U.S. patent application number 11/997135 was filed with the patent office on 2008-12-04 for peptides for diagnostic and therapeutic methods for celiac sprue.
Invention is credited to Chaitan Khosla, Matthew John Siegel, Jiang Xia.
Application Number | 20080299108 11/997135 |
Document ID | / |
Family ID | 37727962 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299108 |
Kind Code |
A1 |
Khosla; Chaitan ; et
al. |
December 4, 2008 |
Peptides for Diagnostic and Therapeutic Methods for Celiac
Sprue
Abstract
Detection of toxic gluten oligopeptides refractory to digestion
and antibodies and T cells responsive thereto can be used to
diagnose Celiac Sprue. Analogs of such oligopeptides are useful in
the inhibition of immune responses.
Inventors: |
Khosla; Chaitan; (Palo Alto,
CA) ; Xia; Jiang; (Stanford, CA) ; Siegel;
Matthew John; (Stanford, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE, SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
37727962 |
Appl. No.: |
11/997135 |
Filed: |
August 3, 2006 |
PCT Filed: |
August 3, 2006 |
PCT NO: |
PCT/US06/30650 |
371 Date: |
July 9, 2008 |
Current U.S.
Class: |
424/94.63 ;
435/7.1; 436/501; 514/1.1; 530/324; 530/325; 530/326; 530/327;
530/328 |
Current CPC
Class: |
A61K 38/482 20130101;
C07K 2299/00 20130101; G01N 2800/202 20130101; G01N 2800/02
20130101; A61P 1/00 20180101; A61K 38/08 20130101; G01N 33/6854
20130101; C07K 14/415 20130101; A61K 47/60 20170801; A61K 39/35
20130101; A61K 38/10 20130101; A61K 38/08 20130101; A61K 2300/00
20130101; A61K 38/10 20130101; A61K 2300/00 20130101; A61K 38/482
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/94.63 ;
530/327; 530/326; 530/325; 530/324; 514/14; 514/13; 514/12;
436/501; 435/7.1; 530/328 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; C07K 14/00 20060101 C07K014/00; A61K 38/08 20060101
A61K038/08; A61K 38/16 20060101 A61K038/16; G01N 33/53 20060101
G01N033/53; A61K 38/48 20060101 A61K038/48; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 2005 |
US |
11/198068 |
Claims
1. A purified oligopeptide of from about 14 amino acids in length
to about 33 amino acids in length, comprising the epitope sequence
(SEQ ID NO:43) PQPEX.sub.1PYPQ, where X.sub.1 is glutamine or
lysine or a conjugate of lysine.
2. The purified oligopeptide of claim 1, wherein said peptide
comprises as the amino terminal sequence, SEQ ID NO:32, LQLQPF.
3. The purified oligopeptide of claim 1, wherein the carboxy
terminal sequence is SEQ ID NO:44 PELPY, or SEQ ID NO:45 PEKPY.
4. The purified oligopeptide of claim 1, wherein said peptide has
the structure: LQLQPFPQPE-X.sub.1-PYPQPE-X.sub.2-PY where X.sub.1
and X.sub.2 are independently selected from lysine; leucine; and
lysine conjugated to a group that provides for steric hindrance of
interactions between the peptide and a cognate receptor.
5. The purified oligopeptide of claim 4, wherein said lysine
residue is conjugated to succinic acid; benzyloxycarbonyl group;
t-Butoxycarbonyl group; 9-fluorenylmethoxycarbonyl group;
phthalimides; or polyethylene glycol.
6. The purified oligopeptide of claim 1, wherein said peptide has
the structure: LQLQPFPQPE-X.sub.1-PYPQPE-X.sub.2-PY where X.sub.1
and X.sub.2 are independently selected from lysine; leucine; and
lysine conjugated a linker, where said linker couples two or more
of said oligopeptides.
7. The purified oligopeptides according to claim 6, wherein said
linker is polyethylene glycol.
8. A pharmaceutical composition comprising the oligopeptides of
claim 1, and a pharmaceutically acceptable excipient.
9. A method for diagnosing Celiac Sprue in an individual, said
method comprising determining whether an oligopeptide of claim 1
stimulates agglutination of anti-gliadin antibodies, anti-tTGase
antibodies, or combinations thereof from said individual, and
correlating an ability to stimulate agglutination with a positive
diagnosis of Celiac Sprue.
10. The method of claim 9, wherein said tissue is a mucosal tissue
selected from the group consisting of oral, nasal, lung, and
intestinal mucosal tissue.
11. The method of claim 9, wherein said bodily fluid is selected
from the group consisting of blood, sputum, urine, phlegm, lymph,
and tears.
12. The method of claim 9, wherein said individual has not consumed
gluten for an extended period of time.
13. The method of claim 12, wherein said extended period of time is
selected from the group consisting of one day, one week, one month,
and one year prior to the performance of the diagnostic method.
14. The method of claim 9, wherein said individual has not had an
endoscopy.
15. The method of claim 9, wherein said individual is the subject
of a therapy intended to treat Celiac Sprue or is in a clinical
trial conducted to evaluate such a therapy.
16. An HLA-binding peptide inhibitor; wherein said inhibitor is an
analog of an immunogenic gluten oligopeptide of at least about 8
residues in length, wherein the immunogenic gluten oligopeptide is
altered by the replacement of one or more amino acids; and wherein
said analog binds tightly to HLA molecules; is proteolytically
stable; and does not activate disease-specific T cells.
17. The HLA-binding peptide inhibitor of claim 16, wherein said
analog comprises one or more naturally occurring amino acids,
non-naturally occurring amino acids, modified amino acids, or amino
acid mimetics.
18. The HLA-binding peptide inhibitor of claim 17, wherein said
analog is further derivatized to reduce the affinity of the analog
for disease-specific T cell receptors.
19. The HLA-binding peptide inhibitor of claim 16, wherein said
immunogenic gluten oligopeptides comprises at least one PXP
motif.
20. The HLA-binding peptide inhibitor of claim 16, wherein said
immunogenic gluten oligopeptides comprises a sequence selected from
the group consisting of: (SEQ ID NO: 36) PQPELPY; PFPQPELPYP, (SEQ
ID NO: 47) PQPELPYPQPQLP, (SEQ ID NO: 48) PQQSFPEQQPP, (SEQ ID NO:
49) VQGQGIIQPEQPAQ, (SEQ ID NO: 50) FPEQPQQPYPQQP, (SEQ ID NO: 51)
FPQQPEQPYPQQP, FSQPEQEFPQPQ; PFPQPQLPY, PQPQLPYPQ, (SEQ ID NO: 17)
PFPQPELPY; (SEQ ID NO: 58) PYPQPELPY and PYPQPQLPY.
21. The HLA-binding peptide inhibitor of claim 16, wherein said
inhibitor comprises the sequence PXPQPELPY, where X is Tyr, Trp,
Arg, Lys, p-iodo-Phe, 3-iodo-Tyr, p-amino-Phe, 3-amino-Tyr,
hydroxylysine, ornithine, Asp or Glu.
22. The HLA-binding peptide inhibitor of claim 21, wherein said
inhibitor is further derivatized to reduce the affinity of the
analog for disease-specific T cell receptors.
23. The HLA-binding peptide inhibitor of claim 16, wherein said
inhibitor is further modified to increase binding potency to an MHC
molecule.
24. The HLA-binding peptide inhibitor of claim 16, wherein said
inhibitor comprises the sequence PFPQX.sub.1ELX.sub.2Y, where
X.sub.1 and X.sub.2 are independently selected from 4-hydroxy-Pro,
4-amino-Pro, or 3-hydroxy-Pro, and proline, with the proviso that
at least one of X.sub.1 and X.sub.2 is a residue other than
proline.
25. The HLA-binding peptide inhibitor of claim 24, wherein said
inhibitor is further derivatized to reduce the affinity of the
analog for disease-specific T cell receptors.
26. The HLA-binding peptide inhibitor of claim 24, wherein said
inhibitor is further modified to increase binding potency to an MHC
molecule.
27. A method of treating Celiac Sprue and/or dermatitis
herpetiformis, the method comprising: administering to a patient an
effective dose of an HLA-binding peptide inhibitor; wherein said
HLA-binding peptide inhibitor attenuates gluten toxicity in said
patient.
28. The method of claim 27, wherein said HLA-binding peptide
inhibitor is administered with a glutenase.
29. The method according to claim 27 wherein said HLA-binding
peptide inhibitor is administered orally.
30. The method according to claim 27, wherein said HLA-binding
peptide inhibitor is contained in a formulation that comprises an
enteric coating.
31. A formulation for use in treatment of Celiac Sprue and/or
dermatitis herpetiformis, comprising: an effective dose of an
HLA-binding peptide inhibitor and a pharmaceutically acceptable
excipient.
32. The formulation according to claim 29, further comprising an
enteric coating.
33. Use of an HLA-binding peptide inhibitor in the treatment of
HLA-DQ2 positive individuals who are either pre-disposed to type I
diabetes or have developed symptoms of type I diabetes.
Description
[0001] 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 prolamine 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. 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 European populations.
[0002] 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.
[0003] Celiac Sprue is generally considered to be an autoimmune
disease and the antibodies found in the serum of the patients
support a theory of an immunological nature of the disease.
Antibodies to tissue transglutaminase (tTG) and gliadin appear in
almost 100% of the patients with active CS, and the presence of
such antibodies, particularly of the IgA class, has been used in
diagnosis of the disease.
[0004] The large majority of patients express the HLA-DQ2
[DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] molecules.
It is believed that intestinal damage is caused by interactions
between specific gliadin oligopeptides and the HLA-DQ2 or DQ8
antigen, which in turn induce proliferation of T lymphocytes in the
sub-epithelial layers. T helper 1 cells and cytokines apparently
play a major role in a local inflammatory process leading to
villous atrophy of the small intestine.
[0005] At the present time there is no good therapy for the
disease, except to completely avoid all foods containing gluten.
Although gluten withdrawal has transformed the prognosis for
children and substantially improved it for adults, some people
still die of the disease, mainly adults who had severe disease at
the outset. An important cause of death is lymphoreticular disease
(especially intestinal lymphoma). It is not known whether a
gluten-free diet diminishes this risk. Apparent clinical remission
is often associated with histologic relapse that is detected only
by review biopsies or by increased EMA titers.
[0006] Gluten is so widely used, for examples in commercial soups,
sauces, ice creams, hot dogs, etc., that patients need detailed
lists of foodstuffs to avoid and expert advice from a dietitian
familiar with celiac disease. Ingesting even small amounts of
gluten may prevent remission or induce relapse. Supplementary
vitamins, minerals, and hematinics may also be required, depending
on deficiency. A few patients respond poorly or not at all to
gluten withdrawal, either because the diagnosis is incorrect or
because the disease is refractory. In the latter case, oral
corticosteroids (e.g., prednisone 10 to 20 mg bid) may induce
response.
[0007] Current diagnostic methods for Celiac Sprue are expensive
and not very accurate. These methods include ELISA-based methods in
which either anti-gliadin or anti-tTG antibodies in the patient's
serum are detected and in which T cell proliferation upon
stimulation with gliadin is observed. Often, however, these methods
are not sensitive enough to detect the diagnostic antibodies in the
blood or, as is the case for T cell proliferation assays, are
deemed to be too expensive for routine use. Typically, even if an
individual tests positive in the diagnostic test, the individual
must be re-challenged with gliadin (typically after maintaining a
gluten-free diet for an extended period of time) and examined by
endoscopy, an invasive and often painful procedure.
[0008] PCT publication No. WO 01/25793, published Apr. 12, 2001,
describes peptides derived from epitope mapping of alpha-gliadin
and methods for diagnosing Celiac Sprue using such peptides. Those
methods, however, do not appear to be significantly more sensitive
than methods currently employed and so do not overcome the
limitations of diagnostic methods currently in use.
[0009] PCT publication No. WO 02/083722 describes HLA-DQ restricted
T cells receptors capable of recognizing prolamine-derived peptides
involved in food-related immune enteropathy.
[0010] There remains a need for better diagnostic methods for
Celiac Sprue, methods that are more sensitive than current methods,
that do not require confirmation by endoscopy, and that do not
require that an individual be challenged with a gluten-containing
diet for accuracy. The present invention addresses this need.
SUMMARY OF THE INVENTION
[0011] Methods are provided for diagnosing Celiac Sprue, and/or
dermatitis herpetiformis, by detecting multivalent toxic gluten
oligopeptides in a patient; antibodies that bind to the toxic
gluten oligopeptides; or T cell proliferation elicited by such
oligopeptides in a patient. Novel peptides are provided, which
interact strongly with gluten reactive T cells and/or HLA
molecules. Certain peptides, particularly modified peptides, are
shown to bind strongly to the HLA molecule, without activating T
cells, thereby blocking reactivity. Such peptides find use in
diagnostic and therapeutic methods.
[0012] In one aspect, the present invention provides methods for
treating Celiac Sprue and/or dermatitis herpetiformis and the
symptoms thereof by administration of an HLA-binding peptide
inhibitor to the patient. In one embodiment, the HLA-binding
peptide inhibitor employed in the method is an analog of an
immunogenic gluten peptide, where an immunogenic gluten peptide is
altered by the replacement of one or more amino acids, where the
replacement may be another naturally occurring amino acid,
non-naturally occurring amino acids, modified amino acids, amino
acid mimetics, and the like. Analogs of immunogenic gluten peptides
that (i) retain the ability to bind tightly to HLA molecules; (ii)
retain the proteolytic stability of these peptides; but (iii) are
unable to activate disease-specific or other T cells, are useful
agents to treat Celiac Sprue.
[0013] In another aspect, the present invention provides novel
HLA-binding peptide inhibitors and methods for treating Celiac
Sprue and/or dermatitis herpetiformis by administering those
compounds.
[0014] In another aspect, the invention provides pharmaceutical
formulations comprising an HLA-binding peptide inhibitor and a
pharmaceutically acceptable carrier. In one embodiment, such
formulations comprise an enteric coating that allows delivery of
the active agent to the intestine, and the agents are stabilized to
resist digestion or acid-catalyzed modification in acidic stomach
conditions. In another embodiment, the formulation also comprises
one or more glutenases, as described in U.S. Provisional
Application 60/392,782 filed Jun. 28, 2002; and U.S. Provisional
Application 60/428,033, filed Nov. 20, 2002, both of which are
incorporated herein by reference. The invention also provides
methods for the administration of enteric formulations of one or
more HLA-binding peptide inhibitors to treat Celiac Sprue.
[0015] These and other aspects and embodiments of the invention and
methods for making and using the invention are described in more
detail in the description of the drawings and the invention, the
examples, the claims, and the drawings that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1B. Brush border membrane catalyzed digestion of
the immunodominant gliadin peptide. FIG. 1A: LC-MS traces of (SEQ
ID NO:1) QLQPFPQPQLPY after digestion with 27 ng/.mu.l rat brush
border membrane (BBM) protein for the indicated time. Reaction
products were separated by reversed phase HPLC and detected by mass
spectroscopy (ion counts m/z=300-2000 g/mol). The indicated peptide
fragments were confirmed by characteristic tandem MS fragmentation
patterns. The SEQ ID NO:2 pyroQLQPFPQPQLPY peak corresponds to an
N-terminally pyroglutaminated species, which is generated during
HPLC purification of the synthetic starting material. FIG. 1B
Abundance of individual digestion products as a function of time.
The peptide fragments in FIG. 1A were quantified by integrating the
corresponding MS peak area (m/z=300-2000 g/mol). The resulting MS
intensities are plotted as a function of digestion time (with BBM
only, colored bars). The digestion experiment was repeated in the
presence of exogenous DPP IV from Aspergillus fumigatus (Chemicon
International, CA, 0.28 .mu.U DPP IV/ng BBM protein) and analyzed
as above (open bars). The relative abundance of different
intermediates could be estimated from the UV.sub.280 traces and
control experiments using authentic standards. The inserted scheme
shows an interpretative diagram of the digestion pathways of (SEQ
ID NO:1) QLQPFPQPQLPY and its intermediates, the BBM peptidases
involved in each step, and the amino acid residues that are
released. The color code for labeling the peptides is similar to
that used in A. The preferred breakdown pathway is indicated in
bold. APN=aminopeptidase N, CPP=carboxypeptidase P, DPP
IV=dipeptidyl dipeptidase IV.
[0017] FIG. 2A-2B. C-terminal digestion of the immunodominant
gliadin peptide by brush border membrane. FIG. 2A: (SEQ ID NO:3)
PQPQLPYPQPQLPY was digested by 27 ng/.mu.l brush border membrane
(BBM) protein preparations for the indicated time and analyzed as
in FIG. 1A. The identity of the starting material and the product
(SEQ ID NO:4) PQPQLPYPQPQLP was corroborated by MSMS fragmentation.
The intrinsic mass intensities of the two peptides were identical,
and the UV.sub.280 extinction coefficient of (SEQ ID NO:4)
PQPQLPYPQPQLP was half of the starting material in accordance with
the loss of one tyrosine. All other intermediates were below
.ltoreq.1%. The scheme below shows the proposed BBM digestion
pathway of (SEQ ID NO:3) PQPQLPYPQPQLPY with no observed N-terminal
processing (crossed arrow) and the removal of the C-terminal
tyrosine by carboxypeptidase P (CPP) in bold. Further C-terminal
processing by dipeptidyl carboxypeptidase (DCP) was too slow to
permit analysis of the subsequent digestion steps (dotted arrows).
FIG. 2B: Influence of dipeptidyl carboxypeptidase on C-terminal
digestion. (SEQ ID NO:3) PQPQLPYPQPQLPY in phosphate buffered
saline:Tris buffered saline=9:1 was digested by BBM alone or with
addition of exogenous rabbit lung DCP (Cortex Biochemicals, CA) or
captopril. After overnight incubation, the fraction of accumulated
SEQ ID NO:4) PQPQLPYPQPQLP (compared to initial amounts of (SEQ ID
NO:3) PQPQLPYPQPQLPY at t=0 min) was analyzed as in FIG. 2A, but
with an acetonitrile gradient of 20-65% in 6-35 minutes.
[0018] FIG. 3. Dose dependent acceleration of brush border mediated
digestion by exogenous endoproteases. As seen from FIG. 2A-2B, the
peptide (SEQ ID NO:4) PQPQLPYPQPQLP is stable toward further
digestion. This peptide was digested with 27 ng/.mu.l brush border
membranes, either alone, with increasing amounts of exogenous
prolyl endopeptidase (PEP, specific activity 28 .mu.U/.mu.g) from
Flavobacterium meningosepticum (US Biological, MA), or with
additional elastase (E-1250, Sigma, MO), bromelain (B-5144, Sigma,
MO) or papain (P-5306, Sigma, MO). After one hour, the fraction of
remaining (SEQ ID NO:4) PQPQLPYPQPQLP (compared to the initial
amount at t=0 min) was analyzed and quantified as in FIG. 1.
[0019] FIG. 4. Products of gastric and pancreatic protease mediated
digestion of .alpha.2-gliadin under physiological conditions.
Analysis was performed by LC-MS. The longest peptides are
highlighted by arrows and also in the sequence of .alpha.2-gliadin
(inset).
[0020] FIG. 5. In vivo brush border membrane digestion of peptides.
LC-UV.sub.215 traces of 25 .mu.M of (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF before perfusion and after
perfusion (residence time=20 min). LC-UV.sub.215 traces of 50 .mu.M
of (SEQ ID NO:1) QLQPFPQPQLPY before perfusion and after perfusion
(residence time=20 min).
[0021] FIG. 6. Alignment of representative gluten and non-gluten
peptides homologous to (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.
[0022] FIG. 7. Breakdown and detoxification of 33-mer gliadin
peptide with PEP. In vitro incubation of PEP (540 mU/ml) with the
33-mer gliadin peptide (100 .mu.M) for the indicated time. In vivo
digestion of the 33-mer gliadin peptide (25 .mu.M) with PEP (25
mU/ml) and the rat's intestine (residence time=20 min).
[0023] FIG. 8. Equilibrium occupancy of individual peptides listed
in Table 1 in the DQ2 binding pocket, as measured by peptide
exchange assays. Measurements were made at (A) pH 5.5 and (B) pH
7.3. 4.7 .mu.M DQ2 was mixed with 0.18 .mu.M fluorescein-conjugated
peptide at 37.degree. C. for 45 h, and the abundance of DQ2 bound
peptide was calculated as a percentage of total peptide.
[0024] FIG. 9A-9B. Stimulation of T cell proliferation by three
peptides, the 33-mer 1 (.DELTA.), peptide 3 ( ), and the 20-mer 5
(.diamond.). Paraformaldehyde-fixed DQ2 cells were used as antigen
presenting cells. (A) Proliferation of a polyclonal T cell line
that recognizes all .alpha. epitopes. (B) Proliferation of a clonal
T cell line that recognizes the .alpha.II epitope (peptide 3).
[0025] FIG. 10. Structures of candidate DQ2 blocking agents
19-22.
[0026] FIG. 11A-11C. Kinetic analysis of exchange of compounds
17-21 in the DQ2 binding pocket. (A) Exchange of compounds 17
(.DELTA.) and 19 ( ) onto DQ2 at pH 5.5 (filled circles) and pH 7.3
(open circles). (B) Exchange of compounds 18 (.DELTA.) and 20 ( )
onto DQ2 at pH 5.5 (filled circles) and pH 7.3 (open circles. (C)
Comparative kinetics of DQ2 binding of compounds 5 ( ), 19
(.DELTA.), 20 (.gradient.), and 21(.diamond.) at pH 5.5 and
37.degree. C.
[0027] FIG. 12. Kinetic analysis of exchange of compound 22
(filled) and 33-mer 1 (filled .DELTA.) at pH 5.5. Data at pH 7.3
was similar; at this pH peptide 1 reaches a maximum occupancy of
28% (45 h), whereas peptide 22 reaches a maximum occupancy of 40%
(20 h).
[0028] FIG. 13. Comparison of T cell proliferation in the presence
of modified peptides 19, 20, 21, and 22. All peptides were tested
at a concentration of 3 .mu.M, except compound 22 was tested at 5
.mu.M. DQ2 APCs were .gamma.-irradiated before incubation with
peptide.
[0029] FIG. 14. T cell response to various concentrations of
antigen peptide 5 co-incubated without any blocker peptide or in
the presence of compound 21 or compound 22 (5 .mu.M each) as
reversible inhibitors of DQ2 antigen presentation. DQ2 APCs were
fixed prior to incubation with peptide.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0030] Celiac Sprue and/or dermatitis herpetiformis are diagnosed
by detecting antibodies that bind to digestion refractory gluten
oligopeptides and/or T-cell proliferation produced by such
oligopeptides in Celiac Sprue individuals. Gluten oligopeptides are
highly resistant to cleavage by gastric and pancreatic peptidases
such as pepsin, trypsin, chymotrypsin, and the like. By providing
for detection of such gluten oligopeptides; of antibodies
specifically reactive thereto; and/or of T-cell proliferation
produced by such oligopeptides in individuals, improved methods of
diagnosing Celiac Sprue and/or dermatitis herpetiformis are
provided.
[0031] Celiac Sprue and/or dermatitis herpetiformis may also be
treated by interfering with HLA binding of immunogenic gluten
peptides. Therapeutic benefit can also be enhanced in some
individuals by increasing the digestion of gluten oligopeptides,
whether by pretreatment of foodstuffs to be ingested or by
administration of an enzyme capable of digesting the gluten
oligopeptides, together with administration of an HLA-binding
peptide inhibitor. Gluten oligopeptides are highly resistant to
cleavage by gastric and pancreatic peptidases such as pepsin,
trypsin, chymotrypsin, and the like, and their prolonged presence
in the digestive tract can induce an autoimmune response. The
antigenicity of gluten oligopeptides and the ill effects caused by
an immune response thereto can be decreased by administration of an
HLA-binding peptide inhibitor. Such inhibitors are analogs of
immunogenic gluten peptides and (i) retain the ability to bind
tightly to HLA molecules; (ii) retain the proteolytic stability of
these peptides; but (iii) are unable to activate disease-specific
or other T cells.
[0032] In some embodiments and for some individuals, the methods of
the invention remove the requirement that abstention from ingestion
of glutens be maintained to keep the disease in remission. The
compositions of the invention include formulations of tTGase
inhibitors that comprise an enteric coating that allows delivery of
the agents to the intestine in an active form; the agents are
stabilized to resist digestion or alternative chemical
transformations in acidic stomach conditions. In another
embodiment, food is pretreated or combined with glutenase, or a
glutenase is co-administered (whether in time or in a formulation
of the invention) with an HLA-binding peptide inhibitor of the
invention.
[0033] The subject methods are useful for both prophylactic and
therapeutic purposes. Thus, as used herein, the term "treating" is
used to refer to both prevention of disease, and treatment of a
pre-existing condition. The treatment of ongoing disease, to
stabilize or improve the clinical symptoms of the patient, is a
particularly important benefit provided by the present invention.
Such treatment is desirably performed prior to loss of function in
the affected tissues; consequently, the prophylactic therapeutic
benefits provided by the invention are also important. Evidence of
therapeutic effect may be any diminution in the severity of
disease, particularly diminution of the severity of such symptoms
as fatigue, chronic diarrhea, malabsorption of nutrients, weight
loss, abdominal distension, and anemia. Other disease indicia
include the presence of antibodies specific for glutens, antibodies
specific for tissue transglutaminase, the presence of
pro-inflammatory T cells and cytokines, and degradation of the
villus structure of the small intestine. Application of the methods
and compositions of the invention can result in the improvement of
any and all of these disease indicia of Celiac Sprue.
[0034] Patients that can benefit from the present invention include
both adults and children. Children in particular benefit from
prophylactic treatment, as prevention of early exposure to toxic
gluten peptides can prevent development of the disease into its
more severe forms. Children suitable for prophylaxis in accordance
with the methods of the invention can be identified by genetic
testing for predisposition, e.g. by HLA typing; by family history,
and by other methods known in the art. As is known in the art for
other medications, and in accordance with the teachings herein,
dosages of the HLA-binding peptide inhibitors of the invention can
be adjusted for pediatric use.
[0035] Because most proteases and peptidases are unable to
hydrolyze the amide bonds of proline residues, the abundance of
proline residues in gliadins and related proteins from wheat, rye
and barley can constitute a major digestive obstacle for the
enzymes involved. This leads to an increased concentration of
relatively stable gluten derived oligopeptides in the gut. These
stable gluten derived oligopeptides, called "immunogenic
oligopeptides" herein, bind to MHC molecules, including HLA HLA-DQ2
or DQ8 molecules, to stimulate an immune response that results in
the autoimmune disease aspects of Celiac Sprue. In some cases the
enzyme tissue transglutaminase selectively deamidates certain
glutamine residues in these peptides, thereby enhancing their
potency for the DQ2 ligand binding pocket.
[0036] Peptides of particular interest for these purposes are
analogs of SEQ ID NO:12 LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. Such
analogs may comprise one or more of the modifications set forth
herein. Analogs may be truncated of 5, 10, 12, 13 or more amino
acids, which truncation may be from the amino or the carboxy
terminus. Analogs may be deamidated at one, two, three or more
glutamine residues, by substitution with glutamic acid at these
positions, where deamidation of residues 10, 17 and 24 are of
particular interest. Analogs may be substituted at one, two three
or more leucine residues for lysine, where substitution of
positions 8, 11, 14, 15 and 18 are of particular interest; and/or
modification of the substituted lysine residues with a sterically
hindered conjugate to the .gamma.-amine group.
[0037] Deamidated, and in some instances truncated and/or modified,
analogs of SEQ ID NO:12 include:
TABLE-US-00001 TABLE 1 ID Number Polypeptide sequence SEQ ID NO:12
1 LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF SEQ ID NO:17 2 PFPQPELPY SEQ ID
NO:18 3 PQPELPYPQ SEQ ID NO:19 4 LQLQPFPQPELPYPQ SEQ ID NO:20 5
LQLQPFPQPELPYPQPELPY SEQ ID NO:21 6 PQPELPYPQPELPY SEQ ID NO:22 7
PQPELPYPQPELPYPQPELPY SEQ ID NO:23 8 PFPQPELPYPQPELPYPQPELPYPQPQP
SEQ ID NO:24 9 LQPFPQPELPYPQPELPYPQPELPYPQPQP SEQ ID NO:25 10
QLQPFPQPELPYPQPELPYPQPELPYPQPQP SEQ ID NO:26 11
LQLQPFPQPELPYPQPQLPYPQPQLPYPQPQPF SEQ ID NO:27 12
LQLQPFPQPQLPYPQPELPYPQPQLPYPQPQPF SEQ ID NO:28 13
LQLQPFPQPQLPYPQPQLPYPQPELPYPQPQPF SEQ ID NO:29 14
LQLQPFPQPELPYPQPELPYPQPQLPYPQPQPF SEQ ID NO:30 15
LQLQPFPQPELPYPQPQLPYPQPELPYPQPQPF SEQ ID NO:31 16
LQLQPFPQPQLPYPQPELPYPQPELPYPQPQPF SEQ ID NO:32 LQLQPF SEQ ID NO:33
LQLQPFPQPEKPYPQPELPY SEQ ID NO:34 LQLQPFPQPELPYPQPEKPY SEQ ID NO:35
LQLQPFPQPEKPYPQPEKPY SEQ ID NO:36 PQPELPY SEQ ID NO:43 PQPEKPYPQ
SEQ ID NO:44 QLQPFPQPELPYP SEQ ID NO:45 PEKPY SEQ ID NO:46
PFPQPELPYP SEQ ID NO:47 PQPELPYPQPQLP SEQ ID NO:48 PQQSFPEQQPP SEQ
ID NO:49 VQGQGIIQPEQPAQ SEQ ID NO:50 FPEQPQQPYPQQP SEQ ID NO:51
FPQQPEQPYPQQP SEQ ID NO:52 FSQPEQEFPQPQ SEQ ID NO:53 PQPQLPY SEQ ID
NO:54 PYPQPELPY SEQ ID NO:55 QLQPFPQPELPY SEQ ID NO:56
GAGSLVPRGSGGGGS SEQ ID NO:58 PYPQPELPY
[0038] It can be seen from these sequences that analogs comprising
various combinations of deamidated residues can be produced (SEQ ID
NO:17-23), and will bind to the HLA molecule DQ2. Additional
modifications include deletion of terminal residues, for example
SEQ ID NO:24-SEQ ID NO:30. The antigenic response of the 33mer is
principally centered around the (SEQ ID NO: 18) PQPELPYPQ epitope
(residues 7-15), but that the N-terminal sequence (SEQ ID NO:32)
LQLQPF as well as a C-terminally located secondary Glu (e.g. E17 or
E24) residue further enhance DQ2 affinity of the 33 mer.
[0039] In one embodiment of the invention, a peptide of at least
about 14 amino acids, at least 18 amino acids, at least 20 amino
acids, at least 22 amino acids, and not more than about 33 amino
acids, not more than about 28, not more than about 26, or not more
than about 24 amino acids is provided, wherein said peptide
comprises the epitope sequence (SEQ ID NO: 18) PQPELPYPQ. The
peptide may optionally comprise as the amino terminal sequence,
(SEQ ID NO:32), LQLQPF. For example, such a peptide may comprise
the sequence of SEQ ID NO:27, and may further comprise a
C-terminally located secondary glu residue. In some embodiments,
the carboxy terminal sequence is SEQ ID NO:44 PELPY, or SEQ ID
NO:45 PEKPY, e.g. (SEQ ID NO:34), LQLQPFPQPELPYPQPEKPY
[0040] In another embodiment, the peptide of interest described
above comprises the epitope sequence (SEQ ID NO:43) PQPEKPYPQ,
wherein the leucine has been replaced with a lysine residue. Such
peptides may optionally comprise as the amino terminal sequence,
SEQ ID NO:32, LQLQPF, for example SEQ ID NO:33
LQLQPFPQPEKPYPQPELPY. Such peptide may optionally comprise a
C-terminally located secondary glu residue. In some embodiments,
the carboxy terminal sequence is SEQ ID NO:44 PELPY, or SEQ ID
NO:45 PEKPY, for example SEQ ID NO:35 LQLQPFPQPEKPYPQPEKPY.
[0041] The .epsilon.-amine of the lysine is generally reactive to
electrophiles, and can readily be modified. The lysine residues in
the peptides described above may be conjugated to a group that
provides for steric hindrance of interactions between the peptide
and a cognate receptor. Such conjugates may include, without
limitation, succinic acid; glutaric acid; .gamma.-aminobutyric
acid; benzyloxycarbonyl group; t-Butoxycarbonyl group;
9-fluorenylmethoxycarbonyl group; phthalimides; polyethylene
glycol; secondary and tertiary amines. Peptides thus modified, for
example peptides 19-21, as shown in FIG. 10, have been found to
bind very well to HLA antigens, but do not activate T cells that
proliferate in response to SEQ ID NO:12. Such peptides therefore
find use as inhibitors of immune responses involved in Celiac Sprue
and/or dermatitis herpetiformis.
[0042] Oligomeric or multimeric peptides can be synthesized using
the lysine substituted or cysteine substituted gliadin peptides
from Table 1 as monomeric units, i.e. linking two of more of such
peptides. Multimeric, e.g. dimeric, trimeric, etc. peptide
constructs can be synthesized using bifunctional crosslinking
reagents such as polyethylene glycols with amine-reactive groups at
both ends, thiol-reactive groups at both ends, or an amine-reactive
group at one end and a thiol-reactive group at the other end. An
example of a residue to be crosslinked is Lys 11 and/or 18 in any
one of SEQ ID NO:33, 34 and 35. Other amino acids to be substituted
at Lys 11 include L-diaminopropionic acid, L-diaminobutyric acid,
or L-ornithine. These lysine residues can also be crosslinked via
bifunctional polyethylene glycol with amine-reactive groups at both
ends to form macrocycles. The size of the macrocycle can be
adjusted by changing the length of polyethylene glycol.
[0043] In some embodiments, the peptide has the structure of SEQ ID
NO:59:
LQLQPFPQPE-X.sub.1-PYPQPE-X.sub.2-PY
[0044] where X.sub.1 and X.sub.2 are independently selected from
lysine; leucine; and lysine conjugated any one of: succinic acid;
glutaric acid; .gamma.-aminobutyric acid; benzyloxycarbonyl group;
a t-Butoxycarbonyl group; a 9-fluorenylmethoxycarbonyl group;
phthalimides; polyethylene glycol; secondary and tertiary amines,
and a linker, where the linker may be flexible or rigid, and may
extend from about 1 to about 100 monomers in length, and where the
linker may couple two or more peptides. Where the lysine is
conjugated to a polyethylene glycol linker, the linker may have the
structure --[OCH.sub.2CH.sub.2].sub.n where n is from about 1 to
100, usually from about 3 to 25, more usually at least about 5 and
not more than about 20.
[0045] Such crosslinked dimers have been found to bind faster and
more stably to HLA-DQ2 than the unmodified or monomeric peptide;
and the resulting complex does not stimulate T cell proliferation
in the presence of antigenic peptides, e.g. SEQ ID NO:12. The
distance between the monomeric units can be optimized by varying
the length of the polyethylene glycols to generate optimal binding
properties of the dimer. Oligomeric peptides can also be
synthesized using standard methods in the literature. Other
dimerization strategies include olefin metathesis, dityrosine
oxidation, and disulfide formation.
[0046] Peptides as described above, including, without limitation,
those comprising or consisting of SEQ ID NO:43, SEQ ID NO:33, SEQ
ID NO:35, may additionally be substituted with cysteine. Sites for
cysteine substitution include the sites of lysine substitution
(residue 11 and 18 with respect to SEQ ID NO:35). Such analogs are
transformed into DQ2 blockers through intramolecular cyclization
via disulfide bond formation. Cyclic DQ2 binding molecules may
further be modified by altering the bridge lengths, e.g. replacing
cysteine with analogues such as homocysteine. More stable analogues
are prepared by replacing disulfide bonds with other flexible
cyclization tethers. Two strategies of interest are replacement of
disulfide bridges with thioether linkages [Robey, F. A. (2000)
Selective and Facile Cyclization of Nchloroacetylated Peptides from
the C4 Domain of HIV Gp120 in LiCl/DMF Solvent Systems. J. Peptide
Res. 56, 115-120; Oligino, L.; Lung, F.-D. T.; Sastry, L.; Bigelow,
J.; Cao, T.; Curran, M.; Burke, T. R., Jr.; Wang, S.; Krag, D.;
Roller, P. P.; King, C. R. (1997) Nonphosphorylated Peptide Ligands
for the Grb2 Src Homology 2 Domain. J. Biol. Chem. 272,
29046-29052] and olefin metathesis [Blackwell, H. E.; Grubbs, R. H.
(1998) Highly Efficient Synthesis of Covalently Cross-linked
Peptide Helices by Ring-Closing Metathesis. Angew. Chem. Int. Ed.
37, 3281-3284; Schafmeister C. E.; Po, J.; Verdine, G. L. (2000) An
All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and
Metabolic Stability of Peptides. J. Am. Chem. Soc. 122, 5891-5892].
Alternatively, a wide range of bis-alkylating agents such as
dibromoketones are used, since the resulting macrocyclic products,
if active, will contain an orthogonal ketone functional group for
further modification
[0047] In another embodiment, an immunogenic gluten oligopeptide
analog is an analog of a peptide that comprises at least about 8
residues, and may comprise at least about 10 residues; at least
about 11 residues, at least about 12 residues, at least about 13
residues, at least about 14 residues, or more, where the term
"residue" refers to naturally occurring amino acids, non-naturally
occurring amino acids, and amino acid mimetics or derivatives; and
where the gluten peptide is altered by the replacement of one or
more amino acids. The replacement may be another naturally
occurring amino acid, non-naturally occurring amino acids, modified
amino acids, amino acid mimetics, and the like; and may further be
derivitized to further reduce the affinity of these ligands for
disease-specific T cell receptors. The sequence of immunogenic
gluten oligopeptides can be determined by one of skill in the art.
Immunogenic gliadin oligopeptides are peptides derived during
normal human digestion of gliadins and related storage proteins as
described above, from dietary cereals, e.g. wheat, rye, barley, and
the like. Such oligopeptides act as antigens for T cells in Celiac
Sprue. For binding to Class II MHC proteins, immunogenic peptides
are usually from about 8 to 20 amino acids in length, more usually
from about 10 to 18 amino acids. Such peptides may include PXP
motifs, such as the motif (SEQ ID NO: 8) PQPQLP. Determination of
whether an oligopeptide is immunogenic for a particular patient is
readily determined by standard T cell activation and other assays
known to those of skill in the art.
[0048] Among gluten proteins with potential harmful effect to
Celiac Sprue patients are included the storage proteins of wheat,
species of which include Triticum aestivum; Triticum aethiopicum;
Triticum baeoticum; Triticum militinae; Triticum monococcum;
Triticum sinskajae; Triticum timopheevii; Triticum turgidum;
Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; etc. A
review of the genes encoding wheat storage proteins may be found in
Colot (1990) Genet Eng (NY) 12:225-41. Gliadin is the
alcohol-soluble protein fraction of wheat gluten. Gliadins are
typically rich in glutamine and proline, particularly in the
N-terminal part. For example, the first 100 amino acids of .alpha.-
and .gamma.-gliadins contain .about.35% and .about.20% of glutamine
and proline residues, respectively. Many wheat gliadins have been
characterized, and as there are many strains of wheat and other
cereals, it is anticipated that many more sequences will be
identified using routine methods of molecular biology. Examples of
gliadin sequences include but are not limited to wheat alpha
gliadin sequences, for example as provided in Genbank, accession
numbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607;
AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307;
U51306; U51304; U51303; U50984; and U08287. A sequence of wheat
omega gliadin is set forth in Genbank accession number
AF280605.
[0049] Among the immunogenic gluten oligopeptides that may be
modified to generate an HLA-binding peptide inhibitor are included
the peptide sequence (SEQ ID NO:44) QLQPFPQPELPYP; the sequence
(SEQ ID NO:36) PQPELPY; the sequence (SEQ ID NO:46) PFPQPELPYP,
(SEQ ID NO:47) PQPELPYPQPQLP, (SEQ ID NO:48) PQQSFPEQQPP, (SEQ ID
NO:49) VQGQGIIQPEQPAQ, (SEQ ID NO:50) FPEQPQQPYPQQP, (SEQ ID NO:51)
FPQQPEQPYPQQP, (SEQ ID NO:52) FSQPEQEFPQPQ and longer peptides
containing such sequences or multiple copies of such sequences.
Gliadins, secalins and hordeins contain several (SEQ ID NO:53)
PQPQLPY sequences or sequences similar thereto rich in Pro-Gln
residues that are high-affinity substrates for tTGase. The tTGase
catalyzed deamidation of such sequences increases their affinity
for HLA-DQ2, the class 11 MHC allele present in >90% Celiac
Sprue patients. Presentation of these deamidated sequences by DQ2
positive antigen presenting cells effectively stimulates
proliferation of gliadin-specific T cells from intestinal biopsies
of most Celiac Sprue patients, providing evidence for the proposed
mechanism of disease progression in Celiac Sprue.
[0050] Analog oligopeptides of the invention comprise at least one
difference in amino acid sequence from a native gluten peptide, by
the replacement of an amino acid with a different amino acid; a
non-naturally occurring amino acid, a peptidomimetics, substituted
amino acid, and the like. An L-amino acid from the native peptide
may be altered to any other one of the 20 L-amino acids commonly
found in proteins, any one of the corresponding D-amino acids, rare
amino acids, such as 4-hydroxyproline, and hydroxylysine, or a
non-protein amino acid, such as .beta.-alanine, ornithine and
homoserine. Also included with the scope of the present invention
are amino acids that have been altered by chemical means such as
methylation (e.g., .alpha.-methylvaline), deamidation, amidation of
the C-terminal amino acid by an alkylamine such as ethylamine,
ethanolamine, and ethylene diamine, and acylation or methylation of
an amino acid side chain function (e.g., acylation of the epsilon
amino group of lysine), deimination of arginine to citrulline,
isoaspartylation, or phosphorylation on serine, threonine, tyrosine
or histidine residues. Importantly, each of these altered amino
acids provide a functional handle, e.g. amine, alcohol, aryl
halide, and the like, which can be regioselectively derivatized to
further reduce the affinity of these ligands for disease-specific T
cell receptors. Peptide analogs may be further derivatized with
substitutions, including, without limitation, ethers, amines,
esters, amides, carbonates, carbamates, carbazates, ureas and C-C
coupled derivatives. Other examples include oxidation of alcohols
to ketones, followed by further modifications of the resulting
carbonyl group, e.g. via preparation of oximes) or the carbon atom
adjacent to the ketone. Such derivatives are encompassed by the
term "analog".
[0051] The proteolytic stability of gluten oligopeptides can be
attributed, at least in part, to the presence of PXP motifs, which
are resistant to enzymatic degradation. Preferred analogs of
immunogenic gluten oligopeptides will comprise one or more proline
residues, and may comprise one or more PXP motifs.
[0052] One inhibitor of interest is an oligopeptide or
peptidomimetic that comprises the sequence PXPQPELPY, where X is
Gly, Ala, Tyr, Trp, Arg, Lys, p-iodo-Phe, 3-iodo-Tyr, p-amino-Phe,
3-amino-Tyr, hydroxylysine, ornithine, Asp, Glu, or any residue
that is substantially bulkier or hydrophilic than Phe. Examples of
suitable modifications include ethers, amines, esters, amides,
carbonates, carbamates, carbazates, ureas and C--C coupled
derivatives. Other examples include oxidation of alcohols to
ketones, followed by further modifications of the resulting
carbonyl group (e.g. via preparation of oximes) or the carbon atom
adjacent to the ketone. The peptide may comprise modifications that
increase binding potency to an MHC molecule, by varying residues
that facilitate peptide docking into the binding cleft. Examples of
such residues include Gln-4, Glu-6, Leu-7, and Tyr-9 (numbering
based on the epitope (SEQ ID NO: 17) PFPQPELPY). Each of these
residues interacts closely with several residues in the DQ2 binding
pocket. By using structure-based molecular design methods, these
interactions can be optimized.
[0053] Another inhibitor of interest is a oligopeptide or
peptidomimetic that comprises the sequence PFPQX.sub.1ELX.sub.2Y,
where X.sub.1 and X.sub.2 are independently selected from
4-hydroxy-Pro (either isomer at C-4), 4-amino-Pro (either isomer
atC-4), or 3-hydroxy-Pro (either isomer atC-3), and proline, with
the proviso that at least one of X.sub.1 and X.sub.2 is a residue
other than proline.
[0054] As described above, the sequence of gluten peptides may be
altered in various ways known in the art to generate targeted
changes in sequence. The sequence changes may be substitutions,
insertions or deletions. Such alterations may be used to alter
properties of the protein, by affecting the stability, specificity,
etc. Techniques for in vitro mutagenesis of cloned genes are known.
Examples of protocols for scanning mutations may be found in Gustin
et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985);
Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et
al., Gene 29:303-13 (1984). Methods for site specific mutagenesis
can be found in Sambrook et al., Molecular Cloning: A Laboratory
Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene
126:35-41 (1993); Sayers et al., Biotechniques 13:592-6 (1992);
Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et
al., Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene
Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4
(1989).
[0055] The peptides may be joined to a wide variety of other
oligopeptides or proteins for a variety of purposes. By providing
for expression of the subject peptides, various post-expression
modifications may be achieved. For example, by employing the
appropriate coding sequences, one may provide farnesylation or
prenylation. The peptides may be PEGylated, where the
polyethyleneoxy group provides for enhanced lifetime in the blood
stream. The peptides may also be combined with other proteins, such
as the Fc of an IgG isotype, which may be complement binding, with
a toxin, such as ricin, abrin, diphtheria toxin, or the like, or
with specific binding agents that allow targeting to specific
moieties on a target cell.
[0056] Modifications of interest that do not alter primary sequence
include chemical derivatization of polypeptides, e.g., acetylation,
acylation, carboxylation, etc. Also embraced are sequences that
have phosphorylated amino acid residues, e.g. phosphotyrosine,
phosphoserine, or phosphothreonine.
[0057] Also included in the subject invention are peptides that
have been modified using ordinary molecular biological techniques
and synthetic chemistry so as to improve their resistance to
proteolytic degradation or to optimize solubility properties or to
render them more suitable as a therapeutic agent. Analogs of such
polypeptides include those containing residues other than naturally
occurring L-amino acids, e.g. D-amino acids or non-naturally
occurring synthetic amino acids. D-amino acids may be substituted
for some or all of the amino acid residues.
[0058] Peptides and peptide analogs may be synthesized by standard
chemistry techniques, including synthesis by automated procedure.
In general, peptide analogs are prepared by solid-phase peptide
synthesis methodology which involves coupling each protected amino
acid residue to a resin support, preferably a
4-methylbenzhydrylamine resin, by activation with
dicyclohexylcarbodiimide to yield a peptide with a C-terminal
amide. Alternatively, a chloromethyl resin (Merrifield resin) may
be used to yield a peptide with a free carboxylic acid at the
C-terminus. After the last residue has been attached, the protected
peptide-resin is treated with hydrogen fluoride to cleave the
peptide from the resin, as well as deprotect the side chain
functional groups. Crude product can be further purified by gel
filtration, HPLC, partition chromatography, or ion-exchange
chromatography.
[0059] If desired, various groups may be introduced into the
peptide during synthesis or during expression, which allow for
linking to other molecules or to a surface. Thus cysteines can be
used to make thioethers, histidines for linking to a metal ion
complex, carboxyl groups for forming amides or esters, amino groups
for forming amides, and the like.
[0060] The polypeptides may also be isolated and purified in
accordance with conventional methods of recombinant synthesis. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. For the
most part, the compositions which are used will comprise at least
20% by weight of the desired product, more usually at least about
75% by weight, preferably at least about 95% by weight, and for
therapeutic purposes, usually at least about 99.5% by weight, in
relation to contaminants related to the method of preparation of
the product and its purification. Usually, the percentages will be
based upon total protein.
[0061] In one embodiment of the invention, the peptide consists
essentially of a polypeptide sequence as set forth in any one of
the SEQ ID NOs provided herein. By "consisting essentially of" in
the context of a polypeptide described herein, it is meant that the
polypeptide is composed of the gluten sequence, which sequence may
be flanked by one or more amino acid or other residues that do not
materially affect the basic characteristic(s) of the
polypeptide.
Interactions with Immune System Receptors
[0062] Preferably, antigenic oligopeptides of interest for use in
the methods of the invention are as described above, and comprise
at least one epitope. As used herein, the term "epitope" refers to
the portion of an antigen bound by an antibody or T cell receptor,
which portion is sufficient for high affinity binding. In
polypeptide antigens, generally a linear epitope for recognition
will be at least about 7 amino acids in length, and may be 8 amino
acids, 9 amino acids, 10 amino acids, or more.
[0063] Antibodies may recognize linear determinants or
conformational determinants formed by non-contiguous residues on an
antigen, and an epitope can therefore require a larger fragment of
the antigen to be present for binding, e.g. a protein domain, or
substantially all of a protein sequence. The binding site of
antibodies typically utilizes multiple non-covalent interactions to
achieve high affinity binding. While a few contact residues of the
antigen may be brought into close proximity to the binding pocket,
other parts of the antigen molecule can also be required for
maintaining a conformation that permits binding. In order to
consider an antibody interaction to be "specific", the affinity
will be at least about 10.sup.-7 M, usually about 10.sup.-8 M to
10.sup.-9 M, and may be up to 10.sup.-11 M or higher for the
epitope of interest. It will be understood by those of skill in the
art that the term "specificity" refers to such a high affinity
binding, and is not intended to mean that the antibody cannot bind
to other molecules as well. One may find cross-reactivity with
different epitopes, due, e.g. to a relatedness of antigen sequence
or structure, or to the structure of the antibody binding pocket
itself.
[0064] The T cell receptor recognizes a more complex structure than
antibodies, and requires both a major histocompatibility antigen
binding pocket and an antigenic peptide to be present. The binding
affinity of T cell receptors is lower than that of antibodies, and
will usually be at least about 1 M, more usually at least about
10.sup.-5 M.
[0065] Affinity and stability are different measures of binding
interaction. The definition of affinity is a thermodynamic
expression of the strength of interaction between a single antigen
binding site and a single antigenic determinant (and thus of the
stereochemical compatibility between them). Affinity does not
change with valency, because it is the measure of interaction
between a single binding site and a single antigenic determinant.
In contrast to affinity, avidity (which relates to the t.sub.1/2 of
an interaction) is defined as the strength of binding, usually of a
small molecule with multiple binding sites by a larger molecule,
and in particular, the binding of a complex antigen by an antibody.
Therefore, it is avidity that takes into account the effect or
multiple interactions, and it is the change in avidity that may
provides the hyperantigenicity observed with the oligopeptide of
SEQ ID NO:12.
[0066] Certain of the gluten oligopeptides analogs described herein
are useful in stimulating T cells from Celiac Sprue patients for
diagnostic purposes, while others are shown to inhibit T cell
stimulation. Such peptides are provided by the present invention in
isolated and highly purified forms. Further, the gluten
oligopeptides analogs described herein are useful in diagnostic
assays for detecting antibodies against such oligopeptides or for
producing antibodies that bind specifically to such oligopeptides
for their detection.
Diagnostic Methods
[0067] The present invention provides a variety of methods for
diagnosing Celiac Sprue. In one embodiment, the diagnosis involves
detecting the presence of a gluten oligopeptides digestion product,
e.g. SEQ ID NO:12; deamidated counterparts there; a tTGase-linked
counterpart thereof; etc., in a tissue, bodily fluid, or stool of
an individual. The detecting step can be accomplished by use of a
reagent, e.g. an antibody, that recognizes the indicated antigen,
or by a cell that proliferates in the presence of the indicated
antigen and suitable antigen presenting cells, wherein said antigen
presenting cells are compatible with the MHC type of the
proliferating cell, e.g. allogeneic cells, autologous cells,
etc.
[0068] In another embodiment, the diagnosis involves detecting the
presence of an antibody, one or more T cells reactive with the
33-mer or a deamidated counterpart thereof, or a tTGase-linked
counterpart thereof in a tissue, bodily fluid, or stool of an
individual. In one embodiment, an antibody is detected by, for
example, an agglutination assay using an antigen provided by the
present invention. In another embodiment, a T cell is detected by
its proliferation in response to exposure to a multivalent gluten
oligopeptide provided by the present invention and presented by
autologous or suitable allogeneic antigen presenting cells.
[0069] In one aspect, the methods and reagents of the present
invention are capable of detecting the toxic oligopeptides of
gluten proteins of wheat, barley, oats and rye remaining after
digestion or partial digestion of the same by a Celiac Sprue
individual. Gluten is the protein fraction in cereal dough, which
can be subdivided into glutenins and prolamines, which can be
further subclassified as gliadins, secalins, hordeins, avenins from
wheat, rye, barley and oat, respectively. For further discussion of
gluten proteins, see the review by Wieser (1996) Acta Paediatr
Suppl. 412:3-9; herein incorporated by reference. Among gluten
proteins of interest are included the storage proteins of wheat,
species of which include Triticum aestivum; Triticum aethiopicum;
Triticum baeoticum; Triticum militinae; Triticum monococcum;
Triticum sinskajae; Triticum timopheevii; Triticum turgidum;
Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; and the
like. A review of the genes encoding wheat storage proteins may be
found in Colot (1990) Genet Eng (NY) 12:225-41.
[0070] Of particular interest is gliadin, which is the
alcohol-soluble protein fraction of wheat gluten. Gliadins are
typically rich in glutamine and proline, particularly in the
N-terminal part. For example, the first 100 amino acids of .alpha.-
and .gamma.-gliadins contain .about.35% and .about.20% of glutamine
and proline residues, respectively. Many wheat gliadins have been
characterized, and as there are many strains of wheat and other
cereals, it is anticipated that many more sequences will be
obtained using routine methods of molecular biology. Examples of
sequenced gliadins include wheat alpha gliadin sequences, for
example as provided in Genbank, accession numbers AJ133612;
AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606;
AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306;
U51304; U51303; U50984; and U08287. A sequence of wheat omega
gliadin is set forth in Genbank accession number AF280605.
[0071] For the purposes of the present invention, toxic gliadin
oligopeptides are peptides derived during normal digestion of
gliadins and related storage proteins as described above, from
dietary cereals, e.g. wheat, rye, barley, and the like, by a Celiac
Sprue individual. Such oligopeptides are believed to act as
antigens for T cells in Celiac Sprue individuals. For binding to
Class II MHC proteins, immunogenic peptides are usually from about
6 to 20 amino acids in length, more usually from about 10 to 18
amino acids, and as demonstrated herein, a particularly stimulatory
toxic gliadin oligopeptide is the multivalent 33-mer described
above. Such peptides include PXP motifs, for example the motif
PQPQLP (SEQ ID NO:8). Determination of whether an oligopeptide is
immunogenic for a particular patient is readily determined by
standard T cell activation assays known to those of skill in the
art. Illustrative toxic gliadin oligopeptides of the invention are
described in Examples 1 and 2 below. The 33-mer gliadin
oligopeptide of Example 2 and its deamidated counterpart formed by
tTGase are preferred toxic gliadin oligopeptides of the
invention.
[0072] Samples may be obtained from patient tissue, which may be a
mucosal tissue, including but not limited to oral, nasal, lung, and
intestinal mucosal tissue, a bodily fluid, e.g. blood, sputum,
urine, phlegm, lymph, and tears. One advantage of the present
invention is that the antigens provided are such potent antigens,
both for antibody-binding and T-cell stimulation, that the
diagnostic methods of the invention can be employed with samples
(tissue, bodily fluid, or stool) in which a Celiac Sprue diagnostic
antibody, peptide, or T cell is present in very low abundance. This
allows the methods of the invention to be practiced in ways that
are much less invasive, much less expensive, and much less harmful
to the Celiac Sprue individual.
[0073] Patients may be monitored for the presence of reactive T
cells, using one or more multivalent oligopeptides as described
above. The presence of such reactive T cells indicates the presence
of an on-going immune response. The antigen used in the assays is a
gluten oligopeptide analog as described above; including, without
limitation, SEQ ID NO:12; deamidated counterparts; tTGase fusions
thereof; and derivatives. Cocktails comprising multiple
oligopeptides; panels of peptides; etc. may be also used.
Overlapping peptides may be generated, where each peptide is
frameshifted from 1 to 5 amino acids, thereby generating a set of
epitopes.
[0074] The diagnosis may determine the level of reactivity, e.g.
based on the number of reactive T cells found in a sample, as
compared to a negative control from a naive host, or standardized
to a data curve obtained from one or more positive controls. In
addition to detecting the qualitative and quantitative presence of
antigen reactive T cells, the T cells may be typed as to the
expression of cytokines known to increase or suppress inflammatory
responses. While not necessary for diagnostic purposes, it may also
be desirable to type the epitopic specificity of the reactive T
cells, particularly for use in therapeutic administration of
peptides.
[0075] T cell may be isolated from patient peripheral blood, lymph
nodes, including peyer's patches and other gut-related lymph nodes,
or from tissue samples as described above. Reactivity assays may be
performed on primary T cells, or the cells may be fused to generate
hybridomas. Such reactive T cells may also be used for further
analysis of disease progression, by monitoring their in situ
location, T cell receptor utilization, MHC cross-reactivity, etc.
Assays for monitoring T cell responsiveness are known in the art,
and include proliferation assays and cytokine release assays. Also
of interest is an ELISA spot assay.
[0076] Proliferation assays measure the level of T cell
proliferation in response to a specific antigen, and are widely
used in the art. In one such assay, recipient lymph node, blood or
spleen cells are obtained at one or more time points after
transplantation. A suspension of from about 10.sup.4 to 10.sup.7
cells, usually from about 10.sup.5 to 10.sup.6 cells is prepared
and washed, then cultured in the presence of a control antigen, and
test antigens, as described above. The cells are usually cultured
for several days. Antigen-induced proliferation is assessed by the
monitoring the synthesis of DNA by the cultures, e.g. incorporation
of .sup.3H-thymidine during the last 18 H of culture.
[0077] T cell cytotoxic assays measure the numbers of cytotoxic T
cells having specificity for the test antigen. Lymphocytes are
obtained at different time points after transplantation.
Alloreactive cytotoxic T cells are tested for their ability to kill
target cells bearing recipient MHC class I molecules associated
with peptides derived from a test antigen. In an exemplary assay,
target cells presenting peptides from the test antigen, or a
control antigen, are labeled with Na.sup.51CrO.sub.4. The target
cells are then added to a suspension of candidate reactive
lymphocytes. The cytotoxicity is measured by quantitating the
release of Na.sup.51CrO.sub.4 from lysed cells. Controls for
spontaneous and total release are typically included in the assay.
Percent specific .sup.51Cr release may be calculated as follows:
100.times.(release by CTL-spontaneous release)/(total
release-spontaneous release).
[0078] Enzyme linked immunosorbent assay (ELISA) and ELISA spot
assays are used to determine the cytokine profile of reactive T
cells, and may be used to monitor for the expression of such
cytokines as IL-2, IL-4, IL-5, .gamma.IFN, etc. The capture
antibodies may be any antibody specific for a cytokine of interest,
where supernatants from the T cell proliferation assays, as
described above, are conveniently used as a source of antigen.
After blocking and washing, labeled detector antibodies are added,
and the concentrations of protein present determined as a function
of the label that is bound.
[0079] In one embodiment of the invention, the presence of reactive
T cells is determined by injecting a dose of the 33-mer peptide, or
a derivative or fragment thereof as described above, subcutaneously
or sub-mucosally into a patient, for example into the oral mucosa
(see Lahteenoja et al. (2000) Am. J. Gastroenterology 95:2880,
herein incorporated by reference). A control comprising medium
alone, or an unrelated protein is usually injected nearby at the
same time. The site of injection is examined after a period of
time, by biopsy or for the presence of a wheal.
[0080] A wheal at the site of injection is compared to that at the
site of the control injection, usually by measuring the size of the
wheal. The skin test readings may be assessed by a variety of
objective grading systems. A positive result for the presence of an
immune response will show an increased diameter at the site of
polypeptide injection as compared to the control.
[0081] Where a biopsy is performed, the specimen is examined for
the presence of increased numbers of immunologically active cells,
e.g. T cells, B cells, mast cells, and the like. For example,
methods of histochemistry and/or immunohistochemistry may be used,
as is known in the art. The densities of cells, including antigen
specific T cells, mast cells, B cells, etc. may be examined. It has
been reported that increased numbers of intraepithelial CD8.sup.+ T
cells may correlate with gliadin reactivity.
[0082] An alternative method relies on the detection of circulating
antibodies in a patient. Methods of detecting specific antibodies
are well-known in the art. Antibodies specific for multivalent
gluten oligopeptides as described above may be used in screening
immunoassays. A sample is taken from the patient. Samples, as used
herein, include biological fluids such as blood, tears, saliva,
lymph, dialysis fluid and the like; organ or tissue culture derived
fluids; and fluids extracted from physiological tissues. Also
included in the term are derivatives and fractions of such fluids.
Blood samples and derivatives thereof are of particular
interest.
[0083] Measuring the concentration of specific antibodies in a
sample or fraction thereof may be accomplished by a variety of
specific assays. In general, the assay will measure the reactivity
between a patient sample, usually blood derived, generally in the
form of plasma or serum. The patient sample may be used directly,
or diluted as appropriate, usually about 1:10 and usually not more
than about 1:10,000. Immunoassays may be performed in any
physiological buffer, e.g. PBS, normal saline, HBSS, dPBS, etc.
[0084] In one embodiment, a conventional sandwich type assay is
used. A sandwich assay is performed by first attaching the peptide
to an insoluble surface or support. The peptide may be bound to the
surface by any convenient means, depending upon the nature of the
surface, either directly or through specific antibodies. The
particular manner of binding is not crucial so long as it is
compatible with the reagents and overall methods of the invention.
They may be bound to the plates covalently or non-covalently,
preferably non-covalently.
[0085] The insoluble supports may be any composition to which
peptides can be bound, which is readily separated from -soluble
material, and which is otherwise compatible with the overall method
of measuring antibodies. The surface of such supports may be solid
or porous and of any convenient shape. Examples of suitable
insoluble supports to which the receptor is bound include beads,
e.g. magnetic beads, membranes and microtiter plates. These are
typically made of glass, plastic (e.g. polystyrene),
polysaccharides, nylon or nitrocellulose. Microtiter plates are
especially convenient because a large number of assays can be
carried out simultaneously, using small amounts of reagents and
samples.
[0086] Before adding patient samples or fractions thereof, the
non-specific binding sites on the insoluble support i.e. those not
occupied by antigen, are generally blocked. Preferred blocking
agents include non-interfering proteins such as bovine serum
albumin, casein, gelatin, and the like. Alternatively, several
detergents at non-interfering concentrations, such as Tween, NP40,
TX100, and the like may be used.
[0087] Samples, fractions or aliquots thereof are then added to
separately assayable supports (for example, separate wells of a
microtiter plate) containing support-bound antigenic peptide.
Preferably, a series of standards, containing known concentrations
of antibodies is assayed in parallel with the samples or aliquots
thereof to serve as controls.
[0088] Generally from about 0.001 to 1 ml of sample, diluted or
otherwise, is sufficient, usually about 0.01 ml sufficing.
Preferably, each sample and standard will be added to multiple
wells so that mean values can be obtained for each. The incubation
time should be sufficient for antibodies molecules to bind the
insoluble antigenic peptide. Generally, from about 0.1 to 3 hr is
sufficient, usually 1 hr sufficing.
[0089] After incubation, the insoluble support is generally washed
of non-bound components. Generally, a dilute non-ionic detergent
medium at an appropriate pH, generally 7-8, is used as a wash
medium. From one to six washes may be employed, with sufficient
volume to thoroughly wash non-specifically bound proteins present
in the sample.
[0090] After washing, a solution containing a second receptor
specific for the patient antibodies is applied. The receptor may be
any compound that binds patient antibodies with sufficient
specificity such that it can be distinguished from other components
present. In a preferred embodiment, second receptors are antibodies
specific for patient antibodies, either monoclonal or polyclonal
sera, e.g. mouse anti-human antibodies, mouse anti-dog antibodies,
rabbit anti-cat antibodies, etc. Such second stage antibodies may
be labeled to facilitate direct, or indirect quantification of
binding. Examples of labels which permit direct measurement of
second receptor binding include radiolabels, such as .sup.3H or
.sup.125I, fluorescers, dyes, beads, chemilumninescers, colloidal
particles, and the like. Examples of labels that permit indirect
measurement of binding include enzymes where the substrate may
provide for a colored or fluorescent product. In a preferred
embodiment, the second receptors are antibodies labeled with a
covalently bound enzyme capable of providing a detectable product
signal after addition of suitable substrate. Examples of suitable
enzymes for use in conjugates include horseradish peroxidase,
alkaline phosphatase, malate dehydrogenase and the like. Where not
commercially available, such antibody-enzyme conjugates are readily
produced by techniques known to those skilled in the art.
Alternatively, the second stage may be unlabeled, and a labeled
third stage is used. Examples of second receptor/second
receptor-specific molecule pairs include antibody/anti-antibody and
avidin (or streptavidin)/biotin. Since the resultant signal is thus
amplified, this technique may be advantageous where only a small
amount of antibodies is present.
[0091] After the second stage has bound, the insoluble support is
generally again washed free of non-specifically bound molecules,
and the signal produced by the bound conjugate is detected by
conventional means. Where an enzyme conjugate is used, an
appropriate enzyme substrate is provided so a detectable product is
formed. More specifically, where a peroxidase is the selected
enzyme conjugate, a preferred substrate combination is
H.sub.2O.sub.2 and is O-phenylenediamine, which yields a colored
product under appropriate reaction conditions. Appropriate
substrates for other enzyme conjugates such as those disclosed
above are known to those skilled in the art. Suitable reaction
conditions as well as means for detecting the various useful
conjugates or their products are also known to those skilled in the
art. For the product of the substrate O-phenylenediamine for
example, light absorbance at 490-495 nm is conveniently measured
with a spectrophotometer.
[0092] Generally the amount of bound antibodies detected will be
compared to control samples from normal patients. The presence of
increased levels of the antigen specific antibodies is indicative
of disease, usually at least about a 5 fold, 10 fold, or 100 fold
increase will be taken as a positive reaction.
[0093] In some cases, a competitive assay will be used. In addition
to the patient sample, a competitor to the antibodies is added to
the reaction mix. The competitor and the antibodies compete for
binding to the antigenic peptide. Usually, the competitor molecule
will be labeled and detected as previously described, where the
amount of competitor binding will be proportional to the amount of
antibodies present. The concentration of competitor molecule will
be from about 10 times the maximum anticipated antibodies
concentration to about equal concentration in order to make the
most sensitive and linear range of detection.
[0094] An alternative protocol is to provide anti-patient
antibodies bound to the insoluble surface. After adding the sample
and washing away non-specifically bound proteins, one or a
combination of the test antigens are added, where the antigens are
labeled, so as not to interfere with binding to the antibodies.
Conveniently, fused proteins may be employed, where the peptide
sequence is fused to an enzyme sequence, e.g.
.beta.-galactosidase.
[0095] It is particularly convenient in a clinical setting to
perform the immunoassay in a self-contained apparatus. A number of
such methods are known in the art. The apparatus will generally
employ a continuous flow-path of a suitable filter or membrane,
having at least three regions, a fluid transport region, a sample
region, and a measuring region. The sample region is prevented from
fluid transfer contact with the other portions of the flow path
prior to receiving the sample. After the sample region receives the
sample, it is brought into fluid transfer relationship with the
other regions, and the fluid transfer region contacted with fluid
to permit a reagent solution to pass through the sample region and
into the measuring region. The measuring region may have bound to
it the antigenic peptide, with a conjugate of an enzyme with an
antibodies specific antibody employed as a reagent, generally added
to the sample before application. Alternatively, the antigenic
peptide may be conjugated to an enzyme, with antibodies specific
antibody bound to the measurement region.
[0096] Thus, in one aspect, the present invention provides a method
for diagnosing Celiac Sprue in an individual who has not consumed
gluten for an extended period of time, such time including but not
limited to one day, one week, one month, and one year prior to the
performance of the diagnostic method. The advantage conferred by
this aspect of the invention is that current diagnosis of a Celiac
Sprue individual typically involves a preliminary diagnosis, after
which the individual is placed on a gluten-free diet. If the
individual's symptoms abate after initiation of the gluten-free
diet, then the individual is challenged with gluten, and another
diagnostic test, such as an endoscopy or T cell proliferation
assay, is performed to confirm the preliminary diagnosis. This
re-challenge with gluten causes extreme discomfort to the Celiac
Sprue individual. One important benefit provided by certain
embodiments of the invention is that such a re-challenge need not
be performed to diagnose Celiac Sprue, because even very low levels
of 33-mer specific antibodies and T cell responders can be
identified using the methods of the invention.
[0097] In another aspect, the present invention provides a method
for diagnosing Celiac Sprue by detecting the presence of a 33-mer
specific antibody or a T cell responder in a bodily tissue or fluid
other than intestinal mucosa. In this aspect of the invention, the
diagnostic methods are performed without recourse to endoscopy or
intestinal biopsy, thus avoiding the discomfort, pain, and expense
attendant on such procedures in common use today.
[0098] The subject methods are useful not only for diagnosing
Celiac Sprue individuals but also for determining the efficacy of
prophylactic or therapeutic methods for Celiac Sprue as well as the
efficacy of food preparation or treatment methods aimed at removing
glutens or similar substances from food sources. Thus, a Celiac
Sprue individual efficaciously treated with a prophylactic or
therapeutic drug or other therapy for Celiac Sprue tests more like
a non-Celiac Sprue individual with the methods of the invention.
Likewise, the antibodies or T cell responders, e.g. T cell lines,
of the invention that detect the toxic gluten oligopeptides of the
invention are useful in detecting gluten and gluten-like substances
in food and so can be used to determine whether a food treated to
remove such substances has been efficaciously treated.
[0099] As used herein, the term "treating" is used to refer to both
prevention of disease, and treatment of pre-existing conditions.
The treatment of ongoing disease, to stabilize or improve the
clinical symptoms of the patient, is of particular interest. Such
treatment is desirably performed prior to loss of function in the
affected tissues. Evidence of therapeutic effect may be any
diminution in the severity of disease, particularly measuring the
severity of such symptoms as fatigue, chronic diarrhea,
malabsorption of nutrients, weight loss, abdominal distension, and
anemia. Other disease indicia include the presence of antibodies
specific for the 33-mer of the invention or its deamidated
counterparts, glutens, antibodies specific for tissue
transglutaminase or tTGase linked to the 33-mer of the invention or
its deamidated counterparts, the presence of pro-inflammatory T
cells and cytokines, histological examination of the villus
structure of the small intestine, and the like. Patients may be
adult or child, where children in particular benefit from
prophylactic treatment, as prevention of early exposure to toxic
gluten peptides may prevent initial development of the disease.
Children suitable for prophylaxis can be identified by genetic
testing for predisposition, e.g. by HLA typing; by family history,
and, preferably, by the diagnostic methods of the present
invention.
[0100] The various methods and reagents of the invention can be
prepared and modified as described below. Although specific methods
and reagents are exemplified in the discussion below, it is
understood that any of a number of alternative methods, including
those described above are equally applicable and suitable for use
in practicing the invention. It will also be understood that an
evaluation of the methods of the invention may be carried out using
procedures standard in the art, including the diagnostic and
assessment methods described above.
[0101] The practice of the present invention may employ
conventional techniques of molecular biology (including recombinant
techniques), microbiology, cell biology, biochemistry and
immunology, which are within the scope of those of skill in the
art. Such techniques are explained fully in the literature, such
as, "Molecular Cloning: A Laboratory Manual", second edition
(Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait,
ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987);
"Methods in Enzymology" (Academic Press, Inc.); "Handbook of
Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.);
"Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M.
P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.
M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain
Reaction" (Mullis et al., eds., 1994); and "Current Protocols in
Immunology" (J. E. Coligan et al., eds., 1991).
[0102] As noted above, the subject methods are useful to monitor
the progress and efficacy of therapies to treat individuals
suffering from Celiac Sprue and/or dermatitis herpetiformis. Such
therapies can involve administration of an effective dose of
glutenase and/or tTGase inhibitor, through a pharmaceutical
formulation, incorporating glutenase into food products,
administering live organisms that express glutenase, and the like.
As these therapies may not have been approved by the FDA or an
equivalent other regulatory agency, the methods of the invention
have application in clinical trials conducted to evaluate the
safety and efficacy of such therapies. Diagnosis of suitable
patients may utilize a variety of criteria known to those of skill
in the art in addition to those methods described herein. A
quantitative increase in antibodies specific for gliadin, and/or
tissue transglutaminase is indicative of the disease. Family
histories and the presence of the HLA alleles HLA-DQ2 [DQ(a1*0501,
b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] are indicative of a
susceptibility to the disease.
[0103] In addition to employing the diagnostic methods of the
invention, the therapeutic effect may be measured in terms of
clinical outcome, or may rely on immunological or biochemical
tests. Suppression of the deleterious T-cell activity can be
measured by enumeration of reactive Th1 cells, by quantitating the
release of cytokines at the sites of lesions, or using other assays
for the presence of autoimmune T cells known in the art.
Alternatively, one may look for a reduction in symptoms of a
disease.
Pharmaceutical Compositions
[0104] The HLA-binding peptide inhibitors are incorporated into a
variety of formulations for therapeutic administration. In one
aspect, the agents are formulated into pharmaceutical compositions
by combination with appropriate, pharmaceutically acceptable
carriers or diluents, and may be formulated into preparations in
solid, semi-solid, liquid or gaseous forms, such as tablets,
capsules, powders, granules, ointments, solutions, suppositories,
injections, inhalants, gels, microspheres, and aerosols. As such,
administration can be achieved in various ways, usually by oral
administration. The HLA-binding peptide inhibitors may be systemic
after administration or may be localized by virtue of the
formulation, or by the use of an implant that acts to retain the
active dose at the site of implantation.
[0105] In pharmaceutical dosage forms, the HLA-binding peptide
inhibitors may be administered in the form of their
pharmaceutically acceptable salts, or they may also be used alone
or in appropriate association, as well as in combination with other
pharmaceutically active compounds. The agents may be combined, as
previously described, to provide a cocktail of activities. The
following methods and excipients are merely exemplary and are in no
way limiting.
[0106] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make 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 carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0107] In one embodiment of the invention, the oral formulations
comprise enteric coatings, so that the active agent is delivered to
the intestinal tract. Enteric formulations are often used to
protect an active ingredient from the strongly acid contents of the
stomach. 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.
[0108] Other enteric formulation comprise engineered polymer
microspheres made of biologically erodable polymers, which display
strong adhesive interactions with gastrointestinal mucus and
cellular linings, 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.
[0109] Formulations are typically provided in a unit dosage form,
where the term "unit dosage form," refers to physically discrete
units suitable as unitary dosages for human subjects, each unit
containing a predetermined quantity of glutenase calculated in an
amount sufficient to produce the desired effect in association with
a pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the unit dosage forms of the present invention
depend on the particular complex employed and the effect to be
achieved, and the pharmacodynamics associated with each complex in
the host.
[0110] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
Methods of Treatment
[0111] The subject methods are used to treat individuals suffering
from Celiac Sprue and/or dermatitis herpetiformis, by administering
an effective dose through a pharmaceutical formulation. Diagnosis
of suitable patients may utilize a variety of criteria known to
those of skill in the art. A quantitative increase in antibodies
specific for gliadin, and/or tissue transglutaminase is indicative
of the disease. Family histories and the presence of the HLA
alleles HLA-DQ2 [DQ(a1*05, b1*02)] and/or DQ8 [DQ(a1*03, b1*0302)]
are indicative of a susceptibility to the disease. Specific peptide
analogs may be administered therapeutically to decrease
inflammation, and/or to induce antigen-specific tolerance to treat
autoimmunity. Methods for the delivery of peptides that are altered
from a native peptide are known in the art. Alteration of native
peptides with selective changes of crucial residues can induce
unresponsiveness or change the responsiveness of antigen-specific
autoreactive T cells.
[0112] The therapeutic effect may be measured in terms of clinical
outcome, or may rely on immunological or biochemical tests.
Suppression of the deleterious T-cell activity can be measured by
enumeration of reactive Th1 cells, by quantitating the release of
cytokines at the sites of lesions, or using other assays for the
presence of autoimmune T cells known in the art. Alternatively, one
may look for a reduction in symptoms of a disease.
[0113] Various methods for administration may be employed. The
dosage of the therapeutic formulation will vary widely, depending
upon the nature of the disease, the frequency of administration,
the manner of administration, the clearance of the agent from the
host, and the like. Such treatment could either be before meals or
on a once-a-day basis or on a once-a-week basis, depending on the
half-life of the inhibitor. A typical dose is at least about 1
.mu.g, usually at least about 10 .mu.g, more usually at least about
0.1 mg, and not more than about 10 mg, usually not more than about
1 mg. Enteric coating of these peptides may also enhance their
lifetimes in the gut, thereby permitting delivery to the proximal
and distal small intestinal tissue. Treatment of other autoimmune
disorders such as Type I diabetes with such ligands may involve
oral, intravenous or intramuscular administration. The initial dose
may be larger, followed by smaller maintenance doses. The dose may
be administered as infrequently as weekly or biweekly, or more
often fractionated into smaller doses and administered daily, with
meals, semi-weekly, etc. to maintain an effective dosage level.
[0114] The HLA-binding peptide inhibitors of the invention may be
administered in the treatment of Type 1 diabetes (IDDM). IDDM and
celiac disease are both immunologic disorders where specific HLA
alleles are associated with disease risk. Transglutaminase
autoantibodies can be found in some patients with IDDM. The
prevalence of transglutaminase autoantibodies is higher in diabetic
patients with HLA DQ2 or DQ8.
[0115] Human type I or insulin-dependent diabetes mellitus (IDDM)
is characterized by autoimmune destruction of the .beta. cells in
the pancreatic islets of Langerhans. The depletion of .beta. cells
results in an inability to regulate levels of glucose in the blood.
Overt diabetes occurs when the level of glucose in the blood rises
above a specific level, usually about 250 mg/dl. In humans a long
presymptomatic period precedes the onset of diabetes. During this
period there is a gradual loss of pancreatic beta cell function.
IDDM is currently treated by monitoring blood glucose levels to
guide injection, or pump-based delivery, of recombinant insulin.
Diet and exercise regimens contribute to achieving adequate blood
glucose control. The inhibitors of the invention may be
administered alone, or in combination with other therapies. The
route of administration may be oral, as described for treatment of
Celiac Sprue, or may be injected, e.g. i.v., i.m., etc.
Administration may be performed during the pre-symptomatic phase,
or in overt diabetes.
[0116] Related applications include U.S. Provisional application
60/357,238 filed Feb. 14, 2002; to U.S. Provisional Application
60/380,761 filed May 14, 2002; to U.S. Provisional Application
60/392,782 filed Jun. 28, 2002; and U.S. Provisional application
no. 60/422,933, filed Oct. 31, 2002, each of which are herein
specifically incorporated by reference.
[0117] 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 what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.) but some experimental
errors and deviations should be accounted for. 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
Immunodominant Peptides of Gliadin are Protease Resistant
[0118] Recent studies have identified a small number of
immunodominant peptides from gliadin, which account for most of the
stimulatory activity of dietary gluten on intestinal and peripheral
T lymphocytes found in Celiac patients. The proteolytic kinetics of
these immunodominant peptides were analyzed at the small intestinal
surface. Using brush border membrane vesicles from adult rat
intestines, it was shown that these proline-glutamine-rich peptides
are exceptionally resistant to enzymatic processing, and that
dipeptidyl peptidase IV and dipeptidyl carboxypeptidase are the
rate-limiting enzymes in their digestion. These results support the
conclusions drawn from the tests described in Example 2 that
incomplete digestion of gliadin, which results in the formation of
the 33-mer oligopeptide and its deamidated counterpart formed by
tTGase action, contributes to the disease symptoms of Celiac Sprue
and can be employed in improved diagnostic methods for Celiac
Sprue.
[0119] To dissect this complex process, liquid chromatography
coupled mass spectroscopy analysis (LC-MS-MS) was utilized to
investigate the pathways and associated kinetics of hydrolysis of
immunodominant gliadin peptides treated with rat BBM preparations.
Because the rodent is an excellent small animal model for human
intestinal structure and function, rat BBM was chosen as a suitable
model system for these studies.
[0120] BBM fractions were prepared from rat small intestinal mucosa
as described by Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35.
Using standard assays, the specific activities of the known BB
peptidases were determined to be 127 .mu.U/.mu.g for Aminopeptidase
N (APN, EC 3.4.11.2), 60 .mu.U/.mu.g for dipeptidyl peptidase IV
(DPP IV, EC 3.4.14.5), and 41 .mu.U/.mu.g for dipeptidyl
carboxypeptidase (DCP, EC 3.4.15.1). No proline aminopeptidase (EC
3.4.11.5) or prolyl endopeptidase activity (PEP, EC 3.4.21.26)
activity was detectable (<5 .mu.U/.mu.g). Alkaline phosphatase
and sucrase were used as control BBM enzymes with activities of 66
.mu.U/.mu.g and 350 .mu.U/.mu.g, respectively.
[0121] BBM fractions were partially purified from the small
intestinal mucosa of adult female rats maintained on an ad libitum
diet of wheat-based standard rodent chow. Total protein content was
determined by a modified method of Lowry with BSA as a standard.
Alkaline phosphatase activity was determined with nitrophenyl
phosphate. Sucrase activity was measured using a coupled glucose
assay.
[0122] DPP IV, proline aminopeptidase and APN were assayed
continuously at 30.degree. C. in 0.1 M Tris-HCl, pH 8.0, containing
1 mM of the p-nitroanilides (.epsilon.=8,800 M.sup.-1 cm.sup.-1)
Gly-Pro-pNA, Pro-pNA or Leu-pNA, the latter in additional 1% DMSO
to improve solubility. DCP activity was measured in a 100 .mu.l
reaction as the release of hippuric acid from Hip-His-Leu. PEP
activity was determined continuously with 0.4 mM Z-Gly-Pro-pNA in
PBS:H.sub.2O:dioxane (8:1.2:0.8) at 30.degree. C. One unit is
defined as the consumption of 1 .mu.mol substrate per minute.
[0123] DPP IV and DCP are both up-regulated by a high proline
content in the diet. However, APN activity using standard
substrates was found to be higher than DPP IV even when fed extreme
proline rich diets. Also, although a higher DCP vs. CPP activity
has been observed with the model peptide Z-GPLAP at saturating
concentrations, a difference in Km values could easily account the
reversed ratio measured in this study. 100 .mu.M was chosen as the
initial peptide concentration, since non-saturating kinetics
(k.sub.cat/K.sub.m) were considered to be physiologically more
relevant than the maximal rates of hydrolysis (k.sub.cat).
[0124] Proteolysis with the BBM preparation was investigated using
the peptide (SEQ ID NO:1) (SEQ ID NO:1) QLQPFPQPQLPY, a product of
chymotryptic digestion of .alpha.-9 gliadin (Arentz-Hansen et al.
(2000) J. Exp. Med. 191, 603-12). This peptide has been shown to
stimulate proliferation of T cells isolated from most Celiac Sprue
patients, and hence is considered to possess an immunodominant
epitope. It was subjected to BBM digestion, followed by LC-MS-MS
analysis. A standard 50 .mu.l digestion mixture contained 100 .mu.M
of synthetic peptide, 10 .mu.M tryptophan and Cbz-tryptophan as
internal standards, and resuspended BBM preparations with a final
protein content of 27 ng/.mu.l and exogenous proteins, as
indicated, in phosphate buffered saline. After incubation at
37.degree. C. for the indicated time, the enzymes were inactivated
by heating to 95.degree. C. for 3 minutes. The reaction mixtures
were analyzed by LC-MS (SpectraSystem, ThermoFinnigan) using a C18
reversed phase column (Vydac 218TP5215, 2.1.times.150 mm) with
water:acetonitrile:formic acid (0.1%):trifluoroacetic acid (0.025%)
as the mobile phase (flow: 0.2 ml/min) and a gradient of 10%
acetonitrile for 3 minutes, 10-20% for 3 minutes, 20-25% for 21
minutes followed by a 95% wash. Peptide fragments in the mass range
of m/z=300-2000 were detected by electrospray ionization mass
spectroscopy using a LCQ ion trap, and their identities were
confirmed by MSMS fragmentation patterns.
[0125] While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY
disappeared with an apparent half time of 35 min. several
intermediates were observed to accumulate over prolonged periods
(FIG. 1A). The MS intensities (m/z=300-2000 g/mol) and UV.sub.280
absorbances of the parent peptides (SEQ ID NO:1) QLQPFPQPQLPY and
(SEQ ID NO:3) PQPQLPYPQPQLPY were found to depend linearly on
concentration in the range of 6-100 .mu.M. The reference peptides
(SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5) QLQPFPQPQLP, (SEQ ID
NO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP were generated
individually by limited proteolysis of the parent peptides with 10
.mu.g/ml carboxypeptidase A (C-0261, Sigma) and/or 5.9 .mu.g/ml
leucine aminopeptidase (L-5006, Sigma) for 160 min. at 37.degree.
C. and analyzed by LC-MS as in FIG. 1.
[0126] Indeed, the subsequent processing of the peptide was
substantially retarded (FIG. 1B). The identities of the major
intermediates were confirmed by tandem MS, and suggested an
unusually high degree of stability of the (SEQ ID NO:8) PQPQLP
sequence, a common motif in T cell stimulating peptides. Based on
this data and the known amino acid preferences of the BBM
peptidases, the digestive breakdown of (SEQ ID NO:1) QLQPFPQPQLPY
was reconstructed, as shown in the insert of FIG. 1B. The preferred
pathway involves serial cleavage of the N-terminal glutamine and
leucine residues by aminopeptidase N (APN), followed by removal of
the C-terminal tyrosine by carboxypeptidase P (CPP) and hydrolysis
of the remaining N-terminal QP-dipeptide by DPP IV. As seen in FIG.
1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed by APN attack
on the first two N-terminal residues) and its derivatives are
increasingly resistant to further hydrolysis. Because the high
proline content seemed to be a major cause for this proteolytic
resistance, digestion was compared with a commercially available
non-proline control peptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St.
Louis, Mo.). Initial hydrolysis was much faster (t.sub.1/2=10 min).
More importantly, digestive intermediates were only transiently
observed and cleared completely within one hour, reflecting a
continuing high specificity of the BBM for the intermediate
peptides.
[0127] Because the three major intermediate products (SEQ ID NO:6)
QPFPQPQLPY, (SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP)
observed during BBM mediated digestion of (SEQ ID NO:1)
QLQPFPQPQLPY are substrates for DPP IV, the experiment was repeated
in the presence of a 6-fold excess activity of exogenous fungal DPP
IV. Whereas the relatively rapid decrease of the parent peptide and
the intermediate levels of (SEQ ID NO:5) QLQPFPQPQLP were largely
unchanged, the accumulation of DPP IV substrates was entirely
suppressed and complete digestion was observed within four hours.
(FIG. 1B, open bars).
[0128] To investigate the rate-limiting steps in BBM mediated
digestion of gliadin peptides from the C-terminal end, another
known immunodominant peptide derived from wheat .alpha.-gliadin,
(SEQ ID NO:3) PQPQLPYPQPQLPY, was used. Although peptides with
N-terminal proline residues are unlikely to form in the small
intestine (none were observed during BBM digestion of (SEQ ID NO:1)
QLQPFPQPQLPY, FIG. 1A), they serve as a useful model for the
analysis of C-terminal processing since the N-terminal end of this
peptide can be considered proteolytically inaccessible due to
minimal proline aminopeptidase activity in the BBM. As shown in
FIG. 2, this peptide is even more stable than (SEQ ID NO:1)
QLQPFPQPQLPY. In particular, removal of the C-terminal tyrosine
residue by carboxypeptidase P (CPP) is the first event in its
breakdown, and more than four times slower than APN activity on
(SEQ ID NO:1) QLQPFPQPQLPY (FIG. 1B). The DCP substrate (SEQ ID
NO:4) PQPQLPYPQPQLP emerges as a major intermediate following
carboxypeptidase P catalysis, and is highly resistant to further
digestion, presumably due to the low level of endogenous DCP
activity naturally associated with the BBM. To confirm the role of
DCP as a rate-limiting enzyme in the C-terminal processing of
immunodominant gliadin peptides, the reaction mixtures were
supplemented with rabbit lung DCP. Exogenous DCP significantly
reduced the accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after
overnight incubation in a dose dependent manner (FIG. 2C).
Conversely, the amount of accumulated (SEQ ID NO:4) PQPQLPYPQPQLP
increased more than 2-fold in the presence of 10 .mu.M of
captopril, a DCP-specific inhibitor, as compared with
unsupplemented BBM.
[0129] Together, the above results demonstrate that (i)
immunodominant gliadin peptides are exceptionally stable toward
breakdown catalyzed by BBM peptidases, and (ii) DPP IV and
especially DCP are rate-limiting steps in this breakdown process at
the N- and C-terminal ends of the peptides, respectively. Because
BBM exopeptidases are restricted to N- or C-terminal processing, it
was investigated if generation of additional free peptide ends by
pancreatic enzymes would accelerate digestion. Of the pancreatic
proteases tested, only elastase at a high (non-physiological)
concentration of 100 ng/.mu.l was capable of hydrolyzing (SEQ ID
NO:3) PQPQLPYPQPQ.sup..dwnarw.LPY. No proteolysis was detected with
trypsin or chymotrypsin.
[0130] The above data demonstrates that proline-rich gliadin
peptides are extraordinarily resistant to digestion by small
intestinal endo- and exopeptidases, and therefore are likely to
accumulate at high concentrations in the intestinal cavity after a
gluten rich meal. The pathological implication of digestive
resistance is strengthened by the observed close correlation of
proline content and celiac toxicity as observed in the various
common cereals (Schuppan (2000) Gastroenterology 119, 234-42).
Example 2
Immunodominant Peptide of Wheat Gliadin
[0131] It has long been known that the principal toxic components
of wheat gluten are a family of closely related Pro-Gln rich
proteins called gliadins. Recent reports have suggested that
peptides from a short segment of .alpha.-gliadin appear to account
for most of the gluten-specific recognition by CD4+T cells from
Celiac Sprue patients. These peptides are substrates of tissue
transglutaminase (tTGase), the primary auto-antigen in Celiac
Sprue, and the products of this enzymatic reaction bind to the
class 11 HLA DQ2 molecule. This example demonstrates, using a
combination of in vitro and in vivo animal and human studies, that
this "immunodominant" region of .alpha.-gliadin is part of an
unusually long proteolytic product generated by the digestive
process that: (a) is exceptionally resistant to further breakdown
by gastric, pancreatic and intestinal brush border proteases; (b)
is the highest specificity substrate of human tissue
transglutaminase (tTGase) discovered to date; (c) contains at least
six overlapping copies of epitopes known to be recognized by
patient derived T cells; (d) stimulates representative T cell
clones that recognize these epitopes with sub-micromolar efficacy;
and (e) has homologs in proteins from all toxic foodgrains but no
homologs in non-toxic foodgrain proteins. In aggregate, these
findings demonstrate that the onset of symptoms upon gluten
exposure can be traced back to a small segment of .alpha.-gliadin.
Finally, it is shown that this "super-antigenic" long peptide can
be detoxified in vitro and in vivo by treatment with bacterial
prolyl endopeptidase, providing a strategy for peptidase therapy
for Celiac Sprue.
[0132] Identification of stable peptides from gastric protease,
pancreatic protease and brush border membrane peptidase catalyzed
digestion of recombinant .alpha.2-gliadin: .alpha.2-gliadin, a
representative .alpha.-gliadin (Arentz-Hansen et al. (2000) Gut
46:46), was expressed in recombinant form and purified from E.
coli. The .alpha.2-gliadin gene was cloned in pET28a plasmid
(Novagen) and transformed into the expression host BL21(DE3)
(Novagen). The transformed cells were grown in 1-liter cultures of
LB media containing 50 .mu.g/ml of kanamycin at 37.degree. C. until
the OD600 0.6-1 was achieved. The expression of .alpha.2-gliadin
protein was induced with the addition of 0.4 mM isopropyl
.beta.-D-thiogalactoside (Sigma), and the cultures were further
incubated at 37.degree. C. for 20 hours. The cells expressing the
recombinant .alpha.2-gliadin were centrifuged at 3600 rpm for 30
minutes. The pellet was resuspended in 15 ml of disruption buffer
(200 mM sodium phosphate; 200 mM NaCl; 2.5 mM DTT; 1.5 mM
benzamidine; 2.5 mM EDTA; 2 mg/L pepstatin; 2 mg/L leupeptin; 30%
v/v glycerol) and lysed by sonication (1 minute; output control set
to 6). After centrifugation at 45000 g for 45 min, the supernatant
was discarded and the pellet containing gliadin protein was
resuspended in 50 ml of 7M urea in 50 mM Tris (pH=8.0). The
suspension was again centrifuged at 45000 g for 45 min and the
supernatant was harvested for purification.
[0133] The supernatant containing .alpha.2-gliadin was incubated
with 1 ml of nickel-nitrilotriacetic acid resin (Ni-NTA; Qiagen)
overnight and then batch-loaded on a column with 2 ml of Ni-NTA.
The column was washed with 7 M urea in 50 mM Tris (pH=8.0), and
.alpha.2-gliadin was eluted with 200 mM imidazole, 7 M urea in 50
mM Tris (pH=4.5). The fractions containing .alpha.2-gliadin were
pooled into a final concentration of 70% ethanol solution, and two
volumes of 1.5 M NaCl were added to precipitate the protein. The
solution was incubated at 4.degree. C. overnight, and the final
precipitate was collected by centrifugation at 45000 g for 30 min.,
rinsed in water, and re-centrifuged to remove the urea. The final
purification step of the .alpha.-2 gliadin was developed with
reverse-phase HPLC. The Ni-NTA purified protein fractions were
pooled in 7 M urea buffer and injected to a Vydac (Hesperia, CA)
polystyrene reverse-phase column (i.d. 4.6 mm.times.25 cm) with the
starting solvent (30% of solvent B: 1:1 HPLC-grade
acetonitrile/isopropanol: 0.1% TFA). Solvent A was an aqueous
solution with 0.1% TFA. The separation gradient extended from
30-100% of solvent B over 120 min. at a flow rate of 0.8
ml/min.
TABLE-US-00002 TABLE 2 Amount of Peptides Digested after 15 hours
33-mer Control A Control B H1P0 <20% >90% >90% H2P0
<20% >61% >85% H3P0 <20% >87% >95% H4P0 <20%
>96% >95% H5P0 <20% >96% >95%
[0134] The purity of the recombinant gliadin was >95%, which
allowed for facile identification and assignment of proteolytic
products by LC-MS/MS/UV. Although many previous studies utilized
pepsin/trypsin treated gliadins, it was found that, among gastric
and pancreatic proteases, chymotrypsin played a major role in the
breakdown of .alpha.2-gliadin, resulting in many small peptides
from the C-terminal half of the protein and a few longer (>8
residues) peptides from the N-terminal half, the most noteworthy
being a relatively large fragment, the 33-mer (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). This peptide
was of particular interest for two reasons: (a) whereas most other
relatively stable proteolytic fragments were cleaved to smaller
fragments when the reaction times were extended, the 33-mer peptide
remained intact despite prolonged exposure to proteases; and (b)
three distinct patient-specific T cell epitopes identified
previously are present in this peptide, namely, PFPQPQLPY,
PQPQLPYPQ (3 copies), and PYPQPQLPY (2 copies).
[0135] To establish the physiological relevance of this peptide,
composite gastric/pancreatic enzymatic digestion of .alpha.2
gliadin was then examined. As expected, enzymatic digestion with
pepsin (1:100 w/w ratio), trypsin (1:100), chymotrypsin (1:100),
elastase (1:500) and carboxypeptidase (1:100) was quite efficient,
leaving behind only a few peptides longer than 9 residues (the
minimum size for a peptide to show class 11 MHC mediated
antigenicity) (FIG. 4). In addition to the above-mentioned 33-mer,
the peptide (SEQ ID NO:10) WQIPEQSR was also identified, and was
used as a control in many of the following studies. The stability
of the 33-mer peptide can also be appreciated, when comparing the
results of a similar experiment using myoglobin (another common
dietary protein). Under similar proteolytic conditions, myoglobin
is rapidly broken down into much smaller products. No long
intermediate is observed to accumulate.
[0136] The small intestinal brush-border membrane (BBM) enzymes are
known to be vital for breaking down any remaining peptides from
gastric/pancreatic digestion into amino acids, dipeptides or
tripeptides for nutritional uptake. Therefore a comprehensive
analysis of gliadin metabolism also required investigations into
BBM processing of gliadin peptides of reasonable length derived
from gastric and pancreatic protease treatment. BBM fractions were
prepared from rat small intestinal mucosa. The specific activities
of known BBM peptidases were verified to be within the previously
reported range. Whereas the half-life of disappearance of (SEQ ID
NO:10) WQIPEQSR was .about.60 min. in the presence of 12 ng/.mu.l
BBM protein, the half-life of (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h.
Therefore, the latter peptide remains intact throughout the
digestive process in the stomach and upper small intestine, and is
poised to act as a potential antigen for T cell proliferation and
intestinal toxicity in genetically susceptible individuals.
[0137] Verification of proteolytic resistance of the 33-mer gliadin
peptide with brush border membrane preparations from human
intestinal biopsies: To validate the above conclusions, derived
from studies with rat BBM preparations, in the context of human
intestinal digestion, BBM preparations were prepared from a panel
of adult human volunteers, one of whom was a Celiac Sprue patient
in remission, while the rest were found to have normal intestinal
histology. (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, (SEQ
ID NO:1) QLQPFPQPQLPY (an internal sequence from the 33-mer used as
a control), (SEQ ID NO:10) WQIPEQSR and other control peptides (100
.mu.M) were incubated with BBM prepared from each human biopsy
(final aminopeptidase N activity of 13 .mu.U/.mu.l) at 37.degree.
C. for varying time periods. While, (SEQ ID NO:1) QLQPFPQPQLPY,
(SEQ ID NO:10) WQIPEQSR and other control peptides were completely
proteolyzed within 1-5 h, the long peptide remained largely intact
for at 19 hours. These results confirm the equivalence between the
rat and human BBM for the purpose of this study.
[0138] Verification of proteolytic resistance of the 33-mer gliadin
peptide in intact animals: The proteolytic resistance of the 33-mer
gliadin peptide, observed in vitro using BBM from rats and humans,
was confirmed in vivo using a perfusion protocol in intact adult
rats (Smithson and Gray (1977) J. Clin. Invest. 60:665). Purified
peptide solutions were perfused through a 15-20 cm segment of
jejunum in a sedated rat with a residence time of 20 min., and the
products were collected and subjected to LC-MS analysis. Whereas
>90% of (SEQ ID NO:1) QLQPFPQPQLPY was proteolyzed in the
perfusion experiment, most of the 33-mer gliadin peptide remained
intact. These results demonstrate that the 33-mer peptide is very
stable as it is transported through the mammalian upper small
intestine.
[0139] The 33-mer gliadin peptide is an excellent substrate for
tTGase, and the resulting product is a highly potent activator of
patient-derived T cells. A number of recent studies have
demonstrated that regiospecific deamidation of immunogenic gliadin
peptides by tTGase increases their affinity for HLA-DQ2 as well as
the potency with which they activate patient-derived
gluten-specific T cells. It has been shown the specificity of
tTGase for certain short antigenic peptides derived from gliadin is
higher than its specificity toward its physiological target site in
fibronectin, for example, the specificity of tTGase for the
.alpha.-gliadin derived peptide (SEQ ID NO: 3) PQPQLPYPQPQLPY is
5-fold higher than that for its target peptide sequence in
fibrinogen, its natural substrate. The kinetics and
regiospecificity of deamidation of the 33-mer .alpha.-gliadin
peptide identified as above were therefore measured. The
k.sub.cat/K.sub.M was higher than that reported for any peptide
studied thus far: kcat/KM=440 min-1 mM-1 for (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min-1 mM-1
for PQPQLPY and kcat/KM=350 min-1 mM-1 for (SEQ ID NO: 3)
PQPQLPYPQPQLPY.
[0140] Moreover, LC-MS-MS analysis revealed that (SEQ ID NO:12)
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated by
tTGase at the underlined residues. Since tTGase activity is
associated with the brush border membrane of intestinal
enterocytes, it is likely that dietary uptake of even small
quantities of wheat gluten will lead to the build-up of sufficient
quantities of this 33-mer gliadin peptide in the intestinal lumen
so as to be recognized and processed by tTGase.
[0141] Structural characteristics of the 33-mer gliadin peptide and
its naturally occurring homologs: Sequence alignment searches using
BLASTP in all non-redundant protein databases revealed several
homologs (E-value<0.001) of the 33-mer gliadin peptide.
Interestingly, foodgrain derived homologs were only found in
gliadins (from wheat), hordeins (from barley) and secalins (from
rye), all of which have been proven to be toxic to Celiac patients.
See FIG. 6. Nontoxic foodgrain proteins, such as avenins (in oats),
rice and maize, do not contain homologous sequences to the 33-mer
gliadin. In contrast, a BLASTP search with the entire
.alpha.2-gliadin sequence identified foodgrain protein homologs
from both toxic and nontoxic proteins. Based on available
information regarding the substrate specificities of gastric,
pancreatic and BBM proteases and peptidases, it is predicted that,
although most gluten homologs to the 33-mer gliadin peptide
contained multiple proteolytic sites and are therefore unlikely to
be completely stable toward digestion, several sequences from
wheat, rye and barley are expected to be comparably resistant to
gastric and intestinal proteolysis. The stable peptide homologs to
the 33-mer .sigma.2-gliadin peptide are (SEQ ID NO:13)
QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from .alpha.1- and
.alpha.6-gliadins); (SEQ ID NO:14)
QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein); (SEQ ID
NO:15) QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from .gamma.-gliadin); (SEQ
ID NO:16) QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from .omega.-secalin).
These stable peptides are all located at the N-terminal region of
the corresponding proteins. The presence of proline residues after
otherwise cleavable residues in these peptides would contribute to
their proteolytic stability.
[0142] The unique primary sequence of the 33-mer gliadin peptide
also had homologs among a few non-gluten proteins. Among the
strongest homologs were internal sequences from pertactin (a highly
immunogenic protein from Bordetella pertussis) and a mammalian
inositol-polyphosphate 5-phosphatase of unknown function. In both
cases available information suggested that the homology could have
biologically relevance. For example, the region of pertactin that
is homologous to the 33-mer gliadin peptide is known to be part of
the immunodominant segment of the protein. In the case of the
homologous phosphatase, the corresponding peptide region of the
phosphatase is known to be responsible for vesicular trafficking of
the phosphatase to the cytoplasmic Golgi. In analogy with the
current picture of how gliadin peptides are presented to HLA-DQ2
via a tTGase mediated pathway, these Pro-Gln-rich segments of both
pertactin and the phosphatase are likely to be good tTGase
substrates. To test this hypothesis, the corresponding peptides
were synthesized, and the selectivity of tTGase for these peptides
was measured. As predicted, both peptides were found to be good
substrates of tTGase. The tTGase enzyme plays a central role in
receptor mediated endocytosis of several biologically important
proteins. The biological activities of both pertactin and the
phosphatase may depend on tTGase mediated trafficking.
[0143] Secondary structural studies using circular dichroism
spectroscopy on the 33-mer gliadin peptide as well as its homologs
from pertactin and the inositol-polyphosphate 5-phosphatase
demonstrate that these peptides have strong type II polyproline
helical character. In addition to reinforcing the proteolytic
resistance of these peptides, the type II polyproline helical
conformation is also likely to enhance their affinity for class II
MHC proteins.
[0144] Although gluten proteins from food grains such as wheat, rye
and barley are central components of a nutritious diet, they can be
extremely toxic for patients suffering from Celiac Sprue. To
elucidate the structural basis of gluten toxicity in Celiac Sprue,
comprehensive proteolytic analysis was performed on a
representative recombinant gliadin under physiologically relevant
conditions. An unusually long and proteolytically stable peptide
product was discovered, whose physiological relevance was confirmed
by studies involving brush border membrane proteins from rat and
human intestines as well as intestinal perfusion assays in live
rats. In aggregate, these data demonstrate that this peptide and
its homologs found in other wheat, rye and barley proteins are the
"root cause" of the initial inflammatory response to dietary wheat
in Celiac Sprue patients in remission.
Example 3
[0145] Human leukocyte antigen DQ2 is a class II major
histocompatibility complex protein that plays a critical role in
the pathogenesis of Celiac Sprue by binding to epitopes derived
from dietary gluten and triggering the inflammatory response of
disease-specific T cells. Inhibition of DQ2 mediated antigen
presentation in the small intestinal mucosa of Celiac Sprue
patients therefore represents a potentially attractive mode of
therapy for this widespread but unmet medical need. Starting from a
pro-inflammatory, proteolytically resistant, 33-residue peptide,
(SEQ ID NO:12) LQLQPFPQPEL PYPQPELPYPQPELPYPQPQPF, we embarked upon
a systematic effort to dissect the relationships between peptide
structure and DQ2 affinity, and to translate these insights into
prototypical DQ2 blocking agents. Three structural determinants
within the first 20 residues of this 33-mer peptide, including a
(SEQ ID NO: 18) PQPELPYPQ epitope, its N-terminal flanking sequence
and a downstream Glu residue, were found to be critical for DQ2
recognition. Guided by the X-ray crystal structure of DQ2, the L11
and L18 residues in the truncated 20-mer analogue were replaced
with sterically bulky groups so as to retain high DQ2 affinity but
abrogate T cell recognition. A dimeric ligand synthesized by
regiospecific coupling of the 20-mer peptide with a bifunctional
linker, was identified as an especially potent DQ2 binding agent.
Two such ligands were able to attenuate the proliferation of
disease-specific T cell lines in response to gluten antigens, and
therefore represent prototypical examples of pharmacologically
suitable DQ2 blocking agents for the potential treatment of Celiac
Sprue.
[0146] Inhibition of antigen presentation by blocking a
disease-specific MHC on antigen presenting cells with peptide (and
occasionally non-peptide) ligands has been previously explored as a
therapeutic strategy for autoimmune diseases such as multiple
sclerosis, rheumatoid arthritis, diabetes, and experimental
autoimmune encephalomyelitis. Such therapy is of particular
interest for the treatment of Celiac Sprue. First, to date no
non-dietary treatment has been developed for this widespread,
lifelong disease; as such, there is an acute unmet need. Second, in
contrast to the organs affected by most other autoimmune diseases,
the small intestine is readily accessible via oral administration
of a therapeutic candidate. Finally, and perhaps most importantly,
among HLA mediated diseases, Celiac Sprue is unique in that an
environmental trigger (dietary gluten) has been identified and
extensively dissected at an immunological level. In turn, these
studies have led to the identification of proteolytically resistant
gluten peptides that are generated by physiological processes and
are efficiently presented to disease associated T cells in a DQ2
restricted fashion. Thus, if these naturally occurring T cell
stimulatory agents can be transformed into inhibitors of DQ2
mediated antigen presentation, they can be considered as
appropriate medicinal leads for Celiac Sprue.
[0147] A Pro- and Gln-rich 33-mer peptide from .alpha.2-gliadin,
(SEQ ID NO:12) LQLQPFPQPELPYPQPE LPYPQPELPYPQPQPF
(transglutaminase-catalyzed Gln->Glu changes underlined), is a
particularly interesting lead peptide for this purpose. Its extreme
resistance to breakdown by luminal proteases and intestinal
brush-border enzymes allows it to persist for a considerable
duration in the upper small intestine, the primary affected region
of the gastrointestinal tract in a Celiac Sprue patient. Not only
does this peptide have a high affinity for HLA-DQ2, it is displayed
on the surface of antigen presenting cells with unusual robustness.
Not surprisingly, it is a potent proliferative trigger of
gluten-responsive T cells from small intestinal biopsy samples of
all DQ2 Celiac Sprue patients tested thus far. Although this
peptide is multivalent (it has 6 overlapping copies of 3 epitopes),
it binds to HLA-DQ2 with a 1:1 stoichiometry. It has a considerably
higher affinity for DQ2 than any of its constituent epitopes (SEQ
ID NO:17) PFPQPELPY, (SEQ ID NO:18) PQPELPYPQ and (SEQ ID NO:58)
PYPQPELPY. Together, these observations led us to hypothesize that
the 33-mer peptide harbors secondary interactions with DQ2 outside
the core antigen binding pocket. Understanding the precise nature
of these interactions would therefore be a critical prerequisite
for exploiting its potential as a medicinal lead in the design of
DQ2 blocking agents.
[0148] In this report we have dissected the structural determinants
of the high-affinity interaction between the 33-mer peptide and
HLA-DQ2. Based on these findings, we designed and synthesized
simple analogues of the 33-mer peptide that retain its strong
affinity for DQ2 but are not recognized by 33-mer responsive T
cells from Celiac biopsies. The ability of these putative blocking
agents to inhibit T cell proliferation in response to gluten
antigens was also demonstrated. These peptides represent the first
prototypical examples of pharmacologically relevant DQ2 blocking
agents for potential treatment of Celiac Sprue.
Experimental Section
[0149] DQ2 expression and purification. Soluble DQ2 molecules were
expressed and purified as previously described. Briefly, the
soluble extracellular domains of the DQ2 .alpha. and .beta. chains
were co-expressed in High Five insect cells using a pAcAB3
baculovirus expression system, and were affinity-purified using the
anti-DQ2 mAb 2.12.E11. The sequence (SEQ ID NO:55) QLQPFPQPELPY was
fused to the N-terminus of the DQ2 .beta.-chain by a 15-residue
linker (SEQ ID NO:56)(GAGSLVPRGSGGGGS), which includes a thrombin
site. A complementary Fos/Jun leucine zipper pair was engineered at
the C-terminal ends of .alpha. and .beta. chains, respectively,
with intervening factor Xa proteolysis sites, to increase the
heterodimer stability during protein expression.
[0150] The concentration of HLA-DQ2 was determined by UV
spectrophotometry at 280 nm using the absorption coefficient factor
75,700 cm.sup.-1M.sup.-1 as calculated from the contents of
tyrosine, tryptophan and cystine in the DQ2 sequence (22). Prior to
use in binding experiments with exogenous ligands, the DQ2-ligand
fusion protein was first treated with .about.2% w/w thrombin in pH
7.3 PBS at 0.degree. C. for 2 h.
[0151] Peptide synthesis, labeling and purification. All peptides
used in this study were synthesized using Boc/HBTU chemistry
starting from N-.alpha.-t-Boc-L-aminoacyl-phenylacetamidomethyl
(PAM) resin. Peptides were labeled at their N-termini while still
attached to the resin with 5- (and 6-) carboxyfluorescein,
1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride
(EDC.HCl), and 1-Hydroxy-7-azabenzotriazole (HOAt) in 1:1:1 ratio
in dimethylformamide as the solvent. Following cleavage of the
peptidyl resin in trifluoroacetic acid/trifluoromethanesulfonic
acid/thioanisole (TFA/TFMSA/thioanisole 10:1:1, v/v/v) for 4 h, the
crude peptides were precipitated in cold ether and dissolved in 1:1
v/v acetonitrile/water. The peptides were purified by reverse-phase
HPLC on a semi-preparative C.sub.18 column using a
water-acetonitrile gradient in 0.1% (v/v) TFA. The identity and
purity of the peptides were confirmed by electrospray mass
spectrometry and analytical reverse-phase HPLC.
[0152] The peptides were lyophilized and stored at -20.degree. C.
Prior to use, peptide stock solutions were prepared in 10 mM PBS
with 0.02% sodium azide, and their concentrations were determined
by UV-Vis spectrophotometry at 495 nm using the absorption
coefficient factor 80,200 cm.sup.-1M.sup.-1 for the
fluorescein-labeled peptide. The integrity of the peptide stocks
was monitored by analytical HPLC every several months.
[0153] Peptide derivatives 17-22 (see FIG. 10) were synthesized
with N-.alpha.-t-Boc-N-.epsilon.-Fmoc-L-lysine.
[0154] The Fmoc-protected side chain of lysine was deprotected
after synthesis of the full-length peptide by washing the resin
twice in 20% piperidine in dimethylformamide for 15 min. Then 1 g
of succinimide anhydride dissolved in 2 ml dimethylformamide was
added to the resin for 30 min. The extent of amide formation was
monitored by the ninhydrin test.
[0155] The dimeric peptide 22 was synthesized from pure monomeric
fluorescein labeled peptide 17. Fluorescein-conjugated 17 was mixed
with bis-d PEG 6 NHS ester (Quanta Biodesign) in 2:1 ratio in
either dimethylformamide with 10% v/v diisopropylethylamine as
base, or in pH 9 phosphate solution. The reaction was monitored by
analytical reverse phase HPLC using a C.sub.18 column. The product
peak eluted 2 min after the monomeric starting material, and was
purified by preparative reverse phase HPLC. Mass spectrometric
analysis revealed that it had the expected molecular weight of
5858.8 (22+Na.sup.+, exp. MW 5858). The concentration of this
fluorescent dimeric peptide was quantified by using the absorption
coefficient factor 160,400 cm.sup.-1M.sup.-1 at 495 nm.
[0156] Peptide exchange assay. For peptide exchange experiments,
the DQ2 heterodimer purified as described above was incubated with
fluorescein-conjugated ligands in a 25:1 ratio (i.e. 4.7 .mu.M DQ2
with 0.18 .mu.M fluorescent peptide). Incubations were performed at
37.degree. C. in a 1:1 mixture of PBS buffer (10 mM Pi, 150 mM
NaCl, pH 7.3, supplemented with 0.02% NaN.sub.3) and McIlvaine's
citrate-phosphate buffers (pH 5 or pH 7) such that the final pH was
either 5.5 or 7.3, respectively. Peptide binding was measured by
high performance size exclusion chromatography (HPSEC) (17). 1
.mu.l of reaction mixture was diluted into 14 .mu.l PBS. 12.5 .mu.l
of the diluted material was injected onto a BioSep 3000 size
exclusion column (Phenomenex), and eluted with PBS buffer at 1
ml/min. The DQ2-peptide complex eluted at 8.5 min, with free
peptides emerging -2 min later. The fluorescence signal was
recorded using an in-line Shimadzu RA35 fluorescent detector with
excitation wavelength set at 495 nm and emission detection set at
520 nm. Peak areas corresponding to the DQ2-peptide complex and the
free peptide were used to calculate the fractional yield of the
DQ2-fluoresceinated peptide complex.
[0157] Peptide dissociation assay. For dissociation experiments,
DQ2-fluoresceinated peptide complexes were prepared by incubating
thrombin treated DQ2 (3-5 .mu.M) with 20-fold excess
fluorescein-conjugated peptides at pH 5.5 for 25 hours. The buffer
composition was a 1:1 mixture of 10 mM PBS buffer and pH 5.1
McIlvaine's citrate-phosphate buffer (24), such that the final pH
was 5.5. Excess free peptide was separated from the complex on a
chilled spin column (Bio-Rad) packed with Sephadex G50 superfine
medium and blocked with 1% BSA solution to minimize the binding of
DQ2 to the column. Spin columns were pre-washed with pH 7.3 PBS
buffer, and the fluorescein-conjugated peptide+DQ2 mixture was
applied to the column. The DQ2-fluoresceinated peptide complex was
eluted in a volume of .about.230 .mu.l in pH 7.3 PBS buffer.
Typically, this DQ2-peptide fraction contained <10% of free
peptide. The solution was immediately adjusted to pH 5.5 or pH 7.3,
and 20 .mu.M of a tight DQ2 binding peptide (SEQ ID NO:57)
(AAIAAVKEEAF) was added to prevent the re-binding of dissociated
fluorescent peptide to DQ2. Kinetic measurements of ligand
dissociation were performed at 37.degree. C., and a time course was
obtained by injecting 20 .mu.l aliquots into HPSEC column.
[0158] T cell proliferation assays. The DQ2 homozygous B-lymphoma
cell line (LCL) VAVY cells were irradiated with 12,000 rads of
.gamma.-irradiation or fixed with 1% paraformaldehyde for 10
minutes as indicated, and incubated with the appropriate peptides
overnight in media containing 10% fetal bovine serum, 2% human
serum, penicillin and streptomycin at a cell density of
2.times.10.sup.6 cells/ml in 96-well plates. The next day, the
volume was doubled to yield a cell density of 1.times.10.sup.6
cells/ml, 50 .mu.l of which was placed into a U-bottom 96-well
plate. An equal volume of T-cells (50 .mu.l of 1.times.10.sup.6
cells/ml) was added to each well, and cells were incubated at
37.degree. C. and 5% CO.sub.2 for 48 h, at which point 0.5
.mu.Ci/well of [methyl-.sup.3H] thymidine (Amersham, TRK120) was
added. Cells were incubated for an additional 12-14 h and then
frozen. After thawing, incorporated thymidine was collected on a
filter mat (Wallac, 1205-401) using a Tomtec cell harvester, and
counted using a Wallac 1205 Betaplate liquid scintillation
counter.
Results
[0159] SAR analysis of the binding of the 33-mer peptide to
HLA-DQ2. Earlier studies have demonstrated that the highly
immunogenic 33-mer peptide from .alpha.2-gliadin, (SEQ ID NO:12)
LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, potently displaces pre-bound
ligands from the DQ2 binding pocket and also has a long
dissociation half-life. These features, together with its natural
proteolytic resistance, make this 33-mer peptide an attractive
target for engineering an HLA-DQ2 blocking agent. To dissect its
structure-activity relationships (SAR), several analogues were
synthesized and initially evaluated in peptide exchange assays over
a period of 45 h (FIG. 1). Although the percentage of bound peptide
is only reported at the 45 h end-point (i.e. at equilibrium), the
half-maximum binding times for several of these peptides were
obtained, and found to be similar. Therefore, the equilibrium
occupancy is representative of DQ2 avidity.
[0160] Several noteworthy observations emerge from the data
summarized in FIG. 8. First, consistent with earlier data, it is
evident that the .alpha.II epitope (SEQ ID NO:18) (PQPELPYPQ) is
the primary binding site in the 33-mer peptide 1. For example, the
minimal .alpha.II epitope bearing peptide 3, has a binding maximum
of 7% at pH 5.5, whereas the minimal .alpha.I epitope bearing
peptide 2 saturates at less than 1% at pH 5.5 and is undetectable
at pH 7.3. Second, comparison of the DQ2 binding properties of the
minimal .alpha.II epitope 3 with longer analogues (e.g. 6 and 7)
reveals that binding efficiency increases as a greater proportion
of the 33-mer sequence is included at the C-terminal side of the
epitope. Similarly, comparison of the binding properties of the
minimal peptide 3 with the N-terminally elongated peptide 4
suggests that the N-terminus of the 33-mer contributes
significantly to its DQ2 binding potency. Together, these findings
argue that structural determinants on both sides of the core
.alpha.II epitope sequence of the 33-mer influence its
immunological characteristics. The importance of flanking sequences
is also reinforced when one compares 21-mer peptide 7 with
analogues that are incrementally extended on both sides (8, 9, 10
and 1). Interestingly, the difference between peptides 7 and 8 is
more pronounced at pH 7 than at pH 5.
[0161] The 33-mer peptide 1 contains three Glu residues
corresponding to three transglutaminase-catalyzed deamidation
sites. To investigate the role of multiple deamidation sites, we
synthesized and tested all possible deamidation analogues of the
33-mer (peptides 11-16). Q10E (11) has higher affinity for DQ2 than
either Q17E (12) or Q24E (13) at both pH 5 and pH 7, arguing that
DQ2 binding of the 33-mer is primarily centered at the N-terminal
.alpha.II epitope, although the other .alpha.II epitopes can also
bind effectively in the MHC pocket. Notably however, introduction
of a second deamidation site (e.g. Q10EQ17E (14) or Q10EQ24E (15))
significantly enhances affinity relative to Q10E (11), suggesting
that a second acidic residue C-terminal to the core .alpha.II
epitope is important for the optimal immunological properties of
the 33-mer. This is also supported by the observation that the
doubly deamidated analogue Q17EQ24E (16) approximates the 33-mer
binding characteristics.
[0162] Taken together, the above data support that a peptide
comprising: (i) the first .alpha.II epitope of the 33-mer, (ii) the
N-terminal sequence, and (iii) adequate C-terminal sequence to
include at least one additional deamidation site, would be the
shortest 33-mer analogue that could capture the essential DQ2
binding characteristics of the natural product. To test this
hypothesis, we synthesized the 20-mer peptide 5, and quantitatively
compared its ability to bind DQ2 in peptide exchange and
dissociation assays. The results, summarized in FIG. 8 show that
peptide 5 is comparable to the 33-mer peptide 1 in all in vitro
assays. The immunological relevance of these biochemical findings
was verified in T cell proliferation assays using a polyclonal
T-cell line that responds to all three .alpha. epitopes equally
well and a clonal T-cell line that responds to the .alpha.II
epitope exclusively. As shown in FIG. 9A, the T cell stimulatory
characteristics of peptides 1 and 5 are very similar, both of which
are substantially more potent T cell antigens than the minimal
.alpha.II epitope peptide 3. To verify that the enhanced T cell
antigenicity of peptides 1 and 5 is due to superior DQ2 binding
affinity rather than the ability of these peptides to stimulate a
larger population of .alpha.-epitope responsive T cells, the
experiment was repeated using a clonal T cell line specific for the
.alpha.II epitope. FIG. 2B shows that peptides 1 and 5 again have a
comparable T cell stimulatory capacity that exceeds that of peptide
3. Thus, in view of its considerably shorter length, peptide 5
serves as a lead for the design of HLA-DQ2 blocking ligands for
treatment of Celiac Sprue.
[0163] Design and in vitro evaluation of DQ2 blocking peptides. A
major goal of this research is to design and characterize
medicinally appropriate ligands that form tight, long-lived
complexes with HLA-DQ2 on the surface of antigen presenting cells
in the Celiac small intestine, but are not recognized by disease
specific T cells. Toward this end we synthesized analogues of the
20-mer peptide 5, and evaluated their DQ2 binding properties. Our
initial design targeted L11 and L18 residues of peptide 5 as
modification sites. L11 was chosen based on the crystal structure
of the .alpha.I-DQ2 complex, which suggested that the residue at
the corresponding position (i.e. the P5 Pro residue) points away
from the DQ2 protein surface. Consistent with this observation,
epitope scanning experiments have also shown that the antigen
binding pocket of DQ2 can accommodate a spectrum of amino acids at
the P5 position. Therefore, the L11K analogue (17) shown in FIG. 10
was synthesized. In addition, since peptide 5 can bind to HLA-DQ2
in the .alpha.I (SEQ ID NO:17) (PFPQPELPY) and .alpha.II (SEQ ID
NO:18) (PQPELPYPQ) epitope registers, we also wished to introduce
modifications in the downstream .alpha.III epitope. For this reason
L18K (18) was also synthesized. (Like the P5 residue, the P7
residue also points away from the DQ2 protein surface.)
[0164] Surprisingly, both 17 and 18 bound poorly to DQ2 at either
pH 5 or pH 7 (FIGS. 11A and 11B). We speculated that this poor
affinity may be due to formation of a salt bridge between the Lys
residue at positions 11 or 18 and the critical Glu residue at the
preceding position. To test this hypothesis, the Lys side chains in
both compounds were extended with a succinyl group, yielding
peptides 19 and 20 (FIG. 3) with a negative charge at residues 11
and 18, respectively. As shown in FIGS. 11A and 11B, peptides 19
and 20 bound to DQ2 with considerably higher affinity. We therefore
also synthesized peptide 21, which possesses the above changes at
both positions 11 and 18. Peptide 21 bound comparably well with DQ2
as peptide 5, both in exchange experiments (FIG. 4C) and
dissociation experiments.
[0165] A number of independent investigations have highlighted the
feasibility of greatly enhancing ligand avidity to a biological
target by the engineering of multivalent ligands. Therefore, in an
attempt to further improve DQ2 affinity, a dimeric ligand (22) was
synthesized by crosslinking monomeric L11 K via a hexa-ethylene
glycol (6-PEG) bifunctional linker through the Lys side chains
(FIG. 10). Remarkably, this ligand has considerably improved
binding affinity for DQ2 as compared to all gluten peptides
evaluated thus far, including the 33-mer peptide 1 (FIG. 12).
[0166] Since the modified side-chains of ligands 19-22 are oriented
toward the T cell face of the DQ2-peptide complex, we anticipated
that these modifications were likely to alter the T cell
recognition properties of these peptides. As an initial test of
this hypothesis, peptides 5 and 19-21 were labeled with biotin at
their N termini and mixed with live VAVY (DQ2 homozygous) antigen
presenting cells. The extent to which these peptides were presented
on the cell surface was visualized using fluorescent streptavidin.
After 24 h, the intensity of staining of the VAVY cell surface was
found to be comparable by confocal microscopy in the presence of
all labeled peptides.
[0167] Next, we wished to investigate the extent to which compounds
19-22 were able to elicit a proliferative response from gluten
responsive polyclonal T cell lines derived from small intestinal
biopsies of Celiac Sprue patients. For this experiment, a T cell
line that is strongly responsive to the 33-mer peptide 1 (or
alternately peptide 5) was used. As shown in FIG. 13, peptide 20
elicited the strongest T cell response, presumably because it
contained an unmodified .alpha.II (SEQ ID NO:18)(PQPELPYPQ)
epitope. A low but measurable T cell response was also observed for
peptides 19 and 22, perhaps because they contained an unmodified
.alpha.III (SEQ ID NO:58)(PYPQPELPY) epitope.
[0168] In contrast, the doubly modified peptide 21, which lacks any
of the intact .alpha.-gliadin epitopes, completely abrogates T cell
recognition. Together, these results suggest that compounds 21 and
22 can be presented on the surface of DQ2 antigen presenting cells,
but are relatively unrecognized by gluten specific T cells found in
the small intestines of Celiac Sprue patients. Given their
intrinsic proteolytic stability, these peptides were therefore
evaluated as prototypical DQ2 blocking agents.
[0169] To assess the DQ2 blocking properties of the most promising
compounds, 21 and 22, paraformaldehyde-fixed VAVY cells were
incubated for 12 hours with varying concentrations of antigenic
peptide 5 in the presence or absence of 5 .mu.M blocker peptide,
and the resulting antigen presenting cells were then mixed with
gluten responsive T cells under appropriate culture conditions
(FIG. 14). A significant increase in EC.sub.50 was observed in the
presence of compounds 21 and 22, indicating that these synthetic
peptides were indeed capable of competitively blocking T cell
proliferation by potent gluten antigens such as peptide 5.
Discussion
[0170] Several recent reports have highlighted the exceptional
pathogenic characteristics of a 33-mer peptide from .alpha.2
gliadin, (SEQ ID NO:12) LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, using
disease specific T cells derived from small intestinal biopsies of
Celiac Sprue patients. The goal of this research is to evaluate the
feasibility of transforming this potent pro-inflammatory natural
product into a fundamentally new type of anti-inflammatory agent
for potential use in Celiac Sprue patients. To this end we have
dissected the structure-function relationships of the 33-mer
peptide, and have harnessed these insights, together with our
recent X-ray crystal structure of HLA-DQ2, to design prototypical
DQ2 blocking ligands.
[0171] The 33-mer peptide is a multivalent antigen possessing at
least six overlapping copies of three distinct epitopes (designated
earlier as .alpha.I, .alpha.II and .alpha.III epitopes,
corresponding to the sequences (SEQ ID NO:17) PFPQPELPY, (SEQ ID
NO:18) PQPELPYPQ and (SEQ ID NO:58) PYPQPELPY, respectively).
However, its predominant mode of DQ2 interaction involves formation
of stable monomeric complexes (1:1 stoichiometry). Detailed
equilibrium binding and kinetic analysis of the 33-mer peptide to
HLA-DQ2 led us to hypothesize that this peptide derives its DQ2
avidity through a combination of interactions between one or more
core epitopes as well as flanking sequences. Therefore, a first
objective of this study was to identify the preferred binding
epitope(s), the structural determinants in the flanking sequences,
and the precise contributions of the three Glu residues, each of
which is generated by post-translational deamidation of a naturally
occurring Gln residue by the human transglutaminase 2 enzyme.
[0172] We synthesized truncated analogues (peptides 2, 3, 6-10) as
well as site-directed variants (peptides 11-16) of the 33-mer
peptide 1 (Table 1), and tested their ability to displace a
pre-existing ligand in the binding pocket of a purified, soluble
form of HLA-DQ2 (FIG. 8). Our findings suggest that the .alpha.II
epitope centered at E10 is the most preferred register for 33-mer
binding to DQ2. This is presumably due to the higher avidity of the
.alpha.II epitope for DQ2 as compared to .alpha.I and .alpha.III
epitopes, as well as the fact that the first .alpha.II epitope in
the 33-mer peptide can uniquely leverage secondary interactions
between the N-terminal (SEQ ID NO:32) LQLQPF sequence and a yet to
be determined binding site on HLA-DQ2. Additionally, a second
deamidation site located in the C-terminal sequence also
facilitates DQ2 binding of this .alpha.II epitope. Thus, the 20-mer
peptide 5 binds to DQ2 equally well as the 33-mer peptide 1,
whereas peptide 6 (which lacks the N-terminal flank) has a
considerably lower affinity for DQ2 than peptide 5 (which has this
flanking sequence). The good correlation between DQ2 binding and T
cell proliferative capacity was also verified by showing that
peptide 5 has comparable T cell antigenicity as 1 (FIG. 9).
[0173] The X-ray crystal structure of HLA-DQ2 bound to the .alpha.I
gliadin epitope had revealed that some amino acid side chains from
the epitope are deeply buried in the DQ2 binding pocket, whereas
others face outward, presumably in the direction of the T cell
receptor. L11 and L18 in peptide 5 are examples of the latter
category of residues, regardless of whether they are recognized by
DQ2 as part of an .alpha.II epitope (where they bind in the P5
binding pocket) or the .alpha.I or .alpha.III epitopes (where they
bind in the P7 pocket). Leu->Lys substitutions at these
positions therefore provided functional handles for further
modification.
[0174] Analogues (FIG. 10, peptides 17-21) of peptide 5 were
evaluated; of these, compounds 19, 20, and 21 had similar affinity
for HLA-DQ2 as unsubstituted peptide 5 (FIG. 11). In T cell assays,
whereas peptides 19 and 20 did retain some ability to stimulate the
proliferation of gluten specific T cells from intestinal biopsies
of Celiac Sprue patients, the doubly modified peptide 21 is unable
to stimulate an .alpha.I, .alpha.II, and .alpha.III epitope
responsive T cell line (FIG. 13). In the presence of 5 .mu.M 21,
the EC.sub.50 of antigenic peptide 5 against gluten specific T
cells is increased.
[0175] An alternative design of potential DQ2 blocking agents
involved dimerization of peptide 17 through the Lys-11 side chain.
This strategy has borne fruit in the study of protein-protein
interactions in other biological contexts. Our prototypical dimeric
peptide 22 with a hexa-ethylene glycol bifunctional linker had a
substantially enhanced affinity for HLA-DQ2 than both peptides 1
and 5 at pH 5 or pH 7. Moreover, compound 22 showed remarkably
enhanced binding kinetics, reaching half maximal DQ2 occupancy at
2.5 hr instead of .about.8 hr for the 33-mer peptide 1 (FIG. 5).
This rapid binding capacity could be a pharmacologically useful
property in the context of inhibiting antigen presentation. In
support of this anti-inflammatory potential, addition of 5 .mu.M
compound 22 also resulted in a significant elevation of the
EC.sub.50 of antigenic peptide 5.
[0176] In summary, our studies reported here have led to the
development of an improved insight into the structure-activity
relationships of the highly immunogenic 33-mer peptide from
.alpha.2-gliadin, and in turn, to the design of peptidic analogues
of this peptide that bind tightly to HLA-DQ2 but are not recognized
by Celiac Sprue associated T cells. The design of future
generations of such DQ2 blocking agents will require in-depth
biological evaluations of these promising synthetic agents.
[0177] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0178] The present invention has been described in terms of
particular embodiments found or proposed by the present 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
protein structure without affecting the biological action in kind
or amount. All such modifications are intended to be included
within the scope of the appended claims.
* * * * *