U.S. patent application number 11/373894 was filed with the patent office on 2007-01-04 for low dose histone peptide treatment for autoimmune disorders.
This patent application is currently assigned to Northwestern University. Invention is credited to Syamal K. Datta, Hee-Kap Kang.
Application Number | 20070003543 11/373894 |
Document ID | / |
Family ID | 22449513 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003543 |
Kind Code |
A1 |
Datta; Syamal K. ; et
al. |
January 4, 2007 |
Low dose histone peptide treatment for autoimmune disorders
Abstract
The present invention provides low dose compositions comprising
peptides and methods of using the same to treat autoimmune
disorders, such as lupus. In particular, the present invention
provides low dose compositions comprising nucleosomal histone
peptide autoepitopes comprising MHC class I and II binding motifs
and methods of using the same to suppress IFN-gamma and/or
autoantibody production. In certain embodiments, the compositions
contain the histone peptides at subnanomolor concentrations, such
as 0.1 nM to 0.9 nM. In other embodiments, the compositions are
adminstered in a low dose, such as 5-75 ug of histone peptides per
kilogram of patient. In certain embodiments, the present invention
provides related diagonstics employing histone peptides.
Inventors: |
Datta; Syamal K.; (Winnetka,
IL) ; Kang; Hee-Kap; (Chicago, IL) |
Correspondence
Address: |
David A. Casimir;MEDLEN & CARROLL, LLP
101 Howard Street
Suite 350
San Francisco
CA
94105
US
|
Assignee: |
Northwestern University
Evanston
IL
|
Family ID: |
22449513 |
Appl. No.: |
11/373894 |
Filed: |
March 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10211069 |
Aug 2, 2002 |
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11373894 |
Mar 13, 2006 |
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09561490 |
Apr 28, 2000 |
6468537 |
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10211069 |
Aug 2, 2002 |
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60131448 |
Apr 28, 1999 |
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60661281 |
Mar 11, 2005 |
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Current U.S.
Class: |
424/131.1 ;
424/144.1 |
Current CPC
Class: |
A61K 38/1709 20130101;
Y10S 514/866 20130101; A61K 38/00 20130101; G01N 2800/24 20130101;
A61K 39/0008 20130101; G01N 2800/104 20130101; G01N 33/564
20130101; Y10S 514/885 20130101 |
Class at
Publication: |
424/131.1 ;
424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
[0002] This invention was funded, in part, under NIH grants R01 AR
39157, RO1-A141985 and R37-AR39157. The government may have certain
rights in the invention.
Claims
1. A method of treating an animal having systemic lupus
erythematosus (SLE) and a SLE-associated manifestation of
nephritis, autoantibodies, and inflammation associated with
autoantibodies, said method comprising administering to said animal
an isolated peptide comprising a portion of a nucleosome histone
protein, wherein said isolated peptide is capable of specifically
binding with a T cell receptor present on a cell, and further
wherein said isolated peptide is capable of promoting immunological
tolerance in an animal, thereby treating said SLE and said
SLE-associated manifestation, wherein said isolated peptide is
administered at a concentration between 5 ug and 75 ug per kilogram
of said animal or at a concentration of between at least 0.1 nM and
no more than 0.9 nM.
2. The method of claim 1, wherein said isolated peptide is
administered in an amount which is from at least about 25
micrograms per kilogram of animal to about 50 micrograms per
kilogram of said animal.
3. The method of claim 1, wherein said peptide comprises the amino
acid sequence shown in SEQ ID NO:3. or SEQ ID NO:1
4. The method of claim 1, wherein said peptide is administered at a
concentration of between at least 0.1 nM and no more than 0.9
nM.
5. The method of claim 1, wherein said peptide comprises a MHC
class I binding motif.
6. The method of claim 1, wherein said administration is conducted
once about every two weeks.
7. The method of claim 1, wherein said administration is
subcutaneous administration.
8. The composition of claim 1, wherein said isolated peptides
comprise an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1-5, 7, 9-10, 14, 18-19, and 22-26.
9. An isolated composition comprising isolated peptides comprising
a portion of a nucleosome histone protein, wherein said isolated
peptides are present in said composition at a concentration of at
least 0.1 nM and no more than 0.9 nM.
10. The composition of claim 9, wherein said nucleosome histone
protein comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-5, 7, 9-10, 14, 18-19, and 22-26.
11. The composition of claim 9, wherein said nucleosomal histone
peptide comprises SEQ ID NO:3.
12. The composition of claim 9, wherien said nucleosomal histone
peptide comprises SEQ ID NO:1.
13. The composition of claim 9, wherein said isolated peptide is
capable of specifically binding with a T cell receptor present on a
cell, and further wherein the isolated peptide is capable of
promoting immunological tolerance in an animal.
14. A system comprising; a) a composition comprising isolated
peptides comprising a portion of a nucleosome histone protein,
wherein said isolated peptides are present in said composition at a
concentration of at least 0.1 nM and no more than 0.9 nM; and b) a
container, wherein said composition is in said container.
15. The system of claim 14, wherein said container is a syringe
bottle configured to allow a needle to withdraw at least a portion
of said composition in a sterile manner.
16. The system of claim 14, wherein said nucleosome histone protein
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1-5, 7, 9-10, 14, 18-19, and 22-26.
17. The system of claim 14, wherien said nucleosomal histone
peptide comprises SEQ ID NO:3.
18. The system of claim 14, wherien said nucleosomal histone
peptide comprises SEQ ID NO:1.
19. The system of claim 14, wherein the isolated peptide is capable
of specifically binding with a T cell receptor present on a cell,
and further wherein the isolated peptide is capable of promoting
immunological tolerance in an animal.
20. A method comprising: (a) contacting a sample from an animal
with a composition comprising an isolated histone peptide complex,
wherein said histone peptide complex comprises: i) a histone
peptide portion, said histone peptide portion comprising no more
than 27 contiguous amino acids and having an amino acid sequence
corresponding to a portion of a nucleosome histone protein; ii) a
fused portion having an amino acid sequence which corresponds to a
portion of a protein selected from the group consisting of a major
histocompatibility class molecule and an immunoglobin; and iii) an
indicator portion, wherein said indicator portion is a molecule
which is capable of producing a detectable chemical signal; and
wherein each of said histone peptide portion, said fused portion,
and said indication portion is covalently linked to at least one
other component of said histone peptide complex; and (b)
identifying in said animal the signal produced by said indicator
portion, whereby the identification of said signal in said animal
is an indication that said animal has systemic lupus erythematosus,
thereby diagnosing systemic lupus erythematosus in said animal.
21. The method of claim 20 wherein said indicator portion is
selected from the group consisting of a flourophore, a chromophore,
a light reactive moiety and a biotin moiety
Description
[0001] The present application is a Continuation-In-Part of pending
U.S. application Ser. No. 10/211,069, filed Aug. 2, 2002, which is
a Continuation of U.S. application Ser. No. 09/561,490, filed Apr.
28, 2000, now U.S. Pat. No. 6,468,537, which claims priority to
U.S. Provisional Application 60/131,448, filed Apr. 28, 1999, all
of which are herein incorporated by reference in their entireties.
The present Application also claims priority to U.S. Provisional
Application 60/661,281, filed Mar. 11, 2005, herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to low dose compositions
comprising peptides and methods of using the same to treat
autoimmune disorders, such as lupus. In particular, the present
invention provides low dose compositions comprising nucleosomal
histone peptide autoepitopes comprising MHC class I and II binding
motifs and methods of using the same to suppress IFN-gamma and/or
autoantibody production. In certain embodiments, the compositions
contain the histone peptides at subnanomolor concentrations, such
as 0.1 nM to 0.9 nM. In other embodiments, the compositions are
adminstered in a low dose, such as 5-75 ug of histone peptides per
kilogram of patient. In certain embodiments, the present invention
provides related diagnostics employing histone peptides.
BACKGROUND OF THE INVENTION
[0004] Nucleosomes, derived from apoptotic cells (Ref. 1), are
major immunogens for initiating cognate interactions between
autoimmune T helper (Th) and B cells in systemic lupus
erythematosus (SLE).sup.3 (Ref. 2). CD4.sup.+ Th cells drive the
production of pathogenic anti-DNA autoantibodies in lupus patients
and lupus-prone SNF1 mice (Refs. 3, 4). Only certain peptides in
nucleosomal histones are immunodominant, and spontaneous priming to
these particular epitopes occurs in preclinical lupus. The five
major autoepitopes for lupus nephritis-inducing Th cells in murine
and human lupus are H1'.sub.22-42, H2B.sub.10-33, H3.sub.85-102,
H4.sub.16-39 and H4.sub.71-94 (Refs. 5-7). These peptide epitopes
are cross-reactively recognized by autoimmune Th cells, CD8.sup.+
T.sub.reg cells, as well as B cells. The peptides accelerate lupus
nephritis upon immunization, but they delay or even reverse disease
upon tolerization in high doses (Refs. 5, 6, 8). These nucleosomal
peptides can be promiscuously presented and recognized in the
context of diverse MHC alleles, behaving like universal epitopes
(Refs. 9, 10).
[0005] Thus, it would be highly desirable if a universally
tolerogenic peptide could be developed for therapy of lupus in
humans despite the high amount of HLA diversity among subjects.
Generation of such a treatment (e.g., treatment with compositions
comprising a tolerogenic peptide) must account for the sensitivity
subjects possess with regard to high doses of peptide provided.
Furthermore, an ideal tolerance therapy would generate long
lasting, TGF-beta producing T regulatory cells without causing
allergic/anaphylactic reactions or generalized
immunosuppression.
SUMMARY OF THE INVENTION
[0006] The present invention provides low dose compositions
comprising peptides and methods of using the same to treat
autoimmune disorders, such as lupus. In particular, the present
invention provides low dose compositions comprising nucleosomal
histone peptide autoepitopes comprising MHC class I and II binding
motifs and methods of using the same to suppress IFN-gamma and/or
autoantibody production. In certain embodiments, the compositions
contain the histone peptides at subnanomolor concentrations, such
as 0.1 nM to 0.9 nM. In other embodiments, the compositions are
adminstered in a low dose, such as 5-75 ug of histone peptides per
kilogram of patient.
[0007] In some embodiments, the present invention provides methods
of treating an animal having systemic lupus erythematosus (SLE) and
a SLE-associated manifestation of nephritis, autoantibodies, and
inflammation associated with autoantibodies, the method comprising
administering to the animal an isolated peptide comprising a
portion of a nucleosome histone protein, wherein the isolated
peptide is capable of specifically binding with a T cell receptor
present on a cell, and further wherein the isolated peptide is
capable of promoting immunological tolerance in an animal, thereby
treating the SLE and the SLE-associated manifestation, wherein the
isolated peptide is administered at a concentration between 5 ug
and 75 ug per kilogram of the animal, or at a concentration of
between at least 0.1 nM and no more than 0.9 nM (e.g. 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9).
[0008] In certain embodiments, the isolated peptide is administered
in an amount which is from at least about 25 micrograms per
kilogram of animal to about 50 micrograms per kilogram of the
animal (e.g. 25-50, 30-45, or 35-40 micrograms per kilogram). In
particular embodiments, the peptide comprises the amino acid
sequence shown in SEQ ID NO:3. In other embodiments, the peptide
comprises the amino acid sequence shown in SEQ ID NO:1. In further
embodiments, the peptide comprises a MHC class I binding motif.
[0009] In particular embodiments, the administration is conducted
once about every two weeks. In further embodiments, the
administration is subcutaneous administration.
[0010] In certain embodiments, the present invention provides
methods of treating an animal having systemic lupus erythematosus
(SLE) and a SLE-associated manifestation of nephritis,
autoantibodies, and inflammation associated with autoantibodies,
the method comprising administering to the animal an isolated
peptide comprising a portion of a nucleosome histone protein
wherein the peptide consists of an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1-5, 7, 9-10, 14, 18-19,
and 22-26, and further wherein the isolated peptide is capable of
specifically binding with a T cell receptor present on a cell, and
further wherein the isolated peptide is capable of promoting
immunological tolerance in an animal, thereby treating the SLE and
the SLE-associated manifestation, wherein the isolated peptide is
administered at a concentration between 5 ug and 75 ug per kilogram
of the animal, or at a concentration of between at least 0.1 nM and
no more than 0.9 nM (e.g. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
or 0.9).
[0011] In other embodiments, the present invention provides methods
of treating a subject, the method comprising administering a
nucleosomal histone peptide at a concentration between 5 ug and 75
ug per kilogram of the subject, wherein the peptide comprises a MHC
class I binding motif. In particular embodiments, the MHC class I
binding motif is selected from the group consisting of: TAMDVVYAL
(SEQ ID NO:62); NIQGITKPA (SEQ ID NO:63); HRKVLRDNI (SEQ ID NO:64);
and KYSDMIVAA (SEQ ID NO:65).
[0012] In some embodiments, the peptide is administered
subcutaneously. In further embodiments, the treating promotes
immunological tolerance in the subject. In other embodiments, the
subject has systemic lupus erythematosus or displays traits of
systemic lupus erythematosus. In certain embodiments, the treating
generates a beneficial T cell response. In other embodiments, the
beneficial T cell response comprises a reduction in IFN-g responses
of lupus T cells and/or reduced antibody production.
[0013] In some embodiments, the present invention provides
compositions comprising isolated peptides comprising a portion of a
nucleosome histone protein, wherein the isolated peptides are
present in said composition at a concentration of at least 0.1 nM
(e.g. 0.1, 0.2, 0.3, and 0.4 nM) and no more than 0.9 nM (e.g. 0.5,
0.6, 0.7, 0.8, and 0.9 nM). In certain embodiments, the composition
is present in a container (e.g. packaged for sale to a hospital).
In other embodiments, the composition is present in a syringe (e.g.
in preparation for administration to a patient). In further
embodiments, the isolated peptide is capable of specifically
binding with a T cell receptor present on a cell. In some
embodiments, the isolated peptide is capable of promoting
immunological tolerance in an animal.
[0014] In particular embodiments, the present invention provides
systems comprising; a) a composition comprising isolated peptides
comprising a portion of a nucleosome histone protein, wherein the
isolated peptides are present in the composition at a concentration
of at least 0.1 nM and no more than 0.9 nM; and b) a container,
wherein the composition is in the container. In further
embodiments, the isolated peptide is capable of specifically
binding with a T cell receptor present on a cell. In some
embodiments, the isolated peptide is capable of promoting
immunological tolerance in an animal.
[0015] In other embodiments, the container is configured to allow a
needle to withdraw at least a portion of the composition in a
sterile manner (e.g., a standard syringe bottle with a rubber type
top with hole for syringe and glass or plastic bottle for holding
the composition). In some embodiments, the container comprises a
syringe.
[0016] In certain embodiments, the nucleosome histone protein
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1-5, 7, 9-10, 14, 18-19, and 22-26. In other
embodiments, the nucleosomal histone peptide comprises or consists
of SEQ ID NO: 1 or 3.
[0017] The present invention provides a composition comprising an
isolated peptide which has an amino acid sequence corresponding to
the amino acid sequence of a portion of a nucleosome histone
protein and, and which is capable of promoting immunological
tolerance in an animal having systemic lupus erythematosus.
[0018] The invention includes an isolated peptide which has an
amino acid sequence corresponding to the amino acid sequence of a
portion of a nucleosome histone protein, wherein the portion of the
nucleosome histone protein corresponds to an autoepitope which is
associated with systemic lupus erythematosus and which is
recognized by one or more of an autoimmune T cell and an autoimmune
B cell.
[0019] In one embodiment, the isolated peptide can correspond in
amino acid sequence to a nucleosome histone protein which is
selected from the group consisting of histone 1 (H1), histone 2A
(H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4).
[0020] In multiple embodiments, the isolated peptide comprises not
more than 27 contiguous amino acids and has an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, and 26.
[0021] In other embodiments, the isolated peptide can further
comprise a covalently attached moiety selected from the group
consisting of a fluorophore, a chromophore, a biotin moiety, a
light reactive group, and an enzyme cleavable group.
[0022] In one aspect, the invention includes a composition
comprising a pharmaceutically acceptable carrier and an isolated
peptide which has an amino acid sequence corresponding to the amino
acid sequence of a portion of a nucleosome histone protein and, and
which is capable of promoting immunological tolerance in an animal
having systemic lupus erythematosus.
[0023] In another aspect, the invention includes an isolated
nucleic acid encoding an isolated peptide which has an amino acid
sequence corresponding to the amino acid sequence of a portion of a
nucleosome histone protein and, and which is capable of promoting
immunological tolerance in an animal having systemic lupus
erythematosus. In this aspect, the isolated nucleic acid can
comprise a nucleotide sequence selected from the group consisting
of SEQ ID NOS.: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and 52. Also
included in this aspect of the invention is a vector comprising the
isolated nucleic acid.
[0024] In multiple embodiments the isolated nucleic acid of the
invention comprises a nucleotide sequence selected from the group
consisting of SEQ ID NOS.: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and
52.
[0025] The invention also includes a cell comprising an isolated
nucleic acid encoding an isolated peptide which has an amino acid
sequence corresponding to the amino acid sequence of a portion of a
nucleosome histone protein and, and which is capable of promoting
immunological tolerance in an animal having systemic lupus
erythematosus.
[0026] In multiple embodiments, the cell comprising the isolated
nucleic acid is selected from the group consisting of a prokaryotic
cell and a eukaryotic cell. In one embodiment, the cell is an
insect cell.
[0027] The invention additionally includes a method of treating an
animal having an autoimmune disorder. This method comprises
administering to the animal an isolated peptide comprising a
portion of a nucleosome histone protein, wherein the isolated
peptide is capable of promoting immunological tolerance in an
animal, thereby treating the autoimmune disorder in the animal.
[0028] In multiple embodiments, the autoimmune disorder is selected
from the group consisting of rheumatoid arthritis, scleroderma, and
systemic lupus erythematosus.
[0029] In other embodiments, the isolated peptide is administered
in an amount which is from at least about 10 micrograms per
kilogram of animal to at least about 1 gram per kilogram of animal,
and from at least about 100 micrograms per kilogram of animal to
about 600 micrograms per kilogram of animal.
[0030] In still other embodiments, the isolated peptide comprises
not more than 27 contiguous amino acids and having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, and 26.
[0031] Alternatively, the method of treating an animal having an
autoimmune disorder comprises administering to the animal a
modified histone peptide, wherein the modified histone peptide is
an altered peptide ligand, and wherein the modified histone peptide
is capable of promoting immunological tolerance in the animal,
thereby treating the autoimmune disorder. In various embodiments of
this method, the autoimmune disorder is selected from the group
consisting of rheumatoid arthritis, scleroderma, and systemic lupus
erythematosus, and the isolated peptide is administered in an
amount which is from at least about 10 micrograms per kilogram of
animal to at least about 1 gram per kilogram of animal, and from at
least about 100 micrograms per kilogram of animal to about 600
micrograms per kilogram of animal.
[0032] In other embodiments, the modified peptide comprises not
more than 27 contiguous amino acids and has an amino acid sequence
which is at least one amino acid different relative to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, and 26.
[0033] The invention includes a method of treating nephritis in an
animal having systemic lupus erythematosus. This method comprises
administering to the animal an isolated peptide comprising a
portion of a nucleosome histone protein, wherein the isolated
peptide is capable of promoting immunological tolerance in an
animal, thereby alleviating the nephritis in the animal.
Alternatively, this method comprises administering to the animal a
modified histone peptide, wherein the modified histone peptide is
an altered peptide ligand, and wherein the modified histone peptide
is capable of promoting immunological tolerance in the animal,
thereby alleviating the nephritis in the animal.
[0034] The invention encompasses a method of reducing the
production of autoantibodies in an animal. This method comprises
administering to the animal an isolated peptide comprising a
portion of a nucleosome histone protein, wherein the isolated
peptide is capable of promoting immunological tolerance in an
animal, and wherein the peptide is administered in an amount
sufficient to promote immunologic tolerance in the animal, thereby
reducing the production of autoantibodies in the animal.
Alternatively, this method comprises administering to said animal a
modified histone peptide, wherein the modified histone peptide is
an altered peptide ligand capable of promoting immunological
tolerance in an animal, and wherein the modified histone peptide is
administered in an amount sufficient to promote immunologic
tolerance in the animal, thereby reducing the production of
autoantibodies in the animal.
[0035] In another aspect, the invention encompasses a method of
treating inflammation in an animal, which inflammation is caused by
the production of autoantibodies in the animal. This method
comprises administering to the animal an isolated peptide
comprising a portion of a nucleosome histone protein, wherein the
isolated peptide is capable of promoting immunological tolerance in
an animal, and wherein the peptide is administered in an amount
sufficient to promote immunological tolerance in the animal,
thereby inhibiting the production of autoantibodies in the animal
and alleviating inflammation in the animal. Alternatively, this
method comprises administering to the animal a modified histone
peptide, wherein the modified histone peptide is an altered peptide
ligand capable of promoting immunological tolerance in an animal,
and wherein the modified histone peptide is administered in an
amount sufficient to promote immunological tolerance in the animal,
thereby inhibiting the production of autoantibodies in the animal
and alleviating inflammation in the animal. In multiple
embodiments, the invention provides a method of diagnosing systemic
lupus erythematosus in an animal.
[0036] This method comprises (a) contacting a sample from the
animal with a composition comprising an isolated histone peptide
complex, wherein said histone peptide complex comprises i) a
histone peptide portion, said histone peptide portion comprising no
more than 27 contiguous amino acids and having an amino acid
sequence corresponding to a portion of a nucleosome histone
protein; ii) a fused portion having an amino acid sequence which
corresponds to a portion of a protein selected from the group
consisting of a major histocompatibility class molecule (e.g. MHC
class I or class II molecule) and an immunoglobin; and compatible
iii) an indicator portion, wherein said indicator portion is a
molecule which is capable of producing a detectable chemical
signal, and which is selected from the group consisting of a
flourophore, a chromophore, a light reactive moiety and a biotin
moiety; and wherein each of said histone peptide portion, said
fused portion, and said indication portion is covalently linked to
at least one other component of the histone peptide complex; and
(b) identifying in the animal the signal produced by the indicator
portion, whereby the identification of the signal in the animal is
an indication that the animal has systemic lupus erythematosus,
thereby diagnosing systemic lupus erythematosus in the animal. In
other embodiments, the invention includes a method of tracking an
autoimmune cell associated with systemic lupus erythematosus in an
animal. This method comprises (a) contacting a sample from the
animal with a composition comprising one or more of a modified
histone peptide and an isolated histone peptide complex, wherein
the modified histone peptide comprises (i) an modified portion
wherein said modified portion is a molecule which is capable of
producing a detectable chemical signal, and which is selected from
the group consisting of a flourophore, a chromophore, a light
reactive moiety and a biotin moiety; and (ii) a peptide portion
comprising not more than 27 contiguous amino acids and having an
amino acid sequence corresponding to a nucleosome histone protein,
and wherein the peptide portion and the modified portion are
covalently linked; and wherein the histone peptide complex
comprises (iii) a histone peptide portion, comprising no more than
27 contiguous amino acids and having an amino acid sequence
corresponding to a portion of a nucleosome histone protein, (iv) a
fused portion having an amino acid sequence which corresponds to a
portion of a protein selected from the group consisting of a major
histocompatibility molecule and an immunoglobin, and (v) an
indicator portion, wherein the indicator portion is a molecule
which is capable of producing a detectable chemical signal, and
which is selected from the group consisting of a flourophore, a
chromophore, a light reactive moiety and a biotin moiety; and
wherein each of the histone peptide portion, the fused portion, and
the indicator portion is covalently linked to at least one other
component of the histone peptide complex; and (b) identifying and
monitoring in the animal the signal produced by the indicator
portion, wherein the identification and monitoring of the signal in
the animal constitutes the identification and monitoring of an
autoimmune cell associated with systemic lupus erythematosus to
which the histone protein complex is bound, thereby tracking the
autoimmune cell associated with systemic lupus erythematosus in the
animal.
BRIEF DESCRIPTION OF THE FIGURES
[0037] FIG. 1 is a diagram depicting the mechanism by which an
autoimmune T helper cell (CD+4 Th) drives the production of
SLE-associated autoantibodies by autoimmune B cells. For
simplicity, costimulatory signaling molecules are not shown. In
this figure, Ia refers to the MHC class II molecule-autoantigenic
peptide complex, and IL refers to interleukins.
[0038] FIG. 2 is a graph illustrating the effect on SLE-associated
nephritis of brief therapy with nucleosomal histone peptides. The
figure illustrates the incidence of severe (4+grade) nephritis in
SNF1 mice that received four injections of either a histone peptide
(i.e. H4 71-94, H2B 10-33, H4 16-39) or saline starting at 12 weeks
age.
[0039] FIG. 3 is a bar graph depicting the ability of CD4+ T cells
obtained from treated mice to facilitate autoantibody production.
Units of IgG autoantibodies produced in the culture supernatants
are expressed as a mean number of antibody units per
deciliter.+-.the standard error of the mean (SEM) from five
experiments. Baseline levels of IgG autoantibodies produced by B
cells cultured alone were: anti-dsDNA, 4.1.+-.0.8; anti-ssDNA,
2.2.+-.0.1; anti-nucleosome, 4.4.+-.1.1; and anti-histone,
3.7.+-..0.6.
[0040] FIG. 4, comprising FIGS. 4A-4C, is a series of graphs
depicting the effect of IL-2 on the ability of T cells obtained
from treated mice to facilitate autoantibody production by
autoimmune B cells. The concentration of rIL-2 used in these
experiments was 100 units per milliliter of culture medium. The
results are expressed as a mean number of antibody units per
deciliter of culture medium.+-.SEM of five experiments.
[0041] FIG. 5 is a graph depicting the effect of peptide therapy on
the ability of B cells obtained from treated mice to receive help
(i.e. facilitation in initiating autoantibody production) from Th
cells. The amount of IgG autoantibodies produced is expressed as a
mean number of units per deciliter of culture medium.+-.SEM of five
experiments. The base line autoantibody levels produced by B cells
cultured alone were: anti-dsDNA, 2.4.+-.0.96; anti-ssDNA,
2.1.+-.0.34; anti-nucleosome, 2.5.+-.1.9; and anti-histone,
1.3.+-.1.0.
[0042] FIG. 6 is a graph depicting the effect of H4 16-39 peptide
treatment on diminishing autoimmune B cells as assessed the absence
of recovery of these cells following stimulation with either
anti-Ig or anti-Ig and IL-4. Autoantibody production is expressed
as a mean number of units per deciliter of culture
supernatant.+-.SEM of five experiments. The base line autoantibody
levels produced by B cells cultured alone were: anti-dsDNA,
2.1.+-.0.72; anti-ssDNA, 1.9.+-.0.1; anti-nucleosome, 1.9.+-.1.0;
and anti-histone, 2.0.+-.1.6.
[0043] FIG. 7 is a graph depicting the effect of soluble CD40
ligand (CD40L) and IL-4 stimulation on B cells obtained from mice
treated with either a saline solution or a solution comprising the
H4 16-39 peptide. The results are expressed as a mean number of
antibody units per deciliter of culture medium.+-.SEM of five
experiments.
[0044] FIG. 8 is a graph depicting the response to
lipopolysaccharide (LPS) by B cells from peptide-injected mice. The
results are expressed as a mean number of antibody units per
deciliter of culture supernatant.+-.SEM of five experiments. The
base line autoantibody levels produced by B cells cultured alone
were the same as those shown in FIG. 6.
[0045] FIG. 9, comprising FIGS. 9A-9D, is a series of graphs
depicting IgG autoantibody levels in the serum of peptide-injected
mice. Sera from SNF1 mice were assayed for levels of IgG
autoantibodies to dsDNA (FIG. 9A), ssDNA (FIG. 9B), nucleosomes
(FIG. 9C), and histones (FIG. 9D). Sera were obtained for these
analyses at the start of peptide injections, and at 36 weeks of
age. The results are expressed as a mean number of antibody units
per deciliter of serum.+-.SEM of five experiments.
[0046] FIG. 10 is a graph depicting the results of experiments in
which nucleosomal peptides were chronically administered to mice
with established SLE-associated nephritis. The data represent the
monitoring of proteinuria in mice until they died of kidney
disease. The results are expressed as the percentage of mice that
survived.
[0047] FIG. 11 depicts a proposed model for the production of
diverse autoantibodies in SLE by multipotent, intermolecular T-cell
help.
[0048] FIG. 12, comprising FIGS. 12A-12D, is a series of graphs
depicting IL-2 production by a pathogenic CD4+ T cell line in
response to selected nucleosomal histone peptides which were
overlapping 15-mers. The cell line was derived from CD4+ T cell
clone, DD2. An autologous EBV-B cell line incubated briefly (i.e.
pulsed) with a selected histone peptide acted as the antigen
presenting cell (APC) line in these experiments. The results
depicted represent the mean of triplicate experiments. In this
figure, values higher than 3 SD (designated by the horizontal line)
above the mean of background were considered stimulatory. The
background production of II-2 by the T cell clone DD2 cultured with
APC without added peptide was on the average 132 picograms of IL-2
per milliliter of culture supernatant. IL-2 production by T cell
clone DD2 in response to anti-CD3 stimulation was 554 picograms per
milliliter. Peptides corresponding to H2A histone protein are shown
in FIG. 12A. Peptides corresponding to H2B histone protein are
shown in FIG. 12B. Peptides corresponding to H3 histone protein are
shown in FIG. 12C. Peptides corresponding to H4 histone protein are
shown in FIG. 12D.
[0049] FIG. 13, comprising FIGS. 13A and 13B, is a pair of graphs
depicting the response of SLE-associated T cells to histone
peptides. FIG. 13A depicts a dose response curve of a
representative SLE-associated T cell line. IL-2 production by this
T cell line was observed during co-culture with autologous EBV-B
cells acting as APCs which were pre-incubated with selected
concentrations of the histone peptide, H4 49-63. FIG. 13B depicts
an example of MHC class II, HLADR-dependent recognition of
nucleosomal peptides by an SLE-associated Th cell line. In this
figure, stimulation with concentrations of peptide from 0.01
micromolar to 100 micromolar exhibited significant differences
(p<0.05 at 0.01 uM, Student's test) relative to background level
of T cells cultured with APC and without peptide. Presentation of
the H4 49-63 peptide by autologous EBV-B cells to the short-term T
cell line L-EB, was inhibited mainly by anti-DR mAb.
[0050] FIG. 14, comprising FIGS. 14A-14D, is a series of graphs
which illustrate the results of a pepscan. These experiments
measure IL-2 production by an SLE-associated, short-term, CD4+ T
cell line, L-SC, in response to histone peptides presented by APCs.
The baseline value for the y-axis is set at 3 standard deviations
(SD) above the mean of background values of IL-2 in picograms per
milliliter of culture medium produced by T cells cultured with APC
alone. Only IL-2 production responses to peptides above this 3 SD
value are shown. Peptides corresponding to H2A histone protein are
shown in FIG. 14A. Peptides corresponding to H2B histone protein
are shown in FIG. 14B. Peptides corresponding to H3 histone protein
are shown in FIG. 14C. Peptides corresponding to H4 histone protein
are shown in FIG. 14D.
[0051] FIG. 15, comprising FIGS. 15A-15D, is a series of graphs
depicting the results of a pepscan. This experiment measures IL-2
production by a short-term CD4+ T cell line from a normal subject
(N-JV) in response to histone peptides using autologous EBV-B cells
obtained from the same subject as APCs. The horizontal line in each
panel demarcates 3 SD above the mean of background values. Peptides
corresponding to H2A histone protein are shown in FIG. 15A.
Peptides corresponding to H2B histone protein are shown in FIG.
15B. Peptides corresponding to H3 histone protein are shown in FIG.
15C. Peptides corresponding to H4 histone protein are shown in FIG.
15D.
[0052] FIG. 16, comprising FIGS. 16A-16D, depicts the collective
data of IL-2 production responses of short-term T cell lines to
nucleosomal peptides in 10 patients with lupus. The results are
expressed as percentages of "maximal" responses of the respective T
cell lines stimulated by anti-CD3 mAb.+-.the standard error of the
mean. Bars represent mean values from the ten lupus T cell lines,
with error bars in one SEM. Values of T cells cultured with
autologous EBV B-cell APCs were considered as background. The
baseline for the y-axis is set at 22% which is 3 SD above the mean
of background values (14.5%). A peptide was considered as
stimulatory when it elicited a positive response above this
baseline. For instance, the average of responses of all 10 T cell
lines to H4 16-30 peptide was 23.+-.2.6. All the stimulatory
peptides shown elicited positive responses in all 10 lupus T cell
lines to levels greater than 3 SD above their respective background
values. Peptides corresponding to H2A histone protein are shown in
FIG. 16A. Peptides corresponding to H2B histone protein are shown
in FIG. 16B. Peptides corresponding to H3 histone protein are shown
in FIG. 16C. Peptides corresponding to H4 histone protein are shown
in FIG. 16D. In FIG. 16D, the bar labeled "Nuc" represents the mean
value of responses for all ten cell lines to a whole nucleosome
preparation.
[0053] FIG. 17 is a table which lists the amino acid sequences of
histone peptides that correspond to SLE-associated autoepitope
regions identified using SLE-associated T-cell lines described
herein, particularly in Examples 2 and 3.
[0054] FIG. 18, comprising FIGS. 18A and 18B, depict representative
examples of two-color intracellular cytokine staining of CD4+ T
cells freshly obtained from two SLE patients, designated R-WG and
R-SC, who were in remission. FIG. 18A illustrates IFN-gamma and
IL-10 production in patient R-WG in response to stimulation by
either anti-CD3, nucleosomes or one of the histone peptides. FIG.
18B depicts IL-2 and IL-4 production in patient R-SC in response to
stimulation by anti-CD3, nucleosomes, or selected histone peptides.
Demarcation of the quadrants was based on background staining of
the T cells cultured in medium alone without stimulation.
[0055] FIG. 19, comprising FIGS. 19A and 19B, is a table which
lists the data from flow cytometry experiments performed using
intracellular cytokine staining of viable, CD4+ T cells of the
peripheral blood from each of twelve SLE patients. A T cell
response to a histone peptide was considered positive when the
percent of positive cells from a given patient sample was two times
greater than the percent of positive cells in the background sample
(i.e. a sample comprised of cells cultured in medium only), and
when at least 0.2% of the total viable CD4+ T cells in the patient
sample were stained positive. A positive response is indicated by
outlined and bold numbers.
[0056] FIG. 20 is a table which lists the data from flow cytometry
analysis of intracellular cytokine staining of CD4+ T cells
obtained from seven normal subjects. A positive response is
indicated by outlined and bold numbers.
[0057] FIG. 21 is a table indicating the frequency of SLE patients
whose T cells responded to histone peptide epitopes. Patients
listed correspond to those listed in FIG. 19. Bold, outlined
numbers indicate histone peptides to which 50% or more of patients
responded. Bold, italicized numbers indicate histone peptides to
which at least 40% of patients responded.
[0058] FIG. 22 is a table which lists human MHC II-{HLA-DR} binding
motifs in histone autoepitopes. The autoepitopes shown in FIG. 21
which were identified as being recurrently recognized by
SLE-associated T cells, were aligned with various known
HLA-DR-binding motifs (Southwood et al., 1998, Immunol.
160:3363-3373; Chicz et al., 1993, Exp. Med. 178:27-47; Chicz et
al., 1992, Nature 358:764-768). Underlining indicates amino acids
which are anchor residues (i.e. amino acids which interact directly
with residues in the MHC II groove. H4 71-94 also contains the
binding motif for HLA-DR alleles, 1, 3, 4, 5, 7, and 8, in addition
to those shown.
[0059] FIG. 23, comprising FIGS. 23A-23C, is a series of graphs
illustrating the identification of naturally processed and
presented histone peptide autoepitopes. FIG. 23A depicts an
analysis using high-performance liquid chromatography (HPLC) of
peptides eluted from mouse I-Ad molecules obtained from a chromatin
pulsed APC line. Peptides having molecular weights of less than
3,000 Daltons were purified by elution through a C-18 column using
acetonitrile and a solution comprising water and 0.1%
trifluoroacetic acid (TFA). FIG. 23B is a graph depicting IL-2
release by a representative, SLE-associated pathogenic Th clone,
L-3A, after stimulation with HPLC-purified histone peptides, using
the A20 cell line as APCs. FIG. 23C depicts a mass spectral
analysis of one of the stimulatory fractions (#23) from HPLC
purification.
[0060] FIG. 24 is a series of graphs which illustrate the
specificity of the naturally processed histone peptide epitope,
EP-3 or H1 22-42. Nested sequences and unrelated sequences found in
the active fractions were synthesized and retested for their
ability to stimulate the nucleosome-specific Th clones derived from
SNF 1 mice. Representative data from a Th cell clone, 3F6, is
shown. In these experiments, Th cells were stimulated with various
synthetic, nested histone peptide sequences by coculture with
B+M.PHI. cells as APCs. The sequences assayed were as follows:
MIAAAIRAEK (SEQ ID NO:53), SASHPTYSEMIAAAIRAEKSR (SEQ ID NO:54),
EMIAAAIRAEKSR (SEQ ID NO:55), IAAAIRAEKSR (SEQ ID NO:56),
AMGIMNSFVNDIFER (SEQ ID NO:57), SEMIAAAIRAEK (SEQ ID NO:58),
SASHPTYSEMIAAAIRAEKS (SEQ ID NO:59), SASHPTYSEMIAAAIR (SEQ ID
NO:60), KPAGPSVTELITK (SEQ ID NO:61).
[0061] FIG. 25, comprising FIGS. 25A-25D, is a series of graphs
depicting an assessment of the ability of selected naturally
processed peptide autoepitopes to stimulate T cell help in support
of SLE-associated autoantibody production by B cells obtained from
lupus-prone mice. This assessment was made by performing helper
assays with each of four Th cell clones in which each Th clone was
cultured with B cells as APCs in either culture medium (T+B),
culture medium and the naturally processed peptide, EP-3 (EP-3), or
culture medium and a whole nucleosome preparation (Nuc). B cells
were incubated alone as a control (B cell). FIG. 25A depicts the
results of a helper assay performed using Th clone 5E9. FIG. 25B
depicts the results of a helper assay performed using Th clone
1D12. FIG. 25C depicts the results of a helper assay performed
using Th clone 3F6. FIG. 25D depicts the results of a helper assay
performed using Th clone 1 G1, which is known to respond only to
nucleosome preparations, not to individual, naturally processed
peptides.
[0062] FIG. 26 is a graph depicting the acceleration of
SLE-associated nephritis following immunization with naturally
processed peptide autoepitopes in adjuvant.
[0063] FIG. 27 is a table which lists the nucleotide sequences (SEQ
ID Nos: 27-52) that encode histone peptides described herein (SEQ
ID Nos: 1-26).
[0064] FIG. 28 shows the beneficial effects of low-dose tolerance
therapy. Incidence of severe lupus nephritis (A), and % survival
(B), of lupus-prone SNF1 mice injected with respective nucleosomal
histone peptide or saline every two wk (nine mice/group). FIG. 28C
shows representative kidney sections from peptide-tolerized (left,
H4.sub.71-94 peptide-treated) or control (right, saline-treated)
SNF1 mice (H&E, X100). The saline control shows marked
perivascular and intersitial infiltrate of mononuclear cells,
dilated tubules with casts and hyalinized, sclerotic glomeruli.
Lower panels (X400) show that in contrast to the peptide-treated
mice (left), kidney from control-treated mice (right) shows
advanced glomerular lesions with sclerosis, crescent formation and
marked thickening of basement membranes, and perivascular,
interstitial infiltrates of mononuclear cells. FIG. 28D shows
representative immunohistochemistry (X200) shows IgG deposits in
glomeruli from both control (right upper panel) and
peptide-tolerized (left upper) SNF1 mice. However, marked
perivascular cellular infiltrates containing IgG.sup.+ plasma cells
(upper right panel), as well as CD4.sup.+ T (lower left panel),
CD8.sup.+ T (lower middle panel) and CD138.sup.+ plasma cells
(lower right panel) were detected only in kidneys of
control-treated mice, as shown. Positive staining is brown. The
results in panels C-D are representative of five mice/group.
[0065] FIG. 29 shows low-dose peptide therapy markedly reduces the
levels of IgG class autoantibodies (A) and their subclasses (B) in
serum. In this sampling, SNF1 mice were bled after 3 months of
treatment (at 6 months of age) and were assayed for levels of IgG
autoantibodies to dsDNA, ssDNA, histone and nucleosome.
Autoantibody levels (mean.+-.SEM mg/dL) are from 9 mice/treatment
group (key within figure). It should be noted that the commercial
antibody reagents used to measure IgG class as a whole vs. IgG
subclasses were different in sensitivity, thus the standard curves
were not comparable.
[0066] FIG. 30 shows low-dose peptide therapy decreases IFN-.gamma.
responses by lupus T cells in ELISPOT. FIG. 30A shows splenic T
cells from saline, H1'.sub.22-42, H4.sub.16-39 or H4.sub.71-94
peptide treated SNF1 mice were challenged with tolerizing peptide
epitope and other relevant epitopes in various concentrations in
vitro. Baseline IFN-.gamma. spots in lupus T+APC cultures without
antigen were 5.+-.3 spots/1.times.10.sup.6 T cells. FIG. 30B shows
low-dose treatment with peptide (H4.sub.71-94-treated group shown
here) also inhibited IFN-.gamma. responses to nucleosomes in vitro
as compared to control SNF1 mice. IFN-.gamma. responses are
expressed in mean.+-.SEM positive spot/1.times.10.sup.6 T cells
from three experiments (five mice/group).
[0067] FIG. 31 shows induction of potent CD4.sup.+CD25.sup.+, and
CD8.sup.+ T.sub.reg cells by low-dose peptide therapy. FIG. 31A
shows CD4.sup.+CD25.sup.+ T cells and CD8.sup.+ T cells from
low-dose peptide tolerized SNF1 mice suppressed anti-dsDNA,
anti-ssDNA and anti-nucleosome autoantibody production by lupus Th
and B cells from 5 month old, unmanipulated SNF1 mice in
nucleosome-stimulated, helper suppression assay (the ratio of
T.sub.reg: lupus Th was 1:1). Baseline levels of IgG autoantibodies
produced by B cells cultured by themselves were: anti-dsDNA,
0.01.+-.0.005, anti-ssDNA, 0.04.+-.0.006, anti-nucleosome,
0.02.+-.0.001, and anti-histone, 0.03.+-.0.002 mg/dl. FIG. 31B
shows T.sub.reg cells induced by peptide treatment also suppressed
directly the IFN-gamma responses of unmanipulated SNF1 lupus T
cells to nucleosomes presented by APC in ELISPOT assay (ratio of
T.sub.reg: lupus Th=1:10). Results are expressed as % suppression
(mean.+-.SEM) from three experiments (five mice/group). Baseline
number of IFN-.gamma. spots produced by lupus T cells+APC cultures
without antigen were 10.+-.4 spots/1.times.10.sup.6 T cells. The
purity of each subset of T cells was >90%.
[0068] FIG. 32 shows adoptive transfer of T.sub.reg cells
suppresses pathogenic autoantibodies in lupus-accelerated SNF1
mice. CD4.sup.+CD25.sup.-T, CD4.sup.+CD25.sup.+ T and CD8.sup.+ T
cells from H4.sub.71-94 (A) or H4.sub.16-39-treated (B) SNF1 mice
were purified by MACS and immediately injected I.V. into 16-wk-old
recipient SNF1 at 1.times.10.sup.6 cells/mouse. One day after
transfer, the recipient mice were immunized with 100 .mu.g of
H1'.sub.22-42 peptide (SEQ ID NO:26) in 0.1 ml of CFA. After
transfer, protein urea were measured every week. One month after
immunization, sera were collected for measuring IgG class
autoantibodies to nuclear antigens (five mice/group). Levels of
autoantibodies in serum of H1'.sub.22-42 immunized SNF1 mice
without adoptive transfer were not significantly different from
that in H1'.sub.22-42 immunized SNF1 mice that had received
CD4.sup.+CD25.sup.-T cells from peptide-tolerized mice (P>0.05).
The purity of each subset of T cells was >90%.
[0069] FIG. 33 shows suppression of IgG autoantibody production by
CD4.sup.+CD25.sup.+ T cells is mediated by TGF-beta and cell
contact, but suppression by CD8.sup.+ T cells is mediated mainly by
TGF-beta. CD4.sup.+CD25.sup.+ or CD8.sup.+ T cells
(5.times.10.sup.5 each) from H4.sub.71-94 or H4.sub.16-39 tolerized
mice were cocultured with T and B cells (1.times.10.sup.6 each)
from 3-4 mo old unmanipulated SNF1 mice in the presence of
nucleosomes and anti-cytokine antibodies (five mice/group). FIG.
33A shows representative helper suppression assay in the presence
of 250 .mu.g/ml of anti-TGF-beta or isotype control. FIG. 33B shows
T.sub.reg cells were separated by membranes from helper assay
mixtures containing nucleosomes plus lupus T and B-cells from
unmanipulated, 3-4 month old SNF1 in Transwell plates. It should be
noted that the helper assay mixture of lupus T and B cells used
here (A & B) came from 1-2 month younger, unmanipulated SNF1
mice than those in FIG. 31A. The purity of each subtype of T cells
were >90%. FIG. 33C shows TGF-beta1 production by
CD4.sup.+CDC25.sup.+ or CD8.sup.+ T cells (1.times.10.sup.6 each)
from H4.sub.71-94 peptide-tolerized mice stimulated with
H4.sub.71-94 or soluble anti-CD3 (1 .mu.g/ml) plus APC. Results are
expressed in mean.+-.SEM from three experiments. Baseline values of
TGF-beta production without stimulation were 318.+-.14 pg/ml for
CD4.sup.+CDC25.sup.+ T cells and 217.+-.20 pg/ml for CD8.sup.+ T
cells from the H4.sub.71-94 peptide-tolerized mice. FIG. 33D shows
the percentage of CD4.sup.+CD25.sup.+ T cells in 1.times.10.sup.6
splenocytes from low-dose peptide tolerized mice and control mice
are shown. This result is a representative of nine separate
experiments.
[0070] FIG. 34 shows low-dose tolerance therapy does not cause
generalized immunosuppression. Control or low-dose peptide
(H4.sub.71-94) tolerized SNF1 mice were immunized with HEL in CFA
and then immune responses to HEL in both groups were compared (five
mice/group). FIG. 34A shows anti-HEL antibody responses that were
analyzed by ELISA. FIG. 34B shows IFN-.gamma. responses to HEL or
anti-CD3 antibody that were measured by ELISPOT. Results are
expressed in mean.+-.SEM from three experiments.
DEFINITIONS
[0071] As used herein, each of the following terms has the meaning
associated with it in this section.
[0072] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0073] As used herein, amino acid residues are represented by the
full name thereof, by the three letter code corresponding thereto,
or by the one-letter code corresponding thereto, as indicated in
the following table: TABLE-US-00001 Three-Letter One-Letter Full
Name Code Code Aspartic Acid Asp D Glutamic Acid Glu E Lysine Lys K
Arginine Arg R Histidine His H Tyrosine Tyr Y Cysteine Cys C
Asparagine Asn N Glutamine Gln Q Serine Ser S Threonine Thr T
Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine
Ile I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan
Trp W
[0074] Unless otherwise indicated, all amino acid sequences listed
in this disclosure are listed in the order from the amino terminus
to the carboxyl terminus.
[0075] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof linked via
peptide bonds, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof. Synthetic
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer.
[0076] The term "protein" typically refers to large
polypeptides.
[0077] The term "peptide" typically refers to short
polypeptides.
[0078] An "isolated nucleic acid", as used herein, refers to a
nucleic acid sequence, segment, or fragment which has been purified
from the sequences which flank it in a naturally occurring state,
e.g., a DNA fragment which has been removed from the sequences
which are normally adjacent to the fragment e.g., the sequences
adjacent to the fragment in a genome in which it naturally occurs.
The term also applies to nucleic acids which have been
substantially purified from other components which naturally
accompany the nucleic acid, e.g., RNA or DNA or proteins which
naturally accompany it in the cell.
[0079] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0080] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. Nucleotide sequences that encode proteins and RNA
may include introns.
[0081] By describing two polynucleotides as "operably linked" is
meant that a single-stranded or double-stranded nucleic acid moiety
comprises the two polynucleotides arranged within the nucleic acid
moiety in such a manner that at least one of the two
polynucleotides is able to exert a physiological effect by which it
is characterized upon the other. By way of example, a promoter
operably linked to the coding region of a gene is able to promote
transcription of the coding region.
[0082] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0083] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses that
incorporate the recombinant polynucleotide.
[0084] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulatory sequence.
In some instances, this sequence may be the core promoter sequence
and in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue specific manner.
[0085] "Complementary" as used herein, refers to the subunit
sequence complementarily between two nucleic acid molecules, e.g.,
two DNA molecules or two RNA molecules. When a subunit position in
both of the two molecules is occupied by a complementary monomeric
subunit, e.g., if one position in each of two DNA molecules is
occupied by adenine and the other is occupied by a thymine, then
they are complementary at that position. Similarly, if one position
in each of two DNA molecules is occupied by guanine and the other
is occupied by a cytosine, then they too are complementary at that
position. The degree of complementarity between two sequences is a
direct function of the number of positions occupied by
complementary bases, e.g., if half (e.g., five positions in a
polymer ten subunits in length) of the positions in two compound
sequences contain complementary bases then the two sequences share
50% complementarity, if 90% of the positions, e.g., 9 of 10,
contain bases complementary to each other, the two sequences share
90% complementarity. By way of example, the DNA sequences
5'ATTGCC3' and 3'GGCGCC5' share 50% complementarity.
[0086] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two peptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between-two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3' ATTGCC 5' and 3'
TATGCG 5' share 50% homology. Any of a variety of known algorithms
may be used to calculate the percent homology between two nucleic
acids or two proteins of interest and these are well-known in the
art.
[0087] An "isolated peptide" is a peptide which has been
substantially separated from components (e.g., DNA, RNA, other
proteins and peptides, carbohydrates and lipids) which naturally
accompany it in a cell.
[0088] As used herein, a "15-mer" refers to an isolated peptide
which comprises 15 contiguous amino acids.
[0089] As used herein, "tracking" an autoimmune T cell or B cell
refers to one or more of identifying and monitoring the autoimmune
T cell or B cell. Tracking as it is used herein refers to both in
vivo and in vitro identification and monitoring.
[0090] A disorder is "alleviated" if one or more of the frequency,
the severity, and the duration of either the disorder or a symptom
of the disorder are reduced.
[0091] The term "pharmaceutically acceptable carrier" means a
chemical composition with which a pharmaceutically active agent can
be combined and which, following the combination, can be used to
administer the agent to a subject (e.g. a mammal such as a
human).
[0092] The term "physiologically acceptable" ester or salt means an
ester or salt form of a pharmaceutically active agent which is
compatible with any other ingredients of the pharmaceutical
composition and which is not deleterious to the subject to which
the composition is to be administered.
[0093] As used herein, the term "SLE-associated T cells" refers to
autoimmune T cells which are involved in a pathogenic process
associated with SLE. "SLE-associated nephritis" refers to
inflammation of the kidney in an animal which occurs as a result of
autoantibody production and which is concurrent with other symptoms
of SLE.
[0094] "Autoimmune T cells" refer to T cells of an animal which are
stimulated by, and provide help to mount an immune response to,
antigens which are native to, or derived, from the animal itself
(i.e. autoantigenic determinants or autoepitopes).
[0095] "Autoimmune B cells" refers to B cells of an animal that
produce autoantibodies directed to antigenic epitopes which are
native to, or derived from, the animal itself (i.e.
autoepitopes).
[0096] As used herein, the term "SLE-associated autoepitope" refers
to an amino acid sequence of a peptide or protein which is native
to, or derived from, the animal itself, and which is recognized by,
elicits a response from, or stimulates the activity of an
autoimmune T cell or B cell which is involved in a pathogenic
process associated with SLE.
[0097] As used herein, the term "help" refers to the interaction
between a T helper cell (i.e. Th cell) and an antigen-specific B
cell which results in antibody production and the recruitment of
other immune cells to assist in mounting an immune response to the
antigen. As an example, an autoimmune Th cell of an animal can help
an autoimmune B cell of the animal which is specific for an
autoantigen (i.e. an antigen which is derived from the animal
itself), resulting in the production of autoantibodies. In
addition, this interaction of the Th cell with B cell results in
the production of cytokines and other chemotactic factors by the Th
cell, which in turn, leads to the recruitment of macrophages and
other immune cells, subsequent inflammation, and perpetuation of
the autoimmune response.
[0098] As used herein, "tolerance" refers to an immune condition
which develops in an animal by, for example, exposure to a given
antigen in a certain form. Tolerance to a given antigen in an
animal is generally characterized by immune responses in the animal
to the antigen which are reduced with respect to one or more of
numbers of recruited immune cells, incidence, intensity, and
duration, relative to an animal which has not developed tolerance
to the antigen.
[0099] The term "tolerogen" refers to an antigenic agent which
promotes the development of tolerance in an animal. The terms
"antigen" and "epitope" may be used somewhat interchangeably herein
to refer to portions of histone proteins to which antibodies are
directed, and which T cells recognize.
[0100] As used herein, to "load" a histone peptide onto a major
histocompatibility complex class (MHC) molecule (e.g. an I-Ad
molecule) in a B cell means to cause an interaction and the
formation of a complex between a histone peptide and an MHC
molecule in the B cell.
DETAILED DESCRIPTION OF THE INVENTION
[0101] The present invention provides low dose compositions
comprising peptides and methods of using the same to treat
autoimmune disorders, such as lupus. In particular, the present
invention provides low dose compositions comprising nucleosomal
histone peptide autoepitopes comprising MHC class I and II binding
motifs and methods of using the same to suppress IFN-gamma and/or
autoantibody production. In certain embodiments, the compositions
contain the histone peptides at subnanomolor concentrations, such
as 0.1 nM to 0.9 nM. In other embodiments, the compositions are
adminstered in a low dose, such as 5-75 ug of histone peptides per
kilogram of patient.
[0102] The invention relates to the discovery that certain peptides
derived from nucleosomal histone proteins are useful for delaying
the onset and progression of nephritis associated with systemic
lupus erythematosus (i.e. lupus or SLE). In particular, the present
invention provides compositions comprising nucleosomal histone
peptide autoepitopes comprising MHC class I binding motifs and
methods of using the same (e.g., in subnanomolar doses administered
subcutanously) to suppress IFN-gamma and/or autoantibody
production. The invention includes a series of peptides which span
specific regions of the histone proteins (i.e. H1, H2A, H2B, H3,
and H4), and additionally encompasses isolated nucleic acids
encoding histone peptides for the production of same, modified
histone peptides, such as biotinylated histone peptides, amino
acid-substituted histone peptides, and altered peptide ligands
(APLs), and histone peptide complexes, such as histone peptide/MHC
(major histocompatability complex) tetramer complexes and histone
peptide/MHC-Ig (Immunoglobin) chimeric dimer complexes, and methods
of using the same (See, e.g., U.S. Pat. No. 6,468,537, herein
incorporated by reference in its entirety for all purposes). The
invention further encompasses pharmaceutical compositions which
comprise one or more of a histone peptide, a modified histone
peptide, a histone peptide complex, and an isolated nucleic acid
encoding a histone peptide. The histone peptides, modified histone
peptides, and histone peptide complexes described herein are
therapeutically useful as compositions for the treatment of SLE and
complications thereof. The isolated nucleic acids encoding the
peptides are useful as described herein for the production of
histone peptides and histone peptide complexes, and for tracking,
in vivo, SLE-associated T and B cells. The compositions and methods
of the present invention also find use in research and drug
screening applications.
[0103] The invention further provides methods of using histone
peptides or modified histone peptides (e.g., in subnanomolar doses
administered subcutanously) to promote tolerance to SLE-associated
autoepitopes in a human or an animal, inhibit production of
autoantibodies in a human or an animal, inhibit an autoimmune
response and associated inflammation in a human or an animal, and
treat disorders in a human or an animal which are related to the
production of autoantibodies and complications thereof, such as
inflammatory diseases, autoimmune disorders, and nephritis. The
invention encompasses methods of using one or more of modified
histone peptides, histone peptide complexes, and isolated nucleic
encoding histone peptides or histone peptide complexes to track
SLE-associated T cells and SLE associated B cells, and to diagnose
SLE in an animal. The invention also provides methods of making
modified histone peptides and histone peptide complexes.
[0104] Additionally, the invention provides systems and kits which
comprise one or more of a histone peptide, a modified histone
peptide, a histone peptide complex, and an instructional material.
The kits provided by the invention are useful for tracking
SLE-associated T and B cells, promoting tolerance to SLE-associated
autoepitopes, inhibiting the production of autoantibodies,
inhibiting an immune response and associated inflammation, treating
SLE or other autoimmune disorders and complications thereof, and in
the diagnosis and prognostication of SLE in an animal. In
particular embodiments, the kits and systems provide necessary
components sufficient for the diagnostics of the present
invention.
[0105] The present invention provides nucleosomal-histone peptide
epitopes suitable for antigen-specific tolerance therapy of lupus.
Nucleosome is one of the major immunogens driving lupus
autoimmunity in murine and human SLE (Refs. 2, 5, 7, 24, 25).
Critical peptide epitopes from nucleosomal histones are recognized
by autoimmune T cells of lupus patients, irrespective of their MHC
haplotypes (Refs. 5-7). The peptide epitopes are derived from a
highly conserved, ubiquitous self-antigen, which is a product of
ongoing apoptosis in generative lymphoid organs. Therefore, any
anaphylactic/allergic reactions with these peptides is not observed
when used either for immuization or for tolerance therapy in over
1000 SNF1 mice. Peptides of the present invention, when
administered S.C. in a very low-dose regimen, generate T.sub.reg
cells that suppress by TGF-beta and/or by cell contact rather than
causing Th2 deviation with allergic reactions seen in the case of
peptide therapy of EAE/MS and NOD diabetes (Refs. 26, 27). The
beneficial effects of the peptides of the present invention outlast
their short half-life by generating longer-lasting T.sub.reg
cells.
[0106] In some embodiments, only about 1 ug of nucleosomal histone
peptide (H1'.sub.22-42, H4.sub.16-39 or H4.sub.71-94) injected S.C.
every two weeks to SNF1 mice with clinically overt lupus, restores
life span to normal (.about.2 yr) by markedly delaying death from
severe nephritis. This S.C. dosage is 300-1000 fold lower than
doses previously used for I.V. nucleosomal peptide therapy (Ref.
8), and others have applied with anti-DNA autoantibody V region and
related peptides (Refs. 28-30). After 3 months of low-dose
H1'.sub.22-42, H4.sub.16-39 or H4.sub.71-94 peptide treatment, IgG
autoantibodies against nuclear antigens were reduced up to
90.about.100% as compared to controls after 3 months therapy,
indicating impairment of pathogenic T cell help. In some
embodiments, the perivascular and interstitial infiltrations of
mononuclear cells containing T, B and plasma cells are markedly
decreased in the peptide-tolerized SNF1 mice in contrast to control
mice. Thus, in some embodiments, very low-dose peptide therapy
prevents local inflammatory damage in kidneys by diminishing
migration and activation of nephritogenic T and B cells, which
might share antigenic specificities with the cells responsible for
autoantibody production in the periphery.
[0107] In some embodiments, the pathogenic lupus T cells responding
to nucleosomal epitopes are IFN-gamma producing Th1 cells (Refs. 5,
6). Each peptide treatment cross-reactively suppressed responses by
lupus T cells to other peptide epitopes in addition to the
tolerizing peptide, as well as to nucleosomes, the major lupus
immunogen containing many autoepitopes. A single peptide from a
histone in the nucleosome can be recognized by multiple autoimmune
T cells with diverse TCRs and conversely, a single autoimmune T
cell can promiscuously recognize multiple nucleosomal peptides that
are structurally different (Refs. 5, 8, 9). Thus, in some
embodiments, a single epitope may tolerize multiple autoimmune Th
cells and tolerizing one set of Th cells would deprive help for a
broad spectrum of autoimmune B cells (tolerance spreading). In
other embodiments, multiple epitopes may be utilized. The
suppression of IFN-gamma response to autoantigens is dose
dependent, demonstrating autoantigen specificity, but it was
overcome at higher doses, as in other systems (Ref. 31). In some
embodiments, peptide therapy generates autoantigen-specific
T.sub.reg cells as evidenced by increased production of TGF-gamma
by T.sub.reg cells on stimulation with nucleosomal peptide, and
lack of suppression of immune response to foreign antigen (HEL)
immunization. In some embodiments, suppression of help in
autoantibody production by the T.sub.reg cells is done in the
presence of nucleosomes (autoantigenic stimulation) in the
helper-suppression assay cultures.
[0108] In some embodiments, low-dose nucleosomal peptide therapy
repairs deficiencies of TGF-gamma producing cells and CD8.sup.+
T.sub.reg function that have been observed in SLE (Refs. 32-36).
Unlike the case in organ-specific autoimmunity (Refs. 15, 31, 37),
the role of CD4.sup.+CD25.sup.+ T.sub.reg in spontaneous SLE is
controversial (Ref. 38), but it is contemplated that they are
potently induced by therapies of the present invention. In some
embodiments, nasal tolerance with one of the autoepitopes, e.g.,
H4.sub.71-94, will delay or treat lupus nephritis in SNF1 mice
(e.g., through generating IL-10 producing T cells (Ref. 39)). Thus,
in some embodiments, IL-10 producing T.sub.reg cells will benefit
lupus with some caveats (Refs. 30, 40).
[0109] In some embodiments, CD4.sup.+CD25.sup.+ T.sub.reg
population contains a subset of T cells that are secondarily
induced to produce TGF-.beta., which suppresses autoimmunity (Refs.
13, 32, 35, 41, 42). CD8.sup.+ T.sub.reg cells induced by low-dose
tolerance were not cytotoxic T lymphocytes (CTL) because they
suppressed across membranes, even though the autoepitope peptides
inducing such T.sub.reg cells contained class I binding motifs.
Thus, the CD8.sup.+ T.sub.reg cells induced by nucleosomal peptides
of the present invention are different from the TCR
clonotype-specific and cytotoxic suppressor cells in other systems
(Refs. 20, 22, 43-45). In some embodiments, TGF-beta produced by
the CD8.sup.+ T cells could induce CD4.sup.+CD25.sup.+ T.sub.reg
cells in the system of the present invention. Indeed, the
suppressive effect of these adaptive T.sub.reg cells were similar
at 1:1, 1:10 or 1:100 ratios (suppressor: target) suggesting
involvement of "infectious tolerance" mechanisms, as in other
systems (Refs. 12, 42).
[0110] Thus, although a mechanism is not needed to practice the
present invention, it is contemplated that tolerance therapy with
select nucleosomal peptides works by generating suppressive
T.sub.reg cells that impair T cell help for production of a broad
spectrum of pathogenic autoantibodies and especially inhibit
inflammatory insults in the lupus kidney. Moreover, these T.sub.reg
cells induced by very low-dose tolerance could possibly suppress
activated DC and other APC in lupus (Refs. 51, 52).
[0111] In some aspects, the present invention relates to the
discovery that certain peptides derived from nucleosomal histone
proteins are useful for delaying the onset and progression of
nephritis associated with systemic lupus erythematosus (i.e. lupus
or SLE). The invention includes a series of peptides which span
specific regions of the histone proteins (i.e. H1, H2A, H2B, H3,
and H4), and additionally encompasses isolated nucleic acids
encoding histone peptides for the production of same, modified
histone peptides, such as biotinylated histone peptides, amino
acid-substituted histone peptides, and altered peptide ligands
(APLs), and histone peptide complexes, such as histone peptide/MHC
(e.g., major histocompatability complex class I or II) tetramer
complexes and histone peptide/MHC-Ig (Immunoglobin) chimeric dimer
complexes. The invention further encompasses pharmaceutical
compositions which comprise one or more of a histone peptide, a
modified histone peptide, a histone peptide complex, and an
isolated nucleic acid encoding a histone peptide. The histone
peptides, modified histone peptides, and histone peptide complexes
described herein are therapeutically useful as compositions for the
treatment of SLE and complications thereof. The isolated nucleic
acids encoding the peptides are useful as described herein for the
production of histone peptides and histone peptide complexes, and
for tracking, in vivo, SLE-associated T and B cells.
[0112] The invention further provides methods of using histone
peptides or modified histone peptides to promote tolerance to
SLE-associated autoepitopes in an animal, inhibit production of
autoantibodies in an animal, inhibit an autoimmune response and
associated inflammation in an animal, and treat disorders in an
animal which are related to the production of autoantibodies and
complications thereof, such as inflammatory diseases, autoimmune
disorders, and nephritis. The invention encompasses methods of
using one or more of modified histone peptides, histone peptide
complexes, and isolated nucleic encoding histone peptides or
histone peptide complexes to track SLE-associated T cells and SLE
associated B cells, and to diagnose SLE in an animal. The invention
also provides methods of making modified histone peptides and
histone peptide complexes.
[0113] Additionally, the invention provides kits which comprise one
or more of a histone peptide, a modified histone peptide, a histone
peptide complex, and an instructional material. The kits provided
by the invention are useful for tracking SLE-associated T and B
cells, promoting tolerance to SLE-associated autoepitopes,
inhibiting the production of autoantibodies, inhibiting an immune
response and associated inflammation, treating SLE or other
autoimmune disorders and complications thereof, and in the
diagnosis and prognostication of SLE in an animal.
[0114] Prior to the investigations described in this disclosure,
the complexity of SLE has permitted only limited understanding of
how the disease is initiated in an otherwise healthy animal.
Further, it was not considered that autoantigen-specific,
immune-based therapy would be effective in combating the disease.
As a result, it has not previously been contemplated that therapy
with histone peptides, which may act to promote tolerance in an
animal to SLE-associated autoepitoes, and thereby inhibit
production of the pathogenic autoantibodies responsible for
SLE-associated nephritis, could be successful in delaying the onset
and progression of SLE and complications thereof.
[0115] The present invention includes substantially any histone
peptide corresponding to and overlapping a portion of the amino
acid sequence of one or more of the histone proteins, including H1,
H2A, H2B, H3, and H4, that make up nucleosomes in an animal cell. A
histone peptide of the present invention may comprise a portion of
a histone protein which corresponds to an SLE-associated
autoepitope that is known or becomes known, and is useful in the
methods described herein.
[0116] A histone peptide of the invention can correspond to the
amino acid sequence of a histone protein from substantially any
animal. Preferrably, the histone peptide used in the methods
described herein has an amino acid sequence corresponding to a
histone protein from the same species of the animal in whom
tolerance is to be promoted. By way of example, a histone peptide
described herein which is administered to a human will preferably
have an amino acid sequence corresponding to a region of a human
histone protein.
[0117] Preferably, a histone peptide of the invention is capable of
promoting tolerance in an animal to an SLE-associated autoepitope,
thereby inhibiting production by an animal of autoantibodies
directed at antigens native to the animal (i.e., autoepitopes),
such as those derived from single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), nucleosomal particles, and histones of
all of the animal's cells. The invention should be understood to
include peptides corresponding to histone protein amino acid
sequences which are known to be associated with, or which become
associated with, an autoimmune disorder. Also preferable is a
histone peptide having an amino acid sequence corresponding to any
of SEQ. ID Nos. 1-26 and any of those listed in FIG. 17.
[0118] The present invention also provides modified histone
peptides (i.e. analogs of histone peptides) which promote tolerance
to SLE-associated autoepitopes in an animal or which are useful for
identifying SLE associated T and B cells. Such modifications should
not render the peptide more immunogenic, but rather, should render
the peptide more tolerogenic. Analogs of histone peptides can
differ from histone peptides described herein by conservative amino
acid sequence differences or by modifications which do not affect
sequence, or by both. For example, conservative amino acid changes
may be made, which although they alter the primary sequence of the
protein or peptide, do not normally alter its function.
Conservative amino acid substitutions typically include
substitutions within the following groups:
glycine, alanine;
valine, isoleucine, leucine;
aspartic acid, glutamic acid;
asparagine, glutamine;
serine, threonine;
lysine, arginine;
phenylalanine, tyrosine.
[0119] Modifications (which do not normally alter primary sequence)
include in vivo, or in vitro chemical derivatization of peptides,
e.g., acetylation, or carboxylation. Also included are sequences
which have phosphorylated amino acid residues, e.g.,
phosphotyrosine, phosphoserine, or phosphothreonine. In addition,
modifications which result in histone peptides that can be useful
as probes to identify SLE-associated immune cells are included.
Examples of this type of peptide modification include covalent
attachment of a flourophore or chromophore, covalent attachment of
a chemical moiety which can specifically bind other probe molecules
or which can be derivatized to produce such binding, and covalent
attachment of a chromogenic, enzyme cleavable moiety. An exemplary
modification of a histone peptide is biotinylation which produces a
biotinylated histone peptide that can be useful for identifying
(i.e. tracking) SLE associated Th and B cells.
[0120] A particular type of modified histone peptide contemplated
in the present invention is an altered peptide ligand or APL. APLs
are peptides which are very similar in amino acid sequence to
native (i.e. wild type) histone autoepitope peptides, but have
different amino acid residues at positions in the autoepitopes that
are known to contact the Th cell receptor (TCR). As the amino acid
sequences of APLs would differ from the wild type histone peptide
sequences only at those residues, it is considered well within the
ability of the skilled artisan to design and synthesize APLs with
single amino acid substitutions which bind an MHC class II or I
molecules, such as the HLA-DR on a human B cell, but fail to
stimulate the SLE-associated Th cells, thereby inhibiting
autoantibody production. APLs which are useful in the methods of
the present invention can therefore be readily prepared based on
the sequences of the histone autoepitopes described herein.
[0121] Also included are peptides which have been modified using
ordinary molecular biological techniques in order to improve their
resistance to proteolytic degradation or to optimize solubility
properties or to render them more suitable as a therapeutic agent.
Analogs of such peptides include those containing residues other
than naturally occurring L-amino acids, e.g., D-amino acids or
non-naturally occurring synthetic amino acids. The peptides of the
invention are not limited to products of any of the specific
exemplary processes listed herein.
[0122] A histone peptide included in the present invention
comprises from at least about three contiguous amino acids to at
least about 50 amino acids, and from at least about twelve
contiguous amino acids to at least about twenty five contiguous
amino acids, wherein the peptide corresponds to portion of the
amino acid sequence of a histone protein. Preferably, a histone
peptide comprising from at least about fifteen contiguous amino
acids to at least about twenty one contiguous amino acids.
[0123] The invention includes histone peptides, modified histone
peptides, and histone peptide complexes that are encoded by an
isolated nucleic acid, as well as those which are produced by
synthetic means. A histone peptide, a modified histone peptide, or
a histone peptide complex described herein can be made, purified,
or both, using any of a variety of techniques known in the art.
Representative techniques include using an automated peptide
synthesizing apparatus, as well as recombinant techniques in which
an isolated nucleic acid encoding a histone peptide or histone
peptide complex is operably linked with transcriptional and
translational regulatory sequences (e.g. using any of a variety of
known and commercially available expression vectors) and expressed
to yield the histone peptide. Alternatively, a naturally-occurring
histone protein can be isolated and cleaved to yield a histone
peptide. A histone peptide complex as described herein can be
prepared by an ordinarily skilled artisan using the nucleotide
sequences of the histone autoepitopes disclosed herein, for
example, in FIG. 27, and either established recombinant DNA
technology (Crawford et al. 1998, Immunity 8:675; Novak et al.
1999, J. Clin. Invest. 104:R63-R67; Kotzin et al. 1999, Proc. Natl.
Acad. Sci. USA. 97:291), or other published technology (Lebowitz et
al. 1999, Cell. Immunol. 192:175; McMichael and Kelleher 1999, J.
Clin. Invest. 104:1669). Likewise, modified histone peptides such
as those described herein, can be prepared by a method described
herein and modified using any suitable modification methods or
manufacturer's protocols.
[0124] The invention further includes an isolated nucleic acid
encoding a histone peptide described herein. The isolated nucleic
acid of the invention may be one which encodes a histone peptide
corresponding to a portion of histone protein, and comprises from
at least about three contiguous amino acids to at least about fifty
amino acids, and preferably, from at least about twelve contiguous
amino acids to at least about twenty five contiguous amino acids.
More preferably, an isolated nucleic acid of the invention encodes
a histone peptide comprising from at least about fifteen contiguous
amino acids to at least about twenty one contiguous amino
acids.
[0125] Alternatively, an isolated nucleic acid included in the
invention can encode a histone peptide complex comprising an amino
acid sequence corresponding to a portion of a histone protein and
an amino acid sequence corresponding to a portion of one or more of
a MHC molecule and an immunoglobin (Ig) molecule. An isolated
nucleic acid described herein can encode a histone peptide complex
comprising a histone peptide fused to all or part of the amino acid
sequence of one or more of a MHC molecule and an immunoglobin (Ig)
molecule. A histone protein complex encoded by an isolated nucleic
acid of the invention can include a histone peptide which comprises
from at least about three contiguous amino acids to at least about
fifty amino acids, and preferably, from at least about twelve
contiguous amino acids to at least about twenty five contiguous
amino acids. More preferably, an isolated nucleic acid of the
invention encodes a histone peptide complex which includes a
histone peptide comprising from at least about fifteen contiguous
amino acids to at least about twenty one contiguous amino acids.
Even more preferably, the invention includes an isolated nucleic
acid comprising any of the nucleotide sequences shown in FIG. 27
and any of SEQ ID Nos: 27-52.
[0126] In various embodiments, the invention includes an isolated
nucleic acid which is a vector comprising an nucleic acid sequence
which encodes a histone peptide of the invention. The vector can be
used to introduce the nucleic acid encoding a histone peptide into
a cell. Substantially, any type of vector known in the art is
suitable for this purpose, including without limitation, plasmid
based vectors, viral based vectors, and non-DNA vectors. Examples
of suitable plasmid based vectors include, without limitation, any
plasmid which comprises sequences capable of facilitating either of
propagation and expression of the desired gene in a prokaryotic or
eukaryotic cell. Examples of suitable viral vectors include, but
are not limited to, retroviral vectors, adenoviral vectors, and
adeno-associated viral vectors. Examples of non-DNA vectors
include, without limitation, polylysine compounds, liposomes, and
the like.
[0127] A vector comprising an nucleic acid sequence which encodes a
histone peptide can comprise a promoter/regulatory sequence capable
of driving histone peptide expression, a translational start codon,
a histone peptide coding region, and a translational stop codon.
Optionally, the vector can comprise expression-enhancing nucleotide
sequences and nucleotide sequences which encode stabilizing amino
acids or peptide sequences.
[0128] An isolated nucleic acid encoding a histone peptide
described herein is assembled into, for example, an expression
vector, using ordinary molecular biology techniques, such as those
described in Sambrook et al. (1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York), in Ausubel et al.
(1997, Current Protocols in Molecular Biology, John Wiley &
Sons, New York), and in Gerhardt et al. eds. (1994, Methods for
General and Molecular Bacteriology, American Society for
Microbiology, Washington, D.C.). Accordingly, the design of
functional elements required to generate a vector capable of
expressing a histone peptide, such as start and stop codons,
stabilizing and expression-enhancing sequences, and any appropriate
restriction endonuclease sites, is understood to be within the
ability of one skilled in the art of molecular biology. Upon
reading the disclosure provided herein, and using standard
reference material, it is a simple matter for the skilled artisan
to construct a vector comprising an isolated nucleic acid encoding
a histone peptide, such that it can be useful in the methods of the
present invention. By simply substituting a desired histone
peptide-encoding nucleotide sequence in the appropriate location of
substantially any commercially available expression vector, it is a
simple matter to construct a vector comprising an isolated nucleic
acid which encodes the desired histone peptide.
[0129] The SLE-associated autoepitopes, which have been localized
(as illustrated herein in the Examples) to the amino acid sequences
of nucleosomal histone proteins, H1, H2A, H2B, H3, and H4, are
released for example, during kidney cell death. The onset of SLE is
marked by, among other things, the development of inflammation in
the kidney (i.e. nephritis) progressing to kidney disease and
kidney failure. The inventors have discovered that administering to
an animal histone peptides corresponding to these SLE-associated
autoepitope regions of histone proteins can promote tolerance in
the animal for these autoepitopes. While not wishing to be bound by
a particular mechanism of action, the histone peptides described
herein are believed to promote tolerance in an animal to
SLE-associated autoepitopes which results in decreased
SLE-associated autoantibody production by the animal, thereby
delaying the onset and progression of SLE-associated nephritis.
Thus, a composition comprising one or more of a histone peptide and
a modified histone peptide can be administered to an animal in
order to treat SLE-associated nephritis.
[0130] It has been discovered that portions of the histone
proteins, H1, H2A, H2B, H3 and H4 are recognized as antigens (i.e.
autoepitopes) by autoimmune Th cells that are essential for driving
autoimmune B cells to produce a class of antibodies herein referred
to as SLE-associated autoantibodies. These SLE-associated Th cells
and autoantibodies occur in an animal having SLE, and are directed
to autoepitopes associated with the onset or progression of SLE in
the animal. The production of autoantibodies as is observed in SLE
can be associated with other autoimmune disorders, and results in
immune responses which can damage and destroy any cells or tissues
of the animal by the formation of immune complexes. The histone
peptides described herein correspond to autoepitope regions of the
histone proteins which have been associated with the initiation and
progression of SLE. Thus, a composition comprising one or more of a
histone peptide and a modified histone peptide can be administered
to an animal in order to promote tolerance in an animal to the
autoepitope corresponding to the amino acid sequence of the histone
peptide administered, and thereby, decrease the production of
SLE-associated autoantibodies and inhibit an inflammation response
associated with the production of SLE-associated autoantibodies. In
addition, as these histone autoepitopes may be recognized by Th
cells and autoantibodies associated with multiple other autoimmune
disorders, compositions comprising one or more of a histone peptide
and a modified histone peptide can be administered to an animal to
treat an autoimmune disorder. Examples of other autoimmune
disorders include, but are not limited to, rheumatoid arthritis and
scleroderma.
[0131] It has further been discovered that an altered peptide
ligand (APL) as described herein is useful for promoting tolerance
to SLE-associated autoepitopes in mice. APLs are able function as
antagonists or as partial agonist ligands for Th cells bearing
receptors which are specific for wild type histone autoepitopes,
analogous to other antigen systems (Evabold et al., 1993, Immunol.
Today. 14:602; Sloan-Lancaster et al., 1994, J. Exp. Med. 180:1195;
DeMagistris et al., 1992, Cell. 68:625; Madrenas et al., 1997, J.
Exp. Med. 185:219; Korb et al., 1999, J. Immunol. 162:6401; DePalma
et al., 1999, J. Immunol. 162:1982). Without wishing to be bound by
a particular mechanism, it appears that T cells stimulated with
partial agonist peptides (i.e. APLs) may enter a state of anergy in
which they are unresponsive to subsequent stimulation with the wild
type histone peptide. Because the binding of these modified
peptides is controlled by fast dissociation kinetics, the APLs are
unable to simultaneously engage a threshold number of Th cell
receptors in a given population (Rabinowitz et al., 1996, Immunity
5:125). Therefore, APLs send a negative signal to (i.e. do not
stimulate) autoimmune Th cells through inefficient binding of Th
cell receptors, which in turn, results in decreased production of
SLE-associated autoantibodies by autoimmune B cells. Thus, this
type of modified histone peptide can be useful for promoting
tolerance to SLE-associated autoepitopes, reducing the level of
autoantibodies in an animal, inhibiting an immune response and
associated inflammation, and treating SLE and other autoimmune
disorders and complications thereof such as, SLE-associated
nephritis.
[0132] The present invention includes modified histone peptides and
histone peptide complexes which are useful for methods of
identifying and tracking SLE-associated T cells and B cells. For
example, the binding of a SLE-associated autoimmune T cell receptor
(SLE-TCR) to its cognate antigenic histone peptide complexed with a
MHC molecule (e.g., a histone peptide/MHC II complex) on the
surface of an autoimmune B cell, could be mimicked by a histone
peptide complex, such as a fluorochrome-conjugated histone
peptide/MHC II complex, which would be useful for binding, and
thereby staining, SLE-associated T cells. By way of alternative
example, a biotinylated histone peptide or histone peptide complex
could be used to stain and track autoimmune B or T cells,
respectively, in an animal with SLE at various stages of disease
activity, including the active, recent remission, and long-term
remission stages. This type of cell staining has previously been
used to facillitate identification and tracking the
antigen-specific T cells (McMichael and Kelleher. 1999, J. Clin.
Invest. 104: 1669).
[0133] Due to the promiscuous nature of SLE-TCR/nucleosomal peptide
interactions, a significant proportion of the SLE-associated T
cells present in an animal, such as a human, may be detected using
a histone peptide complex, such as a histone peptide/DR tetramer.
Thus, a modified histone peptide or a histone peptide complex would
be useful for early detection of SLE-associated T and B cells, and
diagnosis of SLE before any clinical manifestations of the disease.
Further, the onset and progression of SLE could be characterized
with respect to disease manifestations, such as SLE-associated
nephritis, using a modified histone peptide or a histone peptide
complex. An improved clinical picture may result from instituting a
more aggressive course of therapy at an earlier stage of the
disease. Therefore, either a histone peptide complex or a modified
histone peptide described herein can be useful for diagnosis or
prognostication of SLE, and may also improve the therapeutic
outcome of treating SLE and complications thereof by virtue of
their diagnostic and prognostic value.
[0134] As noted elsewhere herein, an isolated nucleic acid encoding
either a histone peptide or a histone peptide complex can be useful
for tracking (i.e. identifying and monitoring) a pathogenic,
SLE-associated T cell or B cell, and for producing useful
quantities of one or more histone peptides or histone peptide
complexes.
[0135] The histone peptides described in this disclosure, such as
any of SEQ ID Nos: 1-26, and isolated nucleic acids which encode
histone peptides and histone peptide complexes, such as an
expression vector expressing a histone peptide or histone peptide
complex, can be incorporated into pharmaceutical compositions for
ethical administration to humans and other animals. Such
pharmaceutical compositions are described elsewhere herein.
[0136] The present invention provides a method of promoting
tolerance in an animal to SLE-associated autoepitopes, a method of
reducing the production of autoantibodies in an animal, and a
method of inhibiting inflammation associated with the production of
autoantibodies. Each of these methods comprises administering to an
animal, preferably a human, one or more of a histone peptide and an
isolated nucleic acid encoding a histone peptide. This
administration preferably results in one or more of the development
of tolerance in the animal, the reduction of autoantibody
production in the animal and the inhibition of inflammation
associated with SLE-associated autoantibody production in the
animal. More preferably, this administration results in the
reduction of SLE-associated autoantibody production or inflammation
derived therefrom.
[0137] As the production of SLE-associated autoantibodies to
histone autoepitope regions may be involved in autoimmune disorders
such as SLE, the present invention encompasses methods of treating
an autoimmune disorder in an animal, such as a human, by
administering a composition comprising a histone peptide or or a
modified histone peptide in an amount sufficient to promote
tolerance in the animal to autoepitopes associated with the
autoimmune disorder, thereby inhibiting the production of
autoantibodies associated with the disorder in the animal. In these
methods, the preferred animal is a human, and the administration of
such a composition promotes tolerance and results in reduced
intermolecular help, less intense immune responses to autoantigens,
and the inhibition of autoantibody production, thereby treating the
autoimmune disorder in the animal. In an alternative embodiment,
the modified histone peptide is an APL.
[0138] The present invention includes a method of tracking one or
more of an SLE-associated T cell and an SLE-associated B cell. This
method comprises administering to a population of immune cells,
either in vivo or in vitro, a composition comprising one or more of
a modified histone peptide, histone peptide complex, and an
isolated nucleic acid encoding a histone peptide complex or
modified histone peptide in an amount sufficient to detect binding
of the histone peptide complex or modified histone peptide to an
SLE-associated T cell or SLE-associated B cell present in the
population. The binding of the peptide or peptide complex to the
SLE-associated B or T cells is assessed by detecting an indicator
portion (i.e. detectable label which emits or is associated with
the emission of a detectable chemical signal) which is covalently
linked (i.e. attached) to the peptide or peptide complex as
described elsewhere herein. Preferably, this method results in the
tracking SLE-associated cells and effectively tagging (i.e.
staining) these cells so that the cells can be monitored (i.e.
tracked) in a population of immune cells either within the body of
an animal (i.e. in vivo), or obtained from the animal (i.e. in
vitro). More preferably, this method employs a histone peptide
complex comprising either a fluorochrome-conjugated histone
peptide/MHC II molecule tetramer or a fluorochrome-conjugated
histone peptide/MHC II-Ig chimeric dimer, or a modified histone
peptide comprising a biotinylated histone peptide.
[0139] An isolated nucleic acid encoding a histone peptide complex,
such as a vector comprising a histone peptide/MHC II gene, can be
used to express a histone peptide complex in a cell, and therefore,
can also be useful in tracking one or more of an SLE-associated T
cell and an SLE-associated B cell. Accordingly, the above method of
tracking one or more of an SLE-associated T cell and an
SLE-associated B cell using an isolated nucleic acid which encodes
a histone peptide complex or modified histone peptide complex
additionally comprises administering the isolated nucleic acid to
an animal or to the cells of an animal in a manner which permits
the expression of a histone peptide complex or modified histone
peptide in an amount sufficient to detect binding of the histone
peptide complex or modified histone peptide to an SLE-associated T
cell or SLE-associated B cell present in the population.
Specific Applications of Pharmaceutical Compositions
[0140] Peptides can be administered orally, intranasally,
intravenously, intraperitoneally, or subcutaneously for tolerance
therapy, and these classical methods have been described using
other protein or peptide antigens over many decades (Wells. 1911,
J. Infect. Dis. 8:147; Chase. 1946, Proc. Soc. Exp. Biol. Med.
61:257; Liblau et al. 1997, Immunol. Today. 18:599; Weiner. 1994,
Proc. Natl. Acad. Sci. USA. 91:10762; Strober et al. 1998, J. Clin.
Immunol. 18:1; Anderton et al. 1999, Immunological Rev. 169:123).
In certain embodiments, relatively high doses of the peptides have
to be administered, although tolerance by administering low doses
of peptides have also been described in other systems (Briner et
al. 1993, Proc. Natl. Acad. Sci. USA. 90:7608). In certain
preferred embodiments, low dose administration is preferred.
[0141] The exact mechanism of tolerization may vary (Liblau et al.
1997, Immunol. Today. 18:599; Zhong et al. 1997, J. Exp. Med.
186:673; Jenkins and Schwartz. 1987, J. Exp. Med. 165:302; Weiner.
1994, Proc. Natl. Acad. Sci. USA. 91:10762). Most commonly, the
peptides are taken up by resting APC, and because the peptides are
in simple soluble form without any inflammatory adjuvant carriers,
the APC are not activated to express any co-stimulatory signaling
molecules. Thus the peptides are presented on MHC molecules of the
APC to their cognate T cells, without any co-stimulatory signals.
The autoimmune T cells receive only signal 1, from the peptide-MHC
binding to their antigen receptors (TCR), but not signal 2
(costimulation), thus rendering them inactive (anergic) or dead
(deleted). As a consequence of this inactivation and/or deletion of
the autoimmune T cells, autoimmune B cells are deprived of T-cell
help, and thus autoantibody production is markedly diminished, as
shown in SLE using our system (see Example 1, below).
[0142] Peptides are usually stored frozen in lyophilized powder
form at -20 degrees C., for the short-term or at -70 degrees C. for
longer storage. For oral tolerance therapy, peptides may be given
in high doses, for example in mice, 300 mg daily feeding for 8 days
(Javed et al. 1995, J. Immunol. 155:1599; Benson et al. 1999, J.
Immunol. 162:6247; Weiner. 1994, Proc. Natl. Acad. Sci USA.
91:10762), or may be given low dose therapy such, as 5-75 ug per
kilogram of subject every two weeks. For oral tolerance, the
peptide may be suspended in 0.15 M bicarbonate buffer together with
soybean trypsin inhibitor (20 mg/ml) also suspended in the same
buffer for protection against proteolysis in the stomach (Javed et
al. 1995, J. Immunol. 155:1599). However others found that peptide
administered in simple PBS buffer is as effective (Baggi et al.
1999, J. Clin. Invest. 104:1287). Our histone peptides carry
charged residues and they go into aqueous solution readily (see
Example 1, below). In humans, oral tolerance, can be achieved with
peptides as a lyophilized powder enclosed in opaque gelatin
capsules which dissolve in the stomach. Oral administration of 300
mg of myelin basic protein daily for one year has been tried in
patients with multiple sclerosis in such a manner (Weiner et al.,
1993, Science. 259:1321).
[0143] For administrations through the other routes mentioned above
(intranasal, intravenous, or subcutaneous), peptides are usually
reconstituted in sterile, phosphate buffered saline solutions
(PBS), just before administration. The dosage may vary from 100 to
300 microgram per day in mice for these alternate routes of
tolerance induction. In certain preferred embodiments, a low dosage
is used, such as between 5 and 75 micrograms per kilogram of
subject, or a composition with a concentration of peptides between
0.1 and 0.9 nM.
Further Improvements in Tolerance Therapy with Overlapping B-cell
and T-cell Autoepitopes
[0144] Among the nucleosomal peptide epitopes for Th cells of SNF1
mice, H4 16-39 exhibited the most beneficial effect in tolerance
therapy (see Example 1). Interestingly, the H4 16-39 peptide was
not the most immunogenic among the nucleosomal autoepitopes in
triggering pathogenic Th cells when administered with CFA into SNF1
mice, nor did it have the highest affinity for MHC class II
molecules (Kaliyaperumal et al. 1996, J. Exp. Med. 183:2459; Shi et
al. 1998, J. Exp. Med. 187:367). But, H4 16-39, administered
intravenously (I.V.) in saline, was able to tolerize both the
autoimmune Th cells and the B cells of lupus, and thus was more
efficient in "tolerance spreading" (Example 1). Autoimmune memory B
cells were probably most affected by this peptide tolerogen, as
they could not be rescued by stimulation with CD40L plus IL-4, or
anti-Ig plus IL-4, or co-culture with autoimmune Th cells (Example
1). Apoptosis of mature B cells with high doses of peptide
intravenously has been observed in the lysozyme system (Shokat and
Goodnow 1995, Nature. 375:334), and it could have contributed to
the tolerogenic effect of H4 16-39 peptide. Moreover, anergic B
cells could be tolerogenic to autoimmune T cells, because they
would present nucleosomal autoantigens without providing
costimulation (Chan and Shlomchik 1998, J. Immunol. 160:51; Mohan
et al. 1995, J. Immunol. 154:1470; Desai-Mehta et al. 1996, J.
Clin. Invest. 97:2063; Eynon and Parker 1992, J. Exp. Med. 175:131;
Mamula et al. 1994, J. Immunol. 152:1453; Buhlmann et al. 1995,
Immunity 2:645).
[0145] Although B cell receptors (antibodies) are generally thought
to recognize conformational determinants (shapes), a relevant
peptide may form crucial parts of such determinants, as has been
shown by antibody binding to peptides in many systems (Tung et al.
1997, Current Opin. Immunol. 9:839; Wucherpfennig et al. 1997, J.
Clin. Invest. 100: 1114; Warren et al. 1995, Proc. Natl. Acad. Sci.
USA. 92:11061). Nevertheless, the histone autoepitope, H4 16-39
falls within the region targeted by lupus autoantibodies. The
overlapping of epitopes for pathogenic Th cells and autoimmune B
cells of lupus makes H4 16-39 a highly efficient tolerogen. An
additional epitope in nucleosomal core histone H3, H3. 85-102, has
been identified to which the splenic T cells of pre-nephritic SNF1
mice spontaneously responded (Kaliyaperumal et al. 1996, J. Exp.
Med. 183:2459). Interestingly, this T-cell epitope was also bound
by spontaneously arising anti-DNA autoantibodies of lupus (Example
1). More recently, by analysing naturally processed peptides, the
H1 22-42 peptide was identified as another dominant autoepitope for
the autoimmune T, as well as B cells of lupus (see Example 3).
[0146] Thus, autoantigen-experienced and presumably memory T and B
cells of lupus can be functionally inactivated in concert, at least
for their ability to produce pathogenic autoantibodies by
tolerogenic therapy with nucleosomal peptides. Importantly, some of
the recurrent T-cell autoepitopes of human lupus that have been
identified so far (Example 2), fall within the regions that might
also be B-cell epitopes, i.e. targeted by lupus autoantibodies.
[0147] To establish this epitope sharing, the peptide epitopes
recognized by T cells of lupus patients that have been identified
(Example 2 and 3), can be tested for competitive inhibition of
binding of autoantibodies in lupus patient's sera, to nucleosomes.
Peptide competition ELISAs for autoantibodies have been described
(Gavalchin et al. 1985, J. Immunol. 134:885; Tung et al. 1997,
Current Opin. Immunol. 9:839; Wucherpfennig et al. 1997, J. Clin.
Invest. 100:1114; Warren et al. 1995, Proc. Natl. Acad. Sci. USA.
92:11061). Anti-DNA autoantibodies from lupus sera can be
affinity-purified on DNA-cellulose columns (Datta et al. 1987, J.
Exp. Med. 165:1252; Shivakumar et al. 1989, J. Immunol. 143:103),
and then tested in similar competition immunoassay. It has been
reported that such pathogenic anti-DNA autoantibodies of lupus mice
(Gavalchin et al. 1985, J. Immunol. 134:885) bind nucleosomes as
strongly as DNA (Mohan et al. 1993, J. Exp. Med. 177:1367).
Tracking Autoimmune B cells
[0148] The histone peptide autoepitopes that are recognized by
autoimmune B cells, in addition to the Th cells may be biotinylated
to study binding of histone peptides to receptors on autoimmune B
cells from peripheral blood of lupus patients, along with staining
for a B-cell marker (CD19 or CD20) for two color flow-cytometry,
using established procedures (Desai-Mehta et al. 1996, J. Clin.
Invest. 97:2063; Shivakumar et al. 1989, J. Immunol. 143:103).
Competitive inhibition by anti-human Ig (Fab'2) may indicate that
binding of the peptides is occuring through a BCR. If this assay
for tracking autoimmune B cells is found to be specific using the
nucleosomal histone peptides, then it may be evaluated in lupus
patients with active disease and those in remission, as well as
other unrelated autoimmune diseases. Thus, this assay for tracking
autoimmune B cells may be useful as a valuable diagnostic and
prognostic tool, complementary to tracking autoimmune Th cells with
tetramers, as described below. Monoclonal antibodies made against
histone peptide-MHC II tetramers that are described below could
also be used to stain and track autoimmune B cells.
Therapy with Analogs of Histone Peptide Epitopes or Altered Peptide
Ligands (APLs)
[0149] APLs with changes in residues that contact the TCR, can
function as antagonists or as partial agonist ligands for T cells
specific for the unaltered (i.e. wild type or wt) peptide epitope
(Evabold et al. 1993, Immunol. Today. 14:602; Sloan-Lancaster et
al. 1994, J. Exp. Med. 180:1195; DeMagistris et al. 1992, Cell.
68:625; Madrenas et al. 1997, J. Exp. Med. 185:219; Korb et al.
1999, J. Immunol. 162:6401; DePalma et al. 1999, J. Immunol.
162:1982). T cells stimulated with partial agonist peptides may
enter a state of anergy in which they are unresponsive to
subsequent stimulation with the agonist (wild type or wt) peptide
(references cited above). APLs send a negative signal to the T cell
because of failure to simultaneously engage a threshold number of
TCRs as a result of fast dissociation kinetics (Rabinowitz et al.
1996, Immunity 5:125). Although, it has been determined that
tolerization with unaltered nucleosomal epitopes in lupus prone
mice can delay and even halt progression of established lupus
nephritis by tolerance spreading (see Example 1), work on other
autoimmune disease models has shown that APLs might be more
effective than wt ligands (Kuchroo et al. 1994, J. Immunol.
153:3326; Karin et al. 1994, J. Exp. Med. 180:2227; Yu et al. 1996,
J. Exp. Med. 183:1771). Moreover, the effect of APLs on the
autoimmune Th cells of lupus is unknown. Therefore, based on the
sequences of the immunodominant, nucleosomal autoepitopes, it may
be feasible to synthesize single amino acid substituted analogs
(i.e. APLs or modified histone peptides) that can still bind to MHC
molecules, such as HLA-DRs in humans, but fail to stimulate the
pathogenic autoantibody-inducing Th cells associated with SLE.
[0150] Fine mapping of MHC and TCR contact residues may be done as
follows. The anti-DNA autoantibody-inducing Th cells of lupus
patients recognize nucleosomal peptide autoepitopes complexed with
HLA-DR (MHC class II) molecules, and the major peptide epitopes
have multiple DR-binding motifs (see Example 2, below). This type
of promiscuous binding and recognition is important for the
striking therapeutic effect of the nucleosomal peptides by
across-the-board tolerance spreading in SNF1 mice with lupus
nephritis (see Example 1). Therefore, to initially map the MHC
contact residues, analogs of the histone peptide autoepitopes can
be synthesized with single amino acid (alanine or glycine)
substitution at each of the putative anchor residues and each
analog peptide can be tested for its ability to bind purified
HLA-DR molecules in a competition immunoassay (Shi et al. 1998, J.
Exp. Med. 187:367). Those substituted peptides which still bind
HLA-DR can then be tested for their ability to stimulate autoimmune
Th cells of SLE for cytokine production and proliferation (as
described in Example 2).
[0151] Following the mapping of MHC contact residues, the TCR
contact residues in the substituted peptides that still bind HLA-DR
can be mapped. These residues would presumably constitute the
residues that point "up" in relation to the MHC contact residues
(Kuchroo et al. 1994, J. Immunol. 153:3326; Evabold et al. 1993,
Immunol. Today. 14:602; Sloan-Lancaster et al. 1994, J. Exp. Med.
180:1195; Karin et al. 1994, J. Exp. Med. 180:2227). After an
initial alanine scanning to localize the TCR contact residues in
the nucleosomal peptides using assays described herein, further
non-conservative substitutions can be performed, such as changes in
charge, size or hydrophobicity. The altered peptides may not be
able to stimulate the T cell clones or lines of human lupus. Such
APLs modified with changes in TCR contact residues but not in MHC
binding, could be tested for induction of anergy or deletion of
autoimmune Th cells of lupus in vitro. More relevant is that the
effect of APLs on the complex T cell response to the native
autoantigen associated with lupus be examined. The ability of the
APLs to antagonize the response of the T cells to whole
nucleosomes, and unrelated wild type peptide epitopes from the
nucleosomes can be tested using the above assays.
[0152] The promiscuity of lupus TCRs and the ability of a single
nucleosomal epitope to cause "tolerance spreading" in murine models
(see Example 1 and 2), suggest that similar features will be found
in human lupus. The functional effect of the APLs is best tested
using the more rigorous T helper assay, rather than using simple
cytokine responses which may or may not be affected. Therefore,
APLs could be assayed for inhibiting the autoantibody-inducing
ability of Th clones or lines from lupus patients in co-culture
systems, as described (Shivakumar et al. 1989, J. Immunol. 143:103;
Rajagopalan et al. 1990, Proc. Natl. Acad. Sci. USA. 87:7020), in
the presence or absence of respective wild type peptide (i.e.
unaltered autoepitope or agonist). Thus, the altered peptide
ligands could be tested for antagonism to autoimmune Th cells of
SLE. Highly efficient APLs should inhibit responses to the wild
type peptide epitopes at equimolar or lower concentrations, but
should not cause MHC blockade by having an affinity for the DR
molecules any higher than the wt peptides. Such APLs can be
prepared based on the sequences of the histone autoepitopes in this
invention.
Histone Peptide-MHC Tetramers for Tracking Autoimmune T Cells
[0153] T cell receptor (TCR) of a particular antigen-specific T
cell binds to its cognate antigenic peptide complexed with MHC
molecule. Therefore, fluorochrome-conjugated, multivalent (i.e.
tetrameric), soluble complexes of the peptide-MHC molecules can
been used to stain and track the antigen-specific T cells
(McMichael and Kelleher. 1999, J. Clin. Invest. 104: 1669).
Recognition of nucleosomal epitopes by T cells from different lupus
patients is HLA-DR dependent (see Example 2); however, the
involvement of CD8.sup.+ T.sub.reg cells indicates that Class I MHC
molecules also have a role in lupus pathogenesis. The major peptide
epitopes appear to have promiscuous DR binding motifs (Example 2),
and two of them, H4 16-39 and H4 71-94 have actually been shown to
bind HLA-DR (Shi et al. 1998, J. Exp. Med. 187:367). Furthermore,
three of the peptides have been shown to comprise Class I epitopes
(Example 4). For these reasons and given the promiscuous nature of
lupus TCR-nucleosomal peptide recognition, a significant proportion
of autoimmune T cells in lupus patients may be detected by
appropriate nucleosomal peptide-MHC tetramers, which can be
prepared by using established recombinant DNA technology (Crawford
et al. 1998, Immunity 8:675; Novak et al. 1999, J. Clin. Invest.
104:R63-R67; Kotzin et al. 1999, Proc. Natl. Acad. Sci. USA.
97:291), by expressing HLA-DR or other MHC genes covalently linked
to any one of the nucleotide sequences of the histone autoepitopes
which are supplied in this invention. Alternatively, chimeric
molecules of histone peptide-MHC dimer-IgG can also be made using
published technology (Lebowitz et al. 1999, Cell. Immunol.
192:175), and again using the nucleic acid encoding the histone
peptide autoepitopes. Another approach is to make empty MHC
molecules in insect cells using appropriate vectors (McMichael and
Kelleher 1999, J. Clin. Invest. 104:1669) and then incubate the MHC
molecules with any of the histone peptide autoepitopes for stable
binding. Similarly, such tetramers of mouse MHC-nucleosomal
peptide, and also chimeric peptide-MHC-Ig dimers, can be made to
track autoimmune T cells in lupus-prone mice. The reason for making
both types of constructs is because sometimes tetramers do not work
for particular TCR-peptide interactions of low avidiy or poor
association kinetics. However, lupus TCR-nucleosomal peptide
interactions might be of high affinity (Shi et al. 1998. J. Exp.
Med. 187:367).
[0154] The biotinylated tetramers or dimers can be used to stain
and track autoimmune T cells in lupus patients at various stages of
disease activity, such as active stage, recent remission and
long-term remission stages. Correlations with particular type of
disease manifestation can be made, particularly in the case of
lupus nephritis. Because the autoimmune Th cells appear long before
the onset of clinical disease, their presence may be used to
predict relapses or flare-up of lupus well in advance, and may
identify patients who are at risk for developing SLE-associated
nephritis. In such patients, more aggressive and early therapy can
be instituted. Therefore, the histone peptide-MHC tetramers or
dimers (i.e. histone peptide complexes) have important diagnostic
and prognostic value.
Pharmaceutical Compositions in General
[0155] Factors and considerations for the application of
compositions and pharmaceutical compositions comprising one or more
of a histone peptide, a modified histone peptide, a histone peptide
complex, and an isolated nucleic acid encoding one or more of a
histone peptide, modified histone peptide, and a histone peptide
complex are now described. As a general rule for induction of
tolerance, peptides have to be administered in a simple soluble
form without any adjuvants or any carriers that might cause
aggregation.
[0156] The invention encompasses the preparation and use of
medicaments and pharmaceutical compositions comprising one or more
of a histone peptide and a modified histone peptide described
herein as an active ingredient. Such a pharmaceutical composition
may consist of the active ingredient alone, in a form suitable for
administration to a subject, or the pharmaceutical composition may
comprise the active ingredient and one or more pharmaceutically
acceptable carriers, one or more additional ingredients, or some
combination of these. Administration of one of these pharmaceutical
compositions to a subject is useful, for example, for alleviating
disorders associated with the production of autoantibodies in the
subject, as described elsewhere in the present disclosure. The
active ingredient may be present in the pharmaceutical composition
in the form of a physiologically acceptable ester or salt, such as
in combination with a physiologically acceptable cation or anion,
as is well known in the art.
[0157] It is important to understand when generating any of the
formulations and dosage units described in this section, that any
of the peptides of the invention are stable in the form of a
lyophilized powder stored at -70 degrees C., or at -20 degrees C.
for shorter periods of time. The peptides must not be aggregated or
conjugated or precipitated (i.e. the peptides or peptide complexes
must be in soluble form in aqueous solution) to be optimally useful
for the tolerance therapy described herein, particularly in
Examples 1-3.
[0158] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of dissolving the active ingredient in an
aqueous solvent carrier or one or more other accessory ingredients,
and then, if necessary or desirable, shaping or packaging the
product into a desired single-or multi-dose unit.
[0159] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates, mammals
including commercially relevant mammals such as cattle, pigs,
horses, sheep, cats, and dogs.
[0160] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for oral, parenteral, (subcontaneous, intravenous or
intraperitoneal) intranasal, or another route of administration.
Other contemplated formulations include immunologically-based
formulations for optimum tolerance induction.
[0161] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0162] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient. A unit dose of a pharmaceutical composition of the
invention will generally comprise from about 1 nanogram to about 1
gram of the active ingredient, and preferably comprises from about
50 nanograms to about 10 milligrams of the active ingredient.
[0163] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology. A formulation of a pharmaceutical
composition of the invention suitable for oral administration may
be prepared, packaged, or sold in the form of a discrete solid dose
unit including, but not limited to, a tablet, a hard or soft
capsule, a cachet, a troche, or a lozenge, each containing a
predetermined amount of the active ingredient. Other formulations
suitable for oral administration include, but are not limited to, a
powdered or granular formulation, an aqueous solution. Soft gelatin
capsules comprising the active ingredient may be made using a
physiologically degradable composition, such as gelatin. Such soft
capsules comprise the active ingredient, which may be mixed with
water.
[0164] Pulsed release technology such as that described in U.S.
Pat. No. 4,777,049 may also be used to administer the active agent
to a specific location within the gastrointestinal tract. Such
systems permit drug delivery at a predetermined time and can be
used to deliver the active agent, optionally together with other
additives that my alter the local microenvironment to promote agent
stability and uptake, without relying on external conditions other
than the presence of water to provide in vivo release.
[0165] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be
prepared, packaged, and sold either in liquid form or in the form
of a dry product intended for reconstitution with water or another
suitable vehicle prior to use. Liquid solutions may further
comprise one or more additional ingredients including, but not
limited to, suspending agents, dispersing or wetting agents,
preservatives, buffers, salts, flavorings, coloring agents, and
sweetening agents. Liquid solutions of the active ingredient in
aqueous solvents may be prepared so that the active ingredient is
dissolved, rather than suspended in the solvent. Aqueous solvents
include, for example, water and isotonic saline.
[0166] Powdered and granular formulations of a pharmaceutical
preparation of the invention may be prepared using known methods.
Such formulations may be administered directly to a subject, used,
for example, to form tablets, to fill capsules, or to prepare an
aqueous solution by addition of an aqueous vehicle thereto. Each of
these formulations may further comprise one or more of dispersing
or wetting agent, a suspending agent, and a preservative.
Additional excipients, such as fillers and sweetening, flavoring,
or coloring agents, may also be included in these formulations.
[0167] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal, intravenous
injection and intravenous, intraarterial, or kidney dialytic
infusion techniques.
[0168] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in
ampules, in multi-dose containers containing a preservative, or in
single-use devices for auto-injection or injection by a medical
practitioner. Formulations for parenteral administration include,
but are not limited to, solutions, or aqueous vehicles, pastes, and
implantable sustained-release formulations. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, stabilizing, or dispersing agents. In one
embodiment of a formulation for parenteral administration, the
active ingredient is provided in dry (i.e. lyophilized powder or
granular) form for reconstitution with a suitable vehicle (e.g.
sterile pyrogen-free water) prior to parenteral administration of
the reconstituted composition.
[0169] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous solution. This
solution may be formulated according to the known art, and may
comprise, in addition to the active ingredient, additional
ingredients such as the dispersing agents, or wetting agents,
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or buffered saline, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, or isotonic sodium chloride solution.
[0170] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
intranasal administration. Such a formulation may comprise dry
particles which comprise the active ingredient and which have a
diameter in the range from about 0.5 to about 7 nanometers, and
preferably from about 1 to about 6 nanometers. Such compositions
are conveniently in the form of dry powders for administration
using a device comprising a dry powder reservoir to which a stream
of propellant may be directed to disperse the powder or using a
self-propelling solvent/powder-dispensing container such as a
device comprising the active ingredient dissolved or suspended in a
low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0171] Low boiling propellants generally include liquid propellants
having a boiling point of below 65 degrees F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0172] Pharmaceutical compositions of the invention formulated for
intranasal delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous solutions optionally
sterile, comprising the active ingredient, and may conveniently be
administered using any nebulization or atomization device. Such
formulations may further comprise one or more additional
ingredients including, but not limited to, a flavoring agent such
as saccharin sodium, a volatile oil, a buffering agent, a surface
active agent, or a preservative such as methylhydroxybenzoate. The
droplets provided by this route of administration preferably have
an average diameter in the range from about 0.1 to about 200
nanometers.
[0173] Another formulation suitable for intranasal administration
is a coarse powder comprising the active ingredient and having an
average particle from about 0.2 to 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken
i.e. by rapid inhalation through the nasal passage from a container
of the powder held close to the nares.
[0174] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of the active ingredient, and may further comprise one
or more of the additional ingredients described herein.
[0175] A pharmaceutical composition of the invention may be
administered to deliver a dose of the active ingredient which is
from about 500 picograms per kilogram body weight per day to about
1 milligram per kilogram body weight per kilogram of an animal.
Preferably the dose is from about 100 micrograms per kilogram of
animal to about 600 micrograms per kilogram body weight. In other
preferred embodiments, the does is about 5 microgram to 75
micrograms per kilogram body weight of a patient.
[0176] As a general rule for induction of tolerance, peptides have
to be administered in a simple soluble form without any adjuvants
or any carriers that might cause aggregation. Peptides can be
administered orally, intranasally, intravenously,
intraperitoneally, or subcutaneously for tolerance therapy, and
these classical methods have been described using other protein or
peptide antigens over many decades (Wells. 1911, J. Infect. Dis.
8:147; Chase. 1946, Proc. Soc. Exp. Biol. Med. 61:257; Liblau et
al. 1997, Immunol. Today. 18:599; Weiner. 1994, Proc. Natl. Acad.
Sci. USA. 91:10762; Strober et al. 1998, J. Clin. Immunol. 18:1;
Anderton et al. 1999, Immunological Rev. 169:123). Usually
relatively high doses of the peptides have to be administered,
although tolerance by administering low doses of peptides have also
been described in other systems (Briner et al. 1993, Proc. Natl.
Acad. Sci. USA. 90:7608). The exact mechanism of tolerization may
vary (Liblau et al. 1997, Immunol. Today. 18:599; Zhong et al.
1997, J. Exp. Med. 186:673; Jenkins and Schwartz. 1987, J. Exp.
Med. 165:302; Weiner. 1994, Proc. Natl. Acad. Sci. USA. 91:10762).
Most commonly, the peptides are taken up by resting APC, and
because the peptides are in simple soluble form without any
inflammatory adjuvant carriers, the APC are not activated to
express any co-stimulatory signaling molecules. Thus the peptides
are presented on MHC molecules of the APC to their cognate T cells,
without any co-stimulatory signals. The autoimmune T cells receive
only signal 1, from the peptide-MHC binding to their antigen
receptors (TCR), but not signal 2 (costimulation), thus rendering
them inactive (anergic) or dead (deleted). As a consequence of this
inactivation and/or deletion of the autoimmune T cells, autoimmune
B cells are deprived of T-cell help, and thus autoantibody
production is markedly diminished, as shown in SLE using our system
(see Example 1, below).
[0177] Peptides are usually stored frozen in lyophilized powder
form at -20 degrees C., for the short-term or at -70 degrees C. for
longer storage. For oral tolerance therapy, peptides are given in
high doses, for example in mice, 300 mg daily feeding for 8 days
(Javed et al. 1995, J. Immunol. 155:1599; Benson et al. 1999, J.
Immunol. 162:6247; Weiner. 1994, Proc. Natl. Acad. Sci USA.
91:10762). For oral tolerance, the peptide may be suspended in 0.15
M bicarbonate buffer together with soybin trypsin inhibitor (20
mg/ml) also suspended in the same buffer for protection against
proteolysis in the stomach (Javed et al. 1995, J. Immunol.
155:1599). However others found that peptide administered in simple
PBS buffer is as effective (Baggi et al. 1999, J. Clin. Invest.
104:1287). Our histone peptides carry charged residues and they go
into aqueous solution readily (see Example 1, below). In humans,
oral tolerance, can be achieved with peptides as a lyophilized
powder enclosed in opaque gelatin capsules which dissolve in the
stomach. Oral administration of 300 mg of myelin basic protein
daily for one year has been tried in patients with multiple
sclerosis in such a manner (Weiner et al., 1993, Science.
259:1321).
[0178] For administrations through the other routes mentioned above
(intranasal, intravenous, or preferably subcutaneous), peptides are
usually reconstituted in sterile, phosphate buffered saline
solutions (PBS), just before administration. The dosage may vary
from 100 to 300 microgram per day, or preferably for low dose about
1 microgram per day in mice for these alternate routes of tolerance
induction or 5-75 ug per kilogram of patient administered about
every two weeks.
[0179] It is understood that the ordinarily skilled physician will
readily determine and prescribe an effective amount of the active
ingredient to alleviate an autoimmune disorder associated with
autoantibody production in an animal. In so proceeding, the
physician may, for example, prescribe a relatively low dose at
first, subsequently increasing the dose until an appropriate
response is obtained. It is further understood, however, that the
specific dose level for any particular subject will depend upon a
variety of factors including the activity of the specific compound
employed, the age, body weight, general health, gender, and diet of
the subject, the time of administration, the route of
administration, the rate of excretion, any drug combination, and
the severity of the disorder being treated.
Kits
[0180] Another aspect of the invention relates to a kit comprising
a pharmaceutical composition of the invention and an instructional
material. As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which is used to communicate the usefulness of the
pharmaceutical composition of the invention for inhibiting the
production of autoantibodies in a subject. The instructional
material may also, for example, describe an appropriate dose of the
pharmaceutical composition of the invention. The instructional
material of the kit of the invention may, for example, be affixed
to a container which contains a pharmaceutical composition of the
invention or be shipped together with a container which contains
the pharmaceutical composition. Alternatively, the instructional
material may be shipped separately from the container with the
intention that the instructional material and the pharmaceutical
composition be used cooperatively by the recipient.
[0181] The invention also includes a kit comprising a
pharmaceutical composition of the invention and a delivery device
for delivering the composition to a subject. By way of example, the
delivery device may be a squeezable spray bottle, a metered-dose
spray bottle, an aerosol spray device, an atomizer, a dry powder
delivery device, a self-propelling solvent or powder-dispensing
device, a syringe, a needle, or a dosage measuring container. The
kit may further comprise an instructional material as described
herein.
[0182] A kit provided by the invention can be useful for, among
other things, tracking SLE-associated T and B cells, promoting
tolerance to SLE-associated autoepitopes, inhibiting the production
of autoantibodies, inhibiting an immune response and associated
inflammation, treating SLE or other autoimmune disorders and
complications thereof, and in the diagnosis and prognostication of
SLE in an animal. Useful applications for a kit included in the
present invention are described below.
EXAMPLES
[0183] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein.
Example 1
Antigen-Specific Therapy of Murine Lupus Nephritis Using
Nucleosomal Peptides
[0184] The experiments of this Example demonstrate that the
pathogenic T and B cells of systemic lupus erythematosus are
susceptible to autoantigen-specific tolerogens, and that
administration of nucleosomal peptides can impair autoimmune T cell
responses and inhibit production of pathogenic autoantibodies.
[0185] The materials and methods used in this Example are now
described.
Mice
[0186] NZB and SWR mice were purchased from The Jackson Laboratory
(Bar Harbor, Me.). (SWR.times.NZB)F.sub.1 SNF.sub.1 hybrid mice
were bred and female progeny mice were used, in accordance with
standard procedures. The lupus-prone SNF.sub.1 mice spontaneously
develop SLE and die from SLE-associated nephritis with age (Datta
and Schwartz, 1976, Nature 263:412; Datta et al. 1978, J. Exp. Med.
147:854).
Antibodies
[0187] The following monoclonal antibodies (mAbs) were used:
anti-I-A.sup.d (HB3), anti-I-A.sup..b,d,q (TIB12O), anti-HSA
(TIB183), anti-Thy1.2 (TIB99), anti-CD8 (TIB211), and anti-CD3
(145-2C11). All antibodies were obtained from American Type Culture
Collection (ATCC, Rockville, Md.).
Synthesis of Peptides
[0188] All peptides were synthesized using FMOC-compatible
protecting group, reagents and procedures by Chiron Mimotopes, (San
Diego, Calif.). Purity of the peptides was verified by the
manufacturer using amino acid analysis. The nucleosomal histone
peptides used in these experiments were H4 16-39, H4 71-94, and H2B
10-33, which correspond to the histone 4 protein (H4), amino acids
16-34 and 71-94, and histone 2B protein, (H2B) amino acids 10-33,
respectively. The peptides were purified by high performance liquid
chromatography (HPLC) using a gradient of water and acetonitrile,
and analyzed by mass spectrometry to verify purity.
Tolerance Induction with Histone Derived Peptides
[0189] Long-term experiments used 12 week-old, autoimmune, but
pre-nephritic SNF.sub.1 female mice. Nine mice per peptide test
group were injected intravenously with either a saline (solution)
or a peptide solution comprising saline and 300 micrograms of one
of H2B 10-33, H4 16-39, and H4 71-94. Each mouse received three
subsequent injections of either the saline solution or one of the
peptide solutions at two-week intervals. The mice were monitored
weekly for proteinuria using an Albustix kit (VWR Scientific,
Chicago, Ill.), and sacrificed upon the development of persistent
proteinuria. Persistent protenuria was identified as an amount of
protein in urine that is equivalent to or greater than 300
milligrams per deciliter, and is a primary indication of severe
kidney disease and damage associated with nephritis. Sera were
collected from each mouse and the IgG anti-nuclear autoantibodies
determined. Blood urea nitrogen (BUN) was measured by Azostix
(Miles, Ekhart, Ind.). Immune complex deposition and
glomerulonephritis in the kidney were detected by staining with
Hematoxylin and Eosin and anti-mouse Ig. These procedures were
carried out as previously described (Mohan et al., 1993, J. Exp.
Med. 177:1367; Mohan et al., 1995, J. Immunol. 154:1470; Eastcott
et al., 1983, J. Immunol. 131:2232; Adams et al., 1991, Proc. Natl.
Acad. Sci. USA 88:11271; Kalled et al., 1998, J. Immunol. 160:2158;
Gaynor et al., 1997, Proc. Natl. Acad. Sci. USA 94:1955).
[0190] Short-term experiments were conducted to test the early
immunological consequences of tolerance therapy. Twelve week old
SNF.sub.1 mice were injected once a week for four weeks with either
a saline solution or a peptide solution comprising saline and 300
micrograms of one of the above described peptides. Mice were
sacrificed at two weeks after the last injection. Presence of
autoimmune T and B cells was analyzed and renal lesions were
graded.
[0191] For experiments investigating chronic therapy of established
glomerulonephritis, 18 month old SNF1 mice exhibiting persistent
proteinuria were used. Six mice per group were injected
intraperitoneally once each month with either a saline solution or
a peptide solution comprising saline and 300 micrograms of one of
the identified peptides. The endpoint of these experiments was the
development of moribund state due to renal failure.
Autoantibody Quantitation
[0192] Levels of IgG class autoantibodies specific for
single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), histones,
and nucleosomes (i.e. histone-DNA complexes), in culture
supernatants or serum, were estimated using an enzyme-linked
immunosorbent assay (ELISA). Anti-DNA mAbs 564 and 205 were used to
generate standard curves as previously described (Mohan et al.,
1993, J. Exp. Med. 177:1367; Kaliyaperumal et al., 1996, J. Exp.
Med. 183:2459; Mohan et al., 1995, J. Immunol. 154:1470; Adams et
al., 1991, Proc. Natl. Acad. Sci. USA 88:11271). Sera were diluted
1:400 and heat-inactivated before use according to standard
procedures. Sera from normal SWR mice were used as the negative
control. Total polyclonal IgG levels were also measured by ELISA as
previously described (Mohan et al., 1993, J. Exp. Med. 177:1367;
Mohan et al., 1995, J. Immunol. 154:1470; Adams et al., 1991, Proc.
Natl. Acad. Sci. USA 88:11271).
Isolation of CD4+ Cells and B Cells
[0193] Splenic CD4+ T cells were isolated as described previously
(Mohan et al., 1993, J. Exp. Med. 177:1367; Mohan et al., 1995, J.
Immunol. 154:1470). Briefly, splenic T cells were purified from 3-4
month old SNF1 mice by applying spleen cell preparations to a nylon
wool column, lysing CD8.sup.+ T cells, and contaminating B cells
using antibody preparations, anti-CD8 (TIB211), ant-I-A.sup.b d,q
(TIB120), anti-HSA (TIB183), and a 1:10 dilution of rabbit and
guinea pig complement mixture (Pel Freeze Biologicals, Rogers,
Ark.). B cells were prepared from SNF1 mice by treating splenocytes
that were applied to nylon wool columns twice with anti-T cell
antibody, anti-Thy1.2 (TIB99) and complement.
Cytokine Assays
[0194] Fresh splenic CD4+ T cells, obtained from each of the
tolerized (i.e. peptide-injected) or control mice were seeded in
flat-bottom 96-well plates (Costar) at a concentration of 1.times.
10.sup.5 cells per well. Cells were seeded in triplicate wells.
Previously irradiated (3000 rads) APCs treated with anti-Thy-1.2
and complement were also seeded in each well at 5.times.10.sup.5
cells per well. Preparation of APCs was described previously (Mohan
et al., 1993, J. Exp. Med. 177:1367; Kaliyaperumal et al., 1996, J.
Exp. Med. 183:2459). A 200 microliter volume of HL-1 serum free
medium (Biowhittaker, Walkersville, Md.) containing varying
concentrations of each test peptide was also added separately to
the wells in triplicate. The cultures were incubated for 24-36
hours. Control cultures contained cells and medium without any
peptide. The culture supernatants were removed from the wells, and
cytokine assays were performed as previously described
(Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459). Anti-IL-2,
anti-IFNgamma, and anti-IL-4, capture and biotinylated antibody
pairs, and their respective standard cytokines (rIL-2, rIL-4,
rIFNgamma.) were purchased from Pharmingen (San Diego, Calif.).
Streptavidin-conjugated horseradish peroxidase (Streptavidin-HRP)
and the substrate, TMB, were purchased from Sigma Chemical Co. (St.
Louis, Mo.). Cytokines were quantitated according to manufacturer
instructions.
Helper Assays for IgG Autoantibody Production
[0195] T-cells from peptide-treated mice used in the short-term
experiments were co-cultured with splenic B cells from
unmanipulated mice. Conversely, T cells from unmanipulated SNF1
mice (i.e. control mice) were co-cultured with splenic B cells from
tolerized (i.e. peptide-injected) mice. Each of the cell types was
seeded in 24-well plates at an initial concentration of
2.5.times.10.sup.6 cells per well. Cultures were maintained for 7
days, as previously described (Datta et al., 1987, J. Exp. Med.
165:1252; Sainis and Datta, 1988, J. Immunol. 140:2215). B cells
from unmanipulated or tolerized animals were cultured alone to
measure baseline autoantibody production. Culture supernatants were
collected, freeze-thawed, and assayed by ELISA for antibodies
against ssDNA, dsDNA, histones and nucleosomes. For studies
involving stimulation of B cells by soluble CD40 ligand, CD40L-CD8
fusion protein (Wortis et al., 1995, Proc. Natl. Acad. Sci. USA
92:3348) was added to either the co-culture wells or the wells with
B cells alone and maintained in the medium for the entire 7-day
culture period.
Assay for Regulatory T Cells
[0196] To determine whether tolerance therapy induced any
regulatory T cell activity, T cells from either unmanipulated SNF1
mice or tolerized (i.e. peptide injected) mice, were co-cultured
with a mixture of splenic B cells and T cells from unmanipulated
SNF1 mice in 24-well plates for 7 days. Culture supernatants were
then collected, freeze-thawed, and assayed for the presence of IgG
antibodies against ssDNA, dsDNA, histones, and nucleosomes using an
ELISA.
The results of the experiments presented in this Example are now
described.
Brief Therapy with Nucleosomal Histone Peptides
[0197] Twelve week old pre-nephritic SNF1 female mice that did not
have proteinuria, or any other evidence of kidney disease, were
each injected intravenously with a peptide solution comprising
Saline and one of the peptides, H2B 10-33, H4 16-39, or H4 71-94.
The control group of mice were injected with a saline solution that
did not contain peptide. Each group of animals received three
additional injections at two week intervals. The mice were
monitored weekly for proteinuria and sacrificed when they developed
severe nephritis. As shown in FIG. 2, the control mice began
developing severe nephritis at 22 weeks. At 28 weeks of age, 55.5%
of the saline control group, and 33.3% (p=0.637, Fishers exact
test) of the H4 71-94 peptide injected group of mice developed
severe nephritis. In contrast, the H2B 10-33 and H4 16-39 injected
mice did not develop disease at this age (p=0.029).
[0198] The largest difference in incidence of severe nephritis
between the peptide injected groups and the control group was seen
at 36-38 weeks of age. At this age, 88.8% of the control group mice
had developed severe nephritis, whereas the H2B 10-33, and H4 71-94
groups of mice each had an incidence of only 33.3% (p=0.05) and the
H4 16-39 group mice exhibited an incidence of only 11.1% (p=0.003).
Mice in all groups, except the H4(16-39) group mice, developed
severe nephritis by 54 weeks of age. The H4 16-39 group mice had a
55.5% incidence of severe nephritis at 54 weeks of age, but in
relationship to the control group (p=0.08), this difference was not
significant.
T Cell Response to Peptides in Treated Animals
[0199] In unmanipulated SNF1 mice, T cells are spontaneously primed
to nucleosomal peptides early in life, and have been shown to
respond to them in vitro (Kaliyaperumal et al., 1996, J. Exp. Med.
183:2459). Therefore, T cells isolated at the time of sacrifice
from peptide-injected or control mice in the long-term experiments,
were co-cultured with APC in the presence of the peptides or whole
nucleosomal particles. The response of these T cells was assessed
by incorporation of .sup.3H-thymidine as an indication of
proliferation, and by ELISA to determine the extent of cytokine
(IL-2, IFN-gamma. and IL-4) production. These mice had already
developed a 4+grade of severe nephritis at the time of testing,
therefore the background levels of proliferation were high. There
was no deviation in cytokine production when the saline treated
group was compared with the peptide-treated groups.
[0200] The assessment of the incidence of nephritis and the grading
of renal pathology in short term experiment mice are shown in Table
1. For these experiments, the mice were sacrificed two weeks after
the last injection of either peptide solution or saline solution.
The mice were 22-23 weeks old when the data were obtained. In these
mice, no consistent differences were detected in cytokine
production levels or cytokine profiles in T cells from the control
group relative to the peptide-injected groups of mice in response
to any of the peptides or to whole nucleosomes. TABLE-US-00002
TABLE 1 Incidence of lupus nephritis at sacrifice in "short-term"
batch of mice Grading of Lupus Nephritis 0 1+ 2+ 3+ 4+ Group
Percent Incidence Saline 33.3 33.3 33.3 H2B(10-33) 66.6 33.3
H4(16-39) 33.3 66.6 H4(71-94) 66.6 33.3
Effect of Peptide Therapy on CD4+ Cell Help for Autoantibody
Production
[0201] The helper assay, used to detect autoantibody-inducing
ability in T cells, is a much more rigorous test for autoimmune T
helper cell (Th) function. Therefore, in further experiments the
helper assay as described above was used to determine the function
of T and B cells in tolerized (i.e. peptide-injected) mice from the
short-term experiments. The helper assay tests the ability of T
cells to functionally help B cells to produce pathogenic
autoantibodies to nuclear antigens. In this assay, CD4+ T cells
were isolated from the peptide-injected mice and co-cultured with B
cells isolated from unmanipulated 16-20 week old SNF mice. The
co-culture supernatants were analyzed for the presence of IgG
antibodies against ssDNA, dsDNA, nucleosomes, and histones using an
ELISA. The results shown in FIG. 3 are the mean values.+-.the
standard error of the mean (SEM) of five experiments. Anti-dsDNA
antibody production was reduced by approximately 50% in the
cultures containing T cells from H4 71-94, and H4 16-39 treated
group of mice as compared with the control group (p=0.03). The H2B
10-33 group mice exhibited a five fold decrease in anti-dsDNA
antibody production relative to the control group mice. Induction
of anti-ssDNA antibody production was also reduced by approximately
55% in the H2B 10-33 and H4 16-39 group mice, relative to the
control group mice (p=0.005 to 0.001). However, reductions of
anti-ssDNA antibody production in the H4(71-94) group mice were not
significant. Anti-nucleosomal antibody production exhibited a
similar pattern. The co-cultures of T and B cells from H2B 10-33,
H4 16-39, and H4 71-94, injected group mice produced 2.5 to 4 times
less anti-nucleosome antibody than the control group mice (p=0.03
to 0.05). The effect on anti-histone antibody induction in all
groups was not significantly different (p=0.1) relative to the
control group.
Addition of rIL-2 to CD4+ Cells for Testing Anergy
[0202] To determine if the diminished help observed in the above
experiments by CD4+ T cells from peptide treated mice was due to
anergy or deletion, rIL-2 was added at the beginning of the 7-day
culture period to the helper assay co-cultures, in a range of
concentrations from 12.5 to 100 units per milliliter. The results
of these experiments are shown in FIG. 4. The saline-injected
control group mice produced 102.+-.15.5 units of anti-dsDNA
antibody per deciliter. The addition of rIL-2 increased this
antibody production by only a small amount. CD4.sup.+T cells from
the H4 71-94, and H2B 10-33 injected mice did not exhibit any
increase in their ability to help in anti-dsDNA antibody production
after the addition of rIL-2. T cells from the H4 16-39 treated
group mice, exhibited only a modest increase of 35% in help for
anti-dsDNA antibody production following the addition of rIL-2
(p=0.05). The H4 16-39 group mice also exhibited a 30% increase in
anti-ssDNA antibodies and a 20% increase in anti-nucleosome
antibodies, but these increases were not significant relative to
the increases in control group mice.
Effect of Peptide Therapy on the Ability of Autoimmune B Cells to
Receive T Cell Help
[0203] The effect of peptide therapy on the immune function of B
cells was determined by co-culturing B cells from tolerized (i.e.
peptide injected) mice with CD4+ T cells from unmanipulated SNF1
mice, and performing the helper assay using these co-cultures. In
the presence of T helper cells, the B cells from the
peptide-injected and control groups of mice did not exhibit any
difference in their ability to produce IgG autoantibodies, with the
exception of the H4 16-39 group mice B cells, which still produced
diminished levels of all autoantibodies in co-culture experiments.
As shown in FIG. 5, the level of anti-dsDNA antibodies produced by
B cells from H4 16-39 injected mice (45.+-.10.1 units per
deciliter) was significantly reduced in comparison to control mice
(160.+-.18.5 units per deciliter) (p=0.03). Production of
anti-ssDNA antibodies exhibited a similar pattern: the control
group B cells produced 170.+-.10.9 units per deciliter compared
with the H4 16-39 group B cells which produced 60.+-.7.9 units
(p=0005). Production of anti-nucleosome antibodies was reduced by
three fold in the H4 16-39 group mice relative to the control group
mice, and anti-histone antibody production was reduced by
approximately 4-fold (p=0.003) in H4 16-39 injected mice relative
to control group mice. In H4 71-94 injected mice, production of
anti-nucleosome (p=0.01) and anti-histone (p=0.003) antibodies, but
not production of either anti-dsDNA or anti-ssDNA antibodies,
remained significantly low relative to control group mice. The
baseline values for antibody production by B cells cultured alone
ranged from 1.3 to 2.5 units per deciliter of culture medium in
these experiments.
Effect of Anti-Ig (Fab').sub.2, and rIL-4 on B Cells from H4
16-39--Treated Group of Mice
[0204] As indicated by the above experimental results, B cells from
H4 16-39 peptide-injected mice were less responsive to help from T
cells of unmanipulated SNF1 mice. In order to assess, whether this
impairment was due to deletion or anergy, purified B cells from the
H4 16-39 group mice were stimulated with anti-Ig (Fab').sub.2 in
the presence or absence of rIL-4, and the production of IgG
antibodies to dsDNA, ssDNA, nucleosomes and histones was measured.
The results of these experiments are shown in FIG. 6. The B cells
from control group mice produced high levels of antibodies, whereas
B cells from the H4 16-39 group mice did not respond by increasing
antibody production, even when stimulated with anti-Ig (Fab').sub.2
in the presence of high levels of rIL-4. This result suggested that
deletion of autoimmune B cells may be partly responsible for
reduced B cell responses. However, B cells from H4 71-94 group mice
could be stimulated to produce autoantibodies at levels similar to
those of the control mice. The basal level of antibody production
by B cells cultured alone in these experiments ranged from 1.9 to
2.1 units per deciliter of culture medium.
Effect of Soluble CD40 Ligand and rIL-4 on B Cells of H4 16-39
Injected Mice
[0205] In order to assess whether other molecules may be involved
in autoantibody production, B cell cultures were incubated in a
culture medium comprising HL-1 serum free medium, and soluble
CD40L-CD8 fusion protein (CD40L) in a ratio of 4 to 1 with or
without rIL-4. As shown in FIG. 7, purified (i.e. T-cell depleted)
B cells from the control group mice produced anti-nuclear
antibodies upon stimulation with CD40L. The addition of rIL-4 to
these cultures enhanced the production of antibodies by B cells
from the control group mice even further. However, a comparison of
FIGS. 5 and 7 reveals that the increase in levels of antibody
production with addition of soluble CD40L and rIL-4 were much less
than with the addition of intact Th cells from autoimmune mice,
indicating that additional molecules might be involved. B cells
from the H4 16-39 group mice did not produce significant amounts of
antibodies, even with the addition of CD40L and rIL-4.
B Cell Response to Lipopolysaccharide (LPS) in Peptide Injected
Mice
[0206] Purified B cells were isolated from peptide-injected mice
and stimulated with the potent mitogen, LPS. As shown in FIG. 8, B
cells from all the groups of mice responded with increases in
antibody production which were comparable to increases observed in
the control group mice (p=0.07 to 0.1).
IgG Anti-nuclear Autoantibody Levels in Sera of Peptide Injected
Mice
[0207] For the brief therapy experiments, sera were collected from
12 week old mice at the start of treatment, again at 36 weeks of
age, and finally, at the peak of the severe nephritis. IgG antibody
levels were measured for anti-dsDNA, anti-ssDNA, anti-nucleosome,
and anti-histone antibodies using a 1:400 dilution of sera from
mice. As shown in FIG. 9, the anti-dsDNA antibody level in the
control group was 5.5.+-.0.3 units, and in the H2B 10-33, H4 16-39,
and H4 71-94 groups mice, the levels of anti-dsDNA were 7.7.+-.0.9,
9.0.+-.2.1, and 5.4.+-.1.1 units, respectively, at the start of
treatment. At 36 weeks of age in the control group, anti-dsDNA
levels rose to 26.+-.3.2 units. This level was 14.0.+-.3.2,
13.0.+-.4.0, 17.1.+-.2.7 units in the H2B 10-33, H4 16-39, and H4
71-94 groups, respectively (p 0.03 to 0.05). Time point comparisons
among the groups of mice revealed a reduction in anti-ssDNA
antibody levels and anti-nucleosome antibody levels in sera from
peptide-injected mice which was comparable to the reduction
observed in their anti-ds-DNA antibody levels (p from 0.04 to
0.05). The levels of anti-histone antibodies varied among the
groups (p=0.3 to p=0.01). At the time of sacrifice, when the mice
had developed severe nephritis, the serum levels of antibodies were
similar among all groups of mice. Total polyclonal IgG levels were
not significantly different in the peptide-injected group mice
relative to the control group mice varying from 8-11 milligrams per
milliliter.
Search for Regulatory T Cells in Peptide-Injected Mice
[0208] In order to determine if any regulatory T cells might have
been generated by peptide injection, the ability of the T cells
from tolerized mice to inhibit autoantibody production in
co-cultures of T and B cells from unmanipulated SNF1 mice was
determined. T cells or purified CD4+ or CD8+ subsets of T cells
from short-term experiment mice in either the tolerized or control
groups were co-cultured with unmanipulated SNF1 splenic B and T
cell mixtures in 24-well plates for 7 days. IgG antibody production
in these cultures against ssDNA, dsDNA, histones, and nucleosomes
was estimated using an ELISA. No significant reduction in antibody
production by the addition of T cells from peptide-injected mice
was observed.
Treatment of Established Glomerulonephritis with Nucleosomal
Peptides
[0209] The results of chronic therapy experiments using 18-month
old mice with established glomerulonephritis are shown in FIG. 10.
The control group mice exhibited rapid progression of disease and
died within two months after the start of the treatment. At this
time point, 66.6% of the mice in the H4 71-94 and H2B 10-33 groups
of mice were alive (p=0.061), whereas none of the mice in H4 16-39
group had died (p=0.002). All the tolerized groups maintained their
starting levels of proteinuria during the course of the experiment,
with the exception of the H4 16-39 group, in which 66.6% of the
animals actually exhibited a reduction in proteinuria levels from
300-1000 milligrams per deciliter to less than 100 milligrams per
deciliter. By 26 months age, all of the H4 16-39 group mice
remained alive. By contrast, all of the H4 71-94 group mice had
succumbed to renal disease (p=0.002), and only one mouse in the H2B
10-33 group survived (p=0.015 as compared with the H4 16-39
group).
[0210] The multipotent and promiscuous helper activity of the
pathogenic Th cells involved in SLE which is evident from these
experiments is modeled in FIG. 11. This proposed model takes into
account that a single lupus T helper clone appears to have been
able to help different B cells which are specific for either dsDNA,
ssDNA, histone, or nucleosomes. This could result from each of
these B cells binding to its respective antigenic determinant on a
whole chromatin particle, taking up and processing the chromatin,
and presenting the relevant histone peptide epitope in the
chromatin to the Th clone (Mohan et al., 1993, J. Exp. Med.
177:1467; Desai-Mehta, 1995, J. Clin. Invest. 95:531; Datta and
Kaliyaperumal, 1997, Annals. New. York Acad. Sci. 815:155). The
result of the interaction is that intermolecular help is provided
to the different B cells by the same Th cell. The multipotent Th
cells associated with SLE cause immediate epitope diversification,
rather than the sequential epitope spreading seen in other types of
inflammatory damage and progression of other autoimmune disease
(Kalled et al., 1998, J. Immunol. 160:2158; Lehman et al., 1992,
Nature 358:155; Craft and Fatenejad, 1997, Arthritis Rheum.
40:1374).
[0211] The experiments described in this Example demonstrate that
despite an intrinsic polyclonal hyperactivity and a lowering of the
threshold of activation observed previously (Datta et al., 1982, J.
Immunol. 129:1539; Klinman and Steinberg, 1987, J. Exp. Med.
165:1755; Chan and Shlomchik, 1998, J. Immunol. 160:51;
Jongstra-Bilen, 1997, J. Immunol. 159:5810; Mohan et al., 1995, J.
Immunol. 154:1470; Desai-Mehta et al., 1996, J. Clin. Invest.
97:2063; Koshy et al., 1996, J. Clin. Invest. 98:826; Liossis et
al., 1996, J. Clin. Invest. 98:2549), pathogenic T and B cells of
established lupus can be functionally downregulated with injection
of peptides corresponding to the appropriate autoepitopes.
[0212] Among the three nucleosomal peptides described herein,
H416-39 exhibited the most beneficial effect. Interestingly, this
peptide is not the most immunogenic among the nucleosomal
autoepitopes in triggering pathogenic Th cells in SNF1 mice
(Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459), nor does it
have the highest affinity for MHC class II molecules (Shi et al.,
1998, J. Exp. Med. 187:367). However, H4 16-39 administered
intravenously, was able to tolerize both the autoimmune Th cells
and the B cells involved in lupus. Autoimmune memory B cells were
probably most affected by this peptide, as they could not be
rescued by CD40L/IL-4, or anti-Ig/IL-4 stimulation. Addition of
autoimmune Th cells from unmanipulated SNF1 mice did increase
autoantibody production by B cells from the H4 16-39 injected mice
above baseline, but not comparably relative to other groups.
[0213] As shown in this Example, the overlapping of epitopes for
pathogenic Th cells and autoimmune B cells of lupus makes H4 16-39
a highly efficient tolerogen, and this principle might be relevant
to other autoimmune diseases as well (Tung et al., 1997, Current
Opin. Immunol. 9:839; Wucherpfennig et al., 1997, J. Clin. Invest.
100:1114). An additional epitope was identified in nucleosomal core
histone H3, H3 85-102, to which splenic T cells of pre-nephritic
SNF1 mice spontaneously responded. (Kaliyaperumal et al., 1996, J.
Exp. Med. 183:2459). Interestingly, this T-cell epitope is also
bound by spontaneously arising anti-DNA autoantibodies of lupus
(Stemmer et al., 1996, J. Biol. Chem. 271:21257). Future studies
should determine if H3 85.102 is also a potent tolerogen for
therapy of lupus nephritis in SNF1 mice. Thus,
autoantigen-experienced and presumably memory T and B cells of
lupus can be functionally inactivated, at least for their ability
to produce pathogenic autoantibodies by tolerogenic therapy with
nucleosomal peptides. Finally, despite tolerance spreading, the
peptide treated mice did not develop any generalized
immunosuppression. They were housed in conventional cages and their
total serum IgG levels were not affected by the therapy.
The Data Presented in this Example can be Summarized as
Follows.
[0214] Using the (SWR.times.NZB)F1 (SNF1) mouse model of lupus, the
critical autoepitopes have been identified. Nephritis-inducing Th
cells are stimulated by autoepitopes present in the core histones
of nucleosomes, at amino acid positions 10-33 of H2B, 85-102 of H3,
and 16-39 and 71-94 of H4. The experiments in this Example
demonstrate that brief therapy with the peptides administered
intravenously to SNF1 mice that were already producing pathogenic
autoantibodies, markedly delayed the onset of severe lupus
nephritis. Strikingly, injection of these peptides into mice with
established glomerulonephritis, resulted in prolonged survival and
halted the progression of renal disease. Remarkably, tolerization
with any one of the nucleosomal peptides impaired autoimmune T-cell
function, thereby inhibiting the production of multiple pathogenic
autoantibodies. Moreover, suppressor T cells were not detected in
the injected mice. The most promising effect was obtained using
nucleosomal peptide H4 16-39, which had a tolerogenic effect not
only on autoimmune Th cells, but on autoimmune B cells as well.
Example 2
Major Peptide Autoepitopes for Nucleosome-Specific T Cells of Human
SLE
[0215] In the experiments presented in this Example, overlapping
peptides spanning the entire length of the core histones of
nucleosomes have been tested for their ability to stimulate
established T helper cell lines and primary T helper cell isolates
from 23 SLE patients. The peptide autoepitopes in nucleosomes that
are recurrently recognized by the autoimmune T cells of patients
with SLE are identified herein. These Th cells are essential for
sustaining the pathogenic autoantibody-producing B cells associated
with SLE (Datta et al. 1987, J. Exp. Med. 165:1252-1268; Shivakumar
et al. 1989, J. Immunol. 143:103-112; Ray et al., 1996, Proc. Natl.
Acad. Sci. USA. 93:2019-2024; Mohan et al., 1995, J. Immunol.
154:1470-1480). The materials and methods used in this Example are
now described.
Patients and Healthy Donors
[0216] The short-term CD4+ T cell lines were derived from a patient
group consisting of five female patients with active SLE ranging in
age from 21-51 years, five female patients in long term SLE
remission, ranging in age from 21-51 years, and normal healthy
subjects, two male and four female, ranging in age from 24-52
years. Peripheral blood mononuclear cells (PBMC) used to study
intracellular cytokine production were obtained from a separate
patient group consisting of eight patients in long term SLE
remission, including two males and six females, ranging in age from
28 to 55 years, and four female patients with active SLE, ranging
in age aged from 22 to 47 years.
[0217] Disease activity by Systemic Lupus Activity Measure (i.e.
SLAM), ranged between 7 and 20 for active patients (Liang et al.,
1989, Arthritis. Rheum. 32:1107-1118). None of the patients in
remission had detectable proteinuria or serum anti-DNA
autoantibodies at the time of testing, and their SLAM ranged
between 0 and 4. The patients in remission had never received any
cytotoxic drugs, and were not receiving any steroids at the time
their blood samples were drawn. Steroids had been discontinued for
several years in the remission patients, except for two patients
who had received a short course of low dose steriods (Prednisone,
10 milligrams per day) 2 months before the assays were
performed.
Antibodies
[0218] A hybridoma (OKT3) which produces Anti-CD3 antibody (i.e.
mAb) was obtained from the American Type Culture Collection
(Rockville, Md.). The hybridoma supernatants were concentrated by
precipitation in a solution comprising 47% saturated ammonium
sulfate, and dialyzed before use. Anti-CD28 antibody (clone 9.3)
containing ascites was provided by Bristol Myers Squibb (Seattle,
Wash.). Purified mAbs used for ELISAs were purchased from
Pharmingen (San Diego, Calif.). PE-conjugated mAb to human IL-10
and FITC-conjugated mAb to human IL-2 were purchased also from
Pharmingen. PE-conjugated mAb to human IL-4, FITC-conjugated mAb to
human IFN-gamma, PerCP-conjugated anti-human CD4 and purified
anti-human CD28 (clone L293) were purchased from Becton-Dickinson
(San Jose, Calif.).
Antigens
[0219] All the peptides used in the experiments presented in this
Example were synthesized by the pin method (Chiron Mimotopes, San
Diego, Calif.), and purity of the peptides was verified by amino
acid analysis (Kaliyaperumal et al., 1996, J. Exp. Med
183:2459-2469). Overlapping peptides comprising 15 amino acids each
were designed to span the entire stretch of all four core histones,
with each peptide overlapping at least one other peptide by 12
residues. Peptides used for intracellular cytokine response studies
were core histone peptides comprising either 15 amino acids or 24
amino acids. The sequences of these peptides (SEQ ID Nos: 1-26), as
well as those used elsewhere herein, are shown in FIG. 17 and
Example 3. Nucleosomes were prepared as described previously (Mohan
et al., 1993, J. Exp. Med. 177:1367-1381; Kaliyaperumal et al.,
1996, J. Exp. Med 183:2459-2469.
Cell Preparation
[0220] PBMCs obtained from either patients or healthy donors were
isolated from heparinized venous blood by density gradient
sedimentation using Ficoll-Hypaque as a gradient matrix (Pharmacia
LKB Biotechnology Inc., Piscataway, N.J.). Isolated PBMCs were
washed twice in RPMI 1640 culture medium, and aliquots of the cells
suspended in media were used to generate both short-term T cell
lines and EBV-transformed B cell lines. Others cells were either
used directly for stimulation with antigens or frozen into liquid
nitrogen.
[0221] To make short-term, CD4+ T cell lines, CD4+ T cells were
purified from PBMC using magnetic beads absorbed with anti-CD4 mAb,
followed by using DetachBeads (Dynal Inc., Oslo, Norway). The CD4+
T cells were expanded by one round of stimulation with plate-bound
anti-CD3 mAb, anti-CD28 (clone 9.3) mAb, and rIL-2 (20 units per
milliliter) in RPMI 1640 medium supplemented by 10% human AB serum
(Pel-Freez Biologicals, Brown Deer, Wis.). The expanded T cells
were frozen in liquid nitrogen within two weeks of culture using
standard protocols and reagents. EBV-B cell lines used as antigen
presenting cells in co culture were generated as described
previously. (Rajagopalan et al., 1990, Proc. Natl. Acad. Sci.
87:7020-7024; Desai-Mehta et al., 1995, J. Clin. Invest. 95:53
1-541). The CD4+ T cell clone DD2 was derived from a patient with
active SLE and associated nephritis (Rajagopalan et al., 1990,
Proc. Natl. Acad. Sci. 87:7020-7024; Desai-Mehta et al., 1995, J.
Clin. Invest. 95:531-541).
Stimulation of Short-Term T Cell Lines
[0222] Short-term T cell lines were rapidly thawed from liquid
nitrogen and expanded in the presence of plate-bound anti-CD3 mAb,
anti-CD28 mAb, and rIL-2 (20 units per milliliter). The expanded
cells were transferred to fresh culture wells and rested by
incubating in fresh medium for 10 to 14 days. For stimulation
assays, 96 well plates were coated with 2 micrograms per milliliter
of goat anti-mouse IgG antibodies overnight at 37 degrees C. After
washing the plates twice with Dulbecco's phosphate-buffered saline
(dPBS), autologous and previously irradiated (3,000 rad) EBV-B
cells (APC) were first added to each well at a concentration of
1.times.10.sup.5 cells per well. The cells (APC) were cultured
together with either a core histone peptide or a control peptide
for 6 hours in HL-1 serum free medium (Bio Whittaker, Maryland).
This procedure was followed by the addition to each well of CD4+ T
cells from the short-term lines at a concentration of
1.times.10.sup.5 cells per well, and the addition of 0.5 micrograms
per milliliter of a mouse monoclonal antibody (mAb) to human CD28.
The final volume of the cultures was 200 microliters per well.
Cells were co-cultured for 24 hours at 37 degrees C. One-half the
volume of supernatant was collected from each well for ELISA
analysis of interleukin levels. Fresh medium (100 microliters) was
added to cells, and the cells were incubated an additional 48
hours. [.sup.3H] thymidine was added, and the cultures were
incubated an additional 18 hours. Incorporated radioactivity was
measured to quantitate T cell proliferation as described previously
(Desai-Mehta et al., 1995, J. Clin. Invest. 95:53 1-541). HLA class
II dependence of antigen-specific responses by the CD4+ T cells was
determined by adding to the co-cultures a blocking antibody from a
panel of mAb to HLA class II molecules, as described previously
(Desai-Mehta et al., 1995, J. Clin. Invest. 95:53 1-541).
[0223] The autoantibody-inducing T cell clone, DD2, from a patient
with SLE-associated nephritis was previously reported (Rajagopalan
et al., 1990, Proc. Natl. Acad. Sci. 87:7020-7024; Desai-Mehta et
al., 1995, J. Clin. Invest. 95:53 1-541). DD2 was stimulated as
described above with autologous EBV-B cell lines which had been
pre-pulsed with the histone peptides.
IL-2 Production Analysis
[0224] IL-2 secreted by CD4+ T cells in short term experiments was
measured using an ELISA. Capture and biotinylated antibody pairs
directed at human IL-2 and standards for use therein were purchased
from PharMingen. ELISAs were performed in 96-well Nunc-Immunoplate
(Maxisorb.TM., Nunc, Denmark). Streptavidin-conjugated horseradish
peroxidase and the substrate 3,3',5,5'-tetramethyl benzidine
dihydrochloride were purchased from Sigma Chemical Co. (St. Louis,
Mo.). ELISAs were performed according to the protocol provided by
manufacturer.
Direct Stimulation of PBMCs
[0225] Measurement of antigen-specific, intracellular cytokine
responses of T cells were performed as described with slight
modifications (Waldrop et al., 1997, J. Clin. Invest. 99:1739-1750;
Openshaw et al., 1995, J. Exp. Med 182:1357-1367; Estcourt et al.,
1997, Clin. Immunol. Immunopathol. 83:60-67). Purified, PBMCs were
placed in 12.times.75 millimeter polystyrene tissue culture tubes
(Becton Dickinson, Lincoln Park, N.J.) at a concentration of
1.times.10.sup.6 cells per tube. A solution comprising 0.5
milliliters of HL-1 serum free medium, 100 units per milliliter of
penicillin, 100 units per milliliter streptomycin, 2 millimolar L
glutamine (Gibco BRL), varying amounts of individual histone
peptides, and 1 unit of anti-CD28 mAb (Becton-Dickinson, Lincoln
Park, N.J.) was added to each tube. Anti-CD3 mAb was added to a
duplicate set of normal PBMC cultures as positive control. Culture
tubes were incubated for 1 hour. Brefeldin A was added to
individual tubes at a concentration of 1 microgram per milliliter,
and the tubes were incubated for an additional 17 hours.
Flow Cytometry Analysis
[0226] The assays were performed as described previously (Waldrop
et al., 1997, J. Clin. Invest. 99:1739-1750; Openshaw et al., 1995,
J. Exp. Med 182:1357-1367; Estcourt et al., 1997, Clin. Immunol.
Immunopathol. 83:60-67). PBMCs stimulated as described above were
harvested by washing the cells twice with a solution comprising
Dulbecco's phosphate-buffered saline (dPBS) and 10 units of
Brefeldin A. These washed cells were fixed by incubation for 10
minutes in a solution comprising 0.5 milliliters of 4%
paraformaldehyde and dPBS. The cells were washed with a solution
comprising dPBS and 2% fetal calf serum (FCS). The cells were then
either used immediately for intracellular cytokine and surface
marker staining or were frozen for no more than three days in
freezing medium, as described (Waldrop et al., 1997, J. Clin.
Invest. 99:1739-1750).
[0227] For intracellular cytokine staining, the cell preparations
were rapidly thawed in a 37.degree. C. water bath and washed once
with dPBS. Cells, either fresh or frozen, were resuspended in 0.5
milliliters of permeabilizing solution (Becton Dickinson
Immunocytometry systems, San Jose, Calif.) and incubated for 10
minutes at room temperature with protection from light.
Permeabilized cells were washed twice with dPBS and incubated with
directly conjugated mAbs for 20 minutes at room temperature with
protection from light. Optimal concentrations of antibodies were
predetermined according to standard methods. After staining, the
cells were washed, refixed by incubation in a solution comprising
dPBS 1% paraformaldehyde, and stored away from light at 4 degrees
C. for flow cytometry analysis.
[0228] Five parameter flow cytometry analyses were performed with a
FACSCalibur (Becton Dickinson Immunocytometry systems, San Jose,
Calif.) using FITC, phycoerythrin (PE), and peridinin chlorophyl
protein (PerCP) as the fluorescence parameters. Methods of
cytometer set up and data acquisition have been described
previously (Shivakumar et al., 1989, J. Immunol. 143:103-112;
Desai-Mehta et al., 1996, J. Clin. Invest. 97:2063-2073; Waldrop et
al., 1997, J. Clin. Invest. 99:1739-1750; Openshaw et al., 1995, J.
Exp. Med 182:1357-1367; Estcourt et al., 1997, Clin. Immunol.
Immunopathol. 83:60-67). For analysis of each cytokine response
against each of the histone peptides, the cytometer was gated on
CD4 expression and 10,000 events were acquired per analysis. A
light scatter gate was also used to identify nonviable lymphocytes
(Shivakumar et al., 1989, J. Immunol. 143:103-112; Desai-Mehta et
al., 1996, J. Clin. Invest. 97:2063-2073; Waldrop et al., 1997, J.
Clin. Invest. 99:1739-1750). Isotype-matched negative control
reagents were used to verify the staining specificity of
experimental antibodies and as a guide for setting markers to
delineate "positive" and "negative" populations.
The results of the experiments presented in this Example are now
described.
Response of an Anti-DNA Autoantibody-Inducing, Lupus Th Cell Clone
To Histone Peptides
[0229] To localize histone peptide epitopes for autoimmune T cells
of human SLE, the response of CD4+ T cell clone DD2, was
characterized. DD2 has previously been shown to induce the
production of the pathogenic variety of anti-DNA autoantibodies
when co-cultured with autologous B cells. In addition, clone DD2
recognizes nucleosomes, particularly its core histone, H4.
(Desai-Mehta et al., 1995, J. Clin. Invest. 95:531-541). In the
present Example, the DD2 clone was tested against the entire panel
of nucleosomal histone peptides.
[0230] As illustrated in FIG. 12, DD2 can be stimulated by a group
of histone H4 peptides located between amino acid positions 67 and
99. The other nucleosomal histone peptides did not stimulate DD2,
except for one peptide, H3 55-69, which stimulated DD2 very weakly.
These results were consistent with previously published
autoantigenic-specificity of DD2 tested with whole autoantigens
(Desai-Mehta et al., 1995, J. Clin. Invest. 95:53 1-541).
Localization of Major Peptide Epitopes For Autoimmune T Cells by
Using Short-Term, CD4+ Cell Lines Derived from Lupus Patients
[0231] To narrow down the regions in nucleosomal core histones that
would contain the major peptide autoepitopes, short-term CD4+ T
lines derived from both lupus patients and healthy donors were
incubated as described above, and were tested for their response to
the histone peptides. Autologous EBV-B cell lines were included
with these T cells as APCs. Dose response curves were determined
after identifying stimulatory histone peptides (i.e. which comprise
autoepitope regions) for the respective T cell lines. A
representative example of a dose response is shown in FIG. 13A.
Based on these studies 10 micromolar peptide concentrations were
used in further experiments.
[0232] Four of the short-term lupus T cell lines were also tested
for HLA-class II dependence for antigen recognition, and were found
to recognize their respective peptide epitopes presented by HLA-DR
molecules. A representative example is shown in FIG. 13B. These
results with histone peptides are consistent with previous
observations using whole nucleosomes (Desai-Mehta et al., 1995, J.
Clin. Invest. 95:53 1-541).
[0233] Altogether, 10 short-term CD4+ T cell lines derived from
lupus patients and 6 healthy donor T cell lines were co-cultured
with synthetic peptides spanning all of the core histone proteins
and autologous EBV-B cell lines. IL-2 produced by these co-cultured
cells was measured. The response by a representative short-term T
cell line L-SC, derived from a lupus patient, is shown in FIG. 14,
and the response by short term T cell line N-JV, derived from a
normal control donor, is shown in FIG. 15. All of the short-term T
cell lines from normal donors exhibited very weak and insignificant
responses to the synthetic histone peptides similar to those shown
in FIG. 15. By contrast, the lupus T cell lines, which were also
polyclonal, responded strongly to peptides corresponding to certain
regions in the core histones as exemplified in FIG. 14.
[0234] Collective data of significant responses by all the
short-term T cell lines derived from the ten SLE patients is shown
in FIG. 16. These pepscan results localize major regions in the
core histone proteins that contain autoepitopes recognized
consistently and recurrently by the autoimmune T cells from
different patients (i.e. SLE-associated autoepitopes). There were
no discernible differences between the responses of T cell lines
derived from patients with active lupus and those derived from
patients in remission. Results of proliferative responses of the T
cells to the nucleosomal peptides were consistent with their IL-2
production.
[0235] Based on the consensus stimulatory autoepitope regions
identified by using the lupus T cell lines and clone DD2 (FIGS.
12-16), a group of 16 peptides, each comprising 15 amino acids,
were selected for further testing with T cells in PBMCs from lupus
patients and normal subjects. These SLE-associated autoepitope
peptide sequences are listed in FIG. 17. In addition, histone
peptides comprising 24 amino acids each and overlapping some of the
amino acid autoepitopes were also used, and were found to be
relevant to SLE-associated Th cells and B cells.
Histone Peptide Epitopes for CD4.sup.+T Cells in PBMC of Lupus
Patients
[0236] It was necessary to confirm that the histone peptide
autoepitopes were also relevant to unmanipulated CD4+ T cells
derived from PBMCs of SLE patients. Although the T cell lines used
were short-term and not biased by any deliberate addition of
histone peptides, they might still be able to undergo
activation-induced cell death and selective expansion in vitro. In
addition, to determine the frequency of CD4+ T cell response to a
particular peptide epitope, it was found that using flow cytometry
for the identification of newly synthesized, intracellular
cytokines was ideal in rapidly detecting antigen-specific responses
(Waldrop et al., 1997, J. Clin. Invest. 99:1739-1750; Openshaw et
al., 1995, J. Exp. Med 182:1357-1367; Estcourt et al., 1997, Clin.
Immunol. Immunopathol. 83:60-67).
[0237] Since a proportion of the autoimmune T cells involved in SLE
are already activated in vivo, particularly in patients with active
SLE, a measure of antigen-specific induction of T-cell activation
markers, such as CD69, CD25 or CD79, is not suitable because of the
high background levels which are generated. The intracellular
cytokine assay measures only newly synthesized cytokines in
response to antigen-specific stimulation, and the secretion of
these cytokines is blocked by BFA. Any cytokines made by
preactivated T cells are not detectable because they are secreted
when BFA is not present in the cultures. Thus, nucleosomal
peptide-specific responses of the autoimmune memory T cells of
lupus that were not already pre-activated in vivo were measured by
this assay. This assay was optimized by providing costimulatory
signal via CD28, which did not cause any non-specific background
stimulation as described previously (Waldrop et al., 1997, J. Clin.
Invest. 99:1739-1750). Moreover, this multiparameter flow-cytometry
method could be applied directly to PBMC without T cell
purification.
[0238] According to the results using lupus patient T-cell lines
and clone DD2 (FIGS. 12-16), the stimulatory histone peptide
epitopes could be localized to include several regions of
nucleosomal histone proteins. Interestingly, these histone
autoepitopes which stimulate autoimmune T cells overlapped with the
nephritis-inducing autoepitopes which were identified for
pathogenic autoantibody-inducing Th cells of lupus-prone mice in
the experiments of Example 1. Therefore, in addition to the sixteen
histone peptides listed in FIG. 17, and the three longer histone
peptides H2B 10-33, H4 16-39, and H4 71-94, that were also found to
be disease-relevant for pathogenic T and B cells of lupus mice
(Kaliyaperumal et al., 1996, J. Exp. Med 183:2459-2469) were tested
again with the PBMC. As an additional background control, the H3
83-97 peptide was used, which, although it is partly within a
stimulatory region in H3, it was not recognized by any of the
T-cell lines or clone DD2.
[0239] Intracellular production of newly synthesized cytokines,
IFN-gamma, IL-2, IL-10 and IL-4, was assayed. The assay results are
depicted in FIG. 18, wherein two representative cytokine staining
profiles from patients R-WG and R-SC are shown. Cells which stained
positive for a particular cytokine formed a discreet population;
single cells producing more than one cytokine were rare in fresh
PBMCs.
[0240] The results of assays for intracellular cytokine production
by CD4+ T cells from 12 SLE patients are shown in FIG. 19. Eight
patients in remission and 4 patients with active disease,
designated by prefixes, R- and A-respectively, were studied. In
FIG. 20, the corresponding results from 7 normal (designated N-)
control subjects are depicted. The total number of CD4+ T cells in
normal subjects was comparable to the total number in lupus
patients in remission. In consideration of previous studies
(Waldrop et al., 1997, J. Clin. Invest. 99:1739-1750; Openshaw et
al., 1995, J. Exp. Med 182:1357-1367; Estcourt et al., 1997, Clin.
Immunol. Immunopathol. 83:60-67), a response to a nucleosomal
peptide is considered to be unequivocally positive when the
frequency of positive cells was greater than 0.2% and the values
were at least 2-fold higher than respective background values (i.e.
cultures without peptide, designated "medium" in FIGS. 19 and 20).
Repeat assays with aliquots of cells from the same sample generated
almost identical results (i.e. SD of less than 5%).
[0241] Some of the nucleosomal peptides caused impressive
stimulation of T cells obtained from SLE patients. In contrast,
positive responses were rare in the normal subjects. IFN-gamma and
IL-4 production levels in response to nucleosomes or histone
peptides were greater than 10 fold above background values, and
corresponding IL-10 production levels were up to 20 fold higher in
some of the SLE patients. These results are highly significant,
because the frequency of autoepitope-specific CD4+ T cells (i.e.
positive responders) was measured in this assay (Waldrop et al.,
1997, J. Clin. Invest. 99:1739-1750). Remarkably, the ability to
respond to the histone peptides was still present in the patients
in long-term remission, indicating that one or more genetically
programmed defects may be involved. In fact, the anti-CD3 response
and responses to SLE-associated autoepitopes as a whole were
considerably attenuated in the PBMCs of some of the patients with
active lupus (A-KJ and A-WB), possibly due to prior
autoantigen-driven activation in vivo and exhaustion or
desensitization. Remarkably, IL-10 production responses to certain
nucleosomal peptides were, in many cases, higher than corresponding
anti-CD3 responses, even in the patients in remission. In some
patients, responses to certain histone peptides were stronger than
to the whole nucleosome particle.
[0242] FIG. 21 provides a summary of the percentage of positive
responders to selected histone peptides among the SLE patients.
Although, some cytokine responses exhibited spreading to many
different histone peptide autoepitopes in a patient, such as R-DS,
R-JE, and A-MG, certain histone peptides turned out to be recurrent
autoepitopes for the autoimmune T cells of most of the SLE patients
tested. Among these SLE patients, the incidence of positive
responders ranged from 50 to 100% to the following autoepitopes:
whole nucleosomes, H2B 10-33, H4 16-39, H4 71-94, H2A 34-48, H3
91-105, H3 100-114, H4 14-28, and H4 49-63.
[0243] Remarkably, the recurrent autoepitopes identified here for
the T cells involved in SLE, namely H2B 10-33, H3 95-105, H4 16-39,
and H4 71-94, are also the major autoepitopes for
nephritis-inducing T cells in lupus-prone SNF1 mice (Kaliyaperumal
et al., 1996, J. Exp. Med 183:2459-2469).
[0244] In the case of patients in remission in which the T cells
have a phenotype comparable to normal subjects, the preferential
IL-10 stimulation by some peptides over anti-CD3 in some of the
patients would suggest nucleosomal autoepitope-specific modulation
of cytokine production. The importance of IL-10 and other Th2
cytokines in the pathogenesis of SLE has also been well documented,
but its mechanism is not known (Ishida et al., 1994, J. Exp. Med
179:305-310; Llorente et al., 1995, J. Exp. Med 18 1:839-844;
Nakajima et al., 1997, J. Immunol. 158:1466-1472). Thus, SLE is not
a straightforward TH1 or Th2 type disease.
[0245] The value of autoepitope mapping for the pathogenic T cells
in autoimmune diseases cannot be overemphasized. The T cell
receptor (TCR) repertoire of the pathogenic Th cells involved in
SLE is heterogeneous, and the autoepitopes they recognize become
diverse with "epitope-spreading" as the disease progresses (Craft,
J., and Fatenejad, May 1997). Arthritis. Rheum. 40:1374-1382).
Individual TCRs of the pathogenic autoantibody-inducing Th cells of
lupus, recognize more than one histone peptide autoepitope in a
promiscuous or degenerate fashion, and in the context of diverse
class II molecules (Kaliyaperumal et al., 1996, J. Exp. Med
183:2459-2469; Shi et al., 1998, J. Exp. Med. 187:367-378).
Peptide-dominant interactions between the lupus TCRs and
MHC-nucleosomal peptide complex due to reciprocally charged
residues probably overcomes the requirement for MHC-restriction,
but not MHC-dependence (Shi et al., 1998, J. Exp. Med.
187:367-378). Structural motifs for these major T-cell epitopes in
the primary, endogenous immunogens of lupus could be used to
identify viral or bacterial mimicry peptides that have sufficient
structural similarity to initiate the activation and expansion of
the pathogenic Th cells of lupus, as described in other autoimmune
diseases (Wucherpfennig and Strominger, 1995, Cell.
80:695-705).
[0246] Peptides corresponding to the major autoepitopes as
described herein could also be used to design peptide-specific
immunotherapy. Remarkably, in murine lupus, any one of the major
autoepitopes identified herein could diminish pathogenic
autoantibody production against multiple lupus antigens (i.e.
tolerance spreading), and markedly delay the development of
SLE-associated nephritis. A single histone peptide with charged
residues could potentially tolerize a spectrum of Th cells whose
promiscuous TCRs could recognize one or two shared residues in
multiple, different peptide autoepitopes (Kaliyaperumal et al.,
1996, J. Exp. Med 183:2459-2469; Shi et al., 1998, J. Exp. Med.
187:367-378). Importantly, the results described herein demonstrate
that the autoepitopes involved in SLE in mice are also involved in
human SLE. Moreover, the pathogenic Th cells of lupus are
multipotent or promiscuous in their helper activity. According to
the model of SLE-associated autoantibody production illustrated in
FIG. 11, a single lupus Th clone can help either a dsDNA-specific,
a ssDNA-specific, a histone-specific, an HMG specific, or a
nucleosome-specific B cell because each of these B cells, by
binding to its respective epitope on the whole chromatin, can take
it up, process and present the relevant peptide epitope in the
chromatin to the Th clone (Desai-Mehta et al., 1995, J. Clin.
Invest. 95:53 1-541; Mohan et al., 1993, J. Exp. Med.
177:1367-1381) resulting in intermolecular help. Tolerization of
such Th cells would obviously deprive multiple autoimmune B cells
of T-cell help. Furthermore, the SLE-associated autoepitopes of
autoimmune T cells fall within the regions of the histone proteins
that are also targeted by SLE-associated autoantibodies (Stemmer et
al., 1997, J. Mol. Biol. 273:52-60; Monestier and Kotzin, 1992,
Rheum. Dis. Clin. N. Am. 18:415-436; Stemmer et al., 1996, J. Biol.
Chem. 271:21257-21261). Indeed, the overlapping of epitopes for
autoimmune Th cells and autoimmune B cells of lupus makes H4 16-39
a highly efficient tolerogen for therapy of murine lupus nephritis,
as shown in Example 1. Moreover, the nucleosomal autoepitopes for
human lupus T-cells have multiple MHC II, DR binding motifs (FIG.
22), suggesting that they could be used widely for tolerogenic
therapy despite the diversity of lupus patients' HLA-DR
alleles.
[0247] The recognition of nucleosomal autoepitopes by the
pathogenic T cells of murine lupus is MHC-dependent but
unrestricted (Shi et al., 1998, J. Exp. Med. 187:367-378), and so
far, susceptibility to lupus nephritis has not been linked to genes
for any particular MHC molecule. Non-MHC genes within the MHC
locus, such as, gene for TNF, or the C4A null allele, are probably
directly responsible for lupus susceptibility (Vyse and Kotzin,
1998, Annu. Rev. Immunol. 16:261-292; Morel and Wakeland, 1998,
Current Opin. Immunol. 10:718-725). Thus, identification of major
nucleosomal peptide autoepitopes for the T cells of human lupus
might be important for understanding how such autoimmune T cells
arise, for tracking such T cells using peptide-MHC tetramers, and
for developing antigen-specific therapy.
[0248] The experiments of this Example identify nucleosomal histone
peptides corresponding to histone regions, H2B 10-33, H4 16-39, H4
14-28, H4 71-94, H3 91-105, and H3 100-114, which were recurrently
recognized by CD4+ T cells from the majority of lupus patients
regardless of disease stage. These same peptides are also the major
epitopes for the Th cells that induce anti-DNA autoantibodies and
subsequent nephritis in lupus-prone mice. Two other recurrent
epitopes for human SLE-associated T cells have been localized to
H2A 34-48 and H4 49-63. All the SLE-associated autoepitopes have
multiple HLA-DR binding motifs and are located in the histone
regions that are targeted by lupus autoantibodies, suggesting a
basis for their immunodominance and potential efficacy as
tolerogens. These major autoepitopes may reveal the mechanism of
autoimmune T cell expansion and lead to antigen-specific therapy of
human lupus.
Example 3
A Potent SLE-Associated Autoepitope Isolated from Naturally
Processed Chromatin Peptides
[0249] In the experiments presented in this Example, several MHC
Class II-associated peptides have been identified and tested for
their ability to stimulate anti-DNA autoantibody production by
established T helper (Th) cell lines and primary T helper cell
isolates. These Th cells are essential for sustaining the
pathogenic autoantibody-producing B cells associated with SLE
(Datta et al., 1987, J. Exp. Med. 165:1252-1268; Ray et al., 1996,
Proc. Natl. Acad. Sci. USA. 93:2019-2024; Mohan et al., 1995, J.
Immunol. 154:1470-1480). The materials and methods presented in
this Example are now described.
Mice
[0250] BALB/c, NZB, SWR, (BALB/c.times.SWR)F1 mice were purchased
from The Jackson Laboratory (Bar Harbor, Me.). Lupus-prone
(SWR.times.NZB)F1 (SNF1) hybrids were bred at the animal facility
of Northwestern University. Female mice were used in each of the
experiments.
Preparation of Cloned Th Cell Lines and Hybridomas
[0251] Cloned Th cell clones and hybridomas used in the experiments
presented in this Example were derived from SNF1 mice with lupus
nephritis (Adams, et al., 1991, Proc. Natl. Acad. Sci. USA
88:11271-11275; Sainis and Datta, 1988, J. Immunol. 140:2215-2224).
The cloned Th cell lines were maintained in co-culture with
irradiated SNF1 spleen cells and complete medium comprising
RPMI-1640, 20 units per milliliter of rIL-2 (Life Technologies,
Inc., Grand Island, N.Y.), 10% heat inactivated FCS, 100 units per
milliliter of penicillin, 100 micrograms per milliliter of
streptomycin, 0.29 millimolar L-glutamine, 10 millimolar HEPES and
5.times.10.sup.-5 molar .beta.-mercaptoethanol. T cell hybridomas
were maintained in complete medium. The B cell lymphoma A20, was
maintained in DMEM with the same supplements as described for
complete medium, and was used as an APC line (Mohan, et al., 1993,
J. Exp. Med. 177:1367-1381).
Antibodies
[0252] The following monoclonal antibodies (mAb) were used:
anti4-Ad (HB3), anti-1-Ab d q (TIB120), anti-HSA (TIB183), and
anti-Thy-1.2 (TIB99), anti-CD8 (TIB211), anti-CD3 (145-2C11). All
the hybridomas were obtained from the American Type Culture
Collection (ATCC, Rockville, Md.). The culture supernatants were
concentrated 10.times. by ammonium sulfate precipitation and
dialysis in PBS, sterile filtered, and stored at -20 degrees C.
ELISAs
[0253] Anti-IL-2, anti-IFN.gamma., and anti-IL-4, capture and
biotinylated antibody pairs, and the recombinant cytokine standards
(rIL-2, rIL-4, rIFN.gamma.) were purchased from Pharmingen (San
Diego, Calif.). Streptavidin-conjugated horseradish peroxidase
(HRP) and its substrate were purchased from Sigma Chemical Co. (St.
Louis, Mo.). The cytokines were quantitated according to the
manufacturer.
Preparation of Chicken Chromatin Containing Polynucleosomes
[0254] The preparation of chromatin containing polynucleosomes and
purification of mononucleosomes were performed as previously
described (Mohan, et al., 1993, J. Exp. Med. 177:1367-1381; Yager,
et al., 1989, Biochem. 28:2271-2281).
Large Scale Cell Culture and Isolation of MHC Class II
Molecules
[0255] B cell hybridomas were made by fusing HAT sensitive A20 B
cell line (Folsom et al., 1984, Proc. Natl. Acad. Sci. USA
81:2045-2049) with the spleen cells from SNF1 mice. A high I-Ad
expressing and nucleosome binding hybridoma (1F2.28) was used for
isolation and purification of MHC class II, either after chromatin
incubation with the hybridoma cells, or without chromatin
incubation with these cells (i.e. control). Hybridoma cells
(1.times.10.sup.10 total) were grown in DMEM supplemented with 10%
horse serum (Mouritsen, et al., 1994, Immunology 82:529-534). Fifty
micrograms per milliliter of chromatin was added to these cultures
approximately 18 hours before the cells were harvested by
centrifugation at 1000.times.g and lysed in presence of a detergent
solution comprising PBS, 1% NP-40, 50 millimolar iodacetamide, 10
millimolar sodium orthovanadate, and 1 millimolar phenyl methyl
sulphonyl fluoride. The solution was cleared of cell debris by an
additional centrifugation at 10,000.times.g and frozen at -70
degrees C.
MHC Class II Purification
[0256] MHC class II (I-Ad) molecule was affinity purified by
application of peptide-MHC complexes to a sepharose 4B column
modified with a I-Ad specific antibody (HB-3) and elution of the
I-Ad molecules as described previously (Mouritsen, et al., 1994,
Immunology 82:529-534). The protein content of the eluates was
determined using a micro bicinoic acid assay (Pierce, Rockford,
Ill.).
Elution and Purification of Peptides from MHC Class II Olecules
[0257] Eluates containing purified MHC class II molecules (700-900
micrograms per milliliter) were concentrated to about 100
microliter using a Centricon 3 filter system (Amicon, Beverly,
Mass.). The volume was adjusted such that the concentration of MHC
Class II molecules was 700-900 micrograms per milliliter. The
affinity purification was repeated five times. The MHC bound
peptide was eluted by addition of 0.1% trifluoroacetic acid (TFA)
in water and incubation at 37 degrees C. for one hour. The
resulting mixture was concentrated by ultrafiltration, and the
flowthrough was collected and again concentrated to 10% of its
original volume. This concentrated solution was stored under
nitrogen at -70 degrees C.
Fractionation of Peptides
[0258] The initial separation of eluted peptides having a molecular
weight of less than 3000 Daltons was performed on a reverse-phase
(C-18) column (RP-HPLC, 1.times.30 cm) using a gradient of
acetonitrile and a solution comprising water and 0.1% TFA over a
time period of 100 minutes. The flow rate was 1 milliliter per
minute. Fractions were collected at 1 minute intervals and assayed
for their ability to stimulate pathogenic autoantibody-inducing,
nucleosome-specific Th clones from SNF1 mice. The active (i.e.
stimulatory) fractions from the initial screening was further
separated by using a gradient of 0.1% heptafluoroacetone and
hexafluoroacetic acid. The final separation was carried out on a
nanobore HPLC and assayed for stimulation of pathogenic Th cell
clones. The active fractions were sequenced by electro-spray
ionization mass spectrometry (ESI-MS/MS).
Synthesis of Peptides
[0259] All the peptides used in the experiments of this Example
were synthesized using FMOC chemistry (Chiron Mimotopes, san Diego,
Calif.). Purity of the peptides was verified by amino acid analysis
by the manufacturer. Peptides corresponding to histone protein
autoepitopes were used in later in vivo experiments for the
evaluation of autoimmune Th cell response and lupus acceleration.
These peptides were synthesized in larger quantities, purified by
HPLC using a gradient of water and acetonitrile, and analyzed by
mass spectrometry for purity.
Preparation of Antigen Presenting Cells (APC)
[0260] The splenic CD4+ T cells were isolated as reported
previously (Mohan, et al., 1995, J. Immunol. 154:1470-1480; Mohan,
et al., 1993, J. Exp. Med. 177:1367-1381) and in Example 1. Splenic
B cell and macrophage (B+M.psi.) APC were prepared from one month
old SNF1 mice by treating splenocytes with anti-Thy 1.2 (TIB99) and
rabbit complement followed by irradiation (3000 rads). The A20 B
lymphoma line was treated with 50 micrograms per milliliter of
mitomycin-C for 30 minutes, washed five times with PBS, then
incubated for 1 hr at 37 degrees C. After two final washes in
complete medium, they were used directly as APC. For peptide
presentation experiments, either the A20 B cell lymphoma or the
splenic B+Mpsi were used as APCs.
Proliferation and Cytokine Assays
[0261] Fresh splenic, CD4+ T cells (5.times.10.sup.5/well) were
co-cultured in triplicate wells at an initial concentration of
5.times.10.sup.5 cells per well, with 10.sup.6 cells per well of
either irradiated B+Mphi or mitomycin-C treated A20 APC. Varying
concentrations of either control or test peptides were added and
the cultures were adjusted to 200 microliters final volume of HL-1
serum-free medium (Hycor Biomedical Inc., Irvine, Calif.) cultures
were incubated for 96 hours in flat bottom 96-well plates, and at
18 hours prior to harvesting the cells, 1 microcurie of
3H-Thymidine was added to each well. The incorporated radioactivity
was measured by scintillation counting. The Stimulation Index (SI)
was calculated by dividing the mean counts per minute (cpm)
incorporated in co-cultures of T cells and APC with test peptide by
the mean cpm incorporated in control peptide co-cultures. In the
case of cytokine assays, experiments were carried out as described
for the proliferation assays except that the culture supernatants
were removed from duplicate co-culture wells after 24-46 hours of
incubation and cytokine assays were performed.
Autoantibody Quantitation
[0262] IgG autoantibodies to ssDNA, dsDNA, histones, and
nucleosomes were quantitated using an ELISA as described previously
(Mohan, et al., 1995, J. Immunol., 154:1470-1480; Mohan, et al.,
1993, J. Exp. Med. 177:1367-1381; Burlingame, et al., 1993, J.
Clin. Invest. 91:1687-1696; Losman, et al., 1992, J. Immunol.,
148:1561-1569). Sera were diluted 1:100 and heat-inactivated before
use. Serum from normal BALB/c mice were used as negative control.
Anti-DNA mAbs 564 and 205 were used to generate standard curves
(Adams, et al., 1991, Proc. Natl. Acad. Sci. USA 88:11281-11275;
Mohan, et al., 1995, J. Immunol., 154:1470-1480; Mohan, et al.,
1993, J. Exp. Med. 177:1367-1381; Sainis and Datta, 1988, J.
Immunol. 140:2215-2224). For IgG autoantibodies to ssDNA, histones
and nucleosomes, 1 unit per milliliter was considered to be
equivalent to the activity of one microgram per milliliter of mAb
564, which recognizes all three autoantigens (Mohan, et al., 1995,
J. Immunol., 154:1470-1480; Mohan, et al., 1993, J. Exp. Med.
177:1367-1381), and for IgG anti-dsDNA, 1 unit per milliliter was
equivalent to the binding of 0.6 micrograms per milliliter of the
mAb 205.
Assessment of Pathogenicity of Histone Derived Peptides
[0263] Pre-nephritic SNF1 females at 12 weeks of age were each
injected with 100 micrograms of one of the eluted peptide
fractions, EP-1, EP-2, EP-3, or a control peptide, OVA 323-336
emulsified in complete freunds adjuvant (CFA). The animals received
three more injections at two weeks interval with 50 micrograms of
either an eluted peptide or the control peptide absorbed on alum
(Pierce Chemical Co., Rockford, Ill.). The mice were monitored
weekly for proteinuria and sacrificed when persistent proteinuria
defined as in Example 1, developed. Autoantibody production and
grading of glomerulonepohritis were performed as described in
Example 1.
The results of the experiments presented in this Example are now
described.
Identification of the Naturally Processed and Presented Th Cell
Epitopes
[0264] The MHC class II molecules were purified from APC cell lines
derived from SNF1 mice after incubating the cells with a chromatin
preparation containing polynucleosomes. Peptides having a molecular
weight of less than 3000 Daltons were separated on a reverse-phase
(C-18) column HPLC (RP-HPLC) using a gradient of acetonitrile and
water containing 0.1% TFA and assayed for stimulating ability. An
example of HPLC purification of the peptide fractions is shown in
FIG. 23A.
[0265] An aliquot of 50 microliters from each HPLC fraction was
assayed for stimulating ability as described above. Four
characterized Th clones were used as indicators of cytokine
secretion (IL-2 and/or IL-4) in response to the peptide fractions,
using A20 or splenic B+Mpsi as APCs (Adams, et al., 1991, Proc.
Natl. Acad. Sci. USA 88:11271-11275; Shi, et al., 1998, J. Exp.
Med. 187:367-378). Eluate fractions from control APC cultures that
were not incubated with chromatin did not stimulate the Th cell
clones, as shown in FIG. 23B. Only two out of one hundred fractions
tested were able to stimulate Th cell clones. These fractions were
analyzed by mass spectrometry (MALDI-TOP-MS) as exemplified in FIG.
23C, which demonstrates that one fraction (#23) was homogenous,
whereas another fraction (#50) revealed five peaks (not shown). The
latter was further separated as described, and tested for active
fractions of HPLC purified peptides. Three naturally processed and
presented, stimulatory epitopes were identified using these
procedures. The sequences of the peptides are as follows:
SQKEEEEGAQREKE (EP-1, SEQ ID NO: 23); DWMEEEHGAQREKE (EP-2, SEQ ID
NO: 24); and SASHPTYSEMIAAAIRAEKSR (EP-3, or H5 22-42, SEQ ID NO:
25). The first two peptides were homologous to sequences in a
transcription factor, BRN-3, which is identical in chicken, mice
and humans (GenBank). A homology search demonstrated EP-2 to be
identical in sequence to aa position 175-185 in BRN-3, except for
one residue. EP-1 is highly homologous to EP-2, but it also matched
a sequence in cytomegalovirus envelope protein at aa position
57-70. Most likely these peptides are derived from some
transcription factor in the chromatin that was incubated with the
APC. The third sequence (EP-3) was derived from histone H5 (aa
position 22-42) in chicken nucleosome, which is homologous to
histone H1 sequence of mice and humans at amino acid position 22-42
(STDHPKYSDMIVAAIQAEKNR, SEQ ID NO:26). The mouse H1 22-42 peptide
was equivalent to the chicken H5 22-42 peptide in stimulating the
pathogenic Th clones from SNF1 lupus mice. Therefore, the mouse H1
22-42 is the autoantigenic peptide in mice and is also referred to
herein as EP-3. Nested sequences and unrelated sequences that were
obtained in the active fractions from the eluates were also
synthesized and re-tested. As shown in FIG. 24, the unrelated
sequences did not stimulate the pathogenic Th clones, thus
indicating epitope specificity and authenticity of EP-3.
Eluted Peptides and Pathogenic Autoantibody Production
[0266] To test whether the naturally processed and presented
peptide could facilitate augmentation of the pathogenic
autoantibody production, pathogenic autoantibody-inducing Th clones
1D12, 5E9, and 3F6 that were nucleosome-specific and also responded
to the EP-3 peptide or the Th clone 1G1 that was also
nucleosome-specific but did not respond to EP-3, were co-cultured
with freshly isolated, splenic B cells from SNF.sub.1 mice in
presence of EP3 (H1 22-42). After seven days of co-culture, the
supernatants were assayed for IgG antibodies to the autoantigens
(ds-DNA, ss-DNA, nucleosome, and histone) by ELISA. The H1 22-42
peptide stimulated the first three pathogenic Th clones and
augmented their ability to help in autoantibody production. The
non-responder Th clone was not stimulated to augment help by this
peptide, but was stimulated by the whole nucleosome. The H1 22-42
peptide augmented autoantibody-inducing help of responder Th clones
5E9 and 3F6, almost as much as the whole nucleosome. In the case of
clone 1D12, augmentation of autoantibody-inducing help by H1 22-42
peptide was 1.5 to 2.5 times higher than that with nucleosome. The
results of these experiments are depicted in FIG. 25.
Acceleration of SLE
[0267] Twelve week old prenephritic SNF1 mice that were immunized
with an eluted peptide, EP-2 or EP-3, in adjuvant (CFA) developed
severe nephritis earlier than age matched SNF1 mice injected with
CFA alone or control OVA peptide in CFA. Eighty percent of the mice
injected with H1 22-42 (i.e. EP-3) succumbed to severe lupus
nephritis within four weeks of the first injection. Indeed, some of
the H1 22-42 immunized animals developed severe nephritis in 2-3
weeks, just after the first booster immunization. By 28 weeks of
age, all the EP-3 immunized mice developed severe nephritis. Eluted
peptide (EP-2) also accelerated SLE in the initial phase; 60% of
the mice developed disease by 20 weeks of age. But later on, the
incidence of severe nephritis in this group of mice was similar to
that of the control group. By contrast, EP-1 immunization did not
result in any increase in the rate of disease relative to the
control group. The results of these experiments are shown in FIG.
26.
[0268] The experiments presented in this Example identify peptide
epitopes that are preferentially associated with MHC class II
molecules and presented as autoantigens after being naturally
processed by antigen presenting cells (APCs). Peptides were eluted
in fractions of MHC class II molecules from APC lines that were
incubated in the presence of crude chromatin. The eluted peptide
fractions were purified by reverse phase HPLC (RP-HPLC) and tested
for the ability to stimulate autoimmune SLE-associated T cell
lines. The stimulatory peptide fractions were analyzed by
Matric-assisted Laser Desorption Time of Flight Mass Spectrometry
(MALDI-TOF-MS). Amino acid sequences of purified peptides in
fractions which were stimulatory were then deduced by electro-spray
ionization mass spectrometry (ESI-MS/MS) at the Harvard
microchemical facility. The peptide sequences thus identified were
synthesized, and the synthetic peptides were tested again for
stimulating pathogenic, SLE-associated Th cells from SNF.sub.1
mice. These naturally processed peptide autoepitope sequences
include the following: EP-1 (SEQ ID NO: 23); EP-2 (SEQ ID NO: 24);
and EP-3, chicken H5 22-42 (SEQ ID NO:25), or H1 22-42 (SEQ ID NO:
26). The APC line was incubated with chicken chromatin in order to
distinguish the peptides derived from the chromatin processing from
any endogenous nucleosomal peptides derived from dying cells in the
cultures. The first two naturally-processed peptide sequences that
stimulate the autoimmune T cells are homologous to sequences in
transcription factor BRN-3 whose sequence is identical in chicken,
mice and humans. The third sequence is homologous to histone H1
sequence at amino acid positions 22-42, which is identical in mouse
and human (STDHPKYSDMIVAAIQAEKNR, H1 22-42). The H1 22-42 peptide
is an extremely potent immunogen when injected with adjuvant, and
it could simultaneously trigger the expansion of autoimmune Th
cells, as well as autoimmune B cells of lupus. These results also
indicate the potential efficacy of this dominant peptide epitope
for designing tolerogens for inhibiting both autoimmune T and B
cell populations in lupus. Identification of these major peptide
epitopes should provide a basis for: (1), elucidating the
endogeneous mechanisms of emergence and expansion of autoimmune T
cells in SLE, and also for identifying molecular mimics in the
environment that could precipitate the disease in susceptible
individuals; (2), tracking the presence of the disease-causing Th
cells for diagnostic and prognostic purposes, because such
peptide-specific T cells appear long before serologic
manifestations of lupus in murine models, and (3), developing
autoantigenic peptide-specific therapy of lupus in humans.
[0269] The experiments presented in this Example demonstrate that
histone peptides corresponding to the HI histone protein can be
used to (a) develop antigen-specific therapy of SLE. Tolerization
of autoimmune T cells and B cells of lupus with H 1 22-42 epitope
given as soluble peptide orally (oral tolerance), subcutaneously,
or intravenously; (b) develop altered peptide ligands for the same
purpose, after mapping T cell receptor and MHC contact residues in
the peptide epitope; and (c) develop tetramers of the nucleosomal
H1 22-42 peptide linked to I-Ad in mice, or to HLA-DR in humans, to
surface stain and track the autoimmune T cells of lupus for
diagnostic and prognostic purposes. The autoimmune T cells can be
present without any detectable anti-DNA autoantibodies in serum,
and thus can predict the development of lupus nephritis many years
in advance of emerging symptoms of the disease.
[0270] The identification of histone peptides which correspond to
major SLE-associated autoepitopes as described in the above
Examples 1-3 is important for developing an antigen-specific
therapy to combat the disease. The following are possible as a
result of the disclosure of Examples 1-3:
[0271] (a) Development of antigen-specific therapies to treat SLE
which involve tolerizing autoimmune T cells and B cells involved in
lupus with nucleosomal peptides given as soluble peptides orally
(i.e. oral tolerance), intranasally, subcutaneously, or
intravenously, or by any other route for inducing tolerance.
Development of altered peptide ligands for the same purpose,
following mapping of the T cell receptor and MHC contact residues
in the nucleosomal peptide epitopes.
[0272] (b) Development of tetramers of HLA-DR and nucleosomal
peptides to stain the surface of autoimmune T cells involved in SLE
for diagnostic and prognostic purposes. Some autoimmune T cells
could be present in serum without any detectable SLE-associated
autoantibodies. Thus staining is useful for predicting the
development of SLE-associated nephritis many years in advance of
emerging symptoms of the disease.
Example 4
Low Dose Histone Peptide Therapy
[0273] This example describes procedures used to establish the
utility of low dose histone peptide therapy.
Materials and General Methods
Mice
[0274] NZB and SWR mice were purchased from The Jackson Laboratory
(Bar Harbor, Me.). Lupus-prone SNF1 hybrids were bred and females
were used, as approved by Animal Care and Use Committee.
Peptides
[0275] All peptides were synthesized by F-moc chemistry and their
purity checked by amino acid analysis by the manufacturer (Chiron
Mimotopes, San Diego, Calif.).
Tolerance Induction with Very Low Doses of Peptides
[0276] For long term experiments, serologically autoimmune, but
pre-nephritic, 12-wk-old SNF1 females (nine mice per group) were
injected S.C. with either H1'.sub.22-42, H2B.sub.10-33,
H2B.sub.59-73, H4.sub.16-39 or H4.sub.71-94 peptide (1 .mu.g/mouse)
in saline every two wk until the animals died. Control group
received only saline. The mice were monitored weekly for
proteinurea using albustix (VWR Scientific, Chicago, Ill.). Sera
were collected every 2Y.sub.2 months for the determination of total
IgG and IgG subclasses of anti-nuclear autoantibodies. A parallel
batch of identically treated mice of each group were followed and
sacrificed at different time points for evaluation of renal
lesions. To test the immunological consequences of the tolerance
therapy early on, another batch of 12-wk-old SNF1 mice (five per
group) were treated as above, but they received a total of three
injections of each peptide at two-wk intervals. Ten days after the
third injection, these short-term batch of mice were sacrificed for
analysis of autoimmune T and B cells, and T.sub.reg cells.
Autoantibody Quantitation
[0277] IgG class autoantibodies to ssDNA, dsDNA, histone and
nucleosome (histone-DNA complex) were measured by ELISA (Refs. 2,
6). Subclasses of IgG autoantibodies were detected by ELISA using
AP-conjugated anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Southern
Biotechology, Birmingham, Ala.).
Cell Isolation
[0278] Total, CD4.sup.+ and CD8.sup.+ T cells from spleens were
purified using appropriate MACS isolation kits using magnetic bead
conjugated antibodies specific to each antigen. CD4.sup.+CD25.sup.+
T cells were purified by mouse regulatory T cell isolation kit
according to manufacturer (Miltenyi Biotec, Auburn, Calif.).
CD28.sup.+ and CD28.sup.- subsets of CD8.sup.+ T cells were
separated by using anti-CD28-PE conjugate and anti-PE Microbeads
(Miltenyi Biotec). Purity of all isolated cell subsets were
>90%.
Adoptive Transfer
[0279] CD4.sup.+CD25.sup.-, CD4.sup.+CD25.sup.+ or CD8.sup.+ T
cells (1.times.10.sup.6 cells/mouse) from low-dose peptide
(H4.sub.71-94 or H4.sub.16-39)-tolerized SNF1 mice were purified by
MACS and then immediately injected I.V. into 4-mo-old SNF1. One day
after transfer, the mice were immunized I.P. with 100 .mu.g of
H1'.sub.22-42 peptide in CFA, which accelerates lupus nephritis.
Recipient mice were monitored for nephritis and IgG autoantibody
levels in serum.
Enzyme-Linked Immunosorbent Spot (ELISPOT) Assay
[0280] ELISPOT assay plates (Cellular Technology Ltd., Cleveland,
Ohio) were coated with capture antibodies against IL-2, IL-4,
IL-10, or IFN-.gamma. (BD Pharmingen, San Diego, Calif.) in PBS at
4.degree. C. overnight. Splenic T cells (1.times.10.sup.6) from
treated mice were cultured with irradiated (3,000 rad) splenic APC
(B cells, macrophages and DC) from one-mo-old SNF1 mice in the
presence of peptides or PBS alone as control. Cells were removed
after 24 h of incubation for INF-.gamma. and IL-2; or 48 h for IL-4
and IL-10 and the reactions were visualized by addition of the
individual anti-cytokine antibody-biotin and subsequent
AP-conjugated streptavidin. Cytokine-expressing cells were detected
by Immunospot scanning and analysis (Cellular Technology).
Cytokine ELISA
[0281] CD4.sup.+ T cells (1.times.10.sup.6) or CD8.sup.+ T cells
(1.times.10.sup.6) from low-dose peptide tolerized or unmanipulated
SNF1 mice were stimulated with H4.sub.71-94 peptide or antibody to
CD3 (1 .mu.g/ml) with splenic APC. Culture supernatants were
collected after 48 h (for TGF-.beta.1, after 72 h). IL-10 was
measured by BD OptEIA.TM. ELISA set (BD Pharmingen). For
TGF-.beta.1, samples were acidified by addition of HCl at 20 mM for
15 min and neutralized by NaOH, and then amount of TGF-.beta.1 was
measured by TGF-.beta.1 Emax ImmunoAssay System (Promega, Madison,
Wis.).
Helper Suppression Assay
[0282] To detect suppression of autoantibody-inducing help, whole T
cells (2.5.times.10.sup.6/well), or purified CD4.sup.+CD25.sup.-,
CD4.sup.+CD25.sup.+ or CD8.sup.+ T cells (0.3, 0.6, 1.25 or
2.5.times.10.sup.6/well) from peptide-tolerized or saline-treated
mice were co-cultured with a helper assay mixture (Ref. 6)
consisting of splenic B cells (5.times.10.sup.6/well) and Th cells
(2.5.times.10.sup.6/well) from 3-5 month old, unmanipulated SNF1
mice in 24 well plates (or in 96 well plates with 1/10 cell
numbers) for 7 days. The co-cultures were performed in the presence
of 10 ug/ml cognate peptides or 1 .mu.g/ml nucleosomes. Culture
supernatants were collected and assayed for IgG antibodies against
dsDNA, ssDNA, histones and nucleosomes as described (6). Helper
suppression assays were also performed in the presence of
anti-IL-10 Ab (10-250 .mu.g/ml), anti-TGF-beta Ab (10-250
.mu.g/ml), or isotype control for each (R&D Systems Inc.,
Minneapolis, Minn.) (11).
Transwell Experiments
[0283] T.sub.reg cells (7.5.times.10.sup.5) from peptide-treated
SNF1 mice plus APC (7.5.times.10.sup.5) were placed in transwell
chambers (12) separated by a 0.4-um permeable membrane (Corning
Costar, Cambridge, Mass.) from a helper assay co-culture of splenic
B cells (7.5.times.10.sup.5/well) from 4-5 month old, and T cells
(7.5.times.10.sup.5/well) from 3-4 mo old, unmanipulated SNF1 mice.
After 7 days of culture, supernatants were assayed for IgG
autoantibodies.
Flow Cytometry
[0284] T cells from tolerized or control mice were stained with
PE-conjugated anti-CD62L, CTLA-4, CD69, PD-1, or 4-1BB (BD
Pharmingen), TGF-.beta.1, LAP or GITR (R&D Systems) at
4.degree. C. for 30 min, as described previously (Refs. 13, 14).
Matched PE-conjugated IgG isotype controls were used. Cells were
then stained with FITC-conjugated anti-CD25 and
Cy-Chrome-conjugated anti-CD4 at 4.degree. C. for 20 min. For
intracellular CTLA-4 staining, cells were first surface-labeled
with FITC-conjugated anti-CD25 and Cy-conjugated anti-CD4 for 20
min at 4.degree. C. Cells were then fixed and permeabilized and
then stained with PE-anti-CTLA-4 or PE-IgG isotype control. Cells
were analyzed using FACScalibur (BD).
Real Time RT-PCR
[0285] RNA from T cell subsets was isolated by Rneasy kit (Qiagen,
Valencia, Calif.) and then cDNA synthesized to measure expression
of Foxp3, as described (Ref. 15).
Examination of Kidneys
[0286] Kidney sections were stained with hematoxylin and eosin
(H&E) and periododic acid-Schiff (PAS), and graded 0 to 4+for
pathologic changes in a blinded fashion, as described (Refs., 6, 8,
16, 17). Immunohistochemistry was done as described (Refs., 6,
17).
ELISA for Anti-HEL Antibodies
[0287] HEL (10 ug/ml) was coated onto 96-well plates (Nunc,
Roskilde, Denmark). After blocking with 1% BSA in PBS, serially
diluted sera were added. Anti-HEL IgG antibodies were detected by
AP-conjugated anti-mouse IgG (Southern Biotechnology).
Statistical Analysis
[0288] Chi squire (.chi..sup.2) test, Log Rank test and the Student
two-tailed t-test were used. Results are expressed as
Mean.+-.S.E.M.
Procedures and Results
Very Low-Dose Peptide Epitope Therapy Postpones Lupus Nephritis and
Prolongs Lifespan
[0289] Twelve wk old prenephritic SNF1 females (nine mice per
group) were injected S.C. every two wk with one of the major
autoepitopes, H1'.sub.22-42, H2B.sub.10-33, H4.sub.16-39 or
H4.sub.71-91, or a non-stimulatory peptide H2B.sub.59-73, each at 1
.mu.g (.about.0.42 nM) of peptide per dose/mouse in saline. A
control group received only saline. The control group started
developing severe nephritis from 20 wk age, as documented by
persistent proteinurea of >100 mg/dl, and a 3-4+renal pathology
(FIG. 28A). At 31 wk of age, 60% of the saline control group, 40%
of the H2B.sub.59-73 peptide-injected group, and 33.3% of
H2B.sub.10-33-injected group of mice developed severe nephritis
(P>0.2), whereas the H1'.sub.22-42, H4.sub.16-39 or H4.sub.71-94
peptide-injected mice did not develop disease at this time point
(P<0.01). The largest differences were at 35-38 wk of age, when
80% of the control mice had severe nephritis, and the
H2B.sub.10-33, H2B.sub.59-73, or H4.sub.16-39 group had an
incidence of 44.4%, 60%, or 40%, respectively (P>0.1->0.2);
whereas in the H1'.sub.22-42 or H4.sub.71-94 group the incidence
was nil (P<0.01). By 47 wk of age, all mice in saline control
group, 88.8% of the H2B.sub.10-33, 80% of the H2B.sub.59-73, and
85.7% of the H4.sub.16-39 groups developed severe nephritis,
whereas only 40% of the H1'.sub.22-42 (P<0.05) and 20% of the
H4.sub.71-94 peptide-injected groups had severe nephritis
(P<0.01).
[0290] The saline-injected mice rapidly died within 12 months (FIG.
28B). At this time, 22.2% of the H2B.sub.10-33, and 20% of the
H2B.sub.59-73 peptide-treated animals were alive (P>0.2),
whereas 57.1% of the H4.sub.16-39-treated (P<0.05), and 100% of
the H1'.sub.22-42 and H4.sub.71-94-treated animals were alive
(P<0.01). At 15 months, 11.1% of H2B.sub.10-33, and 20% of
H2B.sub.59-73-treated mice were alive (P>0.2), whereas 42.9% of
H4.sub.16-39 (P<0.05), 60% of H1'.sub.2242 (P<0.05), and 100%
of H4.sub.71-94 (P<0.01)-treated mice were alive. At 21 mo age,
60% of H4.sub.71-94 peptide-treated animals were alive (P<0.05),
whereas only 20% of H1'.sub.22-42 and 33.3% of H4.sub.16-39-treated
mice were alive; but all other groups were dead. Therefore, very
low-dose therapy with H4.sub.71-94 extended life span longer than
21 mo, in contrast to the control group that all died within 12
months. Log rank test for survival was consistent:
H1'.sub.22-42-treated group (P=0.000315), H2B.sub.10-33 (P=0.153),
H2B.sub.59-73 (P=0.217), H4.sub.16-39 (P=0.0162), and H4.sub.71-94
group (P=0.0000182).
[0291] Three months after the start of therapy, kidney sections
from control mice had an overall score of 3.5.+-.0.5 for nephritis,
whereas H4.sub.71-94, H1'.sub.22-42 or H4.sub.16-39-treated groups
showed 1.4.+-.0.8 overall score (P<0.001; FIG. 28C). Glomerular
IgG deposits were observed in kidneys from both tolerized and
control mice (FIG. 28D), but, perivascular and interstitial
infiltrations of mononuclear cells containing CD4.sup.+ and
CD8.sup.+ T cells and IgG-producing B cells, were markedly reduced
in the peptide treated mice (P<0.001).
Very Low-Dose Peptide Therapy Reduces Anti-Nuclear Autoantibody
Levels in Serum
[0292] Sera were first assayed 2 weeks after fifth injection, i.e.
3 mo after starting therapy (at early nephritic age). H4.sub.71-94
treatment, as compared to H2B.sub.59-73 (FIG. 29A), was very
effective in reducing levels of autoantibodies pathognomonic of
lupus nephritis (Refs., 18, 19). H4.sub.71-94 therapy reduced IgG
class autoantibodies to dsDNA, ssDNA, nucleosomes and histones by
41.5%, 50%, 94.2%, and 98.6% respectively (P<0.01, P<0.001,
P<0.001 and P<0.001), and that of IgG2a subclass
autoantibodies by 54%, 95.3%, 94%, and 98% respectively (P<0.01,
P<0.001, P<0.001 and P<0.001), and that of IgG2b
autoantibodies by 82.9%, 68%, 89%, and 88% respectively (P<0.01,
P<0.01, P<0.02 and P<0.001), and that of IgG3
autoantibodies by 45%, 83.2%, 80% and 99% respectively (all
P<0.001). IgG1 autoantibody levels were already very low in the
controls.
[0293] H4.sub.16-39 therapy also reduced IgG autoantibodies to
nuclear autoantigens, as much as H4.sub.71-94. H1'.sub.22-42
therapy reduced the levels of IgG autoantibodies against dsDNA,
ssDNA and nucleosome effectively, but not against histone.
H2B.sub.10-33 reduced the levels of IgG2a autoantibodies against
dsDNA and ssDNA by 25% (P<0.02) and 85% (P<0.001),
respectively, and that of IgG2b autoantibodies against dsDNA,
ssDNA, and nucleosomes by 93%, 61%, and 82%, respectively
(P<0.001, P<0.001, and P<0.05, respectively), but the
levels of IgG2a against nucleosome and histone actually increased
by 29% and 34%, respectively. The low-dose peptide therapy did not
cause IgG1 isotype shift (Th2 deviation), and total polyclonal IgG
levels were not significantly different among the groups.
T Cells Response to Autoepitopes was Markedly Reduced in
Peptide-Treated Mice
[0294] To test the immunologic consequences of the low-dose peptide
therapy early on, a separate set of 12-14-wk-old SNF1 mice was
injected with the most effective peptide epitope (H4.sub.16-39 or
H4.sub.71-94), or saline or H2B.sub.59-73 every two wk, three times
and then sacrificed. Animals were 18-20 weeks old at this time. T
cells in unmanipulated SNF1 mice are spontaneously primed to the
major nucleosomal peptide epitopes early in life and respond to
them in vitro (Refs., 5, 6). T cells from peptide-treated or
control mice herein were challenged with the epitopes by
coculturing with APC in the presence of the peptides or
nucleosomes, and their cytokine responses were measured (IL-2,
IL-4, IL-10, and IFN-gamma). Only IFN-gamma was detected. T cells
from H1'.sub.22-42, H4.sub.16-39, and H4.sub.71-94-treated mice
showed markedly reduced responses, as compared to the control group
(FIG. 30A). H1'.sub.22-42-treated mice showed the highest reduction
at 1 .mu.g/ml of cognate epitope (P<0.001). The therapy also
reduced responses to other epitopes (H4.sub.16-39, H4.sub.71-94)
cross-reactively (P<0.05). H4.sub.71-94 treatment resulted in
the highest inhibition of response to cognate epitope at 0.1
.mu.g/ml (P<0.01), as well as to the other epitopes,
H1'.sub.22-42 and H4.sub.16-39 (P<0.01), (FIG. 30A).
H4.sub.16-39 treatment also markedly reduced responses to cognate
epitope (optimally at 1 .mu.g/ml, P<0.001), as well as to
H1'.sub.22-42 and H4.sub.71-94 (P<0.001).
[0295] Because low-dose peptide treatment reduced IFN-gamma
responses against histone peptide epitopes cross-reactively (FIG.
30A), T cell responses to whole nucleosomes were also assessed, and
found to be significantly reduced in H4.sub.71-94 treated mice
(FIG. 30B). Similar results were found in H1'.sub.22-42
(P<0.01), and H4.sub.16-39-treated mice.
Very Low-Dose Peptide Therapy Generates CD8.sup.+ and CD4.sup.+
CD25.sup.+ Regulatory T Cells
[0296] Low-dose peptide treatment suppressed autoantibodies without
causing Th1/Th2 deviation, indicating the possibility of T.sub.reg
cell involvement. Using the helper-suppression assay, the ability
of CD4.sup.+CD25.sup.- T cells, CD4.sup.+CD25.sup.+ T cells or
CD8.sup.+ T cells from the peptide-treated mice to suppress
nucleosome-stimulated autoantibody production in cocultures of
lupus T helper (Th) and B cells of unmanipulated SNF1 mice was
determined. CD4.sup.+CD25.sup.+ T cells and CD8.sup.+ T cells from
tolerized mice strongly suppressed the ability of unmanipulated
lupus CD4+ T cells to help B cells to produce IgG autoantibodies
(FIG. 31A). Because help was already optimal in the
nucleosome-stimulated helper assay cultures, the levels of
autoantibodies produced by unmanipulated SNF1 lupus Th and B cells
cultured by themselves did not change significantly upon their
cocultures with the CD4.sup.+ CD25.sup.- T cells from the treated
mice. Compared to those levels, suppressions of autoantibodies to
dsDNA, ssDNA and nucleosomes by CD4.sup.+ CD25+T cells from
H4.sub.16-39-treated mice were 25%, 98.8%, and 83% (P<0.05,
P<0.001, P<0.001) respectively; from H4.sub.71-94-treated
mice 24%, 74%, and 76% (all P<0.01-<0.001); but from
age-matched unmanipulated SNF1 mice were 7%, 6.2% and 17%
(P>0.2) respectively. Similarly, suppressions of autoantibody
production to dsDNA, ssDNA and nucleosome by CD8+ T cells from the
same groups respectively were 41%, 99.8% and 72.6% (all
P<0.001); 55%, 78%, and 90% (all P<0.001); and 15%, 17% and
21.2% (P<0.02). Both sets of T.sub.reg cells from
peptide-treated mice were effective at up to 1:10 ratio in
inhibiting autoantibody production in the helper-suppression
assays.
[0297] Direct suppressing ability of IFN-gamma response to
autoantigen was also determined by co-culturing T.sub.reg cells
from treated mice with T cells from 5.5 mo old unmanipulated SNF1
mice in the presence of nucleosomes (1 ug/ml) (FIG. 31B). Both sets
of T.sub.reg cells from peptide-treated mice were effective at up
to 1:100 ratio (T.sub.reg cells: target lupus T cells) in strongly
inhibiting autoantigen-specific responses of lupus T cells in
ELISPOT assays (P<0.001, FIG. 31B).
[0298] Taken together, CD4.sup.+CD25.sup.+ or CD8.sup.+ T cells
from peptide-treated mice showed 3-16 fold greater suppressive
activity on autoantibody production and nucleosome-specific
responses than equivalent numbers of those cells from age-matched,
control SNF1 mice (FIG. 31, P<0.01-<0.001). Furthermore, no
differences were detected in the suppressive ability of CD28.sup.+
vs CD28.sup.- subsets of CD8.sup.+ T cells, as found in other
systems (Ref. 20).
Adoptively Transferred T.sub.reg Cells Suppress Autoantibody
Production and Nephritis In Vivo
[0299] Each T.sub.reg subset from peptide-treated mice was isolated
and adoptively transferred them into pre-nephritic (4 month old)
SNF1 mice. One day after adoptive cell transfer, recipient SNF1
mice were immunized with pathogenic H1'.sub.22-42 in CFA. SNF1 mice
immunized with H1'.sub.22-42 (100 .mu.g/mouse) in adjuvant (CFA)
developed severe nephritis and produced high levels of
autoantibodies earlier than age-matched SNF1 mice injected with CFA
alone or the non-stimulatory peptide, H2B.sub.59-73 in CFA, as
described (Ref. 6). CD4.sup.+CD25.sup.- T cell transfer did not
affect autoantibody levels in the H1'.sub.22-42 immunized mice, as
they were maximally immunostimulated by autoantigen immunization
(Ref. 6). In comparison to those levels, suppression of serum
autoantibodies to dsDNA, ssDNA, nucleosome and histone by
CD4.sup.+CD25.sup.+ T.sub.reg cells from H4.sub.71-94 treated mice
were 40%, 75%, 94%, 97%, respectively (P<0.025-<0.001); and
from H4.sub.16-39 treated mice were 100%, 80%, 92%, and 94%,
respectively (all P<0.001), (FIG. 32). Suppression of the same
IgG autoantibodies by CD8.sup.+ T.sub.reg cells from H4.sub.71-94
treated group were 45%, 95%, 94% and 97%, respectively
(P<0.025-<0.001), and from H4.sub.16-39-treated mice were
99%, 76%, 97% and 97%, respectively (all P<0.001), (FIG. 32).
Both types of T.sub.reg cells inhibited serum autoantibody levels
for up to 2 months after the one-time adoptive transfer. During
this period, 30% of CD4.sup.+CD25.sup.- T cell recipient group
developed severe nephritis within 6 weeks of immunization with
H1'.sub.22-42, and died a week later, whereas the incidence of
severe nephritis and death in T.sub.reg cell recipient groups were
nil at this time (P<0.05). Because the lupus-prone mice were
maximally stimulated by major autoepitope immunization, incidence
of disease and level of autoantibodies in H1'.sub.22-42-immunized
SNF1 mice without adoptive transfer were not significantly
different from that in H1'.sub.22-42-immunized SNF1 mice that had
received CD4.sup.+CD25.sup.- T cells from peptide-tolerized mice
(FIG. 32). After 21/2 mo post-transfer, all of the mice receiving
CD4.sup.+CD25.sup.+ T.sub.reg cells still survived (P<0.05), but
30% of mice receiving either CD4.sup.+CD25.sup.- or CD8.sup.+ cells
were dead. The one-time recipients of CD4.sup.+CD25.sup.+ T.sub.reg
cells still had higher survival at 31/2 mo post-transfer, as
compared to the latter groups (75% vs. 50%).
Both sets of T.sub.reg Produce TGF-.beta., but Only CD4.sup.+
CD25.sup.+ T.sub.reg Cells are Partially Contact-Dependent
[0300] Antibody to IL-10 (10-250 .mu.g/ml) did not abrogate the
suppression by the T.sub.reg cells in helper-suppression assay.
With 10-250 .mu.g/ml of anti-TGF-beta antibody, CD4.sup.+CD25.sup.+
T cell-mediated suppression of production of autoantibodies to
dsDNA was not affected, but that to ssDNA and nucleosomes was
reduced, but not abrogated. However, the suppression by regulatory
CD8.sup.+ T cells in same helper assay cultures was almost
completely abrogated by anti-TGF-beta antibody (FIG. 33A).
Furthermore, CD8.sup.+ T.sub.reg cells could suppress autoantibody
production across a Transwell.TM. membrane barrier (FIG. 33B),
indicating that soluble TGF-beta from these cells mediates the
immunosuppression. The CD4.sup.+CD25.sup.+ T cells showed reduced
suppression of autoantibody production through the membrane,
indicating their suppression is significantly contact dependent
(P<0.01). We next measured TGF-.beta.1 production by T.sub.reg
cell subsets (FIG. 33C). Both CD4.sup.+CD25+ and CD8.sup.+ T cells
from peptide-treated mice produced increased amount of total
TGF-beta1 upon the stimulation with H4.sub.71-94 or anti-CD3.
Phenotypes of CD4.sup.+CD25.sup.+ and CD8.sup.+ T.sub.reg Cell
Subsets
[0301] Cell surface molecules that are relevant to T.sub.reg cells
(Refs. 13, 14, 21) were analyzed. Peptide therapy increased the
numbers of CD4.sup.+CD25.sup.+ T cells up to 1.8-fold more in SNF1
mice than in controls (P<0.02; FIG. 33D). Total numbers of
CD4.sup.+CD25.sup.+CD62L.sup.+T cells per 1.times.10.sup.6
splenocytes in peptide-tolerized mice were 2.3.times.10.sup.4 and
that from controls were 1.9 fold less (1.2.times.10.sup.4).
CD4.sup.+CD25.sup.+ cells, but not CD8.sup.+ T.sub.reg cells from
H4.sub.71-94 or H4.sub.1639-treated mice showed slightly (1.3 fold)
increased Foxp3 expression than controls (P<0.01). The CD8.sup.+
T cell population was strongly positive for surface expression of
TGF-beta, CD62L and GITR; and the CD4.sup.+CD25.sup.+ T cells were
strongly positive for GITR, CD62L, TGF-beta, LAP, and CTLA-4 (not
shown). Low-dose peptide therapy did not change the overall
phenotypes of CD4.sup.+CD25.sup.+ T or CD8.sup.+ T cells, when
compared with same subsets isolated from control SNF 1 mice,
indicating that a small percentage of autoantigen-specific
T.sub.reg cells are induced.
The Autoepitope Peptides that are Effective in Low-Dose Therapy
Contain class I Epitopes
[0302] The nucleosomal histone peptides having MHC class II
epitopes stimulate CD4.sup.+ autoimmune Th cells of lupus (Refs
5-7, 9). Because CD8.sup.+ T.sub.reg cells were also induced by
these autoepitopes, we looked for MHC class I binding motifs, as
described (Refs. 22,23). Proteasomal cleavage probability and
MHC-peptide binding scores were assigned by computer prediction
("www" followed by "mpiib-berlin.mpg.de/MAPPP/"). We considered
motifs with scores >0.5 as class I epitopes. The highest overall
score for the sequence (bold, underlined) in the peptide containing
the motif for binding to each class I molecule of the H-2.sup.d
haplotype is shown below (the SNF1 mice are H-2.sup.d/q in
haplotype). TABLE-US-00003 H4.sub.71-94 TYTEHAKRKTVTAMDVVYALKRQG
(K.sup.d, and L.sup.d motif) K.sup.d: cleavage probability: 1.0,
binding score: 0.42, overall score: 0.71 L.sup.d: cleavage
probability: 1.0, binding score: 0.32, overall score: 0.66
H4.sub.16-39 KRHRKVLRDNIQGITKPAIRRLAR (K.sup.d motif) K.sup.d:
cleavage probability: 1.0, binding score: 0.34, overall score: 0.67
KRHRKVLRDNIQGITKPAIRRLAR (L.sup.d motif) L.sup.d: cleavage
probability: 1.0, binding score: 0.30, overall score: 0.64
H1'.sub.22-42 STDHPKYSDMIVAAIQAEKNR (K.sup.d motif) K.sup.d:
cleavage probability: 1.0, binding score: 0.53, overall score:
0.76
Low-Dose Therapy Does Not Cause Generalized Immunosuppression
[0303] SNF1 mice were tolerized with H4.sub.71-94 peptide or saline
as control. Ten days after the third injection, the mice were
immunized with hen egg lysozyme (HEL) in CFA (100 .mu.g/mouse)
twice at two wk interval. Seven days after second immunization, the
mice were bled to measure anti-HEL antibody response. Low-dose
peptide-tolerized mice actually produced 2-fold higher titer of
anti-HEL antibody than control mice (FIG. 34). Moreover,
IFN-.gamma. or IL-2 response to in vitro re-challenge with HEL was
actually increased in low-dose peptide-tolerized mice than control
mice (P<0.01), but responses to anti-CD3 were similar in both
groups (P>0.2, FIG. 34).
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Sequence CWU 1
1
71 1 24 PRT Artificial Sequence Synthetic 1 Thr Tyr Thr Glu His Ala
Lys Arg Lys Thr Val Thr Ala Met Asp Val 1 5 10 15 Val Tyr Ala Leu
Lys Arg Gln Gly 20 2 15 PRT Artificial Sequence Synthetic 2 Leu Ile
Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu 1 5 10 15 3 24
PRT Artificial Sequence Synthetic 3 Lys Arg His Arg Lys Val Leu Arg
Asp Asn Ile Gln Gly Ile Thr Lys 1 5 10 15 Pro Ala Ile Arg Arg Ile
Ala Arg 20 4 15 PRT Artificial Sequence Synthetic 4 Gly Ala Lys Arg
His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly 1 5 10 15 5 15 PRT
Artificial Sequence Synthetic 5 Leu Val Gly Leu Phe Glu Asp Thr Asn
Leu Cys Ala Ile His Ala 1 5 10 15 6 15 PRT Artificial Sequence
Synthetic 6 Lys Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg
Gln 1 5 10 15 7 24 PRT Artificial Sequence Synthetic 7 Pro Lys Lys
Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp 1 5 10 15 Gly
Lys Lys Arg Lys Arg Ser Arg 20 8 15 PRT Artificial Sequence
Synthetic 8 Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg Leu Ala
His 1 5 10 15 9 15 PRT Artificial Sequence Synthetic 9 Leu Arg Lys
Gly Asn Tyr Ala Glu Arg Val Gly Ala Gly Ala Pro 1 5 10 15 10 15 PRT
Artificial Sequence Synthetic 10 Asp Asn Lys Lys Thr Arg Ile Ile
Pro Arg His Leu Gln Leu Ala 1 5 10 15 11 15 PRT Artificial Sequence
Synthetic 11 Gln Lys Ser Thr Glu Leu Leu Ile Arg Lys Leu Pro Phe
Gln Arg 1 5 10 15 12 15 PRT Artificial Sequence Synthetic 12 Arg
Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala Ser Glu 1 5 10 15
13 18 PRT Artificial Sequence Synthetic 13 Gln Ser Ser Ala Val Met
Ala Leu Gln Glu Ala Ser Glu Ala Tyr Leu 1 5 10 15 Val Gly 14 15 PRT
Artificial Sequence Synthetic 14 Ala Leu Gln Glu Ala Ser Glu Ala
Tyr Leu Val Gly Leu Phe Glu 1 5 10 15 15 15 PRT Artificial Sequence
Synthetic 15 Thr Glu His Ala Lys Arg Lys Thr Val Thr Ala Met Asp
Val Val 1 5 10 15 16 15 PRT Artificial Sequence Synthetic 16 Leu
Gly Lys Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile 1 5 10 15
17 15 PRT Artificial Sequence Synthetic 17 Gly Val Leu Pro Asn Ile
Gln Ala Val Leu Leu Pro Lys Lys Thr 1 5 10 15 18 15 PRT Artificial
Sequence Synthetic 18 Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Ser
His His Lys Ala 1 5 10 15 19 15 PRT Artificial Sequence Synthetic
19 Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys 1 5
10 15 20 15 PRT Artificial Sequence Synthetic 20 Lys Gln Val His
Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly 1 5 10 15 21 15 PRT
Artificial Sequence Synthetic 21 Arg Asp Ala Val Thr Tyr Thr Glu
His Ala Lys Arg Lys Thr Val 1 5 10 15 22 15 PRT Artificial Sequence
Synthetic 22 Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys Arg
His Arg 1 5 10 15 23 14 PRT Artificial Sequence Synthetic 23 Ser
Gln Lys Glu Glu Glu Glu Gly Ala Gln Arg Glu Lys Glu 1 5 10 24 14
PRT Artificial Sequence Synthetic 24 Asp Trp Met Glu Glu Glu His
Gly Ala Gln Arg Glu Lys Glu 1 5 10 25 21 PRT Artificial Sequence
Synthetic 25 Ser Ala Ser His Pro Thr Tyr Ser Glu Met Ile Ala Ala
Ala Ile Arg 1 5 10 15 Ala Glu Lys Ser Arg 20 26 21 PRT Artificial
Sequence Synthetic 26 Ser Thr Asp His Pro Lys Tyr Ser Asp Met Ile
Val Ala Ala Ile Gln 1 5 10 15 Ala Glu Lys Asn Arg 20 27 47 DNA
Artificial Sequence Synthetic 27 ctgcggaaag gtaactacgc ggagcgggtg
ggggccggag cgcccgt 47 28 46 DNA Artificial Sequence Synthetic 28
gacaacaaga agacgcgcat catcccccgc cacctgcagc tggcca 46 29 45 DNA
Artificial Sequence Synthetic 29 ctgggccgcg tgaccatcgc gcagggcggc
gtcctgccca acatc 45 30 45 DNA Artificial Sequence Synthetic 30
ggcgtcctgc ccaacatcca ggccgtgctg ctgcccaaga agacc 45 31 45 DNA
Artificial Sequence Synthetic 31 caggccgtgc tgctgcccaa gaagaccgag
agccaccaca aggcc 45 32 72 DNA Artificial Sequence Synthetic 32
ccgaagaagg gctccaagaa ggccgtcacc aaggcccaaa agaaggatgg caagaagcgc
60 aagcgcagcc gc 72 33 45 DNA Artificial Sequence Synthetic 33
ggctccaaga aggcggtgac caagacccag aagaagggcg acaag 45 34 45 DNA
Artificial Sequence Synthetic 34 aagcaggtgc accccgacac gggcatctcg
tccaaggcca tgggc 45 35 45 DNA Artificial Sequence Synthetic 35
gacatcttcg agcgcatcgc cggcgaggcg tcgcgcctgg cgcac 45 36 45 DNA
Artificial Sequence Synthetic 36 cagaagtcca cggagctgct gatccgcaag
ctgcccttcc agcgc 45 37 45 DNA Artificial Sequence Synthetic 37
cgcttccaga gctcggccgt catggcgctg caggaggcga gcgag 45 38 53 DNA
Artificial Sequence Synthetic 38 cgagctcggc cgtcatggcg ctgcaggagg
cgagcgaggc ctacctggtg ggg 53 39 45 DNA Artificial Sequence
Synthetic 39 gcgctgcagg aggcgagcga ggcctacctg gtggggctct tcgag 45
40 45 DNA Artificial Sequence Synthetic 40 ctcgtgggtc tgtttgagga
caccaacctg tgcgccatcc acgcc 45 41 45 DNA Artificial Sequence
Synthetic 41 aagggcggga aggggctcgg caagggcggc gccaagcgcc accgc 45
42 45 DNA Artificial Sequence Synthetic 42 ggcgccaagc gccaccgcaa
ggtgctgcgc gacaacatcc agggc 45 43 72 DNA Artificial Sequence
Synthetic 43 aagcgccacc gcaaggtgct gcgcgacaac atccagggca tcaccaagcc
ggccatccgc 60 cgcctggcgc gg 72 44 45 DNA Artificial Sequence
Synthetic 44 ctcatctacg aggagacgcg cggcgtgctc aaggtcttcc tggag 45
45 45 DNA Artificial Sequence Synthetic 45 cgcgacgccg tcacctacac
cgagcacgcc aagaggaaga cggtc 45 46 72 DNA Artificial Sequence
Synthetic 46 acctacaccg agcacgccaa gaggaagacg gtcacggcca tggacgtggt
ctacgcgctc 60 aagcgccagg ga 72 47 45 DNA Artificial Sequence
Synthetic 47 accgagcacg ccaagaggaa gacggtcacg gccatggacg tggtc 45
48 45 DNA Artificial Sequence Synthetic 48 aagacggtca cggccatgga
cgtggtctac gcgctcaagc gccag 45 49 43 DNA Artificial Sequence
Synthetic 49 tcgcagaagg aggaggagga gggcgcgcaa cgtgagaaag agg 43 50
43 DNA Artificial Sequence Synthetic 50 gactggatgg aggaggagga
gggcgcgcaa cgtgagaaag agg 43 51 63 DNA Artificial Sequence
Synthetic 51 tcggcatcgc accccaccta ctcggagatg atcgcggcgg ccatccgtgc
ggaaaagagc 60 cgc 63 52 63 DNA Artificial Sequence Synthetic 52
tccacggacc accccaagta ttcagacatg atcgtggctg ctatccaggc agagaagaac
60 cgt 63 53 10 PRT Artificial Sequence Synthetic 53 Met Ile Ala
Ala Ala Ile Arg Ala Glu Lys 1 5 10 54 21 PRT Artificial Sequence
Synthetic 54 Ser Ala Ser His Pro Thr Tyr Ser Glu Met Ile Ala Ala
Ala Ile Arg 1 5 10 15 Ala Glu Lys Ser Arg 20 55 13 PRT Artificial
Sequence Synthetic 55 Glu Met Ile Ala Ala Ala Ile Arg Ala Glu Lys
Ser Arg 1 5 10 56 11 PRT Artificial Sequence Synthetic 56 Ile Ala
Ala Ala Ile Arg Ala Glu Lys Ser Arg 1 5 10 57 15 PRT Artificial
Sequence Synthetic 57 Ala Met Gly Ile Met Asn Ser Phe Val Asn Asp
Ile Phe Glu Arg 1 5 10 15 58 12 PRT Artificial Sequence Synthetic
58 Ser Glu Met Ile Ala Ala Ala Ile Arg Ala Glu Lys 1 5 10 59 20 PRT
Artificial Sequence Synthetic 59 Ser Ala Ser His Pro Thr Tyr Ser
Glu Met Ile Ala Ala Ala Ile Arg 1 5 10 15 Ala Glu Lys Ser 20 60 16
PRT Artificial Sequence Synthetic 60 Ser Ala Ser His Pro Thr Tyr
Ser Glu Met Ile Ala Ala Ala Ile Arg 1 5 10 15 61 13 PRT Artificial
Sequence Synthetic 61 Lys Pro Ala Gly Pro Ser Val Thr Glu Leu Ile
Thr Lys 1 5 10 62 9 PRT Artificial Sequence Synthetic 62 Thr Ala
Met Asp Val Val Tyr Ala Leu 1 5 63 9 PRT Artificial Sequence
Synthetic 63 Asn Ile Gln Gly Ile Thr Lys Pro Ala 1 5 64 9 PRT
Artificial Sequence Synthetic 64 His Arg Lys Val Leu Arg Asp Asn
Ile 1 5 65 9 PRT Artificial Sequence Synthetic 65 Lys Tyr Ser Asp
Met Ile Val Ala Ala 1 5 66 24 PRT Artificial Sequence Synthetic 66
Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys 1 5
10 15 Pro Ala Ile Arg Arg Leu Ala Arg 20 67 21 PRT Artificial
Sequence Synthetic 67 Ser Thr Asp His Pro Lys Tyr Ser Asp Met Ile
Val Ala Ala Ile Gln 1 5 10 15 Ala Glu Lys Asn Arg 20 68 13 PRT
Artificial Sequence Synthetic 68 Leu Ile Tyr Glu Glu Thr Arg Gly
Val Lys Phe Leu Glu 1 5 10 69 15 PRT Artificial Sequence Synthetic
69 Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala Ser Glu Ala Tyr 1 5
10 15 70 15 PRT Artificial Sequence Synthetic 70 Glu Ala Ser Glu
Ala Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn 1 5 10 15 71 13 PRT
Artificial Sequence Synthetic 71 Leu Ile Tyr Glu Glu Thr Arg Gly
Val Lys Phe Leu Glu 1 5 10
* * * * *