U.S. patent application number 14/852425 was filed with the patent office on 2016-03-17 for aquaporin-4 peptide compositions and methods of use.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Bruce Anthony Campbell Cree, Michel Varrin-Doyer, SCOTT S. ZAMVIL.
Application Number | 20160075748 14/852425 |
Document ID | / |
Family ID | 51165305 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160075748 |
Kind Code |
A1 |
ZAMVIL; SCOTT S. ; et
al. |
March 17, 2016 |
Aquaporin-4 Peptide Compositions and Methods of Use
Abstract
The present disclosure provides human Aquaporin 4 (AQP4)
peptides and peptides having homology to human Aquaporin 4 (AQP4)
peptides. Also provided herein are methods for using human AQP4
peptides and peptides homologous to human AQP4 peptides for
diagnosing and/or treating Neuromyelitis Optica.
Inventors: |
ZAMVIL; SCOTT S.; (Palo
Alto, CA) ; Varrin-Doyer; Michel; (San Francisco,
CA) ; Cree; Bruce Anthony Campbell; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
51165305 |
Appl. No.: |
14/852425 |
Filed: |
September 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14102294 |
Dec 10, 2013 |
9234017 |
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14852425 |
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61735675 |
Dec 11, 2012 |
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Current U.S.
Class: |
424/185.1 ;
435/34; 435/7.24; 530/324; 530/325; 530/326; 530/327; 530/328 |
Current CPC
Class: |
G01N 2469/10 20130101;
A61K 39/0008 20130101; C07K 7/06 20130101; G01N 2800/285 20130101;
G01N 2500/10 20130101; G01N 33/505 20130101; C07K 14/705 20130101;
G01N 2800/28 20130101; G01N 33/56972 20130101; G01N 2333/70514
20130101; C07K 7/08 20130101; G01N 33/5094 20130101; C07K 14/47
20130101; A61K 2039/577 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; G01N 33/50 20060101 G01N033/50; A61K 39/00 20060101
A61K039/00; G01N 33/569 20060101 G01N033/569; C07K 7/06 20060101
C07K007/06; C07K 7/08 20060101 C07K007/08 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This inventions was made with government support under Grant
Nos. AI073737, AI059709, NS063008, and GM073210 awarded by the
National Institutes of Health. The federal government has certain
rights in this invention.
Claims
1. A composition comprising: a peptide comprising a contiguous
stretch of amino acids having the consensus amino acid sequence:
TABLE-US-00007
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
wherein the peptide is up to 50 amino acids in length, and wherein
X.sup.1 is Val (V) or Ile (I), X.sup.2 is Ser (S) or Asp (D),
X.sup.3 is Val (V), Ile (I), or Gly (G), X.sup.4 is Leu (L) or Ile
(I), X.sup.5 is Met (M) or Ser (S), and X.sup.6 is Leu (L), Val
(V), or Ala (A).
2. The composition of claim 1, wherein: X.sup.1 is Val (V), X.sup.2
is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4 is Leu (L),
X.sup.5 is Ser (S), and X.sup.6 is Leu (L).
3. A composition comprising: a peptide comprising a contiguous
stretch of amino acids having the consensus amino acid sequence:
TABLE-US-00008 LPVXMVLISL
wherein the peptide is up to 50 amino acids in length, and wherein
X is Asp or Ser.
4. A composition comprising a peptide comprising a contiguous
stretch of amino acids having the sequence: TABLE-US-00009
GILYLVTPPSVVGGLGVTMV
wherein the peptide is up to 50 amino acids in length.
5. A composition comprising a peptide comprising a contiguous
stretch of amino acids having the sequence: TABLE-US-00010
SMNPARSFGPAVIMGNWENH
wherein the peptide is up to 50 amino acids in length.
6. A composition comprising a peptide comprising a contiguous
stretch of amino acids having the sequence: TABLE-US-00011
AGHGLLVELIITFQL
wherein the peptide is up to 50 amino acids in length.
7. A composition comprising a peptide comprising a contiguous
stretch of amino acids having the sequence: TABLE-US-00012
RFKEAFSKAAQQTKGSYMEV
wherein the peptide is up to 50 amino acids in length.
8. The composition of claim 1, wherein the peptide is 5-30 amino
acids in length.
9. The composition of claim 1, wherein the peptide is 10-20 amino
acids in length.
10. A method of diagnosing Neuromyelitis Optica (NMO) in a subject,
the method comprising: contacting a sample from the subject with a
peptide of claim 1; and measuring the number of T cells, wherein an
increase in the number of T cells as compared to a control
indicates that the subject has NMO.
11. The method of claim 10, wherein the T cells are T helper 17
(Th17) T cells.
12. The method of claim 10, wherein the T cells are CD4.sup.+ T
cells.
13. The method of claim 10, wherein the peptide is 5-30 amino acids
in length.
14. The method of claim 10, wherein the peptide is 10-20 amino
acids in length.
15. A method for screening for candidate agents for inhibiting
proliferation of T cells, the method comprising: contacting a T
cell obtained from a subject having Neuromyelitis Optica with: a
peptide claim 1, and a candidate agent; measuring the number of T
cells, wherein a decrease in the number of T cells as compared to a
control indicates that the candidate agent inhibits proliferation
of T cells.
16. The method of claim 15, wherein the T cells are T helper 17
(Th17) T cells.
17. The method of claim 15, wherein the T cells are CD4.sup.+ T
cells.
18. The method of claim 15, wherein the peptide is 5-30 amino acids
in length.
19. The method of claim 15, wherein the peptide is 10-20 amino
acids in length.
20. A method for inducing immune tolerance to AQP-4 protein and
fragments thereof in a subject, the method comprising:
administering an effective dose of the composition of claim 1 to a
subject, wherein the administering the peptide induces immune
tolerance to AQP-4 protein and fragments thereof in the
subject.
21. The method of claim 20, wherein the peptide is 5-30 amino acids
in length.
22. The method of claim 20, wherein the peptide is 10-20 amino
acids in length.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit to U.S. Provisional
Application Ser. No. 61/735,675, filed Dec. 11, 2012, the
disclosure of which is herein incorporated by reference in its
entirety.
TECHNICAL FIELD
[0003] This present disclosure relates to peptides having homology
to human Aquaporin 4 (AQP4) peptides and peptides having homology
to human Aquaporin-4 (AQP-4) peptides and the use of theses
peptides in diagnosing, and/or treating neuromyelitis optica
(NMO).
INTRODUCTION
[0004] Neuromyelitis optica (NMO), or Device's disease, is an
autoimmune, inflammatory disorder of the optic nerves and spinal
cord. The main symptoms of Device's disease are loss of vision and
spinal cord function. As for other etiologies of optic neuritis,
the visual impairment usually manifests as decreased visual acuity,
although visual field defects, or loss of color vision can occur in
isolation or prior to formal loss of acuity. Spinal cord
dysfunction can lead to muscle weakness, reduced sensation, or loss
of bladder and bowel control. The damage in the spinal cord can
range from inflammatory demyelination to necrotic damage of the
white and grey matter. The inflammatory lesions in Device's disease
have been classified as type II lesions (complement mediated
demyelination).
[0005] Attacks optic neuritis and transverse myelitis suffered by
NMO patients are treated with short courses of high dosage
intravenous corticosteroids such as methylprednisolone IV. When
attacks progress or do not respond to corticosteroid treatment,
plasmapheresis can be used. Commonly used immunosuppressant
treatments include azathioprine (Imuran) plus prednisone,
mycophenolate mofetil plus prednisone, Rituximab, Mitoxantrone,
intravenous immunoglobulin (WIG), and Cyclophosphamide.
[0006] The disease can be monophasic, i.e., a single episode with
permanent remission. However, at least 85% of NMO patients have a
relapsing form of the disease with repeated attacks of transverse
myelitis and/or optic neuritis. In patients with the monophasic
form of NMO, the transverse myelitis and optic neuritis occur
simultaneously or within days of each other. On the other hand,
patients with the relapsing form of NMO are more likely to have
weeks or months between the initial attacks and to have better
motor recovery after the initial transverse myelitis event.
Relapses usually occur early with about 55% of patients having a
relapse in the first year and 90% in the first 5 years.
[0007] Studies have suggested a role for T helper 17 (Th17) cells
and autoantigens, including aquaporin and myelin proteins in the
pathology of NMO. Th17 T cells are a subset of T helper cells,
characterized by their production of interleukin 17 (IL-17). They
are considered developmentally distinct from Th1 and Th2 cells and
excessive amounts of the cells are thought to play a key role in
autoimmune disease.
[0008] Provided herein are peptides, peptide compositions and
methods useful in diagnosing and/or treating NMO.
SUMMARY
[0009] The present disclosure provides human Aquaporin 4 (AQP4)
peptides and peptides having homology to human Aquaporin 4 (AQP4)
peptides. Also provided herein are methods for using human AQP4
peptides and peptides homologous to human AQP4 peptides for
diagnosing and/or treating Neuromyelitis Optica.
[0010] In certain embodiments, a composition is provided. The
composition includes a peptide comprising a contiguous stretch of
amino acids having the consensus amino acid sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6,
wherein the peptide is up to 50 amino acids in length, and wherein
X.sup.1 is Val (V) or Ile (I), X.sup.2 is Ser (S) or Asp (D),
X.sup.3 is Val (V), Ile (I), or Gly (G), X.sup.4 is Leu (L) or Ile
(I), X.sup.5 is Met (M) or Ser (S), and X.sup.6 is Leu (L), Val
(V), or Ala (A). In certain embodiments, X.sup.1 is Val (V),
X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4 is Leu
(L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L).
[0011] In another embodiment, a composition that includes a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence: LPVXMVLISL, wherein the peptide is up to 50
amino acids in length, and wherein X is Asp or Ser, is
provided.
[0012] In another embodiment, a composition that includes a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence: GILYLVTPPSVVGGLGVTMV, wherein the peptide is
up to 50 amino acids in length, is provided.
[0013] In another embodiment, a composition that includes a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence: SMNPARSFGPAVIMGNWENH, wherein the peptide is
up to 50 amino acids in length, is provided.
[0014] In another embodiment, a composition that includes a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence: AGHGLLVELIITFQL, wherein the peptide is up to
50 amino acids in length, is provided.
[0015] In another embodiment, a composition that includes a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence: RFKEAFSKAAQQTKGSYMEV, wherein the peptide is
up to 50 amino acids in length, is provided.
[0016] In certain embodiments, the peptide may be 5-30 amino acids
in length. In some embodiments, the peptide may be 10-20 amino
acids in length.
[0017] In certain embodiments, a method of diagnosing Neuromyelitis
Optica (NMO) in a subject is disclosed. The method may include
contacting a sample from the subject with a peptide as disclosed
herein; and measuring the number of T cells, wherein an increase in
the number of T cells as compared to a control indicates that the
subject has NMO.
[0018] In certain cases, the T cells may be T helper 17 (Th17) T
cells. In certain cases, the T cells may be CD4.sup.+ T cells. The
peptide may be 5-30 amino acids in length. In certain cases, the
peptide may be 10-20 amino acids in length.
[0019] In certain embodiments, a method for screening for candidate
agents for inhibiting proliferation of T cells is provided. The
method includes contacting a T cell obtained from a subject having
Neuromyelitis Optica with a peptide as provided above, and a
candidate agent; measuring the number of T cells, wherein a
decrease in the number of T cells as compared to a control
indicates that the candidate agent inhibits proliferation of T
cells. In certain embodiments, the T cells may be T helper 17
(Th17) T cells. In certain embodiments, the T cells may be
CD4.sup.+ T cells. The peptide may be 5-30 amino acids in length.
In certain cases, the peptide may be 10-20 amino acids in
length.
[0020] A method for inducing immune tolerance to AQP-4 protein and
fragments thereof in a subject is also provided. The method
includes administering an effective dose of the composition set
forth above to a subject, wherein the administering the peptide
induces immune tolerance to AQP-4 protein and fragments thereof in
the subject. The peptide may be 5-30 amino acids in length. In
certain cases, the peptide may be 10-20 amino acids in length.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A-1F shows that T cells from NMO patients recognize
discrete determinants of AQP4.
[0022] FIG. 2A-2C shows that HLA-DR serves as a restriction element
for AQP4-specific T cells.
[0023] FIG. 3A-3E illustrates the cross-reactivity between AQP4
p63-76 and Clostridium perfringens ABC transporter permease
(ABC/TP) p204-217.
[0024] FIG. 4A-4D shows that AQP4 p61-80-specific T cells exhibit a
pro-inflammatory bias.
[0025] FIG. 5A-5C shows CD14+ monocytes from NMO patients exhibit
increased expression of certain co-stimulatory molecules and
production of IL-6.
[0026] FIG. 6 provides the amino acid sequence of human AQP-4
protein (GenBank Accession No. AAH22286.1).
[0027] FIG. 7 provides the amino acid sequence of ABC-transporter
permease protein of Clostridium perfringens (NCBI Reference
Sequence: ZP.sub.--02952885.1).
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0028] The present disclosure provides human Aquaporin 4 (AQP4)
peptides and peptides having homology to human Aquaporin 4 (AQP4)
peptides. Also provided herein are methods for using human AQP4
peptides and peptides homologous to human AQP4 peptides for
diagnosing and/or treating Neuromyelitis Optica.
[0029] Before the present invention and specific exemplary
embodiments of the invention are described, it is to be understood
that this invention is not limited to particular embodiments
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0030] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. To the extent
such publications may set out definitions of a term that conflicts
with the explicit or implicit definition of the present disclosure,
the definition of the present disclosure controls.
[0032] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a sample" includes a plurality of samples
and reference to "a peptide" includes reference to one or more
peptides, and so forth.
[0033] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0034] Definitions
[0035] The phrase "Aquaporin-4 (AQP-4) peptides" as used herein
refers to peptides that are derived from amino acid sequence of
human aquaporin-4. As such, AQP-4 peptides include peptides
comprising a contiguous amino acid sequence that is identical to a
contiguous amino acid sequence found in amino acid sequence of
human aquaporin-4 (GenBank Accession No. AAH22286.1). An AQP-4
peptide may be 5-50 amino acids in length.
[0036] The phrase "AQP-4 homologous peptide" or a grammatical
equivalent thereof, as used herein, refers to peptides that include
a contiguous amino acid sequence that is at least 50%, at least
60%, at least 70%, at least 80%, or at least 90% identical to a
contiguous amino acid sequence found in amino acid sequence of
human AQP-4 protein. For example, a twenty amino acids long peptide
that is at least 50% at least 60%, at least 70%, at least 80%, or
at least 90% identical to a contiguous amino acid sequence found in
amino acid sequence of human aquaporin-4 is an example of an AQP-4
homologous peptide.
[0037] The term "unit dosage form", as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
peptide(s) disclosed herein 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 may depend on the particular peptide employed and
the effect to be achieved, and the pharmacodynamics associated with
each peptide in the host.
[0038] An "immunogen" is capable of inducing an immunological
response against itself on administration to a mammal or due to
autoimmune disease.
[0039] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein refers to
any treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease or symptom from occurring in a
subject which may be predisposed to the disease or symptom but has
not yet been diagnosed as having it; (b) inhibiting the disease
symptom, i.e., arresting its development; or (c) relieving the
disease symptom, i.e., causing regression of the disease or
symptom.
[0040] The terms "individual", "subject", "host", and "patient",
are used interchangeably herein and refer to any mammalian subject
for whom diagnosis, treatment, or therapy is desired, particularly
humans. Mammals other than humans can be advantageously used as
subjects that represent animal models of inflammation. A subject
can be male or female.
[0041] The term "a sample" as used herein refers to any biological
sample that is isolated from a subject. A sample can include,
without limitation, a single cell or multiple cells or cell
fragments, such as, platelets, red blood cells, white blood cells
or leucocytes, such as lymphocytes, e.g., B cell, T cells,
endothelial cells, cell lysates, such as, whole cell lysates or a
fraction of cell lysate; an aliquot of body fluid, whole blood,
serum, plasma, synovial fluid, lymphatic fluid, ascites fluid, and
interstitial or extracellular fluid; tissue biopsies. "Blood
sample" can refer to whole blood or any fraction thereof, including
blood cells, red blood cells, white blood cells or leucocytes,
platelets, serum and plasma. Samples can be obtained from a subject
by means including but not limited to venipuncture, biopsy, needle
aspirate, scraping, surgical incision, or intervention or other
means known in the art.
[0042] An "isolated" peptide is one which has been separated and/or
recovered from a component of the environment in which it was
produced. Contaminant components of its production environment are
materials which would interfere with screening, diagnostic or
therapeutic uses for the peptide, and may include chemicals,
enzymes, hormones, and other proteinaceous or nonproteinaceous
components. In certain cases, the peptides of the present
disclosure are produced synthetically, for example, by chemical
synthesis. In certain embodiments, the peptides are purified using
standard purification procedures known in the art. Ordinarily,
isolated peptide may be prepared by at least one purification
step.
[0043] The term "agent" as used in the context of candidate agent
refers to a chemical compound, a mixture of chemical compounds, a
biological macromolecule, or an extract made from biological
materials.
[0044] The term "effective amount" refers to an amount of a
biologically active molecule or conjugate or derivative thereof
sufficient to exhibit a detectable therapeutic effect without undue
adverse side effects (such as toxicity, irritation and allergic
response) commensurate with a reasonable benefit/risk ratio. The
effective amount for a patient may depend upon the type of patient,
the patient's size and health, the nature and severity of the
condition to be treated, the method of administration, the duration
of treatment, the nature of concurrent therapy (if any), the
specific formulations employed, and the like. The effective amount
for a given subject can be determined by one of ordinary skill in
the art using routine experimentation based on the information
provided herein.
[0045] As used herein, the term "concurrent therapy" is used
interchangeably with the terms "combination therapy" and "adjunct
therapy", and will be understood to mean that the patient in need
of treatment is treated or given another drug for the
disease/disorder in conjunction with the compositions of the
present disclosure. This concurrent therapy can be sequential
therapy where the patient is treated first with one drug and then
the other, or the two drugs are given simultaneously
[0046] The term "pharmaceutically acceptable" refers to peptides
and compositions containing peptides which are suitable for
administration to humans and/or animals without undue adverse side
effects such as toxicity, irritation and/or allergic response
commensurate with a reasonable benefit/risk ratio.
[0047] The compositions comprising peptides disclosed herein may be
administered to a patient by any method known in the art, including
but not limited to, oral, topical, transdermal, parenteral,
subcutaneous, intranasal, intramuscular, intraperitoneal,
intravenous, intraocular, intravitreal, sub-retinal routes,
including both local and systemic applications. In addition, the
peptides disclosed herein may be formulated to provide delayed,
controlled or sustained release using formulation techniques which
are well known in the art.
[0048] Overview
[0049] The present disclosure provides peptides, including AQP-4
peptides derived from human AQP-4 protein and peptides homologous
to AQP-4 peptides. As shown herein, AQP-4 peptide p61-80 includes a
core sequence p63-76 (EKPLPVDMVLISLC) which is a T cell epitope.
Peptides containing this core sequence or a sequence having at
least 60% identity to the amino acid sequence of this core sequence
result in proliferation of AQP-4 specific T cells in NMO
patients.
[0050] Peptides
[0051] In certain embodiments, the present disclosure provides
AQP-4 peptides and peptides homologous to AQP-4 peptides.
[0052] For example, examples of ABC-TP proteins from strains of
Clostridium perfringens, Clostridium scindens, Clostridium
sporogens, and Clostridium hylemonae are aligned in Table 1 over a
region having sequence identity with an example of an AQP4 peptide,
p66-75 (LPVDMVLISL).
TABLE-US-00001 TABLE 1 % Homology with AQP4 p66-75 AqP4 p66-75 L P
V D M V L I S L C. perfringens L P V S M 90% Commensal and V L I S
L pathogenic C. Scindens L P V S M 70% Commensal G L I S V C.
Sporogenes L P V S M 60% Pathogenic I I I M L C. Hylemonae L P I S
M 60% Commensal G L I S A
[0053] Accordingly, AQP-4 peptides comprise a contiguous stretch of
amino acids having the consensus amino acid sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
(Formula I) [0054] wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4,
X.sup.5, and X.sup.6 are any amino acid, and [0055] wherein the
peptide is up to 50 amino acids in length.
[0056] In certain embodiments, X.sup.1 is Val (V) or Ile (I). In
certain embodiments, X.sup.2 is Ser (S) or Asp (D). In certain
embodiments, X.sup.3 is Val (V), Ile (I), or Gly (G). In certain
embodiments, X.sup.4 is Leu (L) or Ile (I). In certain embodiments,
X.sup.5 is Met (M) or Ser (S). In certain embodiments, X.sup.6 is
Leu (L), Val (V), or Ala (A). In certain embodiments, X.sup.1 is
Val (V), X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4
is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L). In certain
embodiments, X.sup.1 is Val (V), X.sup.2 is Asp (D), X.sup.3 is Val
(V), X.sup.4 is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu
(L). In such embodiments, the peptide comprises a contiguous
stretch of amino acids having the consensus amino acid sequence:
Leu-Pro-Val-Asp-Met-Val-Leu-Ile-Ser-Leu.
[0057] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Val-Leu-Ile-Ser-Leu.
[0058] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Val (V). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Gly-Leu-Ile-Ser-Val.
[0059] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Ile (I), X.sup.4 is Ile (I), X.sup.5 is Met (M),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Ile-Ile-Ile-Met-Leu.
[0060] In certain embodiments, X.sup.1 is Ile (I), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Ala (A). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Ile-Ser-Met-Gly-Leu-Ile-Ser-Ala.
[0061] Examples of suitable embodiments include where: X.sup.1 is
Val and X.sup.2 is Ser, X.sup.1 is Val and X.sup.2 is Asp, X.sup.1
is Val and X.sup.3 is Val, X.sup.1 is Val and X.sup.3 is Gly,
X.sup.1 is Val and X.sup.3 is Ile, X.sup.1 is Val and X.sup.4 is
Leu, X.sup.1 is Val and X.sup.4 is Ile, X.sup.1 is Val and X.sup.5
is Ser, X.sup.1 is Val and X.sup.5 is Met, X.sup.1 is Val and
X.sup.6 is Leu, X.sup.1 is Val and X.sup.6 is Val, X.sup.1 is Val
and X.sup.6 is Ala, X.sup.1 is Ile and X.sup.2 is Ser, X.sup.1 is
Ile and X.sup.2 is Asp, X.sup.1 is Ile and X.sup.3 is Val, X.sup.1
is Ile and X.sup.3 is Gly, X.sup.1 is Ile and X.sup.3 is Ile,
X.sup.1 is Ile and X.sup.4 is Leu, X.sup.1 is Ile and X.sup.4 is
Ile, X.sup.1 is Ile and X.sup.5 is Ser, X.sup.1 is Ile and X.sup.5
is Met, X.sup.1 is Ile and X.sup.6 is Leu, X.sup.1 is Ile and
X.sup.6 is Val, X.sup.1 is Ile and X.sup.6 is Ala, X.sup.2 is Ser
and X.sup.3 is Val, X.sup.2 is Ser and X.sup.3 is Gly, X.sup.2 is
Ser and X.sup.3 is Ile, X.sup.2 is Ser and X.sup.4 is Leu, X.sup.2
is Ser and X.sup.4 is Ile, X.sup.2 is Ser and X.sup.5 is Ser,
X.sup.2 is Ser and X.sup.5 is Met, X.sup.2 is Ser and X.sup.6 is
Leu, X.sup.2 is Ser and X.sup.6 is Val, X.sup.2 is Ser and X.sup.6
is Ala, X.sup.2 is Asp and X.sup.3 is Val, X.sup.2 is Asp and
X.sup.3 is Gly, X.sup.2 is Asp and X.sup.3 is Ile, X.sup.2 is Asp
and X.sup.4 is Leu, X.sup.2 is Asp and X.sup.4 is Ile, X.sup.2 is
Asp and X.sup.5 is Ser, X.sup.2 is Asp and X.sup.5 is Met, X.sup.2
is Asp and X.sup.6 is Leu, X.sup.2 is Asp and X.sup.6 is Val,
X.sup.2 is Asp and X.sup.6 is Ala, X.sup.3 is Val and X.sup.4 is
Leu, X.sup.3 is Val and X.sup.4 is Ile, X.sup.3 is Val and X.sup.5
is Ser, X.sup.3 is Val and X.sup.5 is Met, X.sup.3 is Val and
X.sup.6 is Leu, X.sup.3 is Val and X.sup.6 is Val, X.sup.3 is Val
and X.sup.6 is Ala, X.sup.3 is Gly and X.sup.4 is Leu, X.sup.3 is
Gly and X.sup.4 is Ile, X.sup.3 is Gly and X.sup.5 is Ser, X.sup.3
is Gly and X.sup.5 is Met, X.sup.3 is Gly and X.sup.6 is Leu,
X.sup.3 is Gly and X.sup.6 is Val, X.sup.3 is Gly and X.sup.6 is
Ala, X.sup.3 is Ile and X.sup.4 is Leu, X.sup.3 is Ile and X.sup.4
is Ile, X.sup.3 is Ile and X.sup.5 is Ser, X.sup.3 is Ile and
X.sup.5 is Met, X.sup.3 is Ile and X.sup.6 is Leu, X.sup.3 is Ile
and X.sup.6 is Val, X.sup.3 is Ile and X.sup.6 is Ala, X.sup.4 is
Leu and X.sup.5 is Ser, X.sup.4 is Leu and X.sup.5 is Met, X.sup.4
is Leu and X.sup.6 is Leu, X.sup.4 is Leu and X.sup.6 is Val,
X.sup.4 is Leu and X.sup.6 is Ala, X.sup.4 is Ile and X.sup.5 is
Ser, X.sup.4 is Ile and X.sup.5 is Met, X.sup.4 is Ile and X.sup.6
is Leu, X.sup.4 is Ile and X.sup.6 is Val, X.sup.4 is Ile and
X.sup.6 is Ala, X.sup.5 is Ser and X.sup.6 is Leu, X.sup.5 is Ser
and X.sup.6 is Val, X.sup.5 is Ser and X.sup.6 is Ala, X.sup.5 is
Met and X.sup.6 is Leu, X.sup.5 is Met and X.sup.6 is Val, and
X.sup.5 is Met and X.sup.6 is Ala.
[0062] In certain embodiments, the AQP-4 peptide may comprise a
contiguous stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu (Formula II)
[0063] wherein X is any amino acid.
[0064] In some such embodiments, X is Ser (S) or Asp (D). As such,
in some embodiments, X is Ser (S) and the peptide comprises a
contiguous stretch of amino acids having the consensus amino acid
sequence: LPVSMVLISL. In some embodiments, X is Asp (D) and the
peptide comprises a contiguous stretch of amino acids having the
consensus amino acid sequence: LPVDMVLISL.
[0065] In certain embodiments, the peptide may comprise an amino
acid sequence having at least 50%, at least 60%, at least 70%, at
least 80%, or at least 90% sequence identity to a contiguous
stretch of amino acids in the sequence of AQP-4 peptide p63-76
(EKPLPVDMVLISLC), wherein the peptide is up to 50 amino acids in
length. As used herein, p63-76 in the context of AQP-4 peptide
refers to a peptide having the amino acids sequence of amino acid
starting at position 63 and ending at position 76 of human AQP-4
protein sequence shown in FIG. 6.
[0066] In certain embodiments, the peptide may include a contiguous
stretch of ten amino acids having at least 50%, at least 60%, at
least 70%, at least 80%, or at least 90% sequence identity to a
contiguous stretch of ten amino acids in the sequence of AQP-4
peptide p63-76. In certain embodiments, the peptide may include a
contiguous stretch of ten amino acids having at least 70%, or at
least 80% sequence identity to a contiguous stretch of ten amino
acids of AQP-4 peptide p63-76. In certain embodiments, the peptide
may include a contiguous stretch of ten amino acids having at least
90% sequence identity to a contiguous stretch of ten amino acids of
AQP-4 peptide p63-76.
[0067] In certain embodiments, the peptide may include a contiguous
stretch of ten amino acids having at least 70% sequence identity to
the amino acid sequence of AQP-4 peptide p66-75 (LPVDMVLISL). In
certain embodiments, the peptide may include a contiguous stretch
of ten amino acids having at least 80% sequence identity to the
amino acid sequence of AQP-4 peptide p66-75. In certain
embodiments, the peptide may include a contiguous stretch of ten
amino acids having at least 90% sequence identity to the amino acid
sequence of AQP-4 peptide p66-75. As used herein, p66-75 in the
context of AQP-4 peptide refers to a peptide having the amino acids
sequence of amino acid starting at position 66 and ending at
position 75 of human AQP-4 protein sequence shown in FIG. 6.
[0068] In certain embodiments, the peptide may be AQP-4 peptide
including the AQP-4 p61-80 sequence. In certain embodiments, the
peptide may be AQP-4 p61-80 peptide. As used herein, p61-80 in the
context of AQP-4 peptide refers to a peptide having the amino acids
sequence of amino acid starting at position 61 and ending at
position 80 of human AQP-4 protein sequence shown in FIG. 6. In
certain embodiments, the peptide may be AQP-4 p63-76 peptide. In
certain embodiments, the peptide may be AQP-4 p66-75 peptide
(LPVDMVLISL).
[0069] In certain embodiments, the peptide may be derived from ABC
transporter permease (ABC-TP) protein of a Clostridium species. In
certain cases, the peptide may be a peptide derived from ABC-TP
protein from a strain of bacterium in the genus Clostridium (e.g.,
Clostridium perfringens, Clostridium scindens, Clostridium
sporogens, Clostridium hylemonae, and the like). For example, in
certain cases, the peptide may be a peptide derived from ABC-TP
protein having the accession no. ZP.sub.--02952885.1,
ZP.sub.--02638213.1, ZP.sub.--02634520.1, ZP.sub.--02630305.1,
ZP.sub.--02431563, ZP.sub.--02995934, or ZP.sub.--03776873.1). In
certain cases, the peptide may include a sequence of ABC-TP protein
derived peptide p204-217 (FIILPVSMVLISLV). As used herein, p204-217
in the context of AQP-4 peptide refers to a peptide having the
amino acids sequence of amino acid starting at position 204 and
ending at position 217 of protein sequence shown in FIG. 7. In
certain cases, the peptide may include a sequence of ABC-TP protein
derived peptide p207-216 (LPVSMVLISL). As used herein, p207-216 in
the context of AQP-4 peptide refers to a peptide having the amino
acids sequence of amino acid starting at position 207 and ending at
position 216 of protein sequence shown in FIG. 7. In certain cases,
the peptide may be ABC-TP protein derived peptide p204-217
(FIILPVSMVLISLV). In certain cases, the peptide may be ABC-TP
protein derived peptide p207-216 (LPVSMVLISL). In other cases, the
peptide may be derived from the expressed or predicted ABC-TP
protein in Clostridium species, including commensal bacteria C.
scindens and C. hylemonae as well as the pathogenic strain C.
sporogenes.
[0070] In certain embodiments, the peptide may be 5-50 amino acids
long, such as, 5-40 amino acids long, or 5-30 amino acids long, or
5-25 amino acids long, or 5-20 amino acids long, or 5-15 amino
acids long, or 10-20 amino acids long, or 10-15 amino acids long.
For example, the peptide may be 5 amino acids long, or 7 amino
acids long, or 10 amino acids long, or 15 amino acids long, or 20
amino acids long, or 25 amino acids long, or 30 amino acids long,
or 35 amino acids long, or 40 amino acids long, or 45 amino acids
long, or 50 amino acids long.
[0071] In certain embodiments, the peptide may be a peptide up to
50 amino acids in length (for example, up to 40 amino acids long,
30 amino acids long, such as 10-20 amino acids long, or 10-15 amino
acids long) and including a contiguous stretch of amino acids
having: (i) the sequence of p66-75 (LPVDMVLISL) or (ii) a sequence
at least 90% identical to the amino acid sequence LPVDMVLISL.
[0072] In certain embodiments, the peptide may be a peptide up to
50 amino acids in length (for example, up to 40 amino acids long,
30 amino acids long, such as 10-20 amino acids long, or 10-15 amino
acids long) and including a contiguous stretch of amino acids
having: (i) the sequence of p66-75 (LPVDMVLISL), or (ii) the
sequence of p207-216 (LPVSMVLISL), (iii) or the sequence of
p156-170 (AGHGLLVELIITFQL), (iv) or the sequence of p261-280
(RFKEAFSKAAQQTKGSYMEV).
[0073] In certain cases, the peptides may include amino acid
substitutions compared to the sequence of p61-80. In certain
embodiments, these amino acid substitutions may be conservative
substitutions. By conservative substitutions is intended
substitution of an amino acid with a similar amino acid, such as
those from the following groups: 1) gly, ala; 2) val, ile, leu;
asp, glu; 3) asn, gln; 4) ser, thr; 5) lys, arg; and 6) phe, tyr.
In certain embodiments, the amino acids substitutions may be guided
by the amino acid sequence in AQP-4 protein, predicted AQP-4
protein, or AQP-4 like proteins in other species, such as, mouse,
rat, hamster, non-human primates, and macaques. In certain
embodiments, the amino acids substitutions may be guided by the
amino acid sequence in ABC-TP protein or predicted ABC-TP protein
of Clostridium species, such as, Clostridium perfringens, C.
scindens, C. hylemonae, and C. sporogenes, for example. Predicted
AQP-4 proteins or ABC-TP proteins can be identified by using
methods known in the art, such as, by performing sequence search
using Basic Local Alignment Search Tool (BLAST) with the amino acid
sequence of full length or partial AQP-4 or ABC-TP protein.
[0074] In certain cases, the peptide may be an AQP-4 peptide
comprising a contiguous stretch of amino acids of peptide p131-150
having the sequence:
TABLE-US-00002 GILYLVTPPSVVGGLGVTMV
[0075] wherein the peptide is up to 50 amino acids in length.
[0076] In certain cases, the peptide may be an AQP-4 peptide
comprising a contiguous stretch of amino acids of peptide p211-230
having the sequence:
TABLE-US-00003 SMNPARSFGPAVIMGNWENH
[0077] wherein the peptide is up to 50 amino acids in length.
[0078] In certain cases, the peptide may be an AQP-4 peptide
comprising a contiguous stretch of amino acids of peptide p156-170
having the sequence:
TABLE-US-00004 (AGHGLLVELIITFQL),
[0079] wherein the peptide is up to 50 amino acids in length.
[0080] In certain cases, the peptide may be an AQP-4 peptide
comprising a contiguous stretch of amino acids of peptide p261-280
having the sequence:
TABLE-US-00005 (RFKEAFSKAAQQTKGSYMEV),
[0081] wherein the peptide is up to 50 amino acids in length.
[0082] The peptides disclosed herein may be modified covalently or
non-covalently to increase stability of the peptides, such as,
increase in vivo half-life, increase bioavailability, or for
additional functionality. For example, the peptides may include
non-natural amino acids, modifications of the natural amino acids,
covalent attachment of a tag, such as, avidin or biotin, or the
like.
[0083] Also disclosed herein are compositions comprising the
peptide(s) provided herein. The composition may include one or more
peptide as set forth above. In certain cases, the composition may
include a mixture of a plurality of peptides set for the above,
such as, 2, 3, 4, 5, 6, 10, 12, 15, 20, or more peptides. The
composition may be in solid, semi-solid, liquid or gaseous form,
such as, powders, granules, solutions, injections, inhalants, gels,
hydrogels, microspheres, etc. In certain cases, the peptide
composition may include a diluent, buffer, or other components,
such as, water, buffered water, physiological saline, Phosphate
Buffered Saline (PBS), Ringer's solution, dextrose solution, and
Hank's solution.
[0084] In certain cases, a composition comprising a peptide(s) as
disclosed above, may be a pharmaceutical composition.
Pharmaceutical compositions can include, depending on the
formulation desired, pharmaceutically-acceptable, non-toxic
carriers or diluents, which are commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent or carrier is selected so as not to affect the biological
activity of the combination. Examples of such diluents or carriers
are distilled water, buffered water, physiological saline,
Phosphate Buffered Saline (PBS), Ringer's solution, dextrose
solution, and Hank's solution. In certain embodiments, the
pharmaceutical composition or formulation can include other
carriers, or other diluents, or adjuvants, or non-toxic,
nontherapeutic, nonimmunogenic stabilizers, or excipients and the
like. The compositions can also include additional substances to
approximate physiological conditions, such as pH adjusting and
buffering agents, toxicity adjusting agents, wetting agents and
detergents.
[0085] The composition can also include any of a variety of
stabilizing agents, such as an antioxidant. In certain cases, the
peptide can be complexed with various well-known compounds that
enhance the in vivo stability of the peptide, or otherwise enhance
its pharmacological properties (e.g., increase the half-life of the
peptide, reduce its toxicity, enhance solubility or uptake).
Examples of such modifications or complexing agents include
sulfate, gluconate, citrate and phosphate. The peptides of the
composition can also be complexed with molecules that enhance their
in vivo attributes. Such molecules include, for example,
carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,
sodium, potassium, calcium, magnesium, manganese), and lipids.
[0086] Further guidance regarding formulations that are suitable
for various types of administration can be found in Remington's
Pharmaceutical Sciences, Mace Publishing Company, Philadelphia,
Pa., 17th ed. (1985). For a brief review of methods for drug
delivery, see, Langer, Science 249:1527-1533 (1990).
[0087] The components used to formulate the pharmaceutical
compositions are preferably of high purity and are substantially
free of potentially harmful contaminants (e.g., at least National
Food (NF) grade, generally at least analytical grade, and more
typically at least pharmaceutical grade). Moreover, compositions
intended for in vivo use are usually sterile. To the extent that a
given compound must be synthesized prior to use, the resulting
product is typically substantially free of any potentially toxic
agents, particularly any endotoxins, which may be present during
the synthesis or purification process.
[0088] Diagnosis of NMO
[0089] As shown herein, AQP4-specific T cells are present in
patient with NMO. Accordingly, the peptides disclosed herein may be
used to diagnose NMO in a subject.
[0090] In general, a method for diagnosing NMO in a subject
comprises contacting a sample from the subject with a peptide
comprising a contiguous stretch of amino acids having the consensus
amino acid sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
(Formula I)
[0091] wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 are any amino acid, and
[0092] wherein the peptide is up to 50 amino acids in length.
[0093] In certain embodiments, X.sup.1 is Val (V) or Ile (I). In
certain embodiments, X.sup.2 is Ser (S) or Asp (D). In certain
embodiments, X.sup.3 is Val (V), Ile (I), or Gly (G). In certain
embodiments, X.sup.4 is Leu (L) or Ile (I). In certain embodiments,
X.sup.5 is Met (M) or Ser (S). In certain embodiments, X.sup.6 is
Leu (L), Val (V), or Ala (A). In certain embodiments, X.sup.1 is
Val (V), X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4
is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L). In certain
embodiments, X.sup.1 is Val (V), X.sup.2 is Asp (D), X.sup.3 is Val
(V), X.sup.4 is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu
(L). In such embodiments, the peptide comprises a contiguous
stretch of amino acids having the consensus amino acid sequence:
Leu-Pro-Val-Asp-Met-Val-Leu-Ile-Ser-Leu.
[0094] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Val-Leu-Ile-Ser-Leu.
[0095] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Val (V). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Gly-Leu-Ile-Ser-Val.
[0096] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Ile (I), X.sup.4 is Ile (I), X.sup.5 is Met (M),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Ile-Ile-Ile-Met-Leu.
[0097] In certain embodiments, X.sup.1 is Ile (I), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Ala (A). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Ile-Ser-Met-Gly-Leu-Ile-Ser-Ala.
[0098] Examples of suitable embodiments include where: X.sup.1 is
Val and X.sup.2 is Ser, X.sup.1 is Val and X.sup.2 is Asp, X.sup.1
is Val and X.sup.3 is Val, X.sup.1 is Val and X.sup.3 is Gly,
X.sup.1 is Val and X.sup.3 is Ile, X.sup.1 is Val and X.sup.4 is
Leu, X.sup.1 is Val and X.sup.4 is Ile, X.sup.1 is Val and X.sup.5
is Ser, X.sup.1 is Val and X.sup.5 is Met, X.sup.1 is Val and
X.sup.6 is Leu, X.sup.1 is Val and X.sup.6 is Val, X.sup.1 is Val
and X.sup.6 is Ala, X.sup.1 is Ile and X.sup.2 is Ser, X.sup.1 is
Ile and X.sup.2 is Asp, X.sup.1 is Ile and X.sup.3 is Val, X.sup.1
is Ile and X.sup.3 is Gly, X.sup.1 is Ile and X.sup.3 is Ile,
X.sup.1 is Ile and X.sup.4 is Leu, X.sup.1 is Ile and X.sup.4 is
Ile, X.sup.1 is Ile and X.sup.5 is Ser, X.sup.1 is Ile and X.sup.5
is Met, X.sup.1 is Ile and X.sup.6 is Leu, X.sup.1 is Ile and
X.sup.6 is Val, X.sup.1 is Ile and X.sup.6 is Ala, X.sup.2 is Ser
and X.sup.3 is Val, X.sup.2 is Ser and X.sup.3 is Gly, X.sup.2 is
Ser and X.sup.3 is Ile, X.sup.2 is Ser and X.sup.4 is Leu, X.sup.2
is Ser and X.sup.4 is Ile, X.sup.2 is Ser and X.sup.5 is Ser,
X.sup.2 is Ser and X.sup.5 is Met, X.sup.2 is Ser and X.sup.6 is
Leu, X.sup.2 is Ser and X.sup.6 is Val, X.sup.2 is Ser and X.sup.6
is Ala, X.sup.2 is Asp and X.sup.3 is Val, X.sup.2 is Asp and
X.sup.3 is Gly, X.sup.2 is Asp and X.sup.3 is Ile, X.sup.2 is Asp
and X.sup.4 is Leu, X.sup.2 is Asp and X.sup.4 is Ile, X.sup.2 is
Asp and X.sup.5 is Ser, X.sup.2 is Asp and X.sup.5 is Met, X.sup.2
is Asp and X.sup.6 is Leu, X.sup.2 is Asp and X.sup.6 is Val,
X.sup.2 is Asp and X.sup.6 is Ala, X.sup.3 is Val and X.sup.4 is
Leu, X.sup.3 is Val and X.sup.4 is Ile, X.sup.3 is Val and X.sup.5
is Ser, X.sup.3 is Val and X.sup.5 is Met, X.sup.3 is Val and
X.sup.6 is Leu, X.sup.3 is Val and X.sup.6 is Val, X.sup.3 is Val
and X.sup.6 is Ala, X.sup.3 is Gly and X.sup.4 is Leu, X.sup.3 is
Gly and X.sup.4 is Ile, X.sup.3 is Gly and X.sup.5 is Ser, X.sup.3
is Gly and X.sup.5 is Met, X.sup.3 is Gly and X.sup.6 is Leu,
X.sup.3 is Gly and X.sup.6 is Val, X.sup.3 is Gly and X.sup.6 is
Ala, X.sup.3 is Ile and X.sup.4 is Leu, X.sup.3 is Ile and X.sup.4
is Ile, X.sup.3 is Ile and X.sup.5 is Ser, X.sup.3 is Ile and
X.sup.5 is Met, X.sup.3 is Ile and X.sup.6 is Leu, X.sup.3 is Ile
and X.sup.6 is Val, X.sup.3 is Ile and X.sup.6 is Ala, X.sup.4 is
Leu and X.sup.5 is Ser, X.sup.4 is Leu and X.sup.5 is Met, X.sup.4
is Leu and X.sup.6 is Leu, X.sup.4 is Leu and X.sup.6 is Val,
X.sup.4 is Leu and X.sup.6 is Ala, X.sup.4 is Ile and X.sup.5 is
Ser, X.sup.4 is Ile and X.sup.5 is Met, X.sup.4 is Ile and X.sup.6
is Leu, X.sup.4 is Ile and X.sup.6 is Val, X.sup.4 is Ile and
X.sup.6 is Ala, X.sup.5 is Ser and X.sup.6 is Leu, X.sup.5 is Ser
and X.sup.6 is Val, X.sup.5 is Ser and X.sup.6 is Ala, X.sup.5 is
Met and X.sup.6 is Leu, X.sup.5 is Met and X.sup.6 is Val, and
X.sup.5 is Met and X.sup.6 is Ala.
[0099] In certain embodiments, the peptide may comprise a
contiguous stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu (Formula II)
[0100] wherein X is any amino acid.
[0101] In some such embodiments, X is Ser (S) or Asp (D). As such,
in some embodiments, X is Ser (S) and the peptide comprises a
contiguous stretch of amino acids having the consensus amino acid
sequence: LPVSMVLISL. In some embodiments, X is Asp (D) and the
peptide comprises a contiguous stretch of amino acids having the
consensus amino acid sequence: LPVDMVLISL.
[0102] In certain cases, the method comprises contacting a sample
from the subject with a peptide comprising an amino acid sequence
having at least 60% sequence identity to the amino acid sequence of
a human Aquaphorin-4 (AQP-4) peptide: LPVDMVLISL, wherein the
peptide is up to 50 amino acids in length, wherein the sample
comprises T cells; and measuring the number of T cells, wherein an
increase in the number of T cells as compared to a control
indicates that the subject has NMO.
[0103] In certain cases, the subject may be exhibiting symptoms of
NMO. In other cases, the subject may be asymptomatic but may be
predisposed to NMO. In certain embodiments, the subject may have
symptoms that are common to NMO and other diseases, such as,
multiple sclerosis. In certain cases, the subject may be at an
early stage of the disease before all clinical criteria of NMO are
evident. In certain cases, the subject may exhibit optic neuritis,
myelitis, and at least two of three supportive criteria: (i) MRI
evidence of a contiguous spinal cord lesion 3 or more segments in
length, (ii) onset brain MRI nondiagnostic for multiple sclerosis,
and (iii) NMO-IgG seropositivity. CNS involvement beyond the optic
nerves and spinal cord is compatible with NMO (Wingerchuk et al.
(2006) Neurology, May 23; 66(10):1485-9)
[0104] In certain embodiments, the sample may be a body fluid
sample, such as, blood, serum, plasma, cerebrospinal fluid. In
certain cases, the sample may be a sample containing Peripheral
blood mononuclear cells (PBMC) isolated from the subject. In other
cases, the sample may be a sample containing T cells isolated from
the subject. In certain cases, the sample may be a solid tissue
sample, such as, biopsy sample, for example, sample obtained from
brain biopsy, spinal cord biopsy, and the like.
[0105] The contacting may be carried out for a period of time
sufficient to detect proliferation of T cells. In certain cases,
the contacting may be carried out for a period of 1 day to 30 days,
such as, 1 day, or 5 days, or 10 days, or 15 days, or 20 days, or
25 days, or 30 days.
[0106] An increase in T cell proliferation of at least about 5%, or
at least about 10%, or at least about 20%, or at least about 50%,
or at least about 70%, or at least about 80%, or at least about
90%, or more, compared to a negative control may indicate that the
subject has NMO.
[0107] Any suitable negative control may be used for comparison.
Suitable negative controls include samples obtained from healthy
controls, sample from the subject being diagnosed, where the sample
is not contacted with the peptide, for example.
[0108] In certain embodiments, T cell proliferation may be compared
to a threshold value or range. For example, a normal threshold
value or range of T cell proliferation may be determined by
contacting the peptides disclosed herein with samples from healthy
controls. An increase in T cell proliferation of at least about 5%,
or at least about 10%, or at least about 20%, or at least about
50%, or at least about 70%, or at least about 80%, or at least
about 90%, or more, compared to the normal threshold value or range
may indicate that the subject has NMO.
[0109] T cell proliferation may be measured by methods known in the
art, such as, the methods disclosed herein.
[0110] In certain embodiments, the T cell measured in the method
may be a CD4+ T cell. In certain cases, the T cell measured in the
method may be a Th17 T cell.
[0111] Methods of Screening
[0112] A method for screening for candidate agents for inhibiting
proliferation of T cells in response to exposure to a peptide
disclosed herein is also provided. As shown herein, T cells from
NMO patients proliferate when exposed to a peptide comprising an
amino acid sequence having at least 60% sequence identity to the
amino acid sequence of a human Aquaphorin-4 (AQP-4) peptide
LPVDMVLISL. Moreover, an increased frequency of these T cells are
Th17 cells which may lead to NMO pathogenesis. Accordingly, the
identification of agents that may inhibit the AQP-4 peptide or
peptide homologous to AQP-4 peptide mediated proliferation of T
cells may be useful in treating NMO.
[0113] In general, the method for screening for inhibitors of
proliferation of T cells comprises contacting a sample from the
subject with: (i) a peptide comprising a contiguous stretch of
amino acids having the consensus amino acid sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
(Formula I) [0114] wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4,
X.sup.5, and X.sup.6 are any amino acid, and [0115] wherein the
peptide is up to 50 amino acids in length; and [0116] (ii) a
candidate agent and evaluating the number of T cells, [0117]
wherein a decrease in the number of T cells as compared to a
control indicates that the candidate agent inhibits proliferation
of T cells. In certain embodiments, X.sup.1 is Val (V) or Ile (I).
In certain embodiments, X.sup.2 is Ser (S) or Asp (D). In certain
embodiments, X.sup.3 is Val (V), Ile (I), or Gly (G). In certain
embodiments, X.sup.4 is Leu (L) or Ile (I). In certain embodiments,
X.sup.5 is Met (M) or Ser (S). In certain embodiments, X.sup.6 is
Leu (L), Val (V), or Ala (A). In certain embodiments, X.sup.1 is
Val (V), X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4
is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L).
[0118] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Asp
(D), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Asp-Met-Val-Leu-Ile-Ser-Leu.
[0119] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Val-Leu-Ile-Ser-Leu.
[0120] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Val (V). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Gly-Leu-Ile-Ser-Val.
[0121] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Ile (I), X.sup.4 is Ile (I), X.sup.5 is Met (M),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Ile-Ile-Ile-Met-Leu.
[0122] In certain embodiments, X.sup.1 is Ile (I), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Ala (A). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Ile-Ser-Met-Gly-Leu-Ile-Ser-Ala.
[0123] Examples of suitable embodiments include where: X.sup.1 is
Val and X.sup.2 is Ser, X.sup.1 is Val and X.sup.2 is Asp, X.sup.1
is Val and X.sup.3 is Val, X.sup.1 is Val and X.sup.3 is Gly,
X.sup.1 is Val and X.sup.3 is Ile, X.sup.1 is Val and X.sup.4 is
Leu, X.sup.1 is Val and X.sup.4 is Ile, X.sup.1 is Val and X.sup.5
is Ser, X.sup.1 is Val and X.sup.5 is Met, X.sup.1 is Val and
X.sup.6 is Leu, X.sup.1 is Val and X.sup.6 is Val, X.sup.1 is Val
and X.sup.6 is Ala, X.sup.1 is Ile and X.sup.2 is Ser, X.sup.1 is
Ile and X.sup.2 is Asp, X.sup.1 is Ile and X.sup.3 is Val, X.sup.1
is Ile and X.sup.3 is Gly, X.sup.1 is Ile and X.sup.3 is Ile,
X.sup.1 is Ile and X.sup.4 is Leu, X.sup.1 is Ile and X.sup.4 is
Ile, X.sup.1 is Ile and X.sup.5 is Ser, X.sup.1 is Ile and X.sup.5
is Met, X.sup.1 is Ile and X.sup.6 is Leu, X.sup.1 is Ile and
X.sup.6 is Val, X.sup.1 is Ile and X.sup.6 is Ala, X.sup.2 is Ser
and X.sup.3 is Val, X.sup.2 is Ser and X.sup.3 is Gly, X.sup.2 is
Ser and X.sup.3 is Ile, X.sup.2 is Ser and X.sup.4 is Leu, X.sup.2
is Ser and X.sup.4 is Ile, X.sup.2 is Ser and X.sup.5 is Ser,
X.sup.2 is Ser and X.sup.5 is Met, X.sup.2 is Ser and X.sup.6 is
Leu, X.sup.2 is Ser and X.sup.6 is Val, X.sup.2 is Ser and X.sup.6
is Ala, X.sup.2 is Asp and X.sup.3 is Val, X.sup.2 is Asp and
X.sup.3 is Gly, X.sup.2 is Asp and X.sup.3 is Ile, X.sup.2 is Asp
and X.sup.4 is Leu, X.sup.2 is Asp and X.sup.4 is Ile, X.sup.2 is
Asp and X.sup.5 is Ser, X.sup.2 is Asp and X.sup.5 is Met, X.sup.2
is Asp and X.sup.6 is Leu, X.sup.2 is Asp and X.sup.6 is Val,
X.sup.2 is Asp and X.sup.6 is Ala, X.sup.3 is Val and X.sup.4 is
Leu, X.sup.3 is Val and X.sup.4 is Ile, X.sup.3 is Val and X.sup.5
is Ser, X.sup.3 is Val and X.sup.5 is Met, X.sup.3 is Val and
X.sup.6 is Leu, X.sup.3 is Val and X.sup.6 is Val, X.sup.3 is Val
and X.sup.6 is Ala, X.sup.3 is Gly and X.sup.4 is Leu, X.sup.3 is
Gly and X.sup.4 is Ile, X.sup.3 is Gly and X.sup.5 is Ser, X.sup.3
is Gly and X.sup.5 is Met, X.sup.3 is Gly and X.sup.6 is Leu,
X.sup.3 is Gly and X.sup.6 is Val, X.sup.3 is Gly and X.sup.6 is
Ala, X.sup.3 is Ile and X.sup.4 is Leu, X.sup.3 is Ile and X.sup.4
is Ile, X.sup.3 is Ile and X.sup.5 is Ser, X.sup.3 is Ile and
X.sup.5 is Met, X.sup.3 is Ile and X.sup.6 is Leu, X.sup.3 is Ile
and X.sup.6 is Val, X.sup.3 is Ile and X.sup.6 is Ala, X.sup.4 is
Leu and X.sup.5 is Ser, X.sup.4 is Leu and X.sup.5 is Met, X.sup.4
is Leu and X.sup.6 is Leu, X.sup.4 is Leu and X.sup.6 is Val,
X.sup.4 is Leu and X.sup.6 is Ala, X.sup.4 is Ile and X.sup.5 is
Ser, X.sup.4 is Ile and X.sup.5 is Met, X.sup.4 is Ile and X.sup.6
is Leu, X.sup.4 is Ile and X.sup.6 is Val, X.sup.4 is Ile and
X.sup.6 is Ala, X.sup.5 is Ser and X.sup.6 is Leu, X.sup.5 is Ser
and X.sup.6 is Val, X.sup.5 is Ser and X.sup.6 is Ala, X.sup.5 is
Met and X.sup.6 is Leu, X.sup.5 is Met and X.sup.6 is Val, and
X.sup.5 is Met and X.sup.6 is Ala.
[0124] In certain embodiments, the peptide may comprise a
contiguous stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu (Formula II)
[0125] wherein X is any amino acid.
[0126] In some such embodiments, X is Ser (S) or Asp (D). As such,
in some embodiments, X is Ser (S) and the peptide comprises a
contiguous stretch of amino acids having the consensus amino acid
sequence: LPVSMVLISL. In some embodiments, X is Asp (D) and the
peptide comprises a contiguous stretch of amino acids having the
consensus amino acid sequence: LPVDMVLISL.
[0127] In certain cases, the method may comprise contacting a T
cell obtained from a subject having Neuromyelitis Optica with (i) a
peptide comprising an amino acid sequence having at least 60%
sequence identity to the amino acid sequence of a human
Aquaphorin-4 (AQP-4) peptide: LPVDMVLISL wherein the peptide is up
to 50 amino acids in length, and (ii) a candidate agent and
evaluating the number of T cells, wherein a decrease in the number
of T cells as compared to a control indicates that the candidate
agent inhibits proliferation of T cells.
[0128] Candidate agents of interest for screening include
biologically active agents of numerous chemical classes, organic
molecules, inorganic molecules, organometallic molecules,
immunoglobulins, peptides, proteins, genetic sequences, etc. Also
of interest are small organic molecules, which comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, frequently at least
two of the functional chemical groups. The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Candidate agents are also found among
biomolecules, including peptides, polynucleotides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0129] Candidate agents may be compounds. Compounds may be obtained
from a wide variety of sources including libraries of synthetic or
natural compounds. For example, numerous means are available for
random and directed synthesis of a wide variety of organic
compounds, including biomolecules, including expression of
randomized oligonucleotides and oligopeptides. Alternatively,
libraries of natural compounds in the form of bacterial, fungal,
plant and animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and
compounds are readily modified through conventional chemical,
physical and biochemical means, and may be used to produce
combinatorial libraries. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc., to
produce structural analogs.
[0130] A plurality of assays may be run in parallel with different
concentrations to obtain a differential response to the various
concentrations. As known in the art, determining the effective
concentration of an agent typically uses a range of concentrations
resulting from 1:10, or other log scale, dilutions. The
concentrations may be further refined with a second series of
dilutions, if necessary. Typically, one of these concentrations
serves as a negative control, i.e. at zero concentration or below
the level of detection of the agent or at or below the
concentration of agent that does not give a detectable change in
binding.
[0131] A candidate agent is identified as an inhibitor of AQP-4
specific T cell proliferation if it decreases AQP-4 specific T cell
proliferation by at least about 5%, or at least about 10%, or at
least about 20%, or at least about 50%, or at least about 70%, or
at least about 80%, or at least about 90%, or more, compared to
AQP-4 specific T cell proliferation in absence of the candidate
agent, or another negative control.
[0132] A T cell obtained from a NMO patient used in the screening
method, may be present in a blood sample from the NMO patient, or
present in a purified form, such as, isolated from the blood
sample. The contacting with a peptide as provided herein and a
candidate agent may be simultaneous or sequential. The contacting
may be performed for 1 day, or 5 days, or 10 days, or 15 days, or
20 days, or 25 days, or 30 days. T cell proliferation may be
evaluated by methods known in the art.
[0133] Induction of Immune Tolerance to AQP4
[0134] A method for inducing immune tolerance to AQP-4 protein and
fragments thereof in a subject is also provided. As such, the
peptides disclosed herein may be used in a tolerizing therapy to
suppress immune response to AQP-4 protein and fragments thereof. In
general, the method comprises administering an effective dose of a
peptide to a subject, wherein the peptide comprises a contiguous
stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
(Formula I)
[0135] wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 are any amino acid,
[0136] wherein the peptide is up to 50 amino acids in length,
and
[0137] wherein the administering the peptide induces immune
tolerance to AQP-4 protein and fragments thereof in the
subject.
[0138] In certain embodiments, X.sup.1 is Val (V) or Ile (I). In
certain embodiments, X.sup.2 is Ser (S) or Asp (D). In certain
embodiments, X.sup.3 is Val (V), Ile (I), or Gly (G). In certain
embodiments, X.sup.4 is Leu (L) or Ile (I). In certain embodiments,
X.sup.5 is Met (M) or Ser (S). In certain embodiments, X.sup.6 is
Leu (L), Val (V), or Ala (A). In certain embodiments, X.sup.1 is
Val (V), X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4
is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L).
[0139] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Asp
(D), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Asp-Met-Val-Leu-Ile-Ser-Leu.
[0140] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Val-Leu-Ile-Ser-Leu.
[0141] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Val (V). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Gly-Leu-Ile-Ser-Val.
[0142] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Ile (I), X.sup.4 is Ile (I), X.sup.5 is Met (M),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Ile-Ile-Ile-Met-Leu.
[0143] In certain embodiments, X.sup.1 is Ile (I), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Ala (A). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Ile-Ser-Met-Gly-Leu-Ile-Ser-Ala.
[0144] Examples of suitable embodiments include where: X.sup.1 is
Val and X.sup.2 is Ser, X.sup.1 is Val and X.sup.2 is Asp, X.sup.1
is Val and X.sup.3 is Val, X.sup.1 is Val and X.sup.3 is Gly,
X.sup.1 is Val and X.sup.3 is Ile, X.sup.1 is Val and X.sup.4 is
Leu, X.sup.1 is Val and X.sup.4 is Ile, X.sup.1 is Val and X.sup.5
is Ser, X.sup.1 is Val and X.sup.5 is Met, X.sup.1 is Val and
X.sup.6 is Leu, X.sup.1 is Val and X.sup.6 is Val, X.sup.1 is Val
and X.sup.6 is Ala, X.sup.1 is Ile and X.sup.2 is Ser, X.sup.1 is
Ile and X.sup.2 is Asp, X.sup.1 is Ile and X.sup.3 is Val, X.sup.1
is Ile and X.sup.3 is Gly, X.sup.1 is Ile and X.sup.3 is Ile,
X.sup.1 is Ile and X.sup.4 is Leu, X.sup.1 is Ile and X.sup.4 is
Ile, X.sup.1 is Ile and X.sup.5 is Ser, X.sup.1 is Ile and X.sup.5
is Met, X.sup.1 is Ile and X.sup.6 is Leu, X.sup.1 is Ile and
X.sup.6 is Val, X.sup.1 is Ile and X.sup.6 is Ala, X.sup.2 is Ser
and X.sup.3 is Val, X.sup.2 is Ser and X.sup.3 is Gly, X.sup.2 is
Ser and X.sup.3 is Ile, X.sup.2 is Ser and X.sup.4 is Leu, X.sup.2
is Ser and X.sup.4 is Ile, X.sup.2 is Ser and X.sup.5 is Ser,
X.sup.2 is Ser and X.sup.5 is Met, X.sup.2 is Ser and X.sup.6 is
Leu, X.sup.2 is Ser and X.sup.6 is Val, X.sup.2 is Ser and X.sup.6
is Ala, X.sup.2 is Asp and X.sup.3 is Val, X.sup.2 is Asp and
X.sup.3 is Gly, X.sup.2 is Asp and X.sup.3 is Ile, X.sup.2 is Asp
and X.sup.4 is Leu, X.sup.2 is Asp and X.sup.4 is Ile, X.sup.2 is
Asp and X.sup.5 is Ser, X.sup.2 is Asp and X.sup.5 is Met, X.sup.2
is Asp and X.sup.6 is Leu, X.sup.2 is Asp and X.sup.6 is Val,
X.sup.2 is Asp and X.sup.6 is Ala, X.sup.3 is Val and X.sup.4 is
Leu, X.sup.3 is Val and X.sup.4 is Ile, X.sup.3 is Val and X.sup.5
is Ser, X.sup.3 is Val and X.sup.5 is Met, X.sup.3 is Val and
X.sup.6 is Leu, X.sup.3 is Val and X.sup.6 is Val, X.sup.3 is Val
and X.sup.6 is Ala, X.sup.3 is Gly and X.sup.4 is Leu, X.sup.3 is
Gly and X.sup.4 is Ile, X.sup.3 is Gly and X.sup.5 is Ser, X.sup.3
is Gly and X.sup.5 is Met, X.sup.3 is Gly and X.sup.6 is Leu,
X.sup.3 is Gly and X.sup.6 is Val, X.sup.3 is Gly and X.sup.6 is
Ala, X.sup.3 is Ile and X.sup.4 is Leu, X.sup.3 is Ile and X.sup.4
is Ile, X.sup.3 is Ile and X.sup.5 is Ser, X.sup.3 is Ile and
X.sup.5 is Met, X.sup.3 is Ile and X.sup.6 is Leu, X.sup.3 is Ile
and X.sup.6 is Val, X.sup.3 is Ile and X.sup.6 is Ala, X.sup.4 is
Leu and X.sup.5 is Ser, X.sup.4 is Leu and X.sup.5 is Met, X.sup.4
is Leu and X.sup.6 is Leu, X.sup.4 is Leu and X.sup.6 is Val,
X.sup.4 is Leu and X.sup.6 is Ala, X.sup.4 is Ile and X.sup.5 is
Ser, X.sup.4 is Ile and X.sup.5 is Met, X.sup.4 is Ile and X.sup.6
is Leu, X.sup.4 is Ile and X.sup.6 is Val, X.sup.4 is Ile and
X.sup.6 is Ala, X.sup.5 is Ser and X.sup.6 is Leu, X.sup.5 is Ser
and X.sup.6 is Val, X.sup.5 is Ser and X.sup.6 is Ala, X.sup.5 is
Met and X.sup.6 is Leu, X.sup.5 is Met and X.sup.6 is Val, and
X.sup.5 is Met and X.sup.6 is Ala.
[0145] In certain embodiments, the peptide may comprise a
contiguous stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu (Formula II)
[0146] wherein X is any amino acid.
[0147] In some such embodiments, X is Ser (S) or Asp (D). As such,
in some embodiments, X is Ser (S) and the peptide comprises a
contiguous stretch of amino acids having the consensus amino acid
sequence: LPVSMVLISL. In some embodiments, X is Asp (D) and the
peptide comprises a contiguous stretch of amino acids having the
consensus amino acid sequence: LPVDMVLISL.
[0148] In certain embodiments, the peptide may comprise an amino
acid sequence having at least 60% sequence identity to the amino
acid sequence of a human Aquaphorin-4 (AQP-4) peptide having the
amino acid sequence: LPVDMVLISL wherein the peptide is up to 50
amino acids in length, wherein the administering the peptide
induces immune tolerance to AQP-4 protein and fragments thereof in
the subject. The phrase "fragments of AQP-4 protein" as used in the
context of AQP4 fragments that do not induce an immune reaction in
a subject treated with the peptides disclosed herein refer to AQP4
fragments comprising an amino acid sequence homologous to the amino
acid sequence of the peptide used for induction of immune
tolerance. For example, the AQP4 fragments may include a stretch of
amino acids having: the consensus sequence
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu, or having at least 60%
sequence identity to the sequence: LPVDMVLISL
[0149] In certain embodiments, the subject may be predisposed or at
risk of developing NMO. In certain embodiments, the subject may
have been diagnosed with NMO.
[0150] The peptide(s) may be administered to an individual as a
pharmaceutically acceptable composition. Pharmaceutically
acceptable peptide compositions for administering to an individual
may include, for example, sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of components, such as,
carriers, present in pharmaceutically acceptable peptide
compositions include, without limitation, propylene glycol,
polyethylene glycol, vegetable oils, and injectable organic esters.
Aqueous carriers include water, alcohol, saline, and buffered
solutions. Pharmaceutically acceptable carriers can also include
physiologically acceptable aqueous vehicles (e.g., physiological
saline or artificial cerebral-spinal fluid) or other known carriers
appropriate to specific routes of administration. Additional
compounds can be included with the peptide(s) described herein,
such as steroids, mucolytic agents, anti-inflammatory agents,
immunosuppressants, dilators, vasoconstrictors, or combinations
thereof. Preservatives, flavorings, and other additives such as,
for example, anti-microbials, anti-oxidants, chelating agents,
inert gases, and the like may also be present.
[0151] In certain cases, the peptide(s) may be administered at an
amount that is sufficient to suppress induction of an immune
response to AQP4 or fragments thereof. In certain cases, the amount
may be sufficient to suppress production of AQP4 specific
antibodies in the subject. In certain cases, the amount may be
sufficient to suppress production of AQP4 specific T cells in the
subject. In certain cases, the amount may be sufficient to suppress
production of AQP4 specific-T cells and antibodies in the subject.
In certain embodiments, a subject receiving the AQP4 tolerizing
therapy as described above, may show a 5%-90% decrease in the titer
of AQP4 specific antibodies present in the subject as compared the
titer of the antibodies present before the therapy. In certain
embodiments, a subject receiving the AQP4 tolerizing therapy as
described above, may show a 5%-90% decrease in the number of AQP4
specific T cells present in the subject as compared the number of
AQP4 specific T cells present before the therapy. In certain cases,
the AQP4 tolerizing therapy may result in a substantial decrease
(e.g., 5%-90% decrease) in the number of Th17 cells present in the
subject as compared the number present before the therapy.
[0152] In certain cases, the peptide may be administered at a high
dose, such as, 0.1-10 mg/Kg, such as, 0.1 mg/Kg, 0.3 mg/Kg, 1
mg/Kg, 3 mg/Kg, or 10 mg/Kg.
[0153] In certain cases, the peptide may be administered to the
thymus of the subject. In certain cases, the peptide may be orally
administered to the subject. In certain cases, the method may
include repeated administration of the peptide, such as, every 10
days, 20 days, 30 days, 3 months, 6 months, 12 months, 3 years, 6
years, 10 years, and so forth. In certain cases, the initial dose
of the peptide may be higher than the dose of the peptide in
subsequent administration to the subject.
[0154] Kits
[0155] Kits including the peptides described herein are disclosed.
Kits may include one or more peptides comprising a contiguous
stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-X.sup.1-X.sup.2-Met-X.sup.3-X.sup.4-Ile-X.sup.5-X.sup.6
(Formula I)
[0156] wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 are any amino acid, and
[0157] wherein the peptide is up to 50 amino acids in length.
[0158] In certain embodiments, X.sup.1 is Val (V) or Ile (I). In
certain embodiments, X.sup.2 is Ser (S) or Asp (D). In certain
embodiments, X.sup.3 is Val (V), Ile (I), or Gly (G). In certain
embodiments, X.sup.4 is Leu (L) or Ile (I). In certain embodiments,
X.sup.5 is Met (M) or Ser (S). In certain embodiments, X.sup.6 is
Leu (L), Val (V), or Ala (A). In certain embodiments, X.sup.1 is
Val (V), X.sup.2 is Ser (S) or Asp (D), X.sup.3 is Val (V), X.sup.4
is Leu (L), X.sup.5 is Ser (S), and X.sup.6 is Leu (L).
[0159] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Asp
(D), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Asp-Met-Val-Leu-Ile-Ser-Leu.
[0160] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Val (V), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Val-Leu-Ile-Ser-Leu.
[0161] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Val (V). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Gly-Leu-Ile-Ser-Val.
[0162] In certain embodiments, X.sup.1 is Val (V), X.sup.2 is Ser
(S), X.sup.3 is Ile (I), X.sup.4 is Ile (I), X.sup.5 is Met (M),
and X.sup.6 is Leu (L). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Val-Ser-Met-Ile-Ile-Ile-Met-Leu.
[0163] In certain embodiments, X.sup.1 is Ile (I), X.sup.2 is Ser
(S), X.sup.3 is Gly (G), X.sup.4 is Leu (L), X.sup.5 is Ser (S),
and X.sup.6 is Ala (A). In such embodiments, the peptide comprises
a contiguous stretch of amino acids having the consensus amino acid
sequence: Leu-Pro-Ile-Ser-Met-Gly-Leu-Ile-Ser-Ala.
[0164] Examples of suitable embodiments include where: X.sup.1 is
Val and X.sup.2 is Ser, X.sup.1 is Val and X.sup.2 is Asp, X.sup.1
is Val and X.sup.3 is Val, X.sup.1 is Val and X.sup.3 is Gly,
X.sup.1 is Val and X.sup.3 is Ile, X.sup.1 is Val and X.sup.4 is
Leu, X.sup.1 is Val and X.sup.4 is Ile, X.sup.1 is Val and X.sup.5
is Ser, X.sup.1 is Val and X.sup.5 is Met, X.sup.1 is Val and
X.sup.6 is Leu, X.sup.1 is Val and X.sup.6 is Val, X.sup.1 is Val
and X.sup.6 is Ala, X.sup.1 is Ile and X.sup.2 is Ser, X.sup.1 is
Ile and X.sup.2 is Asp, X.sup.1 is Ile and X.sup.3 is Val, X.sup.1
is Ile and X.sup.3 is Gly, X.sup.1 is Ile and X.sup.3 is Ile,
X.sup.1 is Ile and X.sup.4 is Leu, X.sup.1 is Ile and X.sup.4 is
Ile, X.sup.1 is Ile and X.sup.5 is Ser, X.sup.1 is Ile and X.sup.5
is Met, X.sup.1 is Ile and X.sup.6 is Leu, X.sup.1 is Ile and
X.sup.6 is Val, X.sup.1 is Ile and X.sup.6 is Ala, X.sup.2 is Ser
and X.sup.3 is Val, X.sup.2 is Ser and X.sup.3 is Gly, X.sup.2 is
Ser and X.sup.3 is Ile, X.sup.2 is Ser and X.sup.4 is Leu, X.sup.2
is Ser and X.sup.4 is Ile, X.sup.2 is Ser and X.sup.5 is Ser,
X.sup.2 is Ser and X.sup.5 is Met, X.sup.2 is Ser and X.sup.6 is
Leu, X.sup.2 is Ser and X.sup.6 is Val, X.sup.2 is Ser and X.sup.6
is Ala, X.sup.2 is Asp and X.sup.3 is Val, X.sup.2 is Asp and
X.sup.3 is Gly, X.sup.2 is Asp and X.sup.3 is Ile, X.sup.2 is Asp
and X.sup.4 is Leu, X.sup.2 is Asp and X.sup.4 is Ile, X.sup.2 is
Asp and X.sup.5 is Ser, X.sup.2 is Asp and X.sup.5 is Met, X.sup.2
is Asp and X.sup.6 is Leu, X.sup.2 is Asp and X.sup.6 is Val,
X.sup.2 is Asp and X.sup.6 is Ala, X.sup.3 is Val and X.sup.4 is
Leu, X.sup.3 is Val and X.sup.4 is Ile, X.sup.3 is Val and X.sup.5
is Ser, X.sup.3 is Val and X.sup.5 is Met, X.sup.3 is Val and
X.sup.6 is Leu, X.sup.3 is Val and X.sup.6 is Val, X.sup.3 is Val
and X.sup.6 is Ala, X.sup.3 is Gly and X.sup.4 is Leu, X.sup.3 is
Gly and X.sup.4 is Ile, X.sup.3 is Gly and X.sup.5 is Ser, X.sup.3
is Gly and X.sup.5 is Met, X.sup.3 is Gly and X.sup.6 is Leu,
X.sup.3 is Gly and X.sup.6 is Val, X.sup.3 is Gly and X.sup.6 is
Ala, X.sup.3 is Ile and X.sup.4 is Leu, X.sup.3 is Ile and X.sup.4
is Ile, X.sup.3 is Ile and X.sup.5 is Ser, X.sup.3 is Ile and
X.sup.5 is Met, X.sup.3 is Ile and X.sup.6 is Leu, X.sup.3 is Ile
and X.sup.6 is Val, X.sup.3 is Ile and X.sup.6 is Ala, X.sup.4 is
Leu and X.sup.5 is Ser, X.sup.4 is Leu and X.sup.5 is Met, X.sup.4
is Leu and X.sup.6 is Leu, X.sup.4 is Leu and X.sup.6 is Val,
X.sup.4 is Leu and X.sup.6 is Ala, X.sup.4 is Ile and X.sup.5 is
Ser, X.sup.4 is Ile and X.sup.5 is Met, X.sup.4 is Ile and X.sup.6
is Leu, X.sup.4 is Ile and X.sup.6 is Val, X.sup.4 is Ile and
X.sup.6 is Ala, X.sup.5 is Ser and X.sup.6 is Leu, X.sup.5 is Ser
and X.sup.6 is Val, X.sup.5 is Ser and X.sup.6 is Ala, X.sup.5 is
Met and X.sup.6 is Leu, X.sup.5 is Met and X.sup.6 is Val, and
X.sup.5 is Met and X.sup.6 is Ala.
[0165] In certain embodiments, the peptide may comprise a
contiguous stretch of amino acids having the consensus amino acid
sequence:
Leu-Pro-Val-X-Met-Val-Leu-Ile-Ser-Leu (Formula II)
[0166] wherein X is any amino acid.
[0167] In some such embodiments, X is Ser (S) or Asp (D). As such,
in some embodiments, X is Ser (S) and the peptide comprises a
contiguous stretch of amino acids having the consensus amino acid
sequence: LPVSMVLISL. In some embodiments, X is Asp (D) and the
peptide comprises a contiguous stretch of amino acids having the
consensus amino acid sequence: LPVDMVLISL.
[0168] In certain cases, the kits may include a peptide(s) having
at least 60% sequence identity to the amino acid sequence of a
human Aquaphorin-4 (AQP-4) peptide having the amino acid sequence:
LPVDMVLISL, wherein the peptide is up to 50 amino acids in length.
The kit may contain the peptide(s) in a solid, semi-solid, liquid,
or fluid state. The kit may include one or multiple containers
containing specified quantities of the peptide(s). In certain
embodiments, the kit may include multiple containers containing the
subject peptides described herein unit dosage form.
[0169] In certain embodiments, the kit may further include reagents
and instructions for carrying out diagnosing, treating, or
screening methods using the peptides.
EXAMPLES
[0170] The following example is provided to further illustrate the
advantages and features of the present invention, but is not
intended to limit the scope of the invention. While they are
typical of those that might be used, other procedures,
methodologies, or techniques known to those skilled in the art may
alternatively be used.
Materials and Methods
[0171] Patients. Fifteen NMO patients (12 females and 3 males,
44.3+/-13.8 years) fulfilling Mayo Clinic diagnostic criteria
(Wingerchuk D M, et al., Neurology. 2006 May 23; 66(10):1485-9) and
nine HC (5 females and 4 males, 40.8+/-10.7 years) were recruited
from the UCSF MS Center. A majority of NMO patients had been
treated with rituximab (Jacob A, et al., Arch Neurol. 2008
November; 65(11):1443-8), and none had been treated with
azathioprine, mycophenolate mofetil, cyclophosphamide or other
immunosuppressive medications. None of the patients had received
steroids within two months preceding blood draws. Blood was
collected by venipuncture. This study was approved by UCSF
Committee on Human Research (Protocol #10-00650) and written
informed consent was obtained from subjects prior to
enrollment.
[0172] T cell proliferation assays. Peripheral blood mononuclear
cells (PBMC) were isolated by density gradient centrifugation over
Ficoll (Ficoll-Paque PLUS, GE Healthcare) according to
manufacturer's instruction. T cell proliferation was evaluated by
[.sup.3H] thymidine incorporation or CFSE dilution assays. In
thymidine incorporation assays, PBMC were cultured with antigens in
96-well plates at either 1.times.10.sup.5 cells (AQP4 pools--in at
least 10 wells) or 5.times.10.sup.5 cells (individual peptides--in
duplicate) per well for 6 days (d). Cultures were then pulsed with
[.sup.3H] thymidine and harvested 18 h later. Positive wells were
defined as having counts-per-minute (cpm) values greater than
control cpm average values+3 standard deviation (SD) or stimulation
index (SI) greater than 2. Alternatively, PBMC were stained with 1
.mu.M CFSE (Invitrogen), according to manufacturer's instruction.
Cells were cultured in the presence of antigens for 10 d. T cell
proliferation was assessed by flow cytometric evaluation of CFSE
dilution. Proliferation was expressed as the cell division index
(CDI) (defined as the number of CFSE.sup.low T cells cultured with
antigen/number of CFSE.sup.low T cells without antigen). In all
cases, culture medium consisted of X-VIVO 15 (Lonza) supplemented
with penicillin (100 U/ml) and streptomycin (0.1 mg/ml).
[0173] Antigens. Peptides were synthesized by Genemed Synthesis
Inc. with purity greater than 95% by HPLC analysis. Overlapping
AQP4 20-mer peptides were offset by 10 amino acids. Peptides
corresponding to certain hydrophobic AQP4 sequences were
synthesized in overlapping 15-mer peptide pairs. Truncated peptides
within the 61-80 region (p61-78 GTEKPLPVDMVLISLCFG; p61-76
GTEKPLPVDMVLISLC; p61-74 GTEKPLPVDMVLIS; p61-72 GTEKPLPVDMVL;
p63-80 EKPLPVDMVLISLCFGLS; p65-80 PLPVDMVLISLCFGLS; p67-80
PVDMVLISLCFGLS; p69-80 DMVLISLCFGLS), AQP4 p63-76 (EKPLPVDMVLISLC)
and bacterial peptide ABC/TP p204-217 (FIILPVSMVLISLV) were as
quoted. Full length recombinant human (rh) AQP4 (1-323) was
expressed in Pichia pastoris and purified as described (Ho J D, et
al., Proc Natl Acad Sci USA. 2009 May 5; 106(18):7437-42). Tetanus
toxoid was obtained from List Biological Laboratories, Inc.
(Campbell, Calif.).
[0174] Flow cytometry analysis. Single-cell suspensions were
incubated with human serum to prevent nonspecific antibody binding,
then stained with anti-CD3, -CD4, -CD8, -CD25, -MHC Class II,
-CD40, -CD80, and -CD86 (eBioscience and BD Bioscience).
Intracellular cytokine production by CD4.sup.+ T cells and APC was
analyzed by monitoring the expression of IFN-.gamma., IL-17, IL-6,
IL-1.beta. and IL-10 (1:100) (eBioscience). Foxp3 staining was
performed according to the manufacturer's protocol (eBioscience).
For intracellular cytokine staining, T cells were stimulated with
phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) plus ionomycin (500
ng/ml) in the presence of GolgiStop (10/ml) (BD Biosciences).
CD14.sup.+ cells were stimulated with LPS (1 .mu.g/ml;
Sigma-Aldrich) for 4 or 20 h in the presence of GolgiStop. Cells
were analyzed by flow cytometry on a FACS Canto flow cytometer (BD
Biosciences).
[0175] Blocking of HLA alleles with antibodies. Inhibition of the
proliferation of PBMC to AQP4 p61-80 and rhAQP4 was studied by
using mouse monoclonal anti-HLA-DR (clone G46-6; BD Bioscience,
Mississauga, ON, Canada), anti-HLA-DQ (clone HG-38; Abcam),
anti-HLA-DP (clone B7/21; Abcam) and isotype control (clone
G155-178; BD Biosciences). Antibodies (1 .mu.g/ml) were added to
CFSE-stained PBMC cultures 1 hour before addition of antigens.
[0176] Antigen recall experiments. PBMC were initially stimulated
with antigens. After 10 d, cells were restimulated with rhAQP4 (5
.mu.g/ml), AQP4 peptides or bacterial peptide (10 .mu.g/ml), in the
presence of irradiated autologous APC. Following 3 d of
stimulation, cultures were pulsed with [.sup.3H]thymidine and
harvested 18 h later. Stimulation Index (SI) was calculated by
dividing cpm in wells with antigen by cpm in control wells with no
antigen of each assay test group.
[0177] Analyses for protein sequence homology and MHC core binding
motifs. Sequences similarities between AQP4 and other proteins were
addressed using the protein-protein Basic Local Alignment Search
Tool (BLAST) from NCBI. The prediction of the core binding motif
within AQP4 61-80 sequence for HLA-DRB1*0301 and HLA-DRB3*0202 was
performed with netMHCII-1.1 (Nielsen M, et al., BMC Bioinformatics.
2007; 8:238) and net MHCII-2.2 (Nielsen M, et al., BMC
Bioinformatics. 2009; 10:296), programs that utilizes relative
affinities of identified determinants from the immune epitope
database (IEDB).
[0178] HLA Typing. High-resolution HLA typing was performed by the
UCSF Immunogenetics and Transplantation Laboratory (ITL, UCSF
Department of Surgery). The following HLA loci were analyzed using
sequence-based typing: DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1.
Sequence ambiguities outside exon 2 were resolved.
[0179] Statistics. Statistical analysis was performed using either
GraphPad Prism software or STATA. The nonparametric Mann-Whitney U
test was used to compare data. Paired t-tests were performed to
compare cpm values with antigens to control values with no antigens
presented in FIG. 3E. A value of P.ltoreq.0.05 was considered
significant.
EXAMPLE 1
T Cells from NMO Patients Recognize Discrete AQP4 Determinants and
are Restricted by HLA-DR Molecules
[0180] In general, antigen-specific T cells recognize linear
peptide fragments of 10-15 amino acids in association with MHC
(HLA) proteins expressed on APC (Zamvil S S, et al., Nature. 1986
Nov. 20-26; 324(6094):258-60). In order to identify AQP4-specific T
cells in NMO patients, proliferation of peripheral blood
mononuclear cells (PBMC) to a library of 32 synthetic overlapping
15 mer and 20 mer peptides encompassing the 323 amino acid sequence
of full-length human AQP4 (Ml isoform) was initially tested. Here,
separate pools containing five overlapping AQP4 peptides were
studied. By [.sup.3H]thymidine incorporation, more frequent
proliferative responses in primary cultures to AQP4 pools 1-55,
46-100, 126-170, 201-250 and 241-300 was detected (FIG. 1A).
Lymphocytes from healthy controls (HC) also proliferated to some of
these pools, and exhibited comparable responses to tetanus toxoid
(TT).
[0181] Having identified candidate regions of AQP4 containing T
cell determinants, proliferative responses of NMO patients to
individual AQP4 peptides was then tested. T cell determinants were
identified within peptides (p) 21-40, 61-80, 131-150, 156-170 and
211-230 (FIG. 1B), which corresponded to intracellular,
extracellular and transmembrane sequences of AQP4 (FIG. 1C).
Interestingly, three of these AQP4 determinants, p61-80, p131-150
and p211-230, respectively, are located in extracellular A, C and E
loops, AQP4 domains targeted by NMO-IgG (Owens G, et al., Mult
Scler. 2011; 17(10 Suppl):S291-S2). The fluorescent dye
5,6-caroxylfluorescein diacetate succinimidyl ester (CFSE) dilution
assay is considered a more powerful and sensitive method for
detecting proliferation of rare autoantigen-specific human T cells
than the traditional [.sup.3H]thymidine incorporation (Mannering S
I, et al., J Immunol Methods. 2003 December; 283(1-2):173-83).
Using this approach, responses to individual AQP4 peptides
identified in the initial screening, and also to AQP4 T cell
determinants common to mouse strains with distinct MHC haplotypes
was examined (Nelson P A, et al., PLoS One. 2010 November 2010;
5(11):e15050 1-9; Kalluri S R, et al., PLoS ONE. 2011;
6(1):e16083). A robust proliferative T cell response to p61-80,
which is located within the extracellular A loop, was detected in
all NMO patients tested. T cell responses were observed to AQP4
p21-40, p156-170, p11-30 and p261-280 (FIG. 1D), even though
substantial proliferation to the latter two peptides in the initial
[.sup.3H]thymidine incorporation assays was not detected. T cells
from HC also recognized these AQP4 peptides, but again, the
proliferative responses were both lower and less frequent than in
NMO patients. Proliferating AQP4-specific T cells were
predominantly CD4.sup.+, and the proportion of CD4.sup.+ T cells
that responded to AQP4 p61-80 was higher in NMO patients than HC
(FIG. 1E).
[0182] Presentation of native protein antigens by APC generally
requires proteolytic processing (Slavin A J, et al., J Clin Invest.
2001; 108(8):1133-9; Soos J M, et al., J Immunol. 1998 Dec. 1;
161(11):5959-66; Zamvil S, et al., Nature. 1985 Sep. 26-Oct. 2;
317(6035):355-8; Vyas J M, et al., Nat Rev Immunol. 2008 August;
8(8):607-18.). Therefore, it was examined whether the identified
AQP4 peptides contained natural T cell determinants of intact AQP4.
When T cells initially stimulated with rhAQP4 were tested for
recall responses to individual AQP4 peptides, proliferation to AQP4
p21-40 and p61-80 was observed, indicating that these are naturally
processed determinants of AQP4 (FIG. 1F). Among peptides that were
examined, AQP4 p61-80 was clearly immunodominant. Several studies
have identified over-representation of HLA-DPB1*0501, HLA-DRB1*0301
or HLA-DRB3 in NMO patients (Matsushita T, et al., Tissue Antigens.
2009 February; 73(2):171-6; Brum D G, et al., Mult Scler. 2010
January; 16(1):21-9; Deschamps R, et al., Mult Scler. 2011 January;
17(1):24-31), suggesting these MHC II alleles could serve as
restriction elements for CD4.sup.+ T cells in NMO. A high
representation of these HLA alleles in the patient cohort was also
identified, in particular, an overrepresentation of HLA-DRB3*0202
among NMO subjects was noted (Table 2).
TABLE-US-00006 TABLE 2 HLA haplotypes of NMO patients and healthy
controls DRB1*1501.sup.a DQB1*0602.sup.a DRB1*0301.sup.b
DRB3*0202.sup.b DPB1*0501.sup.b NMO 3/15 2/15 7/15 11/15 7/15
Patients 20% 13% 47% 73% 47% Healthy 2/8 1/8 3/8 3/8 3/8 Controls
25% 12.5% 37.5% 37.5% 37.5% .sup.aAlleles associated with multiple
sclerosis susceptibility; .sup.bAlleles associated with
neuromyelitis optica susceptibility
[0183] Using MHC II blocking antibodies, it was observed that T
cell proliferative responses to AQP4 p61-80 were inhibited by
anti-HLA-DR, but were not statistically inhibited by anti-HLA-DQ or
anti-HLA-DP, demonstrating that HLA-DR molecules serve as
restriction elements for T cells that recognize this determinant
(FIG. 2A). Proliferation of AQP4 p61-80-specific T cells from
HLA-matched HC (Table 2) was also inhibited by anti-HLA-DR
antibodies (FIG. 2B). Further, a similar MHC II-restriction profile
was observed after stimulating T cells from NMO patients with
rhAQP4 (FIG. 2C) suggesting that other AQP4 determinants may also
be restricted by HLA-DR molecules.
[0184] FIGS. 1A-1F. T cells from NMO patients recognize discrete
determinants of AQP4. PBMC were tested for proliferation to (Panel
A) pools of AQP4 peptides (n=8 NMO and n=3 HC) and to (Panel B)
individual AQP4 peptides identified from those pools. In (Panel A
and Panel B) PBMC were cultured for 6 d in the presence of AQP4
pools (10 .mu.g/ml) or AQP4 peptides (10 .mu.g/ml), respectively,
then pulsed with [.sup.3H]thymidine and harvested 18 h later. In
(Panel A), positive wells were defined as values>control cpm
average values+3SD. (Panel C) AQP4 determinants are represented
within a human AQP4 topological diagram using Johns S. J., TOPO2,
Transmembrane protein display software (Crane J M, et al.,
Neuroscience. 2010 Jul. 28; 168(4):892-902.). (Panel D and Panel E)
PBMC were examined by CFSE dilution for proliferation to individual
AQP4 peptides (10 .mu.g/ml), rhAQP4 (5 .mu.g/ml) or in (Panel E)
tetanus toxoid (TT) (1 .mu.g/ml) after 10 d of culture. CFSE was
measured in CD3.sup.+, CD4.sup.+ and CD8.sup.+ T cells by FACS and
quantified by cell division index (CDI). CDI>2 (broken lines)
was considered positive. (Panel F) Recall T cell proliferation
([.sup.3H]thymidine incorporation) to individual AQP4 peptides (10
.mu.g/ml) or rhAQP4 (5 .mu.g/ml) was detected after initial
stimulation with rhAQP4 (5 .mu.g/ml) for 10 d. In Panels A and E,
error bars indicate SEM; in B, D and F, horizontal lines indicate
mean values. *P<0.05 Mann-Whitney U test.
[0185] FIG. 2A-2C. HLA-DR serves as a restriction element for
AQP4-specific T cells. (Panels A and B) CFSE-labeled PBMC from NMO
patients were cultured for 10 d with antigens alone or in
combination with anti-HLA-DR, -HLA-DQ, -HLA-DP or isotype control
antibodies. T cell proliferation was evaluated by FACS analysis of
CFSE dilution. Inhibitory effects of blocking antibodies were
examined on proliferating CD4.sup.+ T cells (n=7 NMO in (Panel A)
and n=4 NMO in (Panel B)). T cell proliferation is expressed as
cell division index (CDI). (Panel C) PBMC from HC were similarly
examined after stimulation with AQP4 p61-80 (n=2). Error bars
represent SEM. *P<0.05, Mann-Whitney U test.
EXAMPLE 2
AQP4 P63-76-Specific T Cells Cross-React with C. Perfringens ABC
Transporter Permease P204-217
[0186] In order to characterize the fine specificity of AQP4
p61-80-specific T cells, proliferation to truncated peptides
corresponding to sequences within this region was examined. AQP4
p61-80-specific T cells proliferated in response to p61-78 and
p61-76 but not to p61-74 or p61-72 (FIG. 3A). Shorter AQP4 peptides
truncated from the N-terminal sequence, p65-80, p67-80 and p69-80
also stimulated proliferation of p61-80-specific T cells although
less efficiently than p63-80. Collectively, these findings
indicated that p63-76 contained the core determinant of AQP4 61-80.
In this regard, it was observed that p61-80-specific T cells
responded nearly as efficiently to p63-76 as to p61-80 (FIG. 3B).
Interesting, AQP4 63-76 contains the predicted binding motif for
HLA-DRB1*0301 and HLA-DRB3*0202 (FIG. 3C).
[0187] Immune responses to pathogens may elicit cross-reactivity to
self-antigens that share structural or sequence homology (Fujinami
R S, Oldstone M B. Science. 1985 Nov. 29; 230(4729):1043-5;
Wucherpfennig K W, Strominger J L. Cell. 1995 Mar. 10;
80(5):695-705). This process, known as `molecular mimicry`, is
considered one important potential mechanism in autoimmunity.
Having found that p63-76 contains an immunodominant AQP4 T cell
epitope, whether this sequence might share homology with other
proteins was addressed. 90% homology between AQP4 66-75 and the ten
amino acid sequence 207-216 within conserved ABC transporter
permease (ABC-TP) proteins from several strains of the bacterium
Clostridium perfringens (NCBI protein reference sequences
ZP.sub.--02952885.1, ZP.sub.--02638213.1, ZP.sub.--02634520.1,
ZP.sub.--02630305.1; 90% positives, 90% identities, 0% gaps) was
identified (FIG. 3C). T cells from NMO patients proliferated
significantly to ABC-TP p204-217, although less intensely than to
AQP4 p61-80 and AQP4 p63-76 (FIG. 3D). To directly test for
cross-reactivity, T cells initially stimulated with AQP4 p63-76 or
ABC-TP were tested for recall responses in a reciprocal manner
(FIG. 3E). Importantly, AQP4-primed T cells proliferated to ABC-TP
p204-217 and vice versa, supporting molecular mimicry between this
bacterial transmembrane protein and AQP4. Confirming specificity of
those recall responses, proliferation to p156-170 was not
observed.
[0188] FIG. 3A-3E. Cross-reactivity between AQP4 p63-76 and
Clostridium perfringens ABC transporter permease (ABC/TP) p204-217.
(Panel A) The T cell epitope within AQP4 p61-80 was mapped by
testing recall proliferation of AQP4 p61-80-reactive T cells from
NMO patients to truncated AQP4 peptides (10 .mu.g/ml) in the
presence of irradiated autologous APC. (Panel B) AQP4 p63-76
appeared to contain p61-80 core determinant. Proliferation was
measured by [.sup.3H]thymidine incorporation after 3 d. Data are
representative of three independent experiments. (Panel C) Sequence
homology between AQP4 p63-76 and C. perfringens ABC transporter
permease (ABC-TP) p204-217 was identified using the protein-protein
Basic Local Alignment Search Tool (BLAST) from NCBI. Top bracket
represents the predicted core binding motif for HLA-DRB1*0301 and
HLA-DRB3*0202 within AQP4 p63-76 (netMHCII-1.1 and -2.2 programs).
(Panel D) CFSE-labeled PBMC from 3 NMO patients were stimulated
with antigens (10 .mu.g/ml) and cultured for 10 d before evaluating
proliferation by FACS. (Panel E) PBMC from 4 NMO patients were
initially stimulated for 10 d with AQP4 p63-76 or ABC/TP p204-217
at 10 .mu.g/ml. Recall responses to peptides in the presence of
irradiated autologous APC were evaluated by [.sup.3H]thymidine
incorporation after 3 d. Paired t-tests were performed to compare
counts per minute (cpm) values of each antigen to cpm values of
no-antigen controls, *P<0.05, **P<0.01. In (Panel A), (Panel
B) and (Panel E) data are presented as means of duplicate or
triplicate wells; in (Panels A-E) error bars indicate SEM.
EXAMPLE 3
AQP4 P61-80-Specific T Cells from NMO Patients Exhibit
Pro-Inflammatory Th17 Polarization
[0189] Although indirect, some clinical and histologic data suggest
Th17 cells may participate in NMO pathogenesis (Lucchinetti C F, et
al. Brain. 2002 July; 125(Pt 7):1450-61; Warabi Y, et al., J Neurol
Sci. 2006 Nov. 15; 249(2):145-52). Thus, proinflammatory cytokine
production in proliferating AQP4-specific T cells was examined. In
comparison to HC, significantly higher frequencies of IL-17.sup.+
single- and IL-17.sup.-IFN-.gamma..sup.+ double-positive cells that
recognized p61-80 in NMO patients was observed (FIG. 4A, B). An
increased frequency of Th17 cells from NMO patients was observed
after stimulation with rhAQP4, but was not significant. No Th17
bias was detected in response to AQP4 p156-170, suggesting the Th17
polarization may be epitope-specific. In contrast, IFN-.gamma.
production by AQP4-specific T cells appeared unchanged between the
two groups. Thus, the Th17/Th1 ratio was elevated in NMO patients
in response to the immunodominant determinant AQP4 p61-80, but not
to the other antigens tested. Interestingly, a difference in the
frequency of peripheral blood regulatory T cells (Treg) from NMO
patients and HC was not detected (FIG. 4C). By contrast, the
examination of AQP4-specific T cells revealed a significantly
reduced frequency of Treg in NMO patients in response to rhAQP4,
but not to p61-80 (FIG. 4D).
[0190] FIG. 4A-4D. AQP4 p61-80-specific T cells exhibit a
proinflammatory bias. PBMC were stained with CFSE and cultured for
10 d with AQP4 peptides (10 .mu.g/ml) or rhAQP4 (5 .mu.g/ml).
(Panel A) CD4.sup.+CFSE.sup.low proliferating T cells were analyzed
for IL-17 and IFN-.gamma. production by intracellular staining
after stimulation with PMA/Ionomycin for 5 h. (Panel B) Frequencies
of IL17.sup.+IFN-.gamma..sup.-, IL17.sup.+IFN-.gamma..sup.+ and
IL17.sup.-IFN-.gamma..sup.+ were examined among proliferating
p61-80-specific CD4.sup.+ T cells (n=8 NMO and n=5 HC),
p156-170-specific CD4.sup.+ T cells (n=6 NMO and n=3 HC) and
rhAQP4-specific CD4.sup.+ T cells (n=6 NMO and n=5 HC). Frequencies
of IL-17- and IFN-.gamma.-single positive T cells were used to
calculate Th17/Th1 ratio. (Panel C) PBMC were examined by FACS for
expression of Treg markers including CD4, CD127 and CD25. (Panel D)
CFSE-labeled PBMC were cultured for 10 d with AQP4 p61-80 (10
.mu.g/ml) or rhAQP4 (5 .mu.g/ml). Proliferating CD4.sup.+ T cells
(CDI>2) were examined by FACS for expression of CD25.sup.hugh,
defined as the top half of CD25.sup.+ cells, and Foxp3 (n=8 NMO
p61-80, n=6 HC p61-80, n=7 NMO rhAQP4 and n=5 HC rhAQP4).
Box-and-whisker plots include the median, distribution and range.
**P<0.01 Mann-Whitney U test.
EXAMPLE 4
Monocytes from NMO Patients Exhibit Pro-Inflammatory
Polarization
[0191] Antigen presenting cells (APC), including monocytes and
other myeloid cells, express costimulatory molecules and secrete
specific cytokines that participate in activation and promote
lineage commitment of antigen-specific T cells. In this regard,
IL-6 is critical for Th17 differentiation (Acosta-Rodriguez E V, et
al. Nat Immunol. 2007 September; 8(9):942-9.). Previous studies
have indicated that serum IL-6 levels are elevated in NMO patients
(Uzawa A, et al. Mult Scler. 2010 December; 16(12):1443-52.). Since
AQP4 p61-80-specific T cells from NMO patients exhibited Th17
polarization, whether there were alterations in expression of
costimulatory molecules or increased production of IL-6 by myeloid
APC was addressed. In comparison to HC, there was no evident change
in frequency of peripheral blood monocytes. However, increased
expression of CD40 and CD80 (FIG. 5A; histogram for healthy control
is located in between the histograms for isotype and NMO patient),
costimulatory molecules that can be associated with
pro-inflammatory T cell polarization was observed (Katzman S D, et
al., J Immunol. 2011 Apr. 15; 186(8):4668-73; Kuchroo V K, et al.,
Cell. 1995 Mar. 10; 80(5):707-18). The frequency of IL-6-producing
monocytes was similar in NMO patients and HC. Nevertheless, there
were both relative and absolute increases of intracellular IL-6
production after LPS stimulation in monocytes from NMO patients
(FIG. 5B, C). No such differences were observed in expression of
IL-1.beta. and IL-10. These results indicate that in addition to
the known involvement of adaptive immunity, phenotypic changes of
cells within the innate immune system may also contribute to NMO
pathogenesis.
[0192] FIG. 5A-5C. CD14.sup.- monocytes from NMO patients exhibit
increased expression of certain co-stimulatory molecules and
production of IL-6. (Panel A) PBMC were rested for 4 h at
37.degree. C. Expression of co-stimulatory (CD80, CD86 and CD40)
and MHC class II molecules was analyzed by FACS gating on the
CD14.sup.+ population (n=8 NMO and n=8 HC). Isotype is indicated by
grey histogram; healthy control and NMO patient histograms for
expression of CD86 and HLA-DR were similar; for CD80 and CD40
expression the histogram for healthy control is present in between
the histograms for isotype and NMO patient. As such, NMO patients
exhibited a higher expression of CD80 and CD40 compared to healthy
control. (Panels B and C) PBMC were stimulated with LPS (1
.mu.g/ml) for 4 h. Expression of IL-6 in CD14.sup.- monocytes was
analyzed by ICS, before and after LPS stimulation. In (Panel C),
horizontal lines indicate mean values; in (Panel A) and (Panel B)
error bars represent SEM. *P<0.05, **P<0.01 Mann-Whitney
test.
Sequence CWU 1
1
25110PRTArtificial Sequencesynthetic poypeptide 1Leu Pro Xaa Xaa
Met Xaa Xaa Ile Xaa Xaa 1 5 10 210PRTArtificial sequencesynthetic
poypeptide 2Leu Pro Val Xaa Met Val Leu Ile Ser Leu 1 5 10
320PRTArtificial sequencesynthetic poypeptide 3Gly Ile Leu Tyr Leu
Val Thr Pro Pro Ser Val Val Gly Gly Leu Gly 1 5 10 15 Val Thr Met
Val 20 420PRTArtificial sequencesynthetic poypeptide 4Ser Met Asn
Pro Ala Arg Ser Phe Gly Pro Ala Val Ile Met Gly Asn 1 5 10 15 Trp
Glu Asn His 20 515PRTArtificial sequencesynthetic poypeptide 5Ala
Gly His Gly Leu Leu Val Glu Leu Ile Ile Thr Phe Gln Leu 1 5 10 15
620PRTArtificial sequencesynthetic poypeptide 6Arg Phe Lys Glu Ala
Phe Ser Lys Ala Ala Gln Gln Thr Lys Gly Ser 1 5 10 15 Tyr Met Glu
Val 20 714PRTHomo sapiens 7Glu Lys Pro Leu Pro Val Asp Met Val Leu
Ile Ser Leu Cys 1 5 10 814PRTClostridium perfringens 8Phe Ile Ile
Leu Pro Val Ser Met Val Leu Ile Ser Leu Val 1 5 10 9323PRTHomo
sapiens 9Met Ser Asp Arg Pro Thr Ala Arg Arg Trp Gly Lys Cys Gly
Pro Leu 1 5 10 15 Cys Thr Arg Glu Asn Ile Met Val Ala Phe Lys Gly
Val Trp Thr Gln 20 25 30 Ala Phe Trp Lys Ala Val Thr Ala Glu Phe
Leu Ala Met Leu Ile Phe 35 40 45 Val Leu Leu Ser Leu Gly Ser Thr
Ile Asn Trp Gly Gly Thr Glu Lys 50 55 60 Pro Leu Pro Val Asp Met
Val Leu Ile Ser Leu Cys Phe Gly Leu Ser 65 70 75 80 Ile Ala Thr Met
Val Gln Cys Phe Gly His Ile Ser Gly Gly His Ile 85 90 95 Asn Pro
Ala Val Thr Val Ala Met Val Cys Thr Arg Lys Ile Ser Ile 100 105 110
Ala Lys Ser Val Phe Tyr Ile Ala Ala Gln Cys Leu Gly Ala Ile Ile 115
120 125 Gly Ala Gly Ile Leu Tyr Leu Val Thr Pro Pro Ser Val Val Gly
Gly 130 135 140 Leu Gly Val Thr Met Val His Gly Asn Leu Thr Ala Gly
His Gly Leu 145 150 155 160 Leu Val Glu Leu Ile Ile Thr Phe Gln Leu
Val Phe Thr Ile Phe Ala 165 170 175 Ser Cys Asp Ser Lys Arg Thr Asp
Val Thr Gly Ser Ile Ala Leu Ala 180 185 190 Ile Gly Phe Ser Val Ala
Ile Gly His Leu Phe Ala Ile Asn Tyr Thr 195 200 205 Gly Ala Ser Met
Asn Pro Ala Arg Ser Phe Gly Pro Ala Val Ile Met 210 215 220 Gly Asn
Trp Glu Asn His Trp Ile Tyr Trp Val Gly Pro Ile Ile Gly 225 230 235
240 Ala Val Leu Ala Gly Gly Leu Tyr Glu Tyr Val Phe Cys Pro Asp Val
245 250 255 Glu Phe Lys Arg Arg Phe Lys Glu Ala Phe Ser Lys Ala Ala
Gln Gln 260 265 270 Thr Lys Gly Ser Tyr Met Glu Val Glu Asp Asn Arg
Ser Gln Val Glu 275 280 285 Thr Asp Asp Leu Ile Leu Lys Pro Gly Val
Val His Val Ile Asp Val 290 295 300 Asp Arg Gly Glu Glu Lys Lys Gly
Lys Asp Gln Ser Gly Glu Val Leu 305 310 315 320 Ser Ser Val
10618PRTClostridium perfringens 10Met Ser Lys Glu Arg Lys Gly Gly
Met Gly Gly Pro Met Gly Arg Met 1 5 10 15 Gly Gly Gly Pro Arg Ala
Val Glu Lys Ala Lys Asp Phe Lys Gly Thr 20 25 30 Met Lys Lys Leu
Gly Val Tyr Leu Lys Pro Tyr Ser Leu Ser Ile Ala 35 40 45 Ile Val
Ile Leu Phe Ala Ile Gly Ser Ala Ala Phe Ser Ile Val Gly 50 55 60
Pro Lys Ile Leu Gly Lys Ala Thr Thr Lys Ile Phe Glu Gly Leu Val 65
70 75 80 Gln Lys Ile Thr Gly Val Pro Asp Ala Ser Ile Asp Phe Gly
Tyr Ile 85 90 95 Gly Asn Ile Ala Met Ile Leu Val Ala Leu Tyr Leu
Val Ser Ser Leu 100 105 110 Phe Gly Ile Ile Gln Ser Phe Ile Met Ser
Gly Val Ala Gln Lys Val 115 120 125 Ser Tyr Asn Leu Arg Lys Gln Ile
Ser Glu Lys Met Asp Thr Leu Pro 130 135 140 Leu Asn Tyr Phe Asp Thr
Arg Thr Asn Gly Glu Val Leu Ser Arg Ile 145 150 155 160 Thr Asn Asp
Val Asp Thr Val Asn Gln Thr Leu Asn Gln Ser Leu Ser 165 170 175 Gln
Ile Ile Thr Ser Val Val Thr Leu Ile Gly Val Leu Ile Met Met 180 185
190 Phe Ser Ile Ser Trp Ile Met Thr Leu Ala Thr Phe Ile Ile Leu Pro
195 200 205 Val Ser Met Val Leu Ile Ser Leu Val Val Lys Lys Ser Gln
Lys Tyr 210 215 220 Phe Lys Ser Gln Gln Glu Tyr Leu Gly His Leu Asn
Gly Gln Val Glu 225 230 235 240 Glu Val Tyr Gly Gly His Asn Ile Met
Lys Ala Phe Asn Arg Glu Glu 245 250 255 Ala Ser Thr Lys Asp Phe Asp
Glu Leu Asn Asn Thr Leu Tyr Lys Ser 260 265 270 Ala Trp Lys Ser Gln
Phe Leu Ser Gly Met Met Met Pro Ile Met Ser 275 280 285 Phe Val Gly
Asn Leu Gly Tyr Val Leu Val Ser Ile Leu Gly Gly Trp 290 295 300 Leu
Thr Ile Lys Ser Val Ile Thr Val Gly Asp Ile Gln Ala Phe Ile 305 310
315 320 Gln Tyr Val Arg Ser Phe Asn Gln Pro Ile Ser Gln Met Ala Gln
Val 325 330 335 Ala Asn Ile Met Gln Ser Thr Ala Ala Ala Ala Glu Arg
Val Phe Glu 340 345 350 Phe Leu Asp Glu Glu Asp Glu Val Lys Asp Pro
Val Asn Ser Val Asp 355 360 365 Pro Ser Glu Ile Arg Gly Glu Val Glu
Phe Glu Asp Phe His Phe Gly 370 375 380 Tyr Asn Glu Asp Lys Ile Ile
Ile Asn Asp Phe Ser Val Asp Val Lys 385 390 395 400 Pro Gly Gln Lys
Val Ala Ile Val Gly Pro Thr Gly Ala Gly Lys Thr 405 410 415 Thr Ile
Val Lys Leu Leu Met Arg Phe Tyr Asp Ile Asn Ser Gly Ser 420 425 430
Ile Lys Ile Asp Gly His Asp Ile Arg Asp Phe Lys Arg Ala Asp Leu 435
440 445 Arg Asn Leu Phe Gly Met Val Leu Gln Asp Thr Trp Leu Phe Asn
Gly 450 455 460 Thr Ile Met Glu Asn Leu Arg Tyr Gly Arg Leu Asp Ala
Thr Asp Ala 465 470 475 480 Glu Val Lys Glu Ala Ala Lys Ala Ala His
Val Asp His Phe Val Lys 485 490 495 Thr Leu Pro Asp Gly Tyr Asn Met
Val Leu Asn Glu Glu Ala Ser Asn 500 505 510 Ile Ser Gln Gly Gln Lys
Gln Leu Leu Thr Ile Ala Arg Ala Phe Leu 515 520 525 Lys Asp Pro Lys
Leu Leu Ile Leu Asp Glu Ala Thr Ser Ser Val Asp 530 535 540 Thr Arg
Thr Glu Leu Leu Ile Gln Lys Ala Met Glu Lys Leu Met Glu 545 550 555
560 Gly Arg Thr Ser Phe Ile Ile Ala His Arg Leu Ser Thr Ile Arg Asp
565 570 575 Ala Asp Leu Ile Leu Val Met Lys Asp Gly Asp Ile Val Glu
Gln Gly 580 585 590 Asn His Glu Glu Leu Leu Glu Lys Gly Gly Phe Tyr
Ser Ser Leu Tyr 595 600 605 Asn Ser Gln Phe Glu Gln Ser Ser Ala Ser
610 615 1110PRTHomo sapiens 11Leu Pro Val Asp Met Val Leu Ile Ser
Leu 1 5 10 1210PRTClostridium perfringens 12Leu Pro Val Ser Met Val
Leu Ile Ser Leu 1 5 10 1310PRTClostridium scindens 13Leu Pro Val
Ser Met Gly Leu Ile Ser Val 1 5 10 1410PRTClostridium sporogens
14Leu Pro Val Ser Met Ile Ile Ile Met Leu 1 5 10 1510PRTClostridium
hylemonae 15Leu Pro Ile Ser Met Gly Leu Ile Ser Ala 1 5 10
1610PRTArtificial sequencesynthetic poypeptide 16Leu Pro Xaa Xaa
Met Xaa Xaa Ile Xaa Xaa 1 5 10 1710PRTArtificial sequencesynthetic
poypeptide 17Leu Pro Val Xaa Met Val Leu Ile Ser Leu 1 5 10
1818PRTArtificial sequencesynthetic poypeptide 18Gly Thr Glu Lys
Pro Leu Pro Val Asp Met Val Leu Ile Ser Leu Cys 1 5 10 15 Phe Gly
1916PRTArtificial sequencesynthetic poypeptide 19Gly Thr Glu Lys
Pro Leu Pro Val Asp Met Val Leu Ile Ser Leu Cys 1 5 10 15
2014PRTArtificial sequencesynthetic poypeptide 20Gly Thr Glu Lys
Pro Leu Pro Val Asp Met Val Leu Ile Ser 1 5 10 2112PRTArtificial
sequencesynthetic poypeptide 21Gly Thr Glu Lys Pro Leu Pro Val Asp
Met Val Leu 1 5 10 2218PRTArtificial sequencesynthetic poypeptide
22Glu Lys Pro Leu Pro Val Asp Met Val Leu Ile Ser Leu Cys Phe Gly 1
5 10 15 Leu Ser 2316PRTArtificial sequencesynthetic poypeptide
23Pro Leu Pro Val Asp Met Val Leu Ile Ser Leu Cys Phe Gly Leu Ser 1
5 10 15 2414PRTArtificial sequencesynthetic poypeptide 24Pro Val
Asp Met Val Leu Ile Ser Leu Cys Phe Gly Leu Ser 1 5 10
2512PRTArtificial sequencesynthetic poypeptide 25Asp Met Val Leu
Ile Ser Leu Cys Phe Gly Leu Ser 1 5 10
* * * * *