U.S. patent application number 10/211069 was filed with the patent office on 2003-01-30 for localization of major peptide autoepitopes for nucleosome specific t cells of systemic lupus erythematosus.
This patent application is currently assigned to The Board of Trustees of Northwestern University. Invention is credited to Datta, Syamal K., Kaliyaperumal, Arunan.
Application Number | 20030021797 10/211069 |
Document ID | / |
Family ID | 22449513 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030021797 |
Kind Code |
A1 |
Datta, Syamal K. ; et
al. |
January 30, 2003 |
Localization of major peptide autoepitopes for nucleosome specific
T cells of systemic lupus erythematosus
Abstract
The present invention includes peptides derived from nucleosomal
histone proteins which are useful for delaying the onset and
progression of systemic lupus erythematosus (i.e. lupus or SLE).
The peptides of the invention span the histone proteins (i.e. H1,
H2A, H2B, H3, and H4). The invention additionally encompasses
isolated nucleic acids which encode these histone peptides as well
as pharmaceutical compositions which comprise one or more of a
histone peptide. Further, the invention provides kits which
comprise one or more histone peptides or isolated nucleic acids
encoding histone peptides and an instructional material. The
invention also provides methods of using these compositions and
analogs of histone peptides to inhibit an immune response and
associated inflammation in an animal and to treat disorders in an
animal which are related to the production of autoantibodies and
complications thereof, such as inflammatory diseases, autoimmune
disorders, and nephritis. In addition, the invention includes
methods for developing diagnostic and prognostic reagents using the
histone peptides and isolated nucleic acids encoding the instone
peptides, for the purpose of tracking autoimmune T helper cells and
B cells of SLE are also included.
Inventors: |
Datta, Syamal K.; (Winnetka,
IL) ; Kaliyaperumal, Arunan; (Skokie, IL) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Assignee: |
The Board of Trustees of
Northwestern University
|
Family ID: |
22449513 |
Appl. No.: |
10/211069 |
Filed: |
August 2, 2002 |
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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09561490 |
Apr 28, 2000 |
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6468537 |
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60131448 |
Apr 28, 1999 |
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Current U.S.
Class: |
424/185.1 ;
435/199; 435/320.1; 435/325; 435/69.1; 530/358; 536/23.5 |
Current CPC
Class: |
G01N 2800/104 20130101;
G01N 2800/24 20130101; Y10S 514/885 20130101; A61K 38/00 20130101;
A61K 39/0008 20130101; A61K 38/1709 20130101; Y10S 514/866
20130101; G01N 33/564 20130101 |
Class at
Publication: |
424/185.1 ;
530/358; 435/69.1; 435/320.1; 435/199; 435/325; 536/23.5 |
International
Class: |
A61K 039/00; C07H
021/04; C12N 009/22; C12P 021/02; C12N 005/06 |
Goverment Interests
[0002] This invention was made in part using funds obtained from
the U.S. Government (National Institutes of Health Grant Nos. RO1
AR 39157 and RO1 AI 41985) and the U.S. Government may therefore
have certain rights in the invention.
Claims
What is claimed is:
1. A composition comprising an isolated peptide wherein said
peptide has an amino acid sequence corresponding to the amino acid
sequence of a portion of a nucleosome histone protein and, wherein
said isolated peptide is capable of promoting immunological
tolerance in an animal having systemic lupus erythematosus.
2. The composition of claim 1, wherein said nucleosome histone
protein is selected from the group consisting of histone 1 (H1),
histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4
(H4).
3. The composition of claim 1, said peptide comprising 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.
4. A composition comprising the isolated peptide of claim 1 and a
pharmaceutically acceptable carrier.
5. The composition of claim 1, wherein said portion of said histone
protein corresponds to an autoepitope, which autoepitope is
associated with systemic lupus erythematosus and recognized by one
or more of an autoimmune T cell and an autoimmune B cell.
6. An isolated nucleic acid encoding the peptide of claim 1.
7. The isolated nucleic acid of claim 6, said nucleic acid
comprising 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.
8. A cell comprising the isolated nucleic acid of claim 6.
9. The cell of claim 8, wherein said cell is selected from the
group consisting of a prokaryotic cell and a eukaryotic cell.
10. The cell of claim 9, wherein said cell is an insect cell.
11. A vector comprising the isolated nucleic acid of claim 6.
12. The composition of claim 1, wherein said peptide further
comprises 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.
13. A method of treating an animal having an autoimmune disorder,
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 promoting immunological
tolerance in an animal, thereby treating said autoimmune
disorder.
14. The method of claim 13, wherein said autoimmune disorder is
selected from the group consisting of rheumatoid arthritis,
scleroderma, and systemic lupus erythematosus.
15. The method of claim 14, wherein said autoimmune disorder is
systemic lupus erythematosus.
16. The method of claim 13, wherein said 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.
17. The method of claim 16, wherein said amount is from at least
about 100 micrograms per kilogram of animal to about 600 micrograms
per kilogram of animal.
18. The method of claim 13, wherein said 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.
19. A method of treating nephritis in an animal having systemic
lupus erythematosus, 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
promoting immunological tolerance in an animal, thereby alleviating
said nephritis in said animal.
20. A method of reducing the production of autoantibodies in an
animal, 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 promoting
immunological tolerance in an animal, and wherein said peptide is
administered in an amount sufficient to promote immunologic
tolerance in said animal, thereby reducing the production of
autoantibodies in said animal.
21. A method of treating inflammation in an animal, which
inflammation is caused by the production of autoantibodies in said
animal, 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 promoting
immunological tolerance in an animal, and wherein said peptide is
administered in an amount sufficient to promote immunological
tolerance in said animal, thereby inhibiting the production of
autoantibodies in said animal and alleviating inflammation in said
animal.
22. A method of diagnosing systemic lupus erythematosus in an
animal, said method comprising (a) contacting a sample from said
animal with a composition comprising an isolated histone peptide
complex, wherein said histone peptide complex comprises i) a
histone peptide portion, 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 II 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
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 said animal said 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.
23. A method of tracking an autoimmune cell associated with
systemic lupus erythematosus in an animal, said method comprising
(a) contacting a sample from said animal with a composition
comprising one or more of a modified histone peptide and an
isolated histone peptide complex, wherein said 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, wherein said peptide
portion and said modified portion are covalently linked; and
wherein said histone peptide complex comprises (iii) a histone
peptide portion, 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 class II molecule and an immunoglobin; and (v)
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 indicator portion is covalently linked to at least one
other component of the histone peptide complex; and (b) identifying
and monitoring in said animal said signal produced by said
indicator portion, wherein said identification and monitoring of
said signal in said animal constitutes the identification and
monitoring of an autoimmune cell associated with systemic lupus
erythematosus to which said histone protein complex is bound,
thereby tracking said autoimmune cell associated with systemic
lupus erythematosus in said animal.
24. A method of treating an animal having an autoimmune disorder,
said method comprising administering to said animal a modified
histone peptide, wherein said modified histone peptide is an
altered peptide ligand, and wherein said modified histone peptide
is capable of promoting immunological tolerance in an animal,
thereby treating said autoimmune disorder.
25. The method of claim 24, wherein said autoimmune disorder is
selected from the group consisting of rheumatoid arthritis,
scleroderma, and systemic lupus erythematosus.
26. The method of claim 25, wherein said autoimmune disorder is
systemic lupus erythematosus.
27. The method of claim 24, wherein said 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.
28. The method of claim 27, wherein said amount is from at least
about 100 micrograms per kilogram of animal to about 600 micrograms
per kilogram of animal.
29. The method of claim 24, wherein said 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.
30. A method of treating nephritis in an animal having systemic
lupus erythematosus, said method comprising administering to said
animal a modified histone peptide, wherein said modified histone
peptide is an altered peptide ligand, and wherein said modified
histone peptide is capable of promoting immunological tolerance in
an animal, thereby alleviating said nephritis in said animal.
31. A method of reducing the production of autoantibodies in an
animal, said method comprising administering to said animal a
modified histone peptide, wherein said modified histone peptide is
an altered peptide ligand capable of promoting immunological
tolerance in an animal, and wherein said modified histone peptide
is administered in an amount sufficient to promote immunologic
tolerance in said animal, thereby reducing the production of
autoantibodies in said animal.
32. A method of treating inflammation in an animal, which
inflammation is caused by the production of autoantibodies in said
animal, said method comprising administering to said animal a
modified histone peptide, wherein said modified histone peptide is
an altered peptide ligand capable of promoting immunological
tolerance in an animal, and wherein said modified histone peptide
is administered in an amount sufficient to promote immunological
tolerance in said animal, thereby inhibiting the production of
autoantibodies in said animal and alleviating inflammation in said
animal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to priority pursuant to 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application No.
60/131,448, which was filed on Apr. 28, 1999.
BACKGROUND OF THE INVENTION
[0003] Nucleosome-specific T helper cells (Th cells) initiate and
sustain the production of pathogenic, anti-nuclear autoantibodies
during the onset and progression of systemic lupus erythematosus
(SLE) through cognate interaction with autoimmune B cells (Mohan et
al., 1993, J. Exp. Med. 177:1367; Desai-Mehta et al., 1995, J.
Clin. Invest. 95:53; Kaliyaperumal et al., 1996, J. Exp. Med.
183:2459; Shi et al., 1998, J. Exp. Med. 187:367; Voll et al.,
1997, Arthritis Rheum. 40:2162; Kretz-Rommel, et al., 1997, J.
Clin. Invest. 99:1888). These SLE-associated Th cells are primarily
responsible for driving the pathogenic autoimmune response. Without
the help provided by these Th cells, autoimmune B cells are unable
to produce the disease-causing (pathogenic) autoantibodies
associated with SLE. Some of the critical epitopes (i.e.
autoantigenic determinants) to which these Th cells are directed
have been localized to the histone proteins of nucleosomes (i.e.
DNA-protein complexes in the nuclei of animal cells). For example,
in lupus prone mice, SLE-associated autoepitopes have been
identified at amino acid positions 10-33 of the H2B histone
protein, at amino acid positions 85-102 of the H3 histone protein,
and at amino acid positions 16-39 and 71-94 of the H4 histone
protein (Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459).
[0004] SLE-associated autoimmune B cells recognize and bind
autoantigenic determinants in DNA, histones, or other proteins in
nucleosomes through surface molecules (i.e. B cell receptors, BCRs)
which are antibody (i.e. immunoglobulin or Ig) receptors.
Autoantigenic recognition and binding by an autoimmune B cell is
followed by endocytosis and processing of the entire nucleosome
particle which bears the recognized autoantigen. As illustrated in
FIG. 1, the processed histone autoepitope (i.e. a histone peptide)
is loaded onto a major histocompatibility complex class II molecule
(i.e. a MHC class II molecule or I-A.sup.d molecule) of the B cell,
and is presented as a complex on the surface of the B cell in an
interaction with an autoimmune Th cell which has receptors specific
for nucleosomal histone peptide autoepitopes. Upon receiving this
antigen-specific signal (i.e. signal 1) and other co-stimulatory
signals (i.e. signal 2) from the B cell, the autoimmune Th cell
helps the autoimmune B cell by recruiting intermolecular support
and enabling the B cell to survive and differentiate into an
autoantibody-producing B cell associated with SLE (Datta and
Kaliyaperumal, 1997, Ann. New York Acad. Sci. 815:155; Datta, 2000,
Nature Med. 6:259).
[0005] Autoimmune T cells of lupus-prone mice are spontaneously
primed to SLE-associated autoepitopes early in life before overt
autoantibody production or any clinical manifestations of the
disease are present (Kaliyaperumal et al., 1996, J. Exp. Med.
183:2459). Moreover, immunization of pre-autoimmune mice with
peptides corresponding to SLE-associated nucleosomal autoepitopes
precipitates SLE-associated nephritis by triggering autoimmune T
helper cells of subtype 1 (i.e. Th 1 cells) which, in turn,
initiate anti-nuclear autoantibody production (Kaliyaperumal et
al., 1996, J. Exp. Med. 183:2459). The T helper cell subtypes 2 and
0 (i.e., Th2 and Th0 cells, respectively) are also involved in the
progression of SLE, as these T helper cell subtypes maintain
autoantibody production (Mohan et al., 1993, J. Exp. Med. 177:1367;
Nakajima et al., 1997, J. Immunol. 158:1466).
[0006] Unlike organ-specific autoimmune diseases in which the
autoimmune response targets a restricted set of autoepitoes and is
mediated by a single population of T cells, the autoimmune response
in SLE involves a complex web of polyclonal T cell and B cell
hyperactivity and appears to be directed by multiple susceptibility
genes (Datta et al., 1982, J. Immunol. 129:1539; Klinman and
Steinberg, 1987, J. Exp. Med. 165:1755; Cohen and Eisenberg, 1991,
Ann. Rev. Immunol. 9:243; 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, 1996, J. Clin. Invest.
97:2063; Koshy et al., 1996, J. Clin. Invest. 98:826; Liossis, et
al., 1996, J. Clin. Invest. 98:2549; Wakeland et al., 1997, J.
Clin. Immunol. 17:272; Vyse and Kotzin 1996, Opin. Immunol. 8:843;
Kono and Theofilopoulos, 1996, J. Autoimmunity 9:437).
[0007] Previously, it has not been considered that a brief
tolerogenic regimen of nucleosomal peptides could delay the
development of SLE-associated nephritis. Moreover, chronic
tolerogenic therapy with peptides has not previously been pursued,
as it was not foreseen that such therapy might have the effect of
prolonging survival and slowing SLE progression. There has long
been a need in the art to identify immunololgically specific agents
which could significantly affect the therapeutic outcome of SLE by
delaying or preventing the onset and progression of the disease and
complications thereof. The present invention provides a novel
therapeutic approach to satisfying this need.
SUMMARY OF THE INVENTION
[0008] 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.
[0009] 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.
[0010] 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).
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] In multiple embodiments, the autoimmune disorder is selected
from the group consisting of rheumatoid arthritis, scleroderma, and
systemic lupus erythematosus.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] In multiple embodiments, the invention provides a method of
diagnosing systemic lupus erythematosus in an animal. 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
[0028] i) a histone peptide portion, 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;
[0029] 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 II molecule and an
immunoglobin; and compatible
[0030] 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;
[0031] 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.
[0032] In other embodiments, the invention includes a method of
tracking an autoimmune cell associated with systemic lupus
erythematosus in an animal. This method comprises
[0033] (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
[0034] (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
[0035] (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
[0036] (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,
[0037] (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 class II molecule and an
immunoglobin, and
[0038] (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
[0039] 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
[0040] (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 DRAWINGS
[0041] 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.
[0042] FIG. 2 is a graph illustrating the effect on SLE-associated
nephritis of brief therapy with nucleosomal histone peptides. The
figure illuatrates the incidence of severe (4+ grade) nephritis in
SNF.sub.1 mice that received four injections of either a histone
peptide (i.e. H4.sub.71-94, H2B.sub.10-33, H4.sub.16-39) or saline
starting at 12 weeks age.
[0043] FIG. 3 is a bar graph depicting the ability of CD4.sup.+ 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.
[0044] 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.
[0045] 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.
[0046] FIG. 6 is a graph depicting the effect of H4.sub.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.
[0047] 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.sub.16-39 peptide. The results are expressed as a mean number of
antibody units per deciliter of culture medium.+-.SEM of five
experiments.
[0048] 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.
[0049] FIG. 9, comprising FIGS. 9A-9D, is a series of graphs
depicting IgG autoantibody levels in the serum of peptide-injected
mice. Sera from SNF.sub.1 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.
[0050] 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.
[0051] FIG. 11 depicts a proposed model for the production of
diverse autoantibodies in SLE by multipotent, intermolecular T-cell
help.
[0052] FIG. 12, comprising FIGS. 12A-12D, is a series of graphs
depicting IL-2 production by a pathogenic CD4.sup.+ T cell line in
response to selected nucleosomal histone peptides which were
overlapping 15-mers. The cell line was derived from CD4.sup.+ 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 Il-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.
[0053] 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.sub.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 .mu.M, Student's test) relative to background
level of T cells cultured with APC and without peptide.
Presentation of the H4.sub.49-63 peptide by autologous EBV-B cells
to the short-term T cell line L-EB, was inhibited mainly by anti-DR
mAb.
[0054] 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.sup.+
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.
[0055] 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.sup.+ 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.
[0056] 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 a 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.sub.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.
[0057] 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.
[0058] FIG. 18, comprising FIGS. 18A and 18B, depict representative
examples of two-color intracellular cytokine staining of CD4.sup.+
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.
[0059] 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.sup.+ 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.sup.+ T cells in the
patient sample were stained positive. A positive response is
indicated by outlined and bold numbers.
[0060] FIG. 20 is a table which lists the data from flow cytometry
analysis of intracellular cytokine staining of CD4.sup.+ T cells
obtained from seven normal subjects. A positive response is
indicated by outlined and bold numbers.
[0061] 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.
[0062] 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.sub.71-94 also contains the
binding motif for HLA-DR alleles, 1, 3, 4, 5, 7, and 8, in addition
to those shown.
[0063] 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-A.sup.d 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.
[0064] FIG. 24, comprising FIGS. 24A-24D, is a series of graphs
which illustrate the specificity of the naturally processed histone
peptide epitope, EP-3 or H1.sub.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.sub.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.
[0065] 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 inclubated 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 1G1, which is known to respond only to
nucleosome preparations, not to individual, naturally processed
peptides.
[0066] FIG. 26 is a graph depicting the acceleration of
SLE-associated nephritis following immunization with naturally
processed peptide autoepitopes in adjuvant.
[0067] 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).
DETAILED DESCRIPTION
[0068] 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). 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 II (major histocompatability
complex class II) tetramer complexes and histone peptide/MHC II-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.
[0069] 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.
[0070] 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.
[0071] Definitions
[0072] As used herein, each of the following terms has the meaning
associated with it in this section.
[0073] 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.
[0074] 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:
1 Full Name Three-Letter Code One-Letter 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
[0075] Unless otherwise indicated, all amino acid sequences listed
in this disclosure are listed in the order from the amino terminus
to the carboxyl terminus.
[0076] "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.
[0077] The term "protein" typically refers to large
polypeptides.
[0078] The term "peptide" typically refers to short
polypeptides.
[0079] 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.
[0080] "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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] "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.
[0085] 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.
[0086] "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.
[0087] "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.
[0088] 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.
[0089] As used herein, a "15-mer" refers to an isolated peptide
which comprises 15 contiguous amino acids.
[0090] 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.
[0091] 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.
[0092] 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).
[0093] 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.
[0094] 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.
[0095] "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).
[0096] "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).
[0097] As used herein, the term "SLE-associated autoepitope" refers
to an amino aicd 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] As used herein, to "load" a histone peptide onto a major
histocompatibility complex class II (MHC II) molecule (i.e. an
I-A.sup.d molecule) in a B cell means to cause an interaction and
the formation of a complex between a histone peptide and a MHC II
molecule in the B cell.
[0102] Description
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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:
[0108] glycine, alanine;
[0109] valine, isoleucine, leucine;
[0110] aspartic acid, glutamic acid;
[0111] asparagine, glutamine;
[0112] serine, threonine;
[0113] lysine, arginine;
[0114] phenylalanine, tyrosine.
[0115] 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.
[0116] 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
molecule, 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 II 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 II 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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. Mcd. 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.
[0128] 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 II molecule (i.e. 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).
[0129] 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.
[0130] 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.
[0131] 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.
[0132] Methods
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] Specific Applications of Pharmaceutical Compositions
[0138] 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 class II 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).
[0139] Peptides are usually stored frozen in lyophilized powder
form at -20.degree. C., for the short-term or at -70.degree. 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 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).
[0140] 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.
[0141] Further Improvements in Tolerance Therapy with Overlapping
B-cell and T-cell Autoepitopes
[0142] Among the nucleosomal peptide epitopes for Th cells of SNF1
mice, H4.sub.16-39 exhibited the most beneficial effect in
tolerance therapy (see Example 1). Interestingly, the H4.sub.16-39
peptide was not the most immunogenic among the nucleosomal
autoepitopes in triggering pathogenic Th cells when administered
with CFA into SNF.sub.1 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.sub.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.sub.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).
[0143] 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.sub.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.sub.16-39 a highly efficient tolerogen. An
additional epitope in nucleosomal core histone H3, H3.sub.85-102,
has been identified to which the splenic T cells of pre-nephritic
SNF.sub.1 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'.sub.22-42 peptide was identified as another
dominant autoepitope for the autoimmune T, as well as B cells of
lupus (see Example 3).
[0144] 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.
[0145] 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).
[0146] Tracking Autoimmune B cells
[0147] 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.
[0148] 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
class II 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 SNF.sub.1 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.
[0153] Histone Peptide-MHC Class II Tetramers for Tracking
Autoimmune Th Cells
[0154] 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). The major peptide
epitopes appear to have promiscuous DR binding motifs (Example 2),
and two of them, H4.sub.16-39 and H4.sub.71-94 have actually been
shown to bind HLA-DR (Shi et al.1998, J. Exp. Med. 187:367). 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-DR 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 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-HLA-DR 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 HLA-DR molecules in
insect cells using appropriate vectors (McMichael and Kelleher
1999, J. Clin. Invest. 104:1669) and then incubate the MHC class II
molecules with any of the histone peptide autoepitopes for stable
binding. Similarly, such tetramers of mouse MHC class II or
I-A.sup.d-nucleosomal peptide, and also chimeric
peptide-I-A.sup.d-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).
[0155] 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.
[0156] Pharmaceutical Compositions in General
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.degree. C., or at -20.degree. 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. 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).
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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 class II 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).
[0184] Peptides are usually stored frozen in lyophilized powder
form at -20.degree. C., for the short-term or at -70.degree. 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).
[0185] 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.
[0186] 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.
[0187] Kits
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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
[0192] 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.
[0193] The materials and methods used in this Example are now
described.
[0194] Mice
[0195] 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).
[0196] Antibodies
[0197] 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.).
[0198] Synthesis of Peptides
[0199] 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.sub.16-39, H4.sub.71-94,
and H2B.sub.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.
[0200] Tolerance Induction with Histone Derived Peptides
[0201] 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.sub.10-33, H4.sub.16-39, and H4.sub.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).
[0202] 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.
[0203] For experiments investigating chronic therapy of established
glomerulonephritis, 18 month old SNF.sub.1 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.
[0204] Autoantibody Quantitation
[0205] 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).
[0206] Isolation of CD4.sup.+ T Cells and B Cells
[0207] Splenic CD4.sup.+ 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 SNF.sub.1 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), anti-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
SNF.sub.1 mice by treating splenocytes that were applied to nylon
wool columns twice with anti-T cell antibody, anti-Thy1.2 (TIB99)
and complement.
[0208] Cytokine Assays
[0209] Fresh splenic CD4.sup.+ 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-IFN.gamma., and anti-IL-4, capture and
biotinylated antibody pairs, and their respective standard
cytokines (rIL-2, rIL-4, rIFN.gamma.) 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.
[0210] Helper Assays for IG Autoantibody Production
[0211] 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
SNF.sub.1 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.
[0212] Assay for Regulatory T Cells
[0213] To determine whether tolerance therapy induced any
regulatory T cell activity, T cells from either unmanipulated
SNF.sub.1 mice or tolerized (i.e. peptide injected) mice, were
co-cultured with a mixture of splenic B cells and T cells from
unmanipulated SNF.sub.1 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.
[0214] The results of the experiments presented in this Example are
now described.
[0215] Brief Therapy with Nucleosomal Histone Peptides
[0216] Twelve week old pre-nephritic SNF.sub.1 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.sub.10-33, H4.sub.16-39, or
H4.sub.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.sub.71-94 peptide injected group of mice
developed severe nephritis. In contrast, the H2B.sub.10-33 and
H4.sub.16-39 injected mice did not develop disease at this age
(p=0.029).
[0217] 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.sub.10-33, and
H4.sub.71-94 groups of mice each had an incidence of only 33.3%
(p=0.05) and the H4.sub.16-39 group mice exhibited an incidence of
only 11.1% (p=0.003). Mice in all groups, except the H4.sub.16-39
group mice, developed severe nephritis by 54 weeks of age. The
H4.sub.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.
[0218] T Cell Response to Peptides in Treated Animals
[0219] In unmanipulated SNF.sub.1 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.
[0220] 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.
2TABLE 1 Incidence of lupus nephritis at sacrifice in "short-term"
batch of mice Grading of Lupus Nephritis Group 0 1+ 2+ 3+ 4+
Percent Incidence Saline 33.3 33.3 33.3 H2B.sub.10-33 66.6 33.3
H4.sub.16-39 33.3 66.6 H4.sub.71-94 66.6 33.3
[0221] Effect of Peptide Therapy on CD4.sup.+ T Cell Help for
Autoantibody Production
[0222] 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.sup.+ T
cells were isolated from the peptide-injected mice and co-cultured
with B cells isolated from unmanipulated 16-20 week. old SNF.sub.1
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.sub.71-94,and H4.sub.16-39
treated group of mice as compared with the control group (p=0.03).
The H2B.sub.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.sub.10-33 and H4.sub.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.sub.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.sub.10-33, H4.sub.16-39, and H4.sub.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 signifcantly
different (p=0.1) relative to the control group.
[0223] Addition of rIL-2 to CD4.sup.+ T Cells for Testing
Anergy
[0224] To determine if the diminished help observed in the above
experiments by CD4.sup.+ 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.sub.71-94, and H2B.sub.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.sub.16-39 treated group mice, exhibited only a modest increase
of 35% in help for anti-ds DNA antibody production following the
addition of rIL-2 (p=0.05). The H4.sub.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.
[0225] Effect of Peptide Therapy on the Ability of Autoimmune B
Cells to Receive T Cell Help
[0226] 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.sup.+ T cells from unmanipulated
SNF.sub.1 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.sub.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.sub.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.sub.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.sub.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.sub.16-39 injected mice
relative to control group mice. In H4.sub.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.
[0227] Effect of Anti-Ig (Fab').sub.2, and rIL-4 on B Cells from
H4.sub.16-39-Treated Group of Mice
[0228] As indicated by the above experimental results, B cells from
H4.sub.16-39 peptide-injected mice were less responsive to help
from T cells of unmanipulated SNF.sub.1 mice. In order to assess,
whether this impairement was due to deletion or anergy, purified B
cells from the H4.sub.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.sub.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.sub.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.
[0229] Effect of Soluble CD40 Ligand and rIL-4 on B Cells of
H4.sub.16-39 Injected Mice
[0230] 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.sub.16-39 group mice did not produce significant
amounts of antibodies, even with the addition of CD40L and
rIL-4.
[0231] B Cell Response to Lipopolysaccharide (LPS) in Peptide
Injected Mice
[0232] 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).
[0233] IG Anti-Nuclear Autoantibody Levels in Sera of Peptide
Injected Mice
[0234] 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.sub.10-33,
H4.sub.16-39, and H4.sub.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.sub.10-33, H4.sub.16-39, and H4.sub.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.
[0235] Search for Regulatory T Cells in Peptide-Injected Mice
[0236] 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 SNF.sub.1 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 SNF.sub.1 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.
[0237] Treatment of Established Glomerulonephritis with Nucleosomal
Peptides
[0238] 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.sub.71-94 and H2B.sub.10-33
groups of mice were alive (p=0.061), whereas none of the mice in
H4.sub.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.sub.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.sub.16-39 group mice remained alive. By contrast, all of the
H4.sub.71-94 group mice had succumbed to renal disease (p=0.002),
and only one mouse in the H2B.sub.10-33 group survived (p=0.015 as
compared with the H4.sub.16-39 group).
[0239] The multipotent and promiscuous helper activity of the
pathogenic Th cells involved in SLE which is evident from these
experiments is modelled 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).
[0240] 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.
[0241] Among the three nucleosomal peptides described herein,
H4.sub.16-39 exhibited the most beneficial effect. Interestingly,
this peptide is not the most immunogenic among the nucleosomal
autoepitopes in triggering pathogenic Th cells in SNF.sub.1mice
(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.sub.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 SNF.sub.1 mice did increase
autoantibody production by B cells from the H4.sub.16-39 injected
mice above baseline, but not comparably relative to other
groups.
[0242] As shown in this Example, the overlapping of epitopes for
pathogenic Th cells and autoimmune B cells of lupus makes
H4.sub.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.sub.85-102, to which splenic T
cells of pre-nephritic SNF.sub.1 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.sub.85-102 is
also a potent tolerogen for therapy of lupus nephritis in SNF.sub.1
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.
[0243] The data presented in this Example can be summarized as
follows.
[0244] Using the (SWR.times.NZB)F.sub.1 (SNF.sub.1) 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 SNF.sub.1 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.sub.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
[0245] 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).
[0246] The materials and methods used in this Example are now
described.
[0247] Patients and Healthy Donors
[0248] The short-term CD4.sup.+ 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.
[0249] 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.
[0250] Antibodies
[0251] 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.).
[0252] Antigens
[0253] 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.
[0254] Cell Preparation
[0255] 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.
[0256] To make short-term, CD4.sup.+ T cell lines, CD4.sup.+ T
cells were purified from PBMC using magnetic beads absorbed with
anti-CD4 mAb, followed by using DetachBeads (Dynal Inc., Oslo,
Norway). The CD4.sup.+ 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.
[0257] 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. 95:531-541).
[0258] The CD4.sup.+ 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).
[0259] Stimulation of Short-Term T Cell Lines
[0260] 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.degree. 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.sup.+ 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.degree. 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.sup.+ 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).
[0261] 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.
[0262] IL-2 Production Analysis
[0263] IL-2 secreted by CD4.sup.+ 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.
[0264] Direct Stimulation of PBMCs
[0265] 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.
[0266] Flow Cytometry Analysis
[0267] 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).
[0268] 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.degree.
C. for flow cytometry analysis.
[0269] 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.
[0270] The results of the experiments presented in this Example are
now described.
[0271] Response of an Anti-DNA Autoantibody-Inducing, Lupus Th Cell
Clone to Histone Peptides
[0272] To localize histone peptide epitopes for autoimmune T cells
of human SLE, the response of CD4.sup.+ 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.
[0273] 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.sub.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).
[0274] Localization of Major Peptide Epitopes for Autoimmune T
Cells by Using Short-Term, CD4.sup.+ T Cell Lines Derived from
Lupus Patients
[0275] To narrow down the regions in nucleosomal core histones that
would contain the major peptide autoepitopes, short-term CD4.sup.+
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.
[0276] 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. 95:531-541).
[0277] Altogether, 10 short-term CD4.sup.+ 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.
[0278] 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.
[0279] 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.
[0280] Histone Peptide Epitopes for CD4.sup.+ T Cells in PBMC of
Lupus Patients
[0281] It was necessary to confirm that the histone peptide
autoepitopes were also relevant to unmanipulated CD4.sup.+ 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.sup.+ 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).
[0282] 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.
[0283] 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.sub.10-33, H4.sub.16-39, and H4.sub.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.sub.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.
[0284] 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.
[0285] The results of assays for intracellular cytokine production
by CD4.sup.+ 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.sup.+ 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%).
[0286] 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.sup.+ 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.
[0287] 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.sub.10-33, H4.sub.16-39, H4.sub.71-94,
H2A.sub.34-48, H3.sub.91-105, H3.sub.100-114, H4.sub.14-28, and
H4.sub.49-63.
[0288] Remarkably, the recurrent autoepitopes identified here for
the T cells involved in SLE, namely H2B.sub.10-33, H3.sub.95-105,
H4.sub.16-39, and H4.sub.71-94, are also the major autoepitopes for
nephritis-inducing T cells in lupus-prone SNF.sub.1 mice
(Kaliyaperumal et al., 1996, J. Exp. Med 183:2459-2469).
[0289] 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.
[0290] 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, 5. 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).
[0291] 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.sub.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.
[0292] 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.
[0293] The experiments of this Example identify nucleosomal histone
peptides corresponding to histone regions, H2B.sub.10-33,
H4.sub.16-39H4.sub.14-28, H4.sub.71-94, H3.sub.91-105, and
H3.sub.100-114, which were recurrently recognized by CD4.sup.+ 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.sub.34-48 and
H4.sub.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
[0294] 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. Nati. Acad Sci. USA. 93:2019-2024; Mohan et al., 1995, J.
Immunol. 154:1470-1480).
[0295] The materials and methods presented in this Example are now
described.
[0296] Mice
[0297] BALB/c, NZB, SWR, (BALB/c.times.SWR)F.sub.1 mice were
purchased from The Jackson Laboratory (Bar Harbor, Me.).
Lupus-prone (SWR.times.NZB)F.sub.1 (SNF.sub.1) hybrids were bred at
the animal facility of Northwestern University. Female mice were
used in each of the experiments.
[0298] Preparation of Cloned Th Cell Lines and Hybridomas
[0299] Cloned Th cell clones and hybridomas used in the experiments
presented in this Example were derived from SNF.sub.1 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 SNF.sub.1 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 .quadrature.-mercaptoetha- nol. 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).
[0300] Antibodies
[0301] The following monoclonal antibodies (mAb) were used:
anti-I-A.sup.d (HB3), anti-I-A.sup.b,d,q (TIB120), anti-HSA
(TIB183), and anti-Thy-1.2 (TIB99), anti-CD8 (TIB21 1), 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.degree. C.
[0302] ELISAs
[0303] Anti-IL-2, anti-IFN.quadrature., and anti-IL-4, capture and
biotinylated antibody pairs, and the recombinant cytokine standards
(rIL-2, rIL-4, rIFN.quadrature.) 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.
[0304] Preparation of Chicken Chromatin Containing
Polynucleosomes
[0305] 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).
[0306] Large Scale Cell Culture and Isolation of MHC class II
Molecules
[0307] 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 SNF.sub.1 mice. A high
I-A.sup.d 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.degree. C.
[0308] MHC Class II Purification
[0309] MHC class II (I-A.sup.d) molecule was affinity purified by
application of peptide-MHC complexes to a sepharose 4B column
modified with a I-A.sup.d specific antibody (HB-3) and elution of
the I-A.sup.d 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.).
[0310] Elution and Purification of Peptides from MHC Class II
Molecules
[0311] 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.degree. 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.degree. C.
[0312] Fractionation of Peptides
[0313] 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 SNF.sub.1 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).
[0314] Synthesis of Peptides
[0315] 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.
[0316] Preparation of Antigen Presenting Cells (APC)
[0317] The splenic CD4.sup.+ 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.phi.) APC were prepared from one month
old SNF.sub.1 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.degree. 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+M.phi. were used as APCs.
[0318] Proliferation and Cytokine Assays
[0319] Fresh splenic, CD4.sup.+ 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+M.phi. 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
.sup.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.
[0320] Autoantibody Quantitation
[0321] 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.
[0322] Assessment of Pathogenicity of Histone Derived Peptides
[0323] Pre-nephritic SNF.sub.1 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.sub.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.
[0324] The results of the experiments presented in this Example are
now described.
[0325] Identification of the Naturally Processed and Presented Th
Cell Epitopes:
[0326] The MHC class II molecules were purified from APC cell lines
derived from SNF.sub.1 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.
[0327] 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+M.phi. 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.sub.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.sub.22-42
peptide was equivalent to the chicken H5.sub.22-42 peptide in
stimulating the pathogenic Th clones from SNF.sub.1 lupus mice.
Therefore, the mouse H1.sub.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.
[0328] Eluted Peptides and Pathogenic Autoantibody Production
[0329] 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.sub.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.sub.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.sub.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.sub.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.
[0330] Acceleration of SLE
[0331] Twelve week old prenephritic SNF.sub.1 mice that were
immunized with an eluted peptide, EP-2 or EP-3, in adjuvant (CFA)
developed severe nephritis earlier than age matched SNF.sub.1 mice
injected with CFA alone or control OVA peptide in CFA. Eighty
percent of the mice injected with H1.sub.22-42 (i.e. EP-3)
succumbed to severe lupus nephritis within four weeks of the first
injection. Indeed, some of the H1.sub.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.
[0332] 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.sub.22-42 (SEQ ID NO:25), or H1.sub.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 Hi sequence at amino acid
positions 22-42, which is identical in mouse and human
(STDHPKYSDMIVAAIQAEKNR, H1.sub.22-42). The H1.sub.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.
[0333] The experiments presented in this Example demonstrate that
histone peptides corresponding to the H1 histone protein can be
used to (a) develop antigen-specific therapy of SLE. Tolerization
of autoimmune T cells and B cells of lupus with H1.sub.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.sub.22-42 peptide linked to I-A.sup.d 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.
[0334] 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:
[0335] (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.
[0336] (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.
[0337] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0338] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
61 1 24 PRT Artificial Sequence Histone fragment 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 Histone
fragment 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 Histone fragment 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
Histone fragment 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 Histone fragment
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 Histone fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 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
Histone fragment 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 Histone
fragment 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 Histone
fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 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
Histone fragment 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 Histone
fragment 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 Histone fragment 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 Histone fragment 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 Histone fragment 23 Ser Gln Lys Glu Glu Glu Glu
Gly Ala Gln Arg Glu Lys Glu 1 5 10 24 14 PRT Artificial sequence
Histone fragment 24 Asp Trp Met Glu Glu Glu Glu Gly Ala Gln Arg Glu
Lys Glu 1 5 10 25 21 PRT Artificial sequence Histone fragment 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 Histone
fragment 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 Histone fragment 27 ctgcggaaag gtaactacgc ggagcgggtg
ggggccggag cgcccgt 47 28 46 DNA Artificial sequence Histone
fragment 28 gacaacaaga agacgcgcat catcccccgc cacctgcagc tggcca 46
29 45 DNA Artificial sequence Histone fragment 29 ctgggccgcg
tgaccatcgc gcagggcggc gtcctgccca acatc 45 30 45 DNA Artificial
sequence Histone fragment 30 ggcgtcctgc ccaacatcca ggccgtgctg
ctgcccaaga agacc 45 31 45 DNA Artificial sequence Histone fragment
31 caggccgtgc tgctgcccaa gaagaccgag agccaccaca aggcc 45 32 72 DNA
Artificial sequence Histone fragment 32 ccgaagaagg gctccaagaa
ggccgtcacc aaggcccaaa agaaggatgg caagaagcgc 60 aagcgcagcc gc 72 33
45 DNA Artificial sequence Histone fragment 33 ggctccaaga
aggcggtgac caagacccag aagaagggcg acaag 45 34 45 DNA Artificial
sequence Histone fragment 34 aagcaggtgc accccgacac gggcatctcg
tccaaggcca tgggc 45 35 45 DNA Artificial sequence Histone fragment
35 gacatcttcg agcgcatcgc cggcgaggcg tcgcgcctgg cgcac 45 36 45 DNA
Artificial sequence Histone fragment 36 cagaagtcca cggagctgct
gatccgcaag ctgcccttcc agcgc 45 37 45 DNA Artificial sequence
Histone fragment 37 cgcttccaga gctcggccgt catggcgctg caggaggcga
gcgag 45 38 53 DNA Artificial sequence Histone fragment 38
cgagctcggc cgtcatggcg ctgcaggagg cgagcgaggc ctacctggtg ggg 53 39 45
DNA Artificial sequence Histone fragment 39 gcgctgcagg aggcgagcga
ggcctacctg gtggggctct tcgag 45 40 45 DNA Artificial sequence
Histone fragment 40 ctcgtgggtc tgtttgagga caccaacctg tgcgccatcc
acgcc 45 41 45 DNA Artificial sequence Histone fragment 41
aagggcggga aggggctcgg caagggcggc gccaagcgcc accgc 45 42 45 DNA
Artificial sequence Histone fragment 42 ggcgccaagc gccaccgcaa
ggtgctgcgc gacaacatcc agggc 45 43 72 DNA Artificial sequence
Histone fragment 43 aagcgccacc gcaaggtgct gcgcgacaac atccagggca
tcaccaagcc ggccatccgc 60 cgcctggcgc gg 72 44 45 DNA Artificial
sequence Histone fragment 44 ctcatctacg aggagacgcg cggcgtgctc
aaggtcttcc tggag 45 45 45 DNA Artificial sequence Histone fragment
45 cgcgacgccg tcacctacac cgagcacgcc aagaggaaga cggtc 45 46 72 DNA
Artificial sequence Histone fragment 46 acctacaccg agcacgccaa
gaggaagacg gtcacggcca tggacgtggt ctacgcgctc 60 aagcgccagg ga 72 47
45 DNA Artificial sequence Histone fragment 47 accgagcacg
ccaagaggaa gacggtcacg gccatggacg tggtc 45 48 45 DNA Artificial
sequence Histone fragment 48 aagacggtca cggccatgga cgtggtctac
gcgctcaagc gccag 45 49 43 DNA Artificial sequence Histone fragment
49 tcgcagaagg aggaggagga gggcgcgcaa cgtgagaaag agg 43 50 43 DNA
Artificial sequence Histone fragment 50 gactggatgg aggaggagga
gggcgcgcaa cgtgagaaag agg 43 51 63 DNA Artificial sequence Histone
fragment 51 tcggcatcgc accccaccta ctcggagatg atcgcggcgg ccatccgtgc
ggaaaagagc 60 cgc 63 52 63 DNA Artificial sequence Histone fragment
52 tccacggacc accccaagta ttcagacatg atcgtggctg ctatccaggc
agagaagaac 60 cgt 63 53 10 PRT Artificial sequence Histone fragment
53 Met Ile Ala Ala Ala Ile Arg Ala Glu Lys 1 5 10 54 21 PRT
Artificial sequence Histone fragment 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 Histone fragment 55 Glu Met Ile
Ala Ala Ala Ile Arg Ala Glu Lys Ser Arg 1 5 10 56 11 PRT Artificial
sequence Histone fragment 56 Ile Ala Ala Ala Ile Arg Ala Glu Lys
Ser Arg 1 5 10 57 15 PRT Artificial Sequence Histone fragment 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 Histone fragment 58 Ser Glu Met
Ile Ala Ala Ala Ile Arg Ala Glu Lys 1 5 10 59 20 PRT Artificial
sequence Histone fragment 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 Histone fragment 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 Histone fragment 61 Lys Pro Ala Gly Pro Ser Val Thr Glu
Leu Ile Thr Lys 1 5 10
* * * * *