold greater extent
following
preincubation of the cells with IFN-y. This suggests these peptides may be
used as a
screening tool for celiac sprue, en route to a diagnosis, as well as for other
inflammatory
bowel diseases in which intestinal IFN-y levels and mucosal leakiness of
antigenic peptides
and proteins are elevated.
Materials and Methods
[99] Materials. Peptide synthesis reagents were purchased from Chem-Impex
(Wood
Dale, IL), Peptides International (Louisville, KY), Anaspec (San Jose, CA) and
Novabiochem (San Diego, CA). Cy5-NHS ester was purchased from Amersham
Biosciences (Piscataway, NJ). Isotopelabeled amino acids were purchased from
Cambridge
Isotope Laboratories (Andover, MA). Recombinant IFN-y was purchased from
Peprotech,
Inc. (Rocky Hill, NJ). Cell culture media, antibiotics, human serum, 5'(and
6')
carboxyfluorescein, and fluorescently labeled dextrans were purchased from
Invitrogen
(Carlsbad, CA). Fetal bovine serum was purchased from Atlanta Biologicals
(Lawrenceville,
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GA). Gluten flour was purchased from Bob's Red Mill (Milwaukie, OR). Thrombin
was
purchased from Novagen (Madison, WI). Protease inhibitor cocktail set 1 was
purchased
from Calbiochem (San Diego, CA). Pepsin, trypsin, chymotrypsin, elastase,
carboxypeptidase A, vancomycin, and Na-p-tosyl-L-arginine methyl ester
hydrochloride
(TAME) were purchased from Sigma-Aldrich (St. Louis, MO). Brush border
membranes
were purified from rat intestine as previously described and stored at -80 C
until use. The
recombinant proenzyme precursor of barley endoprotease EP-B234, Flavobacterium
meningosepticum PEP44 and human TG236 were expressed in E. coli and purified
as
previously described. Recombinant soluble DQ2 heterodimer-al gliadin peptide
fusion
molecules were prepared and purified in insect cells as previously described.
[100] Animals. Twelve 8 week old male Wistar rats weighing between 250 and 300
grams
(Charles River Laboratories, Wilmington, MA) were singly housed in standard
polycarbonate shoebox cages measuring 10.5" x 19" x 8" h with wire bar lids
and micro-
isolator tops (Allentown, Inc., Allentown, NJ). Eight 8 week old male Wistar
rats weighing
between 250 and 300 grams (Charles River Laboratories) with indwelling jugular
vein
catheters were housed similarly. Rats were allowed access to rodent chow #5010
(Purina,
Richmond, IN) and water ad libitum prior to the onset of the studies. The room
was
maintained on a 12:12-hr light:dark cycle. The ambient temperature remained
between 64
and 72 OF with a relative humidity of 30-70%. All experimental procedures were
approved
by the Animal Care and Use Committee of the Tulane National Primate Research
Center
(Covington, LA).
[101] Gluten-sensitive juvenile macaque FH45 (4.5 kg, male) was selected from
a
population of rhesus macaques exhibiting clinical diarrhea, intestinal villous
blunting, and
elevated AGA on a gluten-containing diet. Healthy controls H148 (2.65 kg,
male) and HK31
(2.70 kg, male) were selected from a population that exhibited no clinical or
serological
responses to gluten intake. Throughout the study, animals consumed 4% of their
respective
body weights daily of monkey chow #5K63 (Purina) containing 20% (by weight)
crude
protein including oats and gluten sources such as ground wheat. The animals
were housed
under biosafety level two conditions in accordance with the standards of the
Association for
Assessment and Accreditation of Laboratory Animal Care. Investigators adhered
to the
Guide for the Care and Use of Laboratory Animals prepared by the National
Research
Council.
[102] Peptide synthesis, labeling, and purification. Peptides were synthesized
using
Boc/HBTU chemistry on solid-phase as previously described. To prepare isotope-
labeled
peptides for the study of biomarker metabolism in vivo, [1-13C]-Ieucine was
incorporated at
positions 11, 18, and 25 in the HHH-33-mer biomarker (SEQ ID NO:77)
(LQLQPF(PQPHLPY)3PQPQPF) and [5,5,5-D3]- leucine was incorporated at position
8 in
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the myoglobin peptide ((SEQ ID NO:80) KGHHEAELKPL; underlined). For DQ2
binding
assays, peptides were labeled at their amino terminus on solid-phase with
carboxyfluorescein, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and 1-
hydroxy-7-
azabenzotriazole (HOAt) in 1:1:1 ratio and 10% (v/v) diisopropylethylamine in
dimethylformamide/methanol (2:1) as solvent. Following cleavage from the
resin, peptides
were purified over a reverse-phase C18 column by HPLC using a
water/acetonitrile gradient
in the presence of 0.1% trifluoroacetic acid, lyophilized, and stored at -20
C. For
transepithelial translocation assays, purified peptides were labeled at their
amino terminus
with Cy5-NHS ester in DMSO according to the manufacturer's instructions,
repurified by
HPLC, lyophilized, and stored at -20 C. The correct mass of all peptides was
confirmed by
LC-MS. Prior to use, peptides were resuspended in 50 mM sodium phosphate, pH
7.0
supplemented with 0.02% NaN3. The concentrations of unlabeled and Cy5-labeled
peptides
were determined at pH 7.0 by spectrophotometric measurement of A280 (6280 =
3840 M-
'cm-') and A652 (6652 = 250,000 M"'cm"'), respectively. Due to the absence of
aromatic
residues in the myoglobin peptide, the concentration of the unlabeled
myoglobin peptide
was determined by spectrophotometric measurement of A205 (6205 = 27 (mg/ml)-
1cm) as
previously described. The concentrations of fluorescein-labeled peptides were
determined
at pH 9.0 by spectrophotometric measurement of A495 (6495 = 80,200 M-'cm-1).
Working
stocks were stored at 4 C and their integrity confirmed periodically by RP-
HPLC.
[103] Cell culture. T84 epithelial cells from the American Type Culture
Collection
(Manassas, VA) were grown in T84 media (Dulbecco's Modified Eagle Medium:Ham's
F12
(1:1) supplemented with antibiotics (penicillin/streptomycin) and 5% (v/v)
fetal bovine
serum). Media was changed every alternate day, and the cells were split once a
week. DQ2
homozygous antigen-presenting cells (CD114, an Epstein Barr virus-transformed
B
lymphoblastoid cell line) were grown in APC media (RPMI supplemented with
antibiotics
and 5% (v/v) fetal bovine serum). Every other day, CD1 14 were split to 0.4 x
106 cells/ml.
Gluten-specific, DQ2-restricted T cell lines (TCL 432.1.4, TCL 421.1.4, TCL
446.1.3) and T
cell clones (TCC 436.5.3 (DQ2-a-II specific), TCC 430.1.142 (DQ2-a-I and DQ2-a-
III
specific) were isolated from celiac patient intestinal biopsies and expanded
as previously
described. T cell proliferation assays were performed in T cell media (RPMI
1640
supplemented with antibiotics and 10% (v/v) human serum). All cells were grown
and
assayed at 37 C with 5% atmospheric CO2.
[104] Simulated gastrointestinal digests with glutenase supplementation. In
preparation for
HPLC analysis, all buffers and reagents were filtered (0.2 m) prior to use in
digests. To
simulate gastric digestion, peptides (300 M) were incubated at 37 C in a 10
mM sodium
acetate buffer, pH 4.5 with 1:10 (w/w) pepsin (120 g/ml) supplemented with 0,
6, 12, 24,
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WO 2009/139887 PCT/US2009/002997
48, or 120 g/ml of recombinant proEP-B2 glutenase. Samples were collected at
0, 10, 30,
45, and 60 min timepoints. Samples were heat-deactivated at 95 C for 5 min,
diluted 1:5 in
HPLC solvent A (95% H2O, 5% acetonitrile, 0.1% trifluoroacetic acid)
supplemented with an
internal standard (TAME), and analyzed by reverse-phase HPLC. Samples (50 l)
were
separated over a C18 column (Grace Vydac, Hesperia, CA) using a water-
acetonitrile
gradient in the presence of 0.1% TFA. The absorbance at 215 nm was monitored.
The
area-under-the-curve for each intact peptide was calculated and normalized to
the area
under the curve for the internal standard, TAME. Due to the rapid EP-B2-
catalyzed removal
of the N-terminal LQ from 33-mer and biomarkers (Figure 1 and 2), this
minimally-
processed product was included as intact peptide in area-under-curve analyses.
Following
simulated gastric digestion of each peptide (300 M) with pepsin 120 g/ml
proEP-B2,
digests were adjusted to pH 6.0 with sodium phosphate buffer (50 mM, final
concentration)
and commercially available pancreatic proteases trypsin (30 g/ml),
chymotrypsin (30
g/ml), elastase (6 g/ml), and carboxypeptidase A (6 g/ml), as well as 27
g/ml rat
intestinal brush border membrane, were added. Recombinant prolyl endopeptidase
from
Flavobacterium meningosepticum (FM PEP) was supplemented at 1.2 U/ml when
added.
Simulated duodenal digests were performed at 37 C. Samples were collected at
0, 10, 30,
and 60 min and processed for HPLC as described. The area-under-the-curve for
each intact
peptide (together with the minimally processed -LQ product) was calculated and
normalized
to the internal standard. To identify digestion products, select samples were
analyzed by
LC-MS. Samples (50 l) processed for HPLC as described were injected on a
reverse-
phase C18 HPLC system (Waters Corporation, Milford, MA) coupled to a UVNis
detector
and a ZQ single quadrupole mass spectrometer with an electrospray ionization
source.
Samples were eluted with a wateracetonitrile gradient in the presence of 0.1%
formic acid.
Absorbance at 214/254 nm and total ion current were monitored, and spectra
corresponding
to major absorbance peaks were examined.
[105] Transglutaminase deamidation assay. Coupled spectrophotometric assays
for TG2
activity in the presence of each peptide were performed as previously
described36. Briefly,
each peptide (100 M) was added to a 200 mM MOPS, pH 7.2 buffer containing 5
mM
CaCl2, 1 mM Na4EDTA, 10 mM a-ketog I uta rate, and 250 M NADH. Glutamate
dehydrogenase was added to a final concentration of 0.036 U/ l, and this
incomplete
reaction mixture was incubated at room temperature for 10 min to stabilize the
initial
absorbance at 340 nm. Finally, 5 M TG2 was added and A340 was monitored. The
specific activity of the enzyme in the presence of each peptide was calculated
from the rate
of NADH consumption (e340 = 6220 M-1 cm-1).
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[1061 HLA-DQ2 binding assay. Peptide exchange assays for determining the
equilibrium
occupancy of each peptide on HLA-DQ2 were modified from previously described
methods.
Briefly, recombinant DQ2-al-gliadin peptide fusions (35 M) were treated with
0.15 U/ I
thrombin in phosphate-buffered saline (PBS), pH 7.4 supplemented with 0.02%
(w/v) NaN3
for 2 h at 4 C, after which protease inhibitor cocktail was added. Thrombin-
cleaved DQ2-al-
gliadin peptide complexes (9.4 M) were incubated with 0.185 M fluorescein-
conjugated
peptides in a citrate- PBS buffer, pH 5.5 (24 mM sodium citrate, 55 mM
Na2HPO4, 75 mM
NaCl, 0.02% (w/v) NaN3) for 45 h at 37 C. To quantify the equilibrium
occupancy of each
fluorescent peptide on DQ2, binding reactions were diluted 1:5 in PBS, pH 7.4
supplemented with 0.02% (w/v) NaN3 and 12.5 l was injected on an HPSEC system
coupled to a fluorescence detector (Shimadzu, Columbia, MD). DQ2-bound and
unbound
fluorescent peptides were separated using a BioSep 3000 size exclusion column
(Phenomenex, Torrance, CA) with a flow rate of 1 ml/min PBS, pH 7.4
supplemented with
0.02% (w/v) NaN3. The detector was set to monitor excitation at 495 nm and
emission at
520 nm. The bound/free ratio for each peptide was calculated by dividing the
measured
peak height for bound peptide by that for the free peptide.
[107] T cell proliferation assay. The T cell proliferation assay was modified
from. previously
described methods, as follows. Briefly, the 33-mer, NNN-33-mer, and HHH-33-mer
peptide
stocks (250 M) were deamidated by treatment with 100 g/ml TG2 in 100 mM
Tris, pH 7.4
for 2 hours at 37 C in the presence of 2 mM CaCl2. Antigen-presenting cells
(CD114,
60,000 cells/well) were irradiated (80 Gy) and incubated overnight at 37 C in
U-bottom, 96-
well plates with various concentrations of native or TG2-deamidated peptides
in T cell
media. As positive and negative controls, 2 M synthetically deamidated EEE-33-
mer or no
peptide was added to antigen-presenting cells. The next day, three T cell
lines and two T
cell clones isolated from intestinal biopsies of HLA-DQ2+ celiac patients were
thawed and
added (40,000 cells/well) to triplicate wells containing peptide-loaded
antigen-presenting
cells. Cells were co-incubated 48 hours to allow T cells to proliferate in
response to DQ2-
peptide complex stimulation. After 48 hours, 1 Ci [3H]-thymidine was added to
each well
and cells were incubated an additional 16 hours. Finally, DNA was collected on
a filter mat
using a cell harvester and counts-per-minute (cpm) resulting from incorporated
[3H]-
thymidine were measured with a liquid scintillation counter.
[108] Biomarker and glutenase dosing to rats. Procedures for administration of
gluten and
therapeutics to rats were modified from those previously described. The study
comprised 3
days, including fasting, acclimation to the gluten test meal, and
glutenase/biomarker dosing.
Adult rats (n = 4 for each of 3 groups) were fed a commercially available rat
chow until Day
1 of the study. Rats were then fasted for 12 h, while drinking water remained
freely available
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throughout the study. On Day 2, all 3 animal groups were administered a
freshly prepared
gluten-sugar test meal (a dough ball composed of 1 g gluten, 0.6 g white
sugar, 0.6 g brown
sugar, 0.35 g croscarmellose sodium, 2.45 ml water) to acclimate them to
eating this meal
following a fast. After 60 min, the test meal was removed and animals were
once again
fasted for 24 h. On Day 3, animals were administered a freshly prepared gluten-
sugar test
meal supplemented with 0, 10, or 40 mg proEP-B2 (for Groups 1, 2, or 3,
respectively), 19.7
mg [13C3]-HHH-33-mer, 3.3 mg [D3]-myoglobin peptide, and 10 mg vancomycin.
Vancomycin was added as a non-absorbable dosing internal standard, as
previously
described. Animals consumed the test meal completely within 60 min, and
animals were
euthanized 30 min thereafter (i.e. 90 min after meal administration). The
gastric, duodenal,
jejunal, and ileal contents were collected immediately and stored at -80 C as
previously
described. Peripheral plasma samples were collected via cardiac puncture at
the time of
euthanasia and stored at -80 C.
[109] Reverse-phase HPLC analysis of rat gastrointestinal contents. Gastric
samples (100
mg) were thawed on ice and suspended in 190 l 0.01 M HCI and 10 l 10 mM
leupeptin
(an inhibitor of EP-B2, to prevent digestion of material ex vivo). Suspensions
were
incubated 10 min at 37 C, pH 2.5, and then 50 mM sodium phosphate, pH 6.0 and
sodium
hydroxide were added to increase the pH of the suspensions to above 6Ø
Trypsin (0.375
mg/ml) and chymotrypsin (0.375 mg/ml) were added to maximize dissolution of
gluten, and
reactions were incubated 30 min at 37 C. Samples were heat-deactivated for 5
min at 95
C, supplemented with ethanol to a final concentration of 70% (v/v), and
centrifuged for 10
min at 16,100 x g. Syringe-filtered (0.45 m) supernatants (100 l) were
diluted 1:5 in 95%
H2O, 5% acetonitrile, 0.1% trifluoroacetic acid supplemented with an internal
standard
(TAME), and analyzed by reverse-phase HPLC. Samples (50 l) were separated
over a
C18 column (Grace Vydac, Hesperia, CA) using a water acetonitrile gradient in
the
presence of 0.1% TFA. The absorbance at 215 nm was monitored. Intestinal
flushes were
thawed on ice and centrifuged for 10 min at 4 C, 3100 x g. Supernatants were
heat-
deactivated and processed for HPLC as described for gastric contents.
[110] Competitive ELISA on rat gastric contents. Gastric samples (100 mg) were
thawed
on ice, suspended in 950 l 70% (v/v) ethanol and 50 l 10 mM leupeptin,
incubated 10 min
at 37 C, and then centrifuged for 10 min at 16,100 x g. Syringe-filtered
(0.45 m)
supernatants were tested for the gluten sequence QPQLPY using a monoclonal
antibody-
based competitive ELISA modified from previous methods. Briefly, equal volumes
of coating
solution (5 g/ml gliadin (Sigma) in 20 mM PBS, pH 7.2) and 20 mM sodium
bicarbonate,
pH 9.6 were added to 96-well microtiter plates (Nunc Maxisorp), and incubated
1 h at 37 C
and overnight at 4 C. The next day, gliadin-coated plates were washed twice
with washing
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buffer (PBS, pH 7.2 containing 0.05% Tween-20) then blocked with blocking
buffer (5%
(w/v) nonfat milk in PBS, pH 7.2) for 2 h at room temperature. Synthetic 33-
mer standard
(10-5 - 10 g/ml) or gastric supernatants were serially diluted in assay
buffer (3% (w/v)
bovine serum albumin in PBS, pH 7.2). An equal volume of G12-HRP monoclonal
antibody-
horseradish peroxidase conjugate (Biomedal, Seville, Spain) diluted 1:10,000
in assay
buffer was added to each standard or sample dilution. Mixtures were incubated
with gentle
agitation for 2 h at room temperature, and then added to plate wells in
triplicate. After 30
min incubation at room temperature, wells were washed 5 times with washing
buffer, and
TMB liquid substrate solution (Sigma) was added to wells. The reaction was
stopped after a
30 min dark incubation by addition of an equal volume of 1 M sulfuric acid and
the
absorbance at 450 nm was measured. Origin 6.0 (OriginLab, Northampton, MA) was
used
to fit the 33-mer standard curve to the sigmoidal model: A450 = A450min +
(A450max -
A450min)/[1 + (x/IC50)n] where x is the peptide concentration, IC50 is the 33-
mer
concentration at which competition is half-maximal, and n is the Hill slope.
The
concentration of peptides containing the sequence QPQLPY in gastric samples
was
determined by comparison to the linear portion of the 33-mer standard curve.
[111] 3Q LC-MS/MS analysis of intact peptides and biomarker metabolites. The
amounts
of gluten-derived 33-mer, [13C3]-HHH-33-mer, and [D3]-myoglobin peptide
present in rat
gastric contents and plasma were determined using a Micromass Quattro Premier
triple
quadrupole LC MS system. Gastric samples (100 mg) were thawed on ice and
suspended
in 950 l 0.01 M HCI and 50 l 10 mM leupeptin. Suspensions were adjusted to
pH 6.0 and
pancreatic proteases were added as described above to release the 33-mer from
gluten
present in the samples. After 30 min, samples were heat-deactivated for 5 min
at 95 C and
centrifuged for 10 min at 16,100 x g. Prior to 3Q LC-MS/MS analysis, syringe-
filtered (0.45
m) gastric supernatants, or plasma samples, were depleted of larger proteins
by addition
of acetonitrile. Samples were mixed with an equal volume of cold acetonitrile
containing
0.1 % formic acid and 200 nM NNN-33-mer as an internal standard. Samples were
vortexed,
incubated for 2 h at 4 C, and centrifuged for 10 min at 4 C, 16,100 x g.
Supernatants were
mixed with an equal volume of 0.1% formic acid in water to dilute the
acetonitrile
concentration to 25%, and used directly for intact peptide analysis or, for
plasma samples
only, processed for metabolite analysis as below. Mass spectrometry analysis
of intact
peptides was performed as previously described30 with the following
modifications.
Samples were injected in triplicate (80 l each) and eluted with a water-
acetonitrile gradient
in the presence of 0.1% formic acid. For 33-mer detection, positive ion SRM
mode was
used for monitoring the transitions of ions at m/z 978.84+ -* 263.2+ (30V cone
voltage,
27eV collision energy) for the quantification assay and m/z 1304.73+ --p263.2+
(40V cone
33
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WO 2009/139887 PCT/US2009/002997
voltage, 50eV collision energy) as a confirmatory transition. For [13C3]-HHH-
33-mer
detection, the transition monitored was m/z 986.754+ --p263.2+ (45V cone
voltage, 32eV
collision energy) for the quantification assay. For [D3]-myoglobin peptide
detection, the
transitions monitored were m/z 632.72+ --p229.2+ (35V cone voltage, 25eV
collision energy.)
for the quantification assay and m/z 632.72+ --*129.2+ (35V cone voltage, 33eV
collision
energy) as a confirmatory transition. For NNN-33-mer internal standard, the
transitions
monitored were m/z 968.64+ -p263.4+ (32V cone voltage, 32eV collision energy)
for the
quantification assay and m/z 968.64+ -*226.0+ (40V cone voltage, 50eV
collision energy)
as a confirmatory transition. Levels of 33-mer, isotope-labeled biomarker, and
isotope-
labeled myoglobin control peptide in each sample were determined by comparison
of the
area under their transition peaks to the area under the NNN-33-mer internal
standard
transition peak and to a calibration curve corresponding to each peptide. Mass
spectrometry
analysis of biomarker metabolites was performed on plasma samples processed as
above.
Protein-depleted samples (30 l) were dried and resuspended in 300 l 20 mM
ammonium
acetate, pH 5.0 containing 100 nM [D10]-leucine as an internal standard.
Samples were
injected in triplicate (20 l each) and eluted from an Atlantis T3 column (3
m, 2.1x100mm,
Waters) with a 20 mM ammonium acetate, pH 5.0-acetonitrile gradient. For
leucine
quantification, the transitions monitored were m/z 132.2+ -*86.2+ (15V cone
voltage, 10eV
collision energy). For [13C]-leucine quantification, the transitions monitored
were m/z 133.2+
->86.2+ (15V cone voltage, 10eV collision energy). For [D3]-leucine
quantification, the
transitions monitored were m/z 135.2+ -->89.2+ (15V cone voltage, 10eV
collision energy.).
For [D10]-leucine internal standard quantification, the transitions monitored
were m/z 142.2+
-->96.2+ (15V cone voltage, 10eV collision energy.)
[112] Peptide translocation assays. Peptide translocation assays were
performed as
previously described. Briefly, cultured T84 cells were seeded on rat tail
collagen-coated
polycarbonate transwell permeable supports (5 m pore size, 6.5 mm diameter;
Corning
Life Sciences, Lowell, MA) at 5 x 104 cells/well and the media was exchanged
every other
day for 2 weeks while the cells grew to confluence and formed tight junctions.
Following
maturation, cell monolayers were preincubated for 48 h with basolateral media
containing
either 0 or 600 U/ml recombinant IFN-y. After preincubation, the translocation
assay was
performed by replacing media in both the apical and basolateral chambers with
serum-free
T84 media (1:1::Dulbecco's Modified Eagle Medium:Ham's F12 media supplemented
with
antibiotics ), and adding 2 M dextran (3,000 mol. wt.)-Alexa Fluor-488, 2 M
dextran
(70,000 mol. wt.)-Texas Red, and 20 M Cy5-labeled peptide to the apical
chamber.
Labeled dextrans were added to confirm monolayer integrity and size-selective
transport4l.
Samples of the apical and basolateral media were taken at the 0 h time-point,
and
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basolateral samples were taken every hour over a 4 h experiment. Fluorescence
in
collected samples was measured in 96-well format on a Flexstation II 384
(Molecular
Devices, Sunnyvale, CA), monitoring three channels (excitation 490 nm,
emission 525 nm
for Alexa Fluor-488; excitation 585 nm, emission 620 nm for Texas Red;
excitation 640 nm,
emission 675 nm for Cy5). The slope of basolateral fluorescence units versus
time (from 1-4
h) was calibrated to the initial apical fluorescence and divided by the
permeable support
area (0.33 cm2) to yield the transepithelial flux (pmol/cm2/h).
[113] Chromatographic and mass spectrometric analysis of translocated gluten
peptides.
During the peptide translocation assay described above, additional samples
from the apical
and basolateral chambers were collected at 0 and 10 h for analysis of peptide
stability and
intact translocation. Samples (50 L) were analyzed by LC-MS as described for
digests,
except absorbance at 640 nm was monitored. Spectra corresponding to A64o peaks
were
examined. Samples were also analyzed by HPSEC with fluorescence detection as
described for DQ2- peptide binding analysis, except the detector was set to
monitor
excitation at 647 nm and emission at 665 nm.
[114] Biomarker dosing in rats following IFN-gtreatment. Adult catheterized
rats (n = 4 for
each of 2 groups) were administered a daily dose of vehicle (PBS) or 108 U/m2
IFN-
y intravenously via catheter for 2 days. On Day 3, 48 hours after the initial
dose of vehicle or
IFN-y, all animals were administered 0.5 ml water containing 20 mg 33-mer and
20 mg
[13C3]-HHH-33-mer via oral gavage. Animals were euthanized 60 min thereafter
and
peripheral plasma samples were collected, stored, and tested via 3Q LC-MS/MS
for intact
peptides as described above.
[115] Biomarker inoculation in gluten-sensitive and healthy rhesus macaques.
Prior to the
study, gluten-sensitive macaque FH45 was administered a gluten-containing diet
and
exhibited elevated anti-gliadin antibodies, intestinal villous blunting, and
clinical symptoms
indicative of gluten sensitivity. On the day of the study, a dose of 100 mg of
isotopically
labeled biomarker ([13C3]-HHH-33-mer) dissolved in 10 ml of Gatorade was
administered
directly into the fasted stomach of FH45 by intragastric tube. A 0.5 ml sample
of EDTA-
blood was collected from an ear vein at 0, 60, 120, 180, and 240 min following
biomarker
inoculation. The experiment was repeated four months later with FH45 and two
healthy
control macaques, H148 and HK31, on a gluten-containing diet. In this second
experiment,
50 mg of isotopically labeled biomarker was intragastrically administered to
each animal.
Animals were sedated and anesthetized prior to peptide inoculation and blood
collections.
Plasma samples were analyzed for intact biomarker by 3Q LC-MS/MS as described
above.
[116] Statistics. Statistical comparisons were conducted using a two-tailed
Student's t-test
assuming unequal variances. A statistical probability of p < 0.05 was
considered significant.
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Example 2
Method of Determining Treatment Efficacy (I)
[117] In conjunction with a clinical trial for determining efficacy of a study
drug ALVO03 in
mitigating the effects of gluten ingestion in patients with celiac disease.
Adult patients are
selected for inclusion in the study based on biopsy-proven CD in past 5 year.
Subjects are
given the study drug at one of four different dose levels, and following a
meal containing a
small amount (1 gram) of gluten.
[118] In combination with the gluten, the subject is given a dose of unlabeled
peptide
consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF
(HHH-33mer, SEQ ID NO:77), where the dose may be 1; 10; 100 or 500 mg of
peptide.
[119] At four hours after ingestion, a urine sample is obtained from the
subject, which
sample is contacted with a dipstick comprising a dye antibody that selectively
binds to the
peptide; and an immobilized second antibody that selectively binds to the
peptide or the
peptide antibody complex. Upon contact, the presence of the peptide is
determined by
localization of the dye label at the position where the second antibody is
immobilized, which
may for convenience be a shape of a "+", or other easily recognized symbol.
[120] In an alternative determination, a sample of blood is tested for the
presence of
peptide one hour after ingestion.
[121] The presence of the peptide in urine is indicative that intestinal
permeability is
undesirably high, suggesting that the peptide (and, by analogy, dietary
gluten) is
inadequately metabolized by ALVO03 in the gastrointestinal lumen.
Example 3
Method of Determining Treatment Efficacy (II)
[122] In conjunction with a clinical trial for determining efficacy of the
study drug CCX282-
B (an orally active inhibitor of chemokine receptor CCR9) in mitigating the
effects of gluten
ingestion in patients with celiac disease. Alternative efficacy determination
is made by
evaluation of the effect of CCX282-B compared to placebo on the villous
height/crypt depth
ratio of small intestinal biopsy specimens taken from subjects with celiac
disease, before
and after gluten exposure. Adult patients are treated with 250mg capsule,
twice daily, 13
weeks of the study drug.
[123] At selected time periods, the subject is given a dose of unlabeled
peptide consisting
of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-
33mer, SEQ ID NO:77), where the dose may be 1; 10; 100 or 500 mg of peptide.
[124] At four hours after ingestion, a urine sample is obtained from the
subject, which
sample is contacted with a dipstick comprising a dye antibody that selectively
binds to the
peptide; and an immobilized second antibody that selectively binds to the
peptide or the
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peptide antibody complex. Upon contact, the presence of the peptide is
determined by
localization of the dye label at the position where the second antibody is
immobilized, which
may for convenience be a shape of a "+", or other easily recognized symbol.
[125] The presence of the peptide in urine is indicative that the intestinal
permeability is
undesirably high, suggesting that an inflammatory response to gluten persists
in the patient.
Example 4.
Method of Determining Treatment Efficacy (III)
[126] In conjunction with a clinical trial for determining efficacy of the
study drug AT-1001
(Larazotide acetate). AT-1001 is an orally administered octapeptide zonulin
receptor
antagonist that appears to exert its inhibitory effect on gliadin-induced
tight junction
disassembly by blocking putative zonulin receptors on the luminal surface of
the small
intestine. Pretreatment with the peptide fails to inhibit gliadin induced
zonulin release, while
administration of zonulin analogues or gliadin in the presence of AT-1001 fail
to significantly
affect intestinal permeability, confirming the effect of the molecule is
specific to the zonulin
receptor. in mitigating the effects of gluten ingestion in patients with
celiac disease.
[127] Adult celiac patients are given capsules of AT-1001 at doses of 1, 4,
and 8 mg of
drug, with 900 mg of gluten.
[128] At selected time periods, the subject is also given a dose of unlabeled
peptide
consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF
(HHH-33mer, SEQ ID NO:77), where the dose may be 1; 10; 100 or 500 mg of
peptide.
[129] At four hours after ingestion, a urine sample is obtained from the
subject, which
sample is contacted with a dipstick comprising a dye antibody that selectively
binds to the
peptide; and an immobilized second antibody that selectively binds to the
peptide or the
peptide antibody complex. Upon contact, the presence of the peptide is
determined by
localization of the dye label at the position where the second antibody is
immobilized, which
may for convenience be a shape of a "+", or other easily recognized symbol.
[130] The presence of the peptide in urine is indicative that the intestinal
permeability is
undesirably high.
Example 5.
Method of Determining Treatment Efficacy (IV)
[131] In conjunction with a clinical trial for determining efficacy of the
study drug Nexvax2.
NexVax2 is a peptide-based vaccine to treat or prevent celiac disease. The
vaccine will
include the gluten peptides most commonly recognized by T cells in people with
celiac
disease.
[132] Adult celiac patients are given Nexvax2 at the following doses:
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9 micrograms Nexvax2 weekly intra-dermal injection, 3 week duration
30 micrograms Nexvax2 weekly intra-dermal injection, 3 week duration
90 micrograms Nexvax2, weekly intra-dermal injection, 3 week duration
300 micrograms Nexvax2, weekly intra-dermal injection, 3 week duration
900 micrograms Nexvax2, weekly intra-dermal injection, 3 week duration
[133] At selected time periods, the subject is also given a dose of unlabeled
peptide
consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF
(HHH-33mer, SEQ ID NO:77), where the dose may be 1; 10; 100 or 500 mg of
peptide.
[134] At four hours after ingestion, a urine sample is obtained from the
subject, which
sample is contacted with a dipstick comprising a dye antibody that selectively
binds to the
peptide; and an immobilized second antibody that selectively binds to the
peptide or the
peptide antibody complex. Upon contact, the presence of the peptide is
determined by
localization of the dye label at the position where the second antibody is
immobilized, which
may for convenience be a shape of a "+", or other easily recognized symbol.
[135] The presence of the peptide in urine is indicative that the intestinal
permeability is
undesirably high, presumably due to ongoing T cell inflammation by dietary
gluten.
Example 6
Method of ongoing patient monitoring
[136] In conjunction with treatment modality for celiac sprue, the subject is
given a dose of
unlabeled peptide consisting of the amino acid sequence
LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where the
dose may be 1; 10; 100 or 500 mg of peptide to be ingested with normal meals.
[137] At four hours after ingestion, a urine sample is obtained from the
subject, which
sample is contacted with a dipstick comprising a dye antibody that selectively
binds to the
peptide; and an immobilized second antibody that selectively binds to the
peptide or the
peptide antibody complex. Upon contact, the presence of the peptide is
determined by
localization of the dye label at the position where the second antibody is
immobilized, which
may for convenience be a shape of a "+", or other easily recognized symbol.
[138] The presence of the peptide in urine is indicative that the peptide is
not metabolized,
and is indicative that intestinal permeability is undesirably high, and is
indicative that the
patient's diet and/or therapeutic regimen should be adjusted accordingly.
Example 7
Method of ongoing patient monitoring
[139] In conjunction with treatment modality for celiac sprue, the subject is
given a dose of
labeled peptide consisting of the amino acid sequence
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LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where a
highly fluorescent label is conjugated to the amino terminus. The dose may be
1; 10; 100 or
500 mg of peptide to be ingested with normal meals. In an alternative
embodiment the
peptide is conjugated with a fluorescent label and a quencher, such that the
fluorescence is
quenched in the intact peptide, and is unquenched in a proteolytically cleaved
peptide.
[140] At four hours after ingestion, a urine sample is obtained from the
subject. Where the
peptide is labeled with a fluorescent dye and quencher pair, the sample is
analyzed for the
presence of fluorescence. Fluorescence is indicative that the peptide is
cleaved and is
indicative that the treatment is appropriately metabolizing gluten.
[141] Where the peptide is labeled with only a fluorescent dye, the sample is
separated by
chromatography to determine whether the label is associated with free amino
acids or with
peptide.
[142] The presence of the label at a position corresponding to the peptide in
urine is
indicative that the peptide is not metabolized, and is indicative that
intestinal permeability is
undesirably high, and is indicative that the therapeutic regimen should be
adjusted
accordingly.
[143] These and other diagnostic methods of the invention can be practiced
using the
methods provided by the invention.
[144] All publications, patents, and patent applications cited in this
specification are herein
incorporated by reference as if each individual publication, patent, or patent
application
were specifically and individually indicated to be incorporated by reference.
[145] The present invention has been described in terms of particular
embodiments found
or proposed by the inventor to comprise preferred modes for the practice of
the invention. It
will be appreciated by those of skill in the art that, in light of the present
disclosure,
numerous modifications and changes can be made in the particular embodiments
exemplified without departing from the intended scope of the invention.
Moreover, due to
biological functional equivalency considerations, changes can be made in
methods,
structures, and compounds without affecting the biological action in kind or
amount. All such
modifications are intended to be included within the scope of the appended
claims.
39
