U.S. patent application number 10/015540 was filed with the patent office on 2002-07-04 for methods for treatment of multiple sclerosis using peptide analogues of human myelin basic protein.
This patent application is currently assigned to Neurocrine Biosciences, Inc.. Invention is credited to Conlon, Paul J., Gaur, Amitabh, Ling, Nicholas, Steinman, Lawrence.
Application Number | 20020086976 10/015540 |
Document ID | / |
Family ID | 23341698 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086976 |
Kind Code |
A1 |
Ling, Nicholas ; et
al. |
July 4, 2002 |
Methods for treatment of multiple sclerosis using peptide analogues
of human myelin basic protein
Abstract
The present invention is directed toward peptide analogues of
human myelin basic protein. The peptide analogue is at least seven
amino acids long and derived from residues 86 to 99 of human myelin
basic protein. The analogues are altered from the native sequence
at least at positions 91, 95, or 97. Additional alterations may be
made at other positions. Pharmaceutical compositions containing
these peptide analogues are provided. The peptide analogues are
useful for treating multiple sclerosis.
Inventors: |
Ling, Nicholas; (San Diego,
CA) ; Gaur, Amitabh; (San Diego, CA) ; Conlon,
Paul J.; (Solana Beach, CA) ; Steinman, Lawrence;
(Palo Alto, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Neurocrine Biosciences,
Inc.
10555 Science Center Drive
San Diego
CA
92121-1102
|
Family ID: |
23341698 |
Appl. No.: |
10/015540 |
Filed: |
December 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10015540 |
Dec 11, 2001 |
|
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08342408 |
Nov 18, 1994 |
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6329499 |
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Current U.S.
Class: |
530/350 ;
435/183 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 37/04 20180101; A61K 38/00 20130101; C07K 14/4713 20130101;
A61P 25/00 20180101; A61P 37/00 20180101; A61P 25/02 20180101 |
Class at
Publication: |
530/350 ;
435/183 |
International
Class: |
C12N 009/00; C07K
014/435 |
Claims
What is claimed is:
1. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 91, wherein the L-lysine at position 91 is
altered to another amino acid, and one to three L-amino acids
selected from the group consisting of valine at position 86, valine
at position 87, histidine at position 88, threonine at position 95,
threonine at position 98 and proline at position 99 are altered to
an amino acid other than the amino acid present in the native
protein at that position.
2. The peptide analogue of claim 1 wherein L-lysine at position 91
is altered to a non-conservative amino acid.
3. The peptide analogue of claim 1 wherein residue 91 is altered to
D-lysine.
4. The peptide analogue of claim 1 wherein residue 91 is altered to
an amino acid selected from the group consisting of arginine,
asparagine, histidine, leucine, serine, glycine, glutamic acid,
phenylalanine, alanine and D-lysine.
5. The peptide analogue of claim 1 wherein residue 91 is altered to
alanine and residue 87 is altered to D-valine.
6. The peptide analogue of claim 1 wherein residue 91 is altered to
alanine and residue 88 is altered to D-histidine.
7. The peptide analogue of claim 1 wherein residue 91 is altered to
alanine and residue 99 is altered to D-proline.
8. The peptide analogue of claim 1 wherein residue 91 is altered to
alanine, residue 87 is altered to D-valine, and residue 99 is
altered to D-proline.
9. The peptide analogue of claim 1 wherein residue 91 is altered to
alanine, residue 88 is altered to D-histidine, and residue 99 is
altered to D-proline.
10. The peptide analogue of claim 1 wherein residue 88 is altered
to an amino acid selected from the group consisting of serine,
glutamic acid, tyrosine, leucine, D-histidine, glutamine,
phenylalanine and lysine.
11. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 97, wherein the L-arginine at position 97 is
altered to another amino acid and one to three L-amino acids
selected from the group consisting of valine at position 86, valine
at position 87, histidine at position 88, threonine at position 95,
threonine at position 98 and proline at position 99 are altered to
an amino acid other than the amino acid present in the native
protein at that position.
12. The peptide analogue of claim 11 wherein the L-arginine at
position 97 is altered to a non-conservative amino acid.
13. The peptide analogue of claim 11 wherein residue 97 is altered
to D-arginine.
14. The peptide analogue of claim 11 wherein residue 97 is altered
to an amino acid selected from the group of D-alanine, D-arginine,
glycine, lysine, glutamine, glutamic acid, threonine, leucine,
phenylalanine, histidine and alanine.
15. The peptide analogue of claim 11 wherein residue 97 is altered
to alanine and residue 87 is altered to D-valine.
16. The peptide analogue of claim 11 wherein residue 97 is altered
to alanine and residue 88 is altered to D-histidine.
17. The peptide analogue of claim 11 wherein residue 97 is altered
to alanine and residue 99 is altered to D-proline.
18. The peptide analogue of claim 11 wherein residue 97 is altered
to alanine, residue 87 is altered to D-valine, and residue 99 is
altered to D-proline.
19. The peptide analogue of claim 11 wherein residue 97 is altered
to alanine, residue 88 is altered to D-histidine and residue 99 is
altered to D-proline.
20. The peptide analogue of claim 11 wherein residue 88 is altered
to an amino acid selected from the group consisting of serine,
glutamic acid, tyrosine, leucine, D-histidine, glutamine,
phenylalanine and lysine.
21. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 95, wherein the L-threonine at position 95 is
altered to another amino acid and one to three L-amino acids
selected from the group consisting of valine at position 86, valine
at position 87, histidine at position 88, threonine at position 98
and proline at position 99 are altered to an amino acid other than
the amino acid present in the native protein at that position.
22. The peptide analogue of claim 21 wherein the L-threonine at
position 95 is altered to a non-conservative amino acid.
23. The peptide analogue of claim 21 wherein residue 95 is altered
to D-threonine.
24. The peptide analogue of claim 21 wherein residue 95 is altered
to an amino acid selected from the group consisting of alanine,
D-threonine, glycine, isoleucine, tyrosine, glutamine, serine,
lysine, glutamic acid and histidine.
25. The peptide analogue of claim 21 wherein residue 95 is altered
to alanine and residue 87 is altered to D-valine.
26. The peptide analogue of claim 21 wherein residue 95 is altered
to alanine and residue 88 is altered to D-histidine.
27. The peptide analogue of claim 21 wherein residue 95 is altered
to alanine and residue 99 is altered to D-proline.
28. The peptide analogue of claim 21 wherein residue 95 is altered
to alanine, residue 87 is altered to D-valine, and residue 99 is
altered to D-proline.
29. The peptide analogue of claim 21 wherein residue 95 is altered
to alanine, residue 88 is altered to D-histidine, and residue 99 is
altered to D-proline.
30. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 91, wherein the L-lysine at position 91 is
altered to another amino acid and the N-terminal amino acid and the
C-terminal amino acid are altered to another amino acid, such that
upon administration of the peptide analogue in vivo proteolysis is
reduced.
31. The peptide analogue of claim 30 wherein the N-terminal and
C-terminal amino acids are D-amino acids.
32. The peptide analogue of claim 30 wherein L-lysine at position
91 is altered to a non-conservative amino acid.
33. The peptide analogue of claim 30 wherein residue 91 is altered
to D-lysine.
34. The peptide analogue of claim 30 wherein residue 91 is altered
to an amino acid selected from the group consisting of arginine,
asparagine, histidine, leucine, serine, glycine, glutamic acid,
phenylalanine, alanine and D-lysine.
35. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 95, wherein the L-lysine at position 91 is
altered to another amino acid and the N-terminal amino acid and the
C-terminal amino acid are altered to another amino acid, such that
upon administration of the peptide analogue in vivo proteolysis is
reduced.
36. The peptide analogue of claim 35 wherein the N-terminal and
C-terminal amino acids are D-amino acids.
37. The peptide analogue of claim 35 wherein the L-threonine at
position 95 is altered to a non-conservative amino acid.
38. The peptide analogue of claim 35 wherein residue 95 is altered
to D-threonine.
39. The peptide analogue of claim 35 wherein residue 95 is altered
to an amino acid selected from the group consisting of alanine,
D-threonine, glycine, isoleucine, tyrosine, glutamine, serine,
lysine, glutamic acid and histidine.
40. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 97, wherein the L-lysine at position 91 is
altered to another amino acid and the N-terminal amino acid and the
C-terminal amino acid are altered to another amino acid, such that
upon administration of the peptide analogue in vivo proteolysis is
reduced.
41. The peptide analogue of claim 40 wherein the N-terminal and
C-terminal amino acids are D-amino acids.
42. The peptide analogue of claim 40 wherein the L-arginine at
position 97 is altered to a non-conservative amino acid.
43. The peptide analogue of claim 40 wherein residue 97 is altered
to D-arginine.
44. The peptide analogue of claim 40 wherein residue 97 is altered
to an amino acid selected from the group of D-alanine, D-arginine,
glycine, lysine, glutamine, glutamic acid, threonine, leucine,
phenylalanine, histidine and alanine.
45. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 91, wherein the L-lysine at position 91 is
altered to another amino acid.
46. The peptide analogue of claim 45 comprising seven to twelve
amino acids.
47. The peptide analogue of claim 45, further comprising altering
one to three additional residues selected from residues 86-90,
92-96, 98 and 99 to another amino acid.
48. The peptide analogue of claim 45 wherein L-lysine at position
91 is altered to a non-conservative amino acid.
49. The peptide analogue of claim 45 wherein residue 91 is altered
to D-lysine.
50. The peptide analogue of claim 45 wherein residue 91 is altered
to an amino acid selected from the group consisting of arginine,
asparagine, histidine, leucine, serine, glycine, glutamic acid,
phenylalanine, alanine and D-lysine.
51. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 95, wherein the L-threonine at position 95 is
altered to another amino acid.
52. The peptide analogue of claim 45, further comprising altering
one to three additional residues selected from residues 86-90,
92-94, and 96-99 to another amino acid.
53. The peptide analogue of claim 45, further comprising altering
one to three additional residues selected from residues 86-94, 96,
98 and 99 to another amino acid.
54. A peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein,
including residue 97, wherein the L-arginine at position 97 is
altered to another amino acid.
55. The peptide analogue of claim 45, further comprising altering
one to three additional residues selected from residues 86-90,
92-96, 98 and 99 to another amino acid.
56. A pharmaceutical composition comprising a peptide analogue
according to any one of claims 1, 11, 21, 30, 35, 40, 45, 51, and
54 in combination with a physiologically acceptable carrier or
diluent.
57. A method of treating multiple sclerosis, comprising:
administering to a patient a therapeutically effective amount of a
pharmaceutical composition comprising a peptide analogue according
to any one of claims 1, 11, 21, 30, 35, 40, 45, 51, and 54 in
combination with a physiologically acceptable carrier or
diluent.
58. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 91 is
alanine, residue 88 is D-histidine and residue 99 is D-proline.
59. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 91 is
alanine, residue 87 is D-valine and residue 99 is D-proline.
60. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 91 is
alanine and residue 88 is D-histidine.
61. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 91 is
alanine and residue 87 is D-valine.
62. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 91 is
alanine and residue 99 is D-proline.
63. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 95 is
alanine, residue 87 is D-valine, and residue 99 is D-proline.
64. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 95 is
alanine, residue 88 is D-histidine and residue 99 is D-proline.
65. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 95 is
alanine and residue 88 is D-histidine.
66. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 95 is
alanine and residue 99 is D-proline.
67. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 95 is
alanine and residue 87 is D-histidine.
68. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 97 is
alanine, residue 87 is D-valine, and residue 99 is D-proline.
69. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 97 is
alanine, residue 88 is D-histidine and residue 99 is D-proline.
70. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 97 is
alanine and residue 87 is D-valine.
71. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 97 is
alanine and residue 88 is D-histidine.
72. The method of claim 57 wherein the peptide analogue comprises
14 amino acids selected from residues 86 to 90 and residue 97 is
alanine and residue 99 is D-proline.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to methods for
treating multiple sclerosis by using peptide analogues of human
myelin basic protein.
BACKGROUND OF THE INVENTION
[0002] Multiple sclerosis (MS) is a chronic, inflammatory disease
that affects approximately 250,000 individuals in the United
States. Although the clinical course may be quite variable, the
most common form is manifested by relapsing neurological deficits,
in particular, paralysis, sensory deficits, and visual
problems.
[0003] The inflammatory process occurs primarily within the white
matter of the central nervous system and is mediated by T
lymphocytes, B lymphocytes, and macrophages. These cells are
responsible for the demyelination of axons. The characteristic
lesion in MS is called the plaque due to its macroscopic
appearance.
[0004] Multiple sclerosis is thought to arise from pathogenic T
cells that somehow evaded mechanisms establishing self-tolerance,
and attack normal tissue. T cell reactivity to myelin basic protein
may be a critical component in the development of MS. The
pathogenic T cells found in lesions have restricted heterogeneity
of antigen receptors (TCR). The T cells isolated from plaques show
rearrangement of a restricted number of V.alpha. and V.beta. gene
segments. In addition, the TCRs display several dominant amino acid
motifs in the third complementarity determining region (CDR), which
is the major antigen contact site. All together, three CDR3 motifs
have been identified in T cell clones known to recognize an epitope
within amino acids 86-106 of myelin basic protein. These motifs
were found in 44% of rearranged TCR sequences involving one
particular VP gene rearranged in T cells isolated from brain of two
patients with MS.
[0005] A definitive treatment for MS has not been established.
Historically, corticosteroids and ACTH have been used to treat MS.
Basically, these drugs reduce the inflammatory response by toxicity
to lymphocytes. Recovery may be hastened from acute exacerbations,
but these drugs do not prevent future attacks or prevent
development of additional disabilities or chronic progression of MS
(Carter and Rodriguez, Mayo Clinic Proc. 64:664, 1989; Weiner and
Hafler, Ann. Neurol. 23:211, 1988). In addition, the substantial
side effects of steroid treatments make these drugs undesirable for
long-term use.
[0006] Other toxic compounds, such as azathioprine, a purine
antagonist, cyclophosphamide, and cyclosporine have been used to
treat symptoms of MS. Like corticosteroid treatment, these drugs
are beneficial at most for a short term and are highly toxic. Side
effects include increased malignancies, leukopenias, toxic
hepatitis, gastrointestinal problems, hypertension, and
nephrotoxicity (Mitchell, Cont. Clin. Neurol. 77:231, 1993; Weiner
and Hafler, supra). Antibody based therapies directed toward T
cells, such as anti-CD4 antibodies, are currently under study for
treatment of MS. However, these agents may cause deleterious side
effects by immunocompromising the patient.
[0007] More recently, cytokines such as IFN-.gamma. and IFN-.beta.
have been administered in attempts to alleviate the symptoms of MS.
However, a pilot study involving IFN-.gamma. was terminated because
7 of 18 patients treated with this drug experienced a clinical
exacerbation within one month after initiation of treatment.
Moreover, there was an increase in the specific response to MBP
(Weiner and Hafler, supra).
[0008] Betaseron, a modified beta interferon, has recently been
approved for use in MS patients. Although Betaseron treatment
showed some improvement in exacerbation rates (Paty et al.,
Neurology 43:662, 1993), there was no difference in the rate of
clinical deterioration between treated and control groups (IFNB MS
Study Group, Neurology 43:655, 1993; Paty et al., supra). Side
effects were commonly observed. The most frequent of such side
effects were fever (40%-58% of patients), flu-like symptoms (76% of
patients), chills (46% of patients), mylagias (41% of patients),
and sweating (23% of patients). In addition, injection site
reactions (85%), including inflammation, pain, hypersensitivity and
necrosis, were common (IFNB MS Study Group, supra; Connelly, Annals
of Pharm. 28:610, 1994).
[0009] In view of the problems associated with existing treatments
of MS, there is a compelling need for improved treatments which are
more effective and are not associated with such disadvantages. The
present invention exploits the use of peptide analogues which
antagonize a T cell response to human myelin basic protein to
effectively treat MS, while providing other related advantages.
SUMMARY OF THE INVENTION
[0010] The present invention provides peptide analogues comprising
at least 7 amino acids selected from residues 86 to 99 of human
myelin basic protein in which either L-lysine at position 91,
L-threonine at position 95, or L-arginine at position 97 is altered
to another amino acid. In one embodiment, L-lysine at position 91
is altered and one to three additional L-amino acids selected from
residues 86, 87, 88, 95, 98 or 99 are altered to another amino
acid. In a second embodiment, L-threonine at position 95 is altered
and one to three additional amino acids selected from residues 86,
87, 88, 91, 98 and 99 or 86, 87, 88, 97, 98, and 99 are altered to
another amino acid. In a third related embodiment, L-arginine at
position 97 is altered and one to three additional amino acids
selected from residues 86, 87, 88, 95, 98 or 99 are altered to
another amino acid. The peptide analogues preferably contain double
or triple alterations. In preferred aspects of the invention, the
peptide analogues have altered residues 91, 95 or 97 to alanine and
the additional amino acids are altered to the corresponding D-form
amino acid.
[0011] In other embodiments, peptide analogues comprise at least
seven amino acids selected from residues 86 to 99 of human myelin
basic protein in which either L-lysine at position 91, L-threonine
at position 95, or L-arginine at position 97 is altered to another
amino acid, and in addition the N-terminal and C-terminal amino
acids are altered in order to reduce proteolysis upon
administration of the peptide analogue. In a preferred aspect, the
N- and C-terminal amino acids are of the D-form.
[0012] In other embodiments, the peptide analogues comprise at
least seven amino acids selected from residues 86 to 99 of human
myelin basic protein in which either L-lysine at position 91,
L-threonine at position 95, or L-arginine at position 97 is altered
to another amino acid and in addition up to three other amino acid
alterations are made. Any residue within 86-99 may be altered
except that in a peptide analogue in which residue 91 is altered,
residue 97 may not be altered. Likewise, in a peptide analogue in
which residue 97 is altered, residue 91 may not be altered.
[0013] Other embodiments provide peptide analogues comprising at
least seven amino acids selected from residues 86 to 99 of human
myelin basic protein in which either L-lysine at position 91,
L-threonine at position 95, or L-arginine at position 97 is altered
to another amino acid. In preferred aspects, residue 91, 95 or 97
are altered to either alanine or the corresponding D-amino
acid.
[0014] Further aspects of the present invention provide a
pharmaceutical composition comprising a peptide analogue according
to the embodiments set out above in which the peptide analogue is
contained in a physiologically acceptable carrier or diluent.
[0015] Further aspects of the present invention provide methods of
treating multiple sclerosis by administering to a patient a
therapeutically effective amount of a pharmaceutical composition
comprising a peptide analogue comprising at least seven amino acids
selected from residues 86 to 99 of human myelin basic protein in
which residues 91, 95 or 97 are altered to another amino acid.
Additionally, one to three additional amino acids may be altered or
the N- and C-ends are altered to reduce proteolysis upon
administration.
[0016] These and other aspects of the invention will become evident
upon reference to the following detailed description and attached
drawings. In addition, various references are set forth below which
describe in more detail certain procedures or compositions. Each of
these references are incorporated herein by reference in their
entirety as if each were individually noted for incorporation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 depicts DNA and predicted amino acid sequence for
human myelin basic protein.
[0018] FIG. 2 depicts the response of draining lymph node cells
from Lewis rats immunized 9-10 days previously with MBP (87-99) to
10 .mu.M of MBP (87-99), medium, the unrelated peptide motilin, and
six different MBP analogues. A, alanine; k, D-lysine; t,
D-threonine; r, D-arginine.
[0019] FIG. 3 is a graph displaying the proliferative response of
the T cell line NBI to residue 91-substituted analogues of human
myelin basic protein (87-99). Ten different substitutions were
tested. The proliferative response of the rat T cell line in
response to concentrations of peptide analogues ranging from 0 to
150 .mu.M was determined. The extent of proliferation is shown as
counts per minute; standard errors of the mean were less than
.+-.10%. MOT, motilin, a peptide unrelated to MBP; MBP (87-99),
human myelin basic protein residues 87-99; K, lysine; R, arginine;
N, asparagine; H, histidine; L, leucine; S, serine; G, glycine; k,
D-Lysine; E, glutamic acid; F, phenylalanine; A, alanine.
[0020] FIG. 4 is a graph displaying the proliferative response of
the T cell line NBI to residue 95-substituted analogues of human
myelin basic protein (87-99). Ten different substitutions were
tested. The proliferative response of the rat T cell line in
response to concentrations of peptide analogues ranging from 0 to
150 .mu.M was determined. The extent of proliferation is shown as
counts per minute; standard errors of the mean were less than
.+-.10%. MOT, motilin, a peptide unrelated to MBP; MBP (87-99),
human myelin basic protein residues 87-99; T, threonine; A,
alanine; t, D-threonine; G, glycine; I, isoleucine; Y, tyrosine; Q,
glutamine; S, serine; K, lysine; E, glutamic acid; H,
histidine.
[0021] FIG. 5 is a graph displaying the proliferative response of
the T cell line NBI to residue 97-substituted analogues of human
myelin basic protein. Eleven different substitutions were tested.
The proliferative response of the T cells to concentrations of
peptide analogues ranging from 0 to 150 .mu.M was determined. The
extent of proliferation is displayed as counts per minute. MBP
87-99, myelin basic protein (87-99); R, arginine; a, D-alanine; r,
D-arginine; G, glycine; K, lysine; Q, glutamine; E, glutamic acid;
T, threonine; L, leucine; F, phenylalanine; H, histidine; A,
alanine.
[0022] FIG. 6 is a graph illustrating the ability of peptide
analogues of MBP to inhibit proliferation of rat T cells that are
reactive to MBP. The proliferative response of draining lymph node
cells from rats immunized with MBP (87-99) to 16.7, 50, or 150
.mu.M of each analogue, or 5 .mu.M of MBP (87-99) is displayed.
Analogues were added in the presence of 5 .mu.M MBP (87-99). The
extent of proliferation is shown as counts per minute. Controls
consisted of MBP (87-99) only at 5 .mu.M and medium only. h88/A91
refers to a representative peptide analogue of MBP (87-99) with
D-histidine at residue 88 and alanine at residue 91; h88/A91/p99
refers to another representative peptide analogue of MBP (87-99)
with D-histidine at 88, alanine at residue 91, and D-proline at
residue 99.
[0023] FIG. 7 is a graph demonstrating the inhibition of EAE
induction in Lewis rats following injection of MBP (87-99). Arrows
indicate days that either PBS (control) or h88/A91 peptide analogue
were administered. EAE was recorded as 0, no symptoms; 1, tail
paralysis; 2, hind limb weakness; 3, hind limb paralysis; 4, hind
and front limb paralysis.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0025] "Human myelin basic protein" ("MBP") refers to a protein
found in the cytoplasm of human oligodendroglial cells. The
nucleotide sequence and predicted amino acid sequence of human MBP
are presented in FIG. 1 (SEQ. ID Nos.______ and ______). Although
not depicted in FIG. 1, different molecular forms of human myelin
basic protein generated by differential splicing or
post-translational modification are also within the scope of this
invention.
[0026] "Peptide analogues" of myelin basic protein are at least 7
amino acids in length and contain at least one difference in amino
acid sequence between the analogue and native human myelin basic
protein, one of which is a difference at residue 91, 95 or 97.
Unless otherwise indicated, a named amino acid refers to the
L-form. An L-amino acid from the native peptide may be altered to
any other one of the 20 L-amino acids commonly found in proteins,
any one of the corresponding D-amino acids, rare amino acids, such
as 4-hydroxyproline, and hydroxylysine, or a non-protein amino
acid, such as .beta.-alanine and homoserine. Also included with the
scope of the present invention are amino acids which have been
altered by chemical means such as methylation (e.g.,
.alpha.-methylvaline), amidation of the C-terminal amino acid by an
alkylamine such as ethylamine, ethanolamine, and ethylene diamine,
and acylation or methylation of an amino acid side chain function
(e.g, acylation of the epsilon amino group of lysine). "Residue
91," "residue 95," and "residue 97" (also called position 91,
position 95, and position 97, respectively), refer to amino acids
91, 95, and 97 of human myelin basic protein as displayed in FIG. 1
or the amino acid at a comparative position. More specifically, the
numbering system for these residues relates to the amino acid
position within the native human protein, regardless of the length
of the peptide or the amino acid position within that peptide.
[0027] Peptide Analogues of Myelin Basic Protein
[0028] As noted above, the present invention provides peptide
analogues comprising at least 7 amino acids selected from residues
86-99 of human myelin basic protein and including an alteration of
the naturally occurring L-lysine at position 91, L-threonine at
position 95, or L-arginine at position 97, to another amino acid.
In one aspect, the peptide analogue includes additional alteration
of one to three L-amino acids at positions 86, 87, 88, 91, 95, 97,
98 and/or 99 of human myelin basic protein as long as 91 and 97 are
not both altered in the same peptide analogue. In another aspect,
the peptide analogue additionally has the N-terminal and C-terminal
residues altered to an amino acid such that proteolysis is reduced
upon administration to a patient compared to a peptide analogue
without these additional alterations. In a further aspect, the
peptide analogue of MBP comprises at least seven amino acids
selected from residues 86-99 and has one of the residues at
position 91, 95 or 97 altered to an amino acid not present in
native MBP protein. In addition to such single alterations, one to
three additional alterations of residues 86 to 99 may be made, as
long as residues 91 and 97 are not altered in the same peptide
analogue.
[0029] The peptide analogues are preferably 7 to 16 amino acids,
and usually not longer than 20 amino acids. Particularly preferred
peptide analogues are 14 aminoacids in length. Residues 91, 95, and
97, which are L-lysine, L-threonine, and L-arginine, respectively,
in the native human protein, are the key residues. Within the
subject invention, analogues must have an amino acid other than
L-lysine at position 91, an amino acid other than L-threonine at
position 95, or an amino acid other than L-arginine at position
97.
[0030] As noted above, any amino acid alteration at position 91 is
within the scope of this invention. Preferred peptide analogues
include alteration of L-lysine to any one of the following amino
acids: D-lysine, alanine, glycine, glutamic acid, phenylalanine,
arginine, asparagine, histidine, leucine or serine. These amino
acids include both conservative (similar charge, polarity,
hydrophobicity, and bulkiness) and non-conservative amino acids.
Although typically one might expect that only non-conservative
amino acid alterations would provide a therapeutic effect,
unexpectedly even conservative changes (e.g., arginine) greatly
affect the function of the peptide analogue as compared to the
native peptide. Such diversity of substitution is further
illustrated by the fact that the preferred amino acids noted above
are hydrophobic and hydrophilic, charged and uncharged, polar and
non-polar.
[0031] In addition, any amino acid substitution at residue 95 is
also within the scope of this invention. Preferred peptide
analogues contain alterations of L-threonine to any one of the
following amino acids: D-threonine, alanine, glycine, isoleucine,
tyrosine, glutamine, serine, lysine, glutamic acid and histidine.
Other preferred alterations are to non-conservative amino acids.
Particularly preferred alterations are to alanine or
D-threonine.
[0032] Similarly, any amino acid alteration at position 97 is
within the scope of this invention. Preferred peptide analogues
include alteration of L-arginine to D-alanine, D-arginine, glycine,
lysine, glutamine, glutamic acid, threonine, leucine,
phenylalanine, histidine or alanine. Other preferred alterations
are to non-conservative amino acids. Particularly preferred
alterations are to alanine and D-arginine.
[0033] In addition, in certain embodiments at least one other amino
acid selected from residues 86, 87, 88, 95, 98, or 99 is altered.
If two other amino acids are changed, one is preferably selected
from residues 86, 87, or 88, and the other is selected from
residues 98 or 99. Alternatively, up to three alterations at any
positions may be made.
[0034] With these general considerations in mind, peptide analogues
within the scope of the invention have an alteration of residue 91,
residue 95, or of residue 97. One set of preferred peptide
analogues have double alterations. In one embodiment, residue 91 is
altered as noted above, residue 87 is altered to D-valine, residue
88 to D-histidine or residue 99 to D-proline. Similarly, in another
embodiment, residue 97 is altered as noted above, and either
residue 87 is altered to D-valine, residue 88 to D-histidine or
residue 99 to D-proline. In yet another embodiment, residue 95 is
altered as noted above and residue 87 is altered to D-valine,
residue 88 to D-histidine or residue 99 to D-proline.
[0035] A second set of preferred peptide analogues have triple
substitutions. In one embodiment, residue 91 is altered to alanine,
residue 87 is altered to D-valine or residue 88 is altered to
D-histidine and residue 99 is altered to D-proline. In another
embodiment, residue 97 is altered to alanine, residue 88 is altered
to D-histidine and residue 99 to D-proline. In yet another
embodiment, residue 95 is altered to alanine, residue 88 is altered
to D-histidine and residue 99 to D-proline.
[0036] Peptide analogues may be synthesized by standard chemistry
techniques, including synthesis by automated procedure. In general,
peptide analogues are prepared by solid-phase peptide synthesis
methodology which involves coupling each protected amino acid
residue to a resin support, preferably a 4-methyl-benzhydrylamine
resin, by activation with dicyclohexylcarbodimide to yield a
peptide with a C-terminal amide. Alternatively, a chloromethyl
resin (Merrifield resin) may be used to yield a peptide with a free
carboxylic acid at the C-terminus. Side-chain functional groups are
protected as follows: benzyl for serine, threonine, glutamic acid,
and aspartic acid; tosyl for histidine and arginine;
2-chlorobenzyloxycarbonyl for lysine and 2,6-dichlorobenzyl for
tyrosine. Following coupling, the t-butyloxycarbonyl protecting
group on the alpha amino function of the added amino acid is
removed by treatment with trifluoroacetic acid followed by
neutralization with di-isopropyl-ethylamine. The next protected
residue is then coupled onto the free amino group, propagating the
peptide chain. After the last residue has been attached, the
protected peptide-resin is treated with hydrogen fluoride to cleave
the peptide from the resin, as well as deprotect the side chain
functional groups. Crude product can be further purified by gel
filtration, HPLC, partition chromatography, or ion-exchange
chromatography.
[0037] Peptide analogues within the present invention should (a)
compete for the binding of MBP (87-99) to MHC; (b) not cause
proliferation of an MBP (87-99)-reactive T cell line; and (c)
inhibit induction of experimental allergic encephalomyelitis (EAE)
by MBP (87-99) in rodents.
[0038] Thus, candidate peptide analogues may be screened for their
ability to treat MS by (1) an assay measuring competitive binding
to MHC, (2) an assay measuring a T cell proliferation, and (3) an
assay assessing induction inhibition of EAE. Those analogues that
inhibit binding of the native peptides, do not stimulate
proliferation of MBP-reactive cell lines, and inhibit the
development of EAE by native human MBP (87-99), are useful
therapeutics. Although not essential, a further safety assay may be
performed to demonstrate that the analogue does not itself induce
EAE.
[0039] Binding of peptides to MHC molecules may be assayed on whole
cells. Briefly, Lewis rat spleen cells are cultured for 3 hours to
allow adherent cells to stick to polystyrene petri dishes.
Non-adherent cells are removed. Adherent cells, which contain cells
expressing MHC class II molecules, are collected by scraping the
dishes. The binding of peptide analogues to cells is measured by a
fluorescence assay. In this assay, splenic adherent cells are mixed
with different concentrations of peptide analogues and incubated
for 1 hour at 37.degree. in a CO.sub.2 incubator. Following
incubation, biotin-labeled MBP (87-99) is added to the culture
wells. The cells are incubated for another hour and then washed
three times in medium. Phycoerythrin-conjugated or
fluorescein-conjugated streptavidin is added along with a
fluorochrome-labeled OX-6 or OX-17 monoclonal antibody, which
reacts with rat MHC Class II I-A and I-E, respectively. The cells
are washed twice before analysis by flow cytometry. Fluorescence
intensity is calculated by subtracting the fluorescence value
obtained from cells stained with phycoerythrin-streptavidin alone
(control staining) from the fluorescence value obtained from
biotin-labeled MBP (87-99) plus phycoerythrin-streptavidin
(experimental staining). Staining without analogue establishes a
100% value. Percent inhibition is calculated for each analogue and
expressed as IC.sub.50 values. A peptide analogue with an IC.sub.50
value of less than 100 .mu.M is suitable for further
screenings.
[0040] Candidate peptide analogues are further tested for their
property of causing or inhibiting proliferation of T cell lines.
Two different assays may be used as alternatives. The first
measures the ability of the analogue to cause proliferation of T
cells in a direct fashion. The second measures the ability of the
peptide analogue to inhibit proliferation of T cells induced by
native MBP (87-99) peptide.
[0041] In the direct proliferation assay, MBP (87-99) reactive T
cell lines may be used as target cells. T cell lines are
established from lymph nodes taken from rats injected with MBP
(87-99). Lymph node cells are isolated and cultured for 5 to 8 days
with MBP (87-99) and IL-2 as a source of T cell growth factors.
Viable cells are recovered and a second round of stimulation is
performed with MBP (87-99) and irradiated splenocytes as a source
of growth factors. After 5 to 6 passages in this manner, the
proliferative potential of the cell lines are determined.
MBP-reactive lines are used in the proliferation assay. In this
assay, T cell lines are cultured for three days with various
concentrations of peptide analogues and irradiated, autologous
splenocytes. After three days, 0.5-1.0 .mu.Ci of
[.sup.3H]-thymidine is added for 12-16 hours. Cultures are
harvested and incorporated counts determined. Mean CPM and standard
error of the mean are calculated from triplicate cultures.
[0042] As an alternative to the use of T cell lines as described
above, draining lymph node cells from Lewis rats injected with MBP
(87-99) may be used. Preferably, this assay is used in combination
with the proliferation assay using T cell lines. Briefly, Lewis
rats are injected subcutaneously with MBP (87-99) peptide in
complete Freund's adjuvant. Nine to ten days later, draining lymph
node cells are isolated and single-cell suspensions are prepared.
Lymph node cells are incubated with various concentrations of
peptide analogues for three days in a humidified air chamber
containing 6.5% CO.sub.2. After incubation, the cultures are pulsed
with 1-2 .mu.Ci of [.sup.3H]-thymidine for 12-18 hours. Cultures
are harvested on fiberglass filters and counted in a scintillation
counter. Mean CPM and the standard error of the mean are calculated
from data determined in triplicate cultures. Peptide analogues
yielding results that are more than three standard deviations of
the mean response with a comparable concentration of MBP (87-99)
are considered non-stimulatory. Peptide analogues which do not
stimulate proliferation at concentrations of less than or equal to
50 .mu.M are suitable for further screenings.
[0043] The second or alternative assay is a competition assay for T
cell proliferation. In this assay, antigen presenting spleen cells
are first irradiated and then incubated with native MBP (87-99)
peptide for 24 hours. These cells are then washed and further
cultured with T cells reactive to MBP (87-99). Various
concentrations of candidate peptide analogues are included in
cultures for an additional 3 days. Following this incubation
period, each culture is pulsed with 1 .mu.Ci of [.sup.3H]-thymidine
for an additional 12-18 hours. Cultures are then harvested on
fiberglass filters and counted as above. Mean CPM and standard
error of the mean are calculated from data determined in triplicate
cultures. Peptide analogues which inhibit proliferation to
approximately 25% at a concentration of 50 .mu.M or greater are
suitable for further screening.
[0044] Candidate peptides that compete for binding of MBP (87-99)
to MHC and do not cause direct proliferation of T cell line or can
inhibit proliferation by MBP (87-99), are further tested for their
ability to inhibit the induction of EAE by MBP (87-99). Briefly,
500 .mu.g of MBP (87-99) is injected as an emulsion in complete
Freund's adjuvant supplemented with heat killed Mycobacterium
tuberculosis (H37Ra). Rats are injected subcutaneously at the base
of the tail with 200 .mu.l of the emulsion. Rats are divided into
two groups. Approximately 2 days prior to disease induction
(usually 10 days following injection of MBP (87-99)) rats are
injected intraperitoneally either with PBS or peptide analogues in
PBS. Animals are monitored for clinical signs on a daily basis by
an observer blind to the treatment protocol. EAE is scored on a
scale of 0-4: 0, clinically normal; 1, flaccid tail paralysis; 2,
hind limb weakness; 3, hind limb paralysis; 4, front and hind limbs
affected. Peptide analogues injected at 5 mg/kg or less
(approximately 1 mg per rat) are considered to inhibit the
development of EAE if there is a 50% reduction in the mean
cumulative score over seven days following onset of disease
symptoms in the control group.
[0045] In addition, as a safety measure, but not essential to this
invention, suitable peptide analogues may be tested for direct
induction of EAE. As described in detail in Example 2, various
amounts of peptide analogues are injected at the base of the tail
of rats, and the rats examined daily for signs of EAE. A peptide
analogue which is not considered to cause EAB has a mean cumulative
score of less than or equal to 1 over seven days when 1 mg (5
mg/kg) in complete Freund's adjuvant is injected.
[0046] Treatment and Prevention of Multiple Sclerosis
[0047] As noted above, the present invention provides methods for
treating and preventing multiple sclerosis by administering to the
patient a therapeutically effective amount of a peptide analogue of
human myelin basic protein as described herein. Patients suitable
for such treatment may be identified by criteria establishing a
diagnosis of clinically definite MS as defined by the workshop on
the diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983).
Briefly, an individual with clinically definite MS has had two
attacks and clinical evidence of either two lesions or clinical
evidence of one lesion and paraclinical evidence of another,
separate lesion. Definite MS may also be diagnosed by evidence of
two attacks and oligoclonal bands of IgG in cerebrospinal fluid or
by combination of an attack, clinical evidence of two lesions and
oligoclonal band of IgG in cerebrospinal fluid. Slightly lower
criteria are used for a diagnosis of clinically probable MS.
[0048] Effective treatment of multiple sclerosis may be examined in
several different ways. Satisfying any of the following criteria
evidences effective treatment. Three main criteria are used: EDSS
(extended disability status scale), appearance of exacerbations or
MRI (magnetic resonance imaging).
[0049] The EDSS is a means to grade clinical impairment due to MS
(Kurtzke, Neurology 33:1444, 1983). Eight functional systems are
evaluated for the type and severity of neurologic impairment.
Briefly, prior to treatment, patients are evaluated for impairment
in the following systems.: pyramidal, cerebella, brainstem,
sensory, bowel and bladder, visual, cerebral, and other. Follow-ups
are conducted at defined intervals. The scale ranges from 0
(normal) to 10 (death due to MS). A decrease of one full step
defines an effective treatment in the context of the present
invention (Kurtzke, Ann. Neurol. 36:573-79, 1994).
[0050] Exacerbations are defined as the appearance of a new symptom
that is attributable to MS and accompanied by an appropriate new
neurologic abnormality (IFNB MS Study Group, supra). In addition,
the exacerbation must last at least 24 hours and be preceded by
stability or improvement for at least 30 days. Briefly, patients
are given a standard neurological examination by clinicians.
Exacerbations are either mild, moderate, or severe according to
changes in a Neurological Rating Scale (Sipe et al., Neurology
34:1368, 1984). An annual exacerbation rate and proportion of
exacerbation-free patients are determined. Therapy is deemed to be
effective if there is a statistically significant difference in the
rate or proportion of exacerbation-free patients between the
treated group and the placebo group for either of these
measurements. In addition, time to first exacerbation and
exacerbation duration and severity may also be measured. A measure
of effectiveness as therapy in this regard is a statistically
significant difference in the time to first exacerbation or
duration and severity in the treated group compared to control
group.
[0051] MRI can be used to measure active lesions using
gadolinium-DTPA-enhanced imaging (McDonald et al. Ann. Neurol.
36:14, 1994) or the location and extent of lesions using
T.sub.2-weighted techniques. Briefly, baseline MRIs are obtained.
The same imaging plane and patient position are used for each
subsequent study. Positioning and imaging sequences are chosen to
maximize lesion detection and facilitate lesion tracing. The same
positioning and imaging sequences are used on subsequent studies.
The presence, location and extent of MS lesions are determined by
radiologists. Areas of lesions are outlined and summed slice by
slice for total lesion area. Three analyses may be done: evidence
of new lesions, rate of appearance of active lesions, percentage
change in lesion area (Paty et al., Neurology 43:665, 1993).
Improvement due to therapy is established when there is a
statistically significant improvement in an individual patient
compared to baseline or in a treated group versus a placebo
group.
[0052] Candidate patients for prevention may be identified by the
presence of genetic factors. For example, a majority of MS patients
have HLA-type DR2a and DR2b. The MS patients having genetic
dispositions to MS who are suitable for treatment fall within two
groups. First are patients with early disease of the relapsing
remitting type. Entry criteria would include disease duration of
more than one year, EDSS score of 1.0 to 3.5, exacerbation rate of
more than 0.5 per year, and free of clinical exacerbations for 2
months prior to study. The second group would include people with
disease progression greater than 1.0 EDSS unit/year over the past
two years.
[0053] Efficacy of the peptide analogue in the context of
prevention is judged based on the following criteria: frequency of
MBP reactive T cells determined by limiting dilution, proliferation
response of MBP reactive T cell lines and clones, cytokine profiles
of T cell lines and clones to MBP established from patients.
Efficacy is established by decrease in frequency of reactive cells,
a reduction in thymidine incorporation with altered peptide
compared to native, and a reduction in TNF and IFN-.alpha..
Clinical measurements include the relapse rate in one and two year
intervals, and a change in EDSS, including time to progression from
baseline of 1.0 unit on the EDSS which persists for six months. On
a Kaplan-Meier curve, a delay in sustained progression of
disability shows efficacy. Other criteria include a change in area
and volume of T2 images on MRI, and the number and volume of
lesions determined by gadolinium enhanced images.
[0054] Peptide analogues of the present invention may be
administered either alone, or as a pharmaceutical composition.
Briefly, pharmaceutical compositions of the present invention may
comprise one or more of the peptide analogues described herein, in
combination with one or more pharmaceutically or physiologically
acceptable carriers, diluents or excipients. Such compositions may
comprise buffers such as neutral buffered saline, phosphate
buffered saline and the like, carbohydrates such as glucose,
mannose, sucrose or dextrans, mannitol, proteins, polypeptides or
amino acids such as glycine, antioxidants, chelating agents such as
EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and
preservatives. In addition, pharmaceutical compositions of the
present invention may also contain one or more additional active
ingredients, such as, for example, cytokines like
.beta.-interferon.
[0055] Compositions of the present invention may be formulated for
the manner of administration indicated, including for example, for
oral, nasal, venous, intracranial, intraperitoneal, subcutaneous,
or intramuscular administration. Within other embodiments of the
invention, the compositions described herein may be administered as
part of a sustained release implant. Within yet other embodiments,
compositions of the present invention may be formulized as a
lyophilizate, utilizing appropriate excipients which provide
stability as a lyophilizate, and subsequent to rehydration.
[0056] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease. Within particularly
preferred embodiments of the invention, the peptide analogue or
pharmaceutical compositions described herein may be administered at
a dosage ranging from 5 to 50 mg/kg, although appropriate dosages
may be determined by clinical trials. Patients may be monitored for
therapeutic effectiveness by MRI, EDSS, and signs of clinical
exacerbation, as described above.
[0057] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLE 1
Preparation of Peptides
[0058] The peptides were synthesized by solid phase methodology on
a peptide synthesizer (Beckman model 990). Peptides with an
amidated carboxyl-terminus were prepared with a
p-methylbenzhydrylamine resin (MBHA resin); for peptides with a
free carboxyl-terminus, a Merrifield resin coupled with the
appropriately protected amino acid was used. Both resins were
obtained from Bachem Fine Chemicals (Torrance, Calif.). Derivatized
amino acids (Bachem Fine Chemicals) used in the synthesis were of
the L-configuration unless specified otherwise, and the
N-alpha-amino function protected exclusively with the
t-butyloxycarbonyl group. Side-chain functional groups were
protected as follows: benzyl for serine, threonine, glutamic acid,
and aspartic acid; tosyl for histidine and arginine;
2-chlorobenzyloxycarbonyl for lysine and 2,6-dichlorobenzyl for
tyrosine. Coupling of the carboxyl-terminal amino acid to the MBHA
resin was carried out with dicyclohexylcarbodiimide and the
subsequent amino acids were coupled with dicyclohexylcarbodiimide
according to Ling et al. (Proc. Natl. Acad. Sci. USA 81:4302,
1984). After the last amino acid was incorporated, the
t-butyoxycarbonyl protecting group was removed and the
peptide-resin conjugate treated with a mixture of 14 ml
hydrofluoric acid (HF), 1.4 ml anisole, and 0.28 ml methylethyl
sulfide per gram of resin conjugate at -20.degree. C. for 0.5 hr
and at 0.degree. C. for 0.5 hr. HF was removed in vacuum at
0.degree. C., and the resulting peptide and resin mixture was
washed twice with diethyl ether and twice with chloroform and
diethyl ether alternately. The peptide was extracted five times
with 2 M acetic acid, and the extract lyophilized. The lyophilized
product was first purified on a column of Sephadex G-25 fine
(Pharmacia-LKB, Piscataway, N.J.) developed in 30% acetic acid to
remove the truncated fragments and inorganic salts (Ling et al.,
1984). Next, peptides were further purified by CM-32
carboxymethylcellulose cation-exchange chromatography (Ling et al.,
1984). Final purification was achieved by partition chromatography
on Sephadex G-25 fine (Ling et al., 1984). The synthetic product
was characterized by amino acid analysis, mass spectrometric
analysis, and reversed-phase HPLC.
EXAMPLE 2
Immunizations and EAE Induction
[0059] MBP peptide and peptide analogues were dissolved in
phosphate-buffered saline (PBS) and emulsified with an equal volume
of incomplete Freund's adjuvant supplemented with 4 mg/ml
heat-killed Mycobacterium tuberculosis H37Ra in oil (Difco
Laboratories, Inc., Detroit, Mich.). Rats were immunized
subcutaneously at the base of the tail with 0.2 ml containing 500
.mu.g of peptide in the emulsion and were monitored for clinical
signs daily. EAE was scored on a scale of 0-4, as follows: 0,
clinically normal; 1, flaccid tail; 2, hind limb weakness; 3, hind
limb paralysis; 4, front and hind limbs affected.
EXAMPLE 3
Long-term T Cell Lines
[0060] Antigen specific long-term T cell lines were derived using
the method developed by Ben-Nun et al. (Eur. J Immunol. 11:195,
1981). Lewis rats were injected with MBP (87-99) as described
above. Nine to ten days later draining lymph node cells were
cultured (10.sup.7/ml) for 5-8 days in stimulation medium together
with 10-20 .mu.M of the MBP (87-99) peptide and 15 .mu./ml IL-2.
After 5 to 8 days of culture, viable cells were collected after
Ficoll-Hypaque separation and washed three times. These cells were
recultured at 1.times.10.sup.7 cells/ml in medium with
5.times.10.sup.5 irradiated (3000 rad) autologous splenocytes as
accessory cells and 10-20 .mu.M of MBP (87-99). After 5 to 6
stimulation cycles, plates were screened by the ability of cells to
proliferate in response to MBP (87-99). Positive lines were
transferred to 24-well flat bottom plates and restimulated.
EXAMPLE 4
MHC Binding Assay
[0061] The ability of MBP peptides and peptide analogues to bind
MHC was measured. An assay which characterizes the binding of
peptides to MHC molecules on antigen presenting cells (APC) was
employed (Mozes et al., EMBO J. 8:4049, 1989; Gautam et al., PNAS
91:767, 1994). Spleen cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (Hyclone
Laboratories, Logan, Utah) in standard polystyrene petri dishes
(100.times.15 mm) in a 37.degree. C. incubator containing 6.5%
CO.sub.2 for 3 hours. Thereafter, non-adherent cells were removed,
and the plates were washed three times with PBS. Adherent cells
were collected using a cell scraper. The binding of MBP (87-99)
analogues was measured using a fluorescence assay. Briefly,
5.times.10.sup.5 splenic adherent cells in staining buffer (PBS
containing 0.1% bovine serum albumin) were mixed with different
concentrations ranging from 0-400 .mu.M of MBP (87-99) analogues in
individual wells of U-shape 96-well microculture plates and
incubated for 1 hr at 37.degree. C. in a 6.5% CO.sub.2 incubator.
Following incubation, 10 .mu.M of biotin-labeled MBP (87-99) was
added to culture wells for 1 h. Cells were washed three times with
the staining buffer. Phycoerythrin-conjugated or
fluoroscein-conjugated streptavidin (Becton Dickinson, San Jose,
Calif.) was added as a second step reagent (1 .mu.g/well) along
with 1 .mu.g/well of fluorochrome-labeled OX-6 or OX-17 monoclonal
antibody (Pharmingen, San Diego, Calif.), which reacts with rat MHC
class II I-A or I-E, respectively. The cells were washed twice
before cytofluorographic analysis on a FACScan (Becton Dickinson).
Fluorescence intensity for each sample was calculated by
subtracting the fluorescence obtained from OX positive cells
stained with phycoerythrin-streptavidin alone (control staining)
from the fluorescence obtained from OX positive cells stained with
biotin-labeled MBP (87-99) plus phycoerythrin-streptavidin. Percent
inhibition was calculated for each analogue and expressed as
IC.sub.50 values.
[0062] The peptide analogue, h88/A91, which contains D-histidine at
position 88 and alanine at position 91 competed as effectively as
MBP (87-99) for MHC against MBP (87-99). At 200 .mu.M, MBP (87-99)
inhibited binding by 68.4% and h88/A91 inhibited binding by
67.64%.
EXAMPLE 5
Antigen-specific Lymph Node Cell Proliferation Assay
[0063] Female Lewis rates, approximately six weeks old, were
purchased from Harlan Sprague, Indianapolis, Ind. MBP peptides were
dissolved in phosphate-buffered saline (PBS) and emulsified with an
equal volume of complete Freund's adjuvant (Difco Laboratories,
Inc., Detroit, Mich.) supplemented with 2 mg/ml of heat-killed
Myobacterium tuberculosis H37Ra in oil (Difco). Rats were immunized
subcutaneously in the base of the tail with 0.1 ml containing 100
.mu.g of the peptide in the emulsion. Nine to ten days following
immunization, rats were sacrificed, their draining lymph node
removed and a single cell suspension made. Cells were resuspended
to 5.times.10.sup.6 cells per ml in stimulation medium containing
Dulbecco's modified Eagle's medium (Gibco BRL, Gaithersburg, Md.)
supplemented with 2 mercaptoethanol (5.times.10.sup.-5 M),
L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100
.mu.g/ml), streptomycin (100 .mu.g/ml), and 1% normal rat
serum.
[0064] For the assay, 100 .mu.l of the lymph node cell suspension
was added to 96-well flat-bottom wells in the presence of an equal
volume of medium containing 10 .mu.M of various peptides
(including: motilin as a negative control; MBP87-99; medium only or
alanine or D-amino acid substituted at position 91, 95, or 97).
Cultures were then incubated at 37.degree. C. in humidified air
containing 7.5% CO.sub.2. After 3 days of incubation, 1.0 .mu.Ci of
tritiated thymidine (20 Ci/mM; New England Nuclear) was added to
each well and the plates reincubated for an additional 12-16 hours.
The plates were then harvested with a Matrix filtermate harvester
(Packard) and counted using an Automatic Direct Beta Counter
(Packard). Mean cpm and the standard error of the mean were
calculated from triplicate wells.
[0065] As seen in FIG. 2, MBP (87-99) stimulated lymph node cells
in contrast to the peptide analogues. Alanine alterations at
positions 95 and 97 and D-amino acid alterations at residues 91,
95, and 97 failed to stimulate cells above the control peptide,
motilin.
EXAMPLE 6
Antigen-specific T Cell Line Proliferation Assays
[0066] Assays for the antigen-specific proliferation assay of T
cell lines were performed in 96-well flat bottom microtiter plates
as described (Zamvil et al., 1985; Offner et al., 1992; Gold et
al., 1992). T cell lines were established as described in Example
3. An initial 1:10 dilution of a 1.5 mM stock solution of MBP or
the peptide analogues were added into tissue culture medium. The
samples were diluted by threefold serial dilutions (final volume
100 .mu.l). The responding continuous T cell lines were resuspended
to 4.times.10.sup.5 cells per ml and 50 .mu.l aliquots added to
each well (5.times.10.sup.4 cells per well). Approximately
1.times.10.sup.6 irradiated (3000R) splenocyte feeder cells were
also added to each well. Cultures were then incubated at 37.degree.
C. in humidified air containing 7.5% CO.sub.2 for 3 days. Twelve to
sixteen hours prior to harvesting, 0.5-1.0 .mu.Ci of
[.sup.3H]-thymidine (20 Ci/mM; New England Nuclear) was added to
each well and the cultures reincubated. Plates were then harvested
with a Matrix filtermate harvester (Packard) and counted using an
Automatic Direct Beta Counter (Packard). Mean cpm and the standard
error of the mean were calculated from triplicate wells.
[0067] As can be seen in FIGS. 3, 4, and 5 a peptide analogue with
any substitution of position 91, 95, or 97 failed to stimulate
proliferation of a MBP (87-99)-reactive T cell line. The effect was
dramatic as even 150 .mu.M of peptide analogue was 1 to 2 logs less
effective at causing proliferation.
EXAMPLE 7
Antagonism of T Cell Proliferation Assay
[0068] T cell antagonism was detected in a prepulsed proliferation
assay as described by De Magistris et al. (Cell 58:625, 1992) with
minor modifications. Antigen presenting spleen cells were
.gamma.-irradiated (3000 rad) and incubated with shaking at a
concentration of 107 cells/well with 0.2-2.0 .mu.M of the native
peptide MBP (87-99) in stimulation medium in 10 ml tissue culture
plates for 2.5 hours at 37.degree. C. in humidified air containing
6.5% CO.sub.2. Spleen cells were then washed and re-cultured at a
concentration of 5.times.10.sup.5 cells/well in U-shape 96-well
microculture plates together with 5.times.10.sup.4 resting MBP
(87-99) reactive T cells. Various concentrations of antagonist
peptides, ranging from 5-150 .mu.M, were added for an additional 72
hours. Each well was pulsed with 0.5-1 .mu.Ci of
[.sup.3H]-thymidine (specific activity 10 Ci/mmol) for the final
12-16 hours. The cultures were then harvested on fiberglass filters
and the proliferative response expressed as CPM.+-.SEM.
[0069] The data presented in FIG. 6 demonstrates that the double
altered peptide analogue, h88/A91, and the triple altered peptide
analogue, h88/A91/p99, significantly inhibited proliferation of a
MBP reactive T cell line. The triple altered analogue caused
inhibition at 50 .mu.M and higher concentration, while the double
altered analogue caused inhibition at 150 .mu.M.
EXAMPLE 8
Treatment of 87-99 Induced EAE in Lewis Rats
[0070] Female Lewis rats, which were 6-8 weeks old, were injected
with 500 .mu.g of MBP (87-99) in CFA containing 500 .mu.g of
Mycobacterium tuberculosis at the base of the tail in 200 .mu.l
volume. Rats were divided in groups of 5. The control group
received 0.5 ml of PBS and the treatment group received the h88/A91
peptide analogue (1 mg/0.5 ml PBS) intraperitoneally, twice, on
days 9 and 10 after immunization. Animals were monitored for
disease symptoms on a daily basis. EAE was recorded on the
following scale: 0, no symptoms; 1, tail paralysis; 2, hind limb
weakness; 3, hind limb paralysis; 4, hind and front limbs
affected.
[0071] Data from two different experiments was obtained as mean
cumulative score of 5 animals (FIG. 7). Untreated control animals
went on to develop high level of disease whereas h88/A91 analogue
of the MBP peptide 87-99 was effective in preventing significantly
the development of EAE in two experiments. Though the analogue was
given just before the onset of overt symptoms, it was able to
arrest the development of EAE.
EXAMPLE 9
Induction of EAE by Peptide Analogue
[0072] The ability of peptide analogues to cause EAE is assessed in
vivo. Rats were injected with MBP (87-99) or h88/A91 peptide
analogue as described in Example 2. Animals were monitored daily
for evidence of EAE. Rats receiving MBP (87-99) had 100% incidence
(18/18 rats) of EAE with a mean maximum clinical score of
2.4.+-.0.2. In contrast, 0/12 rats receiving the peptide analogue
h88/A91 had EAE. Therefore, this peptide analogue does not induce
EAE.
[0073] From the foregoing, it will be evident that although
specific embodiments of the invention have been described herein
for the purpose of illustrating the invention, various
modifications may be made without deviating from the spirit and
scope of the invention.
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