U.S. patent application number 08/425175 was filed with the patent office on 2002-03-28 for synthetic peptide for treatment of autoimmune arthritis.
Invention is credited to KANG, ANDREW H., MYERS, LINDA K., SEYER, JEROME M..
Application Number | 20020037844 08/425175 |
Document ID | / |
Family ID | 21826827 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037844 |
Kind Code |
A1 |
MYERS, LINDA K. ; et
al. |
March 28, 2002 |
SYNTHETIC PEPTIDE FOR TREATMENT OF AUTOIMMUNE ARTHRITIS
Abstract
Peptides for suppressing autoimmune arthritis by disrupting
formation of trimolecular complexes which stimulate T cells.
Inventors: |
MYERS, LINDA K.; (MEMPHIS,
TN) ; SEYER, JEROME M.; (MEMPHIS, TN) ; KANG,
ANDREW H.; (MEMPHIS, TN) |
Correspondence
Address: |
HOWARD M. EISENBERG,ESQ.
CHERNOFF,VILHAUER,MCCLUNG & STENCEL,LLP
1600 ODS TOWER
601 S.W. SECOND AVENUE
PORTLAND
OR
972043157
|
Family ID: |
21826827 |
Appl. No.: |
08/425175 |
Filed: |
April 20, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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08425175 |
Apr 20, 1995 |
|
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08025570 |
Mar 3, 1993 |
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Current U.S.
Class: |
514/16.6 ;
514/825; 530/326; 530/327; 530/328; 530/329; 530/806 |
Current CPC
Class: |
Y10S 514/825 20130101;
A61K 38/00 20130101; C07K 14/78 20130101; Y10S 424/81 20130101;
Y10S 530/806 20130101 |
Class at
Publication: |
514/12 ; 530/326;
530/327; 530/328; 530/329; 530/806; 514/13; 514/14; 514/15; 514/16;
514/17; 514/825 |
International
Class: |
A61K 038/00; C07K
005/00; C07K 007/00; C07K 016/00; C07K 017/00; A61K 038/04 |
Claims
What is claimed is:
1. A peptide which suppresses autoimmune arthritis comprising an
analog of a CII peptide fragment having a T-cell epitope, wherein
the analog peptide disrupts formation of trimolecular complexes of
autoimmune antigenic peptide, MHC and T-cell receptor and does not
provoke a material immunogenic response.
2. The peptide as recited in claim 1 wherein the analog is an
ananlog of CII 245-270.
3. The peptide as recited in claim 2 wherein the analog is an
analog of CII 260-270 peptide.
4. The peptide as recited in claim 2 wherein the analog peptide is
CII 245-270 [s260, 261, 263].
5. A peptide which suppresses autoimmune arthritis by disrupting
formation of trimolecular complexes of autoimmune antigenic
peptide, MHC and T cell receptors comprising:
12 260 265 Ala - 4Hyp - Gly - Asn - Lys - Gly -;
6. A peptide which suppresses autoimmune arthritis by disrupting
formation of trimolecular complexes of autoimmune antigenic
peptide, MHC and T cell receptors comprising:
13 260 265 270 Ala - 4Hyp - Gly - Asn - Lys - Gly - Glu- Gln - Gly
- Pro - Lys.
7. A peptide as recited in claim 6 comprising:
14 245 250 Pro - Thr - Gly - Pro - Leu - Gly - Pro - Lys - Gly- 255
260 Gln - Thr - Gly - Glx - Leu - Gly 265 4Hyp - Gly - Asn - Lys -
Gly - Glx - Gln - 270 Gly - Pro - Lys.
8. A peptide comprising:
15 245 250 Pro - Thr - Gly - Pro - Leu - Gly - Pro - Lys - Gly -
255 260 Gln - Thr - Gly - Glx - Leu - Gly - Ala - 265 4Hyp - Gly -
Asn - Lys - Gly - Glx - Gln - 270 Gly - Pro - Lys.
Description
FIELD OF THE INVENTION
[0001] The present invention provides peptides for suppressing
autoimmune arthritis that do not provoke a material immunogenic
response from T cells.
BACKGROUND OF THE INVENTION
[0002] Autoimmune arthritis afflicts a large number of people and
takes many forms including, rheumatoid arthritis, juvenile
arthritis, psoriatic arthritis, spondylo arthritis, relapsing
polychondritis and other connective tissue diseases. These
arthritic conditions occur in mammals when T cells are activated by
particular antigens or complexes containing antigens. When such
activation occurs, proteolytic enzymes are produced which degrade
tissues of the person or mammal afflicted by arthritis. The tissue
targets of autoimmune arthritis are constituents of connective
tissues in joints and tendons of mammals and ordinarily include
type II collagen. Indeed autoimmune arthritis can be induced in
mice, humans and other mammal by immunizing them with type collagen
II derived from cartilage of the same or different mammals. See,
Andriopoulos N A, Mestecky J. Miller E J, Bradley E L: Antibodies
to native and denatured collagen in sera of patients with
rheumatoid arthritis. Arth. Rheum. 19:613-617, 1976; Wooley P H,
Luthra S. Singh S. Huse A, Stuart J M, David C S: Passive transfer
of arthritis in mice by human anti-type II collagen antibody. Mayo
Clinic Proc. 59:737-743, 1984.
[0003] Autoimmune arthritis in mammals develops when T cells are
activated by immunogenic complexes referred to as trimolecular
complexes. These complexes are formed between antigenic peptides
and major histocompatibility complex molecules (MHC). Buus, S., A.
Sette, and H. M. Grey, (1987) "The interaction between
protein-derived immunogenic peptides and Ia". Immuno. Rev. 98:115.
These complexes then are recognized by the T cell receptors of
antigen-specific T cells to form the tri-molecular complexes which
result in the activation and subsequent functioning of T cells and
in the development of arthritis.
[0004] Native type II collagen (CII) can induce arthritis in
susceptible individuals. Certain fragments of native CII also
induce an immunogenic response. Some of those immunogenic fragments
and some of their analogs may also suppress the disease. Frequently
this suppression occurs because T cell tolerance is developed. That
is, the T cells are disabled from responding to the antigen or
trimolecular complex containing the antigen. This immunogenic
response (T cell tolerance) limits the therapeutic potential for
the native polypeptide fragments and many of their analogs because
the body develops immunity to the fragment after its first use.
Subsequent treatments with the native fragments of CII are
therefore expected to be ineffective. It would therefore be
desirable to develop peptides that suppress autoimmune arthritis
without inducing a material immunogenic response or, more
preferably, without inducing any immunogenic response at all.
[0005] Peptides have been identified which may be capable of
inhibiting specific T cell responses by blocking formation of the
trimolecular complex in some way rather than by disabling the T
cells. Babbitt, B. P., G. Matsueda, E. Haber, E. R. Unanue, and P.
M. Allen, 1986, Antigenic Competition at the Level of Peptide-Ia
Binding. Proc. Natl. Acad. Sci. USA 83:4509; Adorini, L. and Z. A.
Nagy, 1990, Peptide Competition for Antigen Presentation. Immuno.
Today. 11:21. Peptides have been used to suppress or prevent murine
experimental autoimmune encephalomyelitis (EAE). Wraith, D. C., D.
E. Smilek, D. J. Mitchell, L. Steinman, and H. O. McDevitt, 1989,
Antigen Recognition in Autoimmune Encephalomyelitis and the
Potential for Peptide-Mediated Immunotherapy. Cell 59:247; Lamont,
A. G., A. Sette, R. Fujinami, S. M. Colon, C. Miles, and H. M.
Grey, 1990, Inhibition of Experimental Autoimmune Encephalomyelitis
Induction in SJL/J Mice By Using a Peptide with High Affinity for
I-As Molecules. J. Immunol. 145:1687; Salao. K., S. S. Zamvil, D.
J. Mitchell, S. Hodgkinson, J. B. Rothbard, and L. Steinman, 1989,
Prevention of Experimental Encephalomyelitis with Peptides that
Block Interaction of T Cells with Major Histocompatibility Complex
Protein. Proc. Natl. Acad. Sci. USA. 86:9470. Investigators, using
the EAE animal model, have demonstrated inhibition of the induction
of experimental encephalomyelitis with synthetic peptides. When
mice bearing the H-2.sup.u haplotype were co-immunized with an
analog peptide and an encephalogenic peptide (amino acid residues
1-9 of myelin basic protein), disease did not develop. An unrelated
peptide, known to bind to I-A.sup.S, was used to inhibit the
development of encephalomyelitis by the EAE-inducing antigen.
Lamont, A. G., A. Sette, R. Fujinami, S. M. Colon, C. Miles, and H.
M. Grey, 1990, Inhibition of Experimental Autoimmune
Encephalomyelitis Induction in SJL/J Mice by Using a Peptide with
High Affinity for I-As molecules. J. Immunol. 145:1687. The ability
of some peptides to "compete" for binding to class II MHC molecules
in vitro has been demonstrated. Werdelin, O, 1982, Chemically
Related Antigens Compete for Presentation by Accessory Cells to T
Cells. J. Immunol. 129:1883; Rock, K. L. and B. Benacerraf, 1984,
Selective Modification of a Private I-A Allostimulating
Determinant(s) Upon Association of Antigen With An
Antigen-Presenting Cell. J. Exp. Med. 159:1238; Babbitt, B. P., G.
Matsueda, E. Haber, E. R. Unanue, and P. M. Allen, 1986, Antigenic
Competition at the Level of Peptide-Ia Binding. Proc. Natl. Acad.
Sci. USA 83:4509.
[0006] The goal of providing peptides that block formation of
trimolecular complexes without inducing material antigenic
responses, however, is not always obtainable nor is success in
obtaining that goal easily predictable. The strategy of developing
a synthetic analog peptide having such a combination of features is
not known to be a consistently reliable technique for developing
therapeutically useful peptides in all autoimmune diseases or for
autoimmune arthritis specifically. Two parameters that affect the
ability of synthetic peptides to compete for antigen presentation
are: 1) the relative affinity of antigenic and competitor peptides
for the MHC molecule, and 2) the avidity of T cells for the
activating ligand. One can not be reasonably assured of being able
to develop a peptide which will have the required affinity and
avidity for MHC yet that does not illicit a material immunogenic
response from T cells.
[0007] In addition, use of analog peptides may make autoimmune
arthritis worse rather than suppressing it in some instances. This
problem occurs primarily when the analog stimulates T cell
immunity. The resulting tolerance can subsequently break down. The
disease then worsens and administration of the analog can not
suppress it. This problem is particularly a concern with analogs of
CII because the native CII fragments are known to be quite
immunogenic and their analogs tend to also have a high level of
immunogenicity. This makes more difficult and unlikely the
development of analog peptides that suppress autoimmune arthritis
without prompting an undesirable immunogenic T cell response.
SUMMARY OF THE INVENTION
[0008] The present invention provides analog peptides of fragments
of CII protein, which contain a T cell antigen, which analog
peptides suppress autoimmune arthritis. The analogs disrupt
formation of trimolecular complexes of autoimmune antigenic
peptide, MHC and T-cell receptor but do not provoke a material
immunogenic response.
[0009] The present invention includes analogues of CII 245-270 and,
more specifically, analogs of CII 260-270 peptide and of CII
245-270 [s 260, 261, 2631] peptide.
[0010] Moreover, the present invention provides the following
peptides
1 Ala - 4Hyp - GLY - Asn - Lys - Gly; Ala - 4Hyp - Gly - Asn - Lys
- Gly - Glu - Gln - Gly - Pro - Lys; and Pro - Thr - Gly - Pro -
Leu - Gly - Pro - Lys - Gly - Gln - Thr - Gly - Glx - Leu - Gly -
Ala - 4Hyp - Gly - Asn - Lys - Gly - Glx - Gln - Gly - Pro -
Lys.
DETAILED DESCRIPTION OF THE INVENTION
[0011] Applicants have now provided analog peptides to a fragment
of type II collagen, (CII) that suppress autoimmune arthritis
without inducing a material immunogenic response. The peptides
appear to function as competitive inhibitors by binding to the
I-A.sup.q molecule of MHC and in this way to interfere with or
disrupt formation of the tri-molecular complex. The analogs of the
present invention therefore suppress autoimmune arthritis by
disrupting formation of trimolecular complexes. In addition to
suppressing arthritis peptides of the present invention do not
provoke a material immunogenic response. Peptides of the present
invention are therefore useful therapeutic agents for suppressing
autoimmune arthritis. They are expected to be useful in treatment
of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis,
spondylo arthritis, relapsing polychondritis and other connective
tissue diseases.
[0012] To develop and test the efficacy of synthetic peptides in
suppression of autoimmune disease, native CII peptide, which is
known to be immunogenic, was obtained. Fragments of the native
peptide were then synthesized. Native CII was solubilized from the
sterna of adult chickens by limited pepsin digestion. Stuart, J.
M., M. A. Cremer, A. H. Kang, and A. S. Townes, 1979,
Collagen-induced Arthritis in Rats: Evaluation of Early Immunologic
Events, Arthritis Rheum. 22:1344. The disclosure of this article is
incorporated by reference. Purified m.alpha.l(II) chains, obtained
by thermally denaturing the CII, were subjected to non-enzymatic
cleavage with cyanogen bromide and the resulting peptides isolated
as described by Miller in Miller, E. J., 1971, Isolation and
Characterization of the Cyanogen Bromide Peptides From the .alpha.1
(II) Chain of Chick Cartilage Collagen. Biochemistry. 10:3030. The
disclosure of this article is incorporated herein by reference.
[0013] From the larger CII native peptide, a fragment identified as
CII 245-270 has been identified as an important region of type II
collagen (CII) in mice bearing the I-A.sup.q haplotype. The native
amino acid sequence is:
2 245 250 255 Pro - Thr - Gly - Pro - Leu - Gly - Pro - Lys - Gly -
Gin - Thr - Gly - 260 265 270 Glx - Leu - Gly - Ile - Ala - Gly -
Phe - Lys - Gly - Glx - Gln - Gly - Pro - Lys.
[0014] This sequence is referred to herein as CII 245-270 (Sequence
No. 1). This fragment is the same as to a comparable immunogenic
fragment of human CII collagen with two exceptions. The comparable
human sequence has alanine at position 245 and 4-hydroxyproline at
position 258. There are no differences in the 260-270 region.
Administration of CII 245-270 suppresses arthritis when used as a
neonatal tolerogen. Myers, L. K., J. M. Stuart, J. M. Seyer, and A.
H. Kang, (1989), "Identification of an Immunosuppressive Epitope of
Type II Collagen that Confers Protection Against Collagen-Induced
Arthritis" J. Exp. Med. 170:1999. The disclosure of that article is
incorporated herein by reference. More recently, five residues,
those numbered 260-265, have been identified which are important
for T cell responses and tolerance. Myers, L. K., K. Terato, J. M.
Seyer, J. M. Stuart, and A. H. Kang, 1992, "Characterization of a
Tolerogenic T Cell Epitope of Type II Collagen and its Relevance to
Collagen-induced Arthritis" J. Immunol. 149:1439. The disclosure of
that article is incorporated herein by reference.
[0015] A number of analog peptides have been tested for their
ability to competitively inhibit antigen presentation in vitro, and
to prevent the development of collagen induced arthritis (CIA) in
vivo. One preferred peptide competitively inhibits the T cell
response to CII and significantly suppresses the development of
arthritis when administered to DBA/I mice simultaneously with CII.
The sequence of that preferred peptide, numbered to correspond to
the native CII fragment disclosed above, is:
3 245 250 Pro - Thr - Gly - Pro - Leu - Gly - Pro - Lys - Gly - 255
260 Gln - Thr - Gly - Glx - Leu - Gly - Ala - 265 4Hyp - Gly - Asn
- Lys - Gly - Glx - Gln - 270 Gly - Pro - Lys.
[0016] This peptide is referred to as CII 245-270 [s260, 261, 263].
(Sequence No. 2) Smaller fragments of the foregoing peptide are
expected to work in the same manner as long as a sufficient number
of residues are present to inhibit formation of the trimolecular
complex. In particular, the following peptides are expected to
exhibit functional characteristics substantially identical to or
very similar to the 26 residue analog disclosed above.
4 260 265 ala - 4Hyp - Gly - Asn - Lys - Gly -; and 260 265 270
4Hyp - Gly - Asn - Lys - Gly - Glu- Gln - Gly - Pro - Lys.
[0017] The shortest of the above sequences may also be referred to
as CII 260-265 [s 260, 261, 263]. (Sequence No. 3) The larger of
the two immediately proceeding sequences is also referred to as CII
260-270 [s 260, 261, 263]. (Sequence No. 4)
[0018] In the development of the present invention, oligopeptides
containing sequences corresponding to known sequences of
.alpha.1(II)-CB11 were chemically synthesized by a solid-phase
procedure described previously using an Applied Biosystem (model
(430) peptide synthesizer. Seyer, J. M., K. A. Hasty, and A. H.
Kang. (1989) "Covalent Structure of Collagen. Amino Acid Sequence
of an Arthritogenic Cyanogen Bromide Peptide from Type II Collagen
of Bovine Cartilage" Eur. J. Biochem. 181:151. Kanomi, H., J. M.
Seyer, Y. Ninomiya, and B. R. Olsen. (1986) "Peptide-Specific
Antibodies Identify the .alpha.2 Chain as the Proteoglycan Subunit
of Type II Collagen" J. Biol. Chem. 261:6742. The sequence
.varies.1(II)-CBII is a large fragment of CII which includes the
native fragment identified above as residues numbered CII 245-270,
The disclosures of these two articles are incorporated by
reference. The sequence of the chick CII gene was obtained and was
used to deduce the entire CII protein sequence.
[0019] For peptide synthesis, protected tBoc amino acids were
purchased from Applied Biosystems, Inc. (Foster City, Calif.) and
coupled sequentially to a benzhydrylamine resin with a PAM linker.
Deprotection was achieved with trifluoroacetic acid (25% in
dichloromethane) and coupling was obtained in the presence of
dicyclohexyl-carbodiimide. The completed synthetic peptide was
cleaved from the resin and the side-chain protecting groups removed
by treatment with liquid HF at 0.degree. C. The desired peptide was
initially purified by filtration through a Sephadex G-25 column
(4.0.times.60 cm) previously equilibrated with 0.1 M acetic acid.
The effluent was collected in fractions of 10 ml. and aliquots
taken from fluorescamine analysis. Fractions containing peptides
were pooled, lyophilized, and further purified by reverse phase
high pressure liquid chromatography on a Whatman ODS-3 (1
cm.times.25 cm) semipreparative column. Peptides were applied to
the column in 0.05% trifluoroacetic acid and eluted over 30 min.
with a gradient of 20-30% acetonitrile containing 0.05%
trifluoroacetic acid at a flow rate of 2.0 ml/min. The effluent was
monitored at 230 nm and the presence of peptides in relevant
fractions confirmed by reaction with fluorescamine. The amino acid
composition of the final peptide was determined using an automatic
amino acid analyzer (Applied Biosystems, model 420A), and amino
acid sequences were confirmed by automatic Edman degradation
(Applied Biosystems, model 477). The amino acid composition found
was .+-.5% theoretical, and the amino acid sequence analysis
confirmed the peptide structure.
EXAMPLE I
[0020] Five specific residues of CII 245-270 have been identified
as being particularly important for the stimulation of
I-A.sup.q-restricted T cells and the induction of tolerance. These
are the residues numbered 260-270 above. Myers, L. K., K. Terato,
J. M. Seyer, J. M. Stuart, and A. H. Kang, 1992, Characterization
of a Tolerogenic T Cell Epitope of Type II Collagen and its
Relevance to Collagen-Induced Arthritis. J. Immunol. 149:1439. To
determine whether synthetic peptides containing amino acid
substitutions at these positions might function as competitive
inhibitors of antigen presentation to T cells, their ability to
stimulate CII-primed T cells was examined. Four hexacosopeptides,
analogs of CII 245-270, synthesized in the manner described above
contained substitutions based on the type I collagen sequences or
alanine substitutions for proline, as shown in Table I.
[0021] Table I. Amino Acid Sequence of Synthetic Peptides
5TABLE I Amino acid sequence of synthetic peptides Peptide.sctn.
246 250 254 258 262 266 270 CII 245-270 P T G P L G P K G Q T G E L
G T A G F K G E Q G P K CII 245-270 [s 248, 249] -- -- -- A B -- --
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- CII
245-270 -- -- -- A -- -- A -- -- -- -- -- -- -- -- -- -- -- -- --
-- -- -- -- A -- [s 248, 251, 269] CII 245-270 -- -- -- -- -- -- --
-- -- -- -- -- -- -- -- A B -- N -- -- -- -- -- -- -- [s 260, 261,
263] CII 245-270 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
-- -- -- -- D T -- A -- [s 266, 267, 269] Type I 245-270 -- S -- A
B -- -- -- -- N S -- -- B -- A B -- N -- -- D T -- A -- .sctn.A
"--" indicates identity at that position with native type peptide
CII 245-270. Amino acid residues are represented by the single
letter code. The letter "A" denotes Alanine, "B" denotes
hydroxyproline, "D" denotes aspartic acid, "N" denotes asparagine
and "S" denotes serine. Amino acid numbering system is based on the
sequence of native CII.
[0022] Type I collagen has a primary structure similar to CII, but
immunization with type I collagen does not elicit (CIA). Nowarck
H., E. Hahn, R. Timple, 1976, "Requirements for T Cells in the
Antibody Response of Mice to Calf Skin Collagen," J. Immunol.
30:29. Each peptide was cultured with pooled spleen and lymph node
cells from CII-immunized mice, and culture supernatant fluids were
tested for the presence of .gamma.-interferon as an indicator of T
cell stimulation. T cell hybridomas were established by
polyethylene glycol-induced fusion of lymph node cells with the T
cell receptor .alpha..sup.-/.beta..sup.-thymo- ma line, BW5147.
White, M., M. Blackman, J. Bill, J. Kappler, P. Marrack, D. P.
Gold, and W. Born, 1989, Two Better Cell Lines for Making
Hybridomas Expressing Specific T Cell Receptors. J. Immunol.
143:1822. Marrack, P., 1982, Production of Antigen-Specific H-2
Restricted T Cell Hybridomas In "Isolation, Characterization, and
Utilization of T Lymphocyte Clones", C. G. Fathman, and F. Fitch,
eds. Academic Press, New York, N.Y., p. 508. The disclosures of
each of these articles are incorporated by reference.
[0023] Lymph node cells were obtained from DBA/1 mice immunized
with .alpha.1(II)-CB11 emulsified with complete Freund's adjuvant
and cultured in vitro with .alpha.1(II)-CB11 for five days, and in
the presence of IL-2 for three days before fusion. Hybridoma cells
reactive to CB11 [CII 245-270] and CII were cloned by limiting
dilution to 0.3 cells/well. Antigen presentation experiments were
performed in 96 well microliter plates in a total volume of 0.3 ml
containing 4.times.10.sup.5 syngenic spleen cells and 10.sup.5
T-hybridoma cells. For competitive inhibition assays, spleen cells
were pulsed with various ratios of inhibitor to indicator peptide
and washed several times prior to addition to the antigen
presentation culture. Cell cultures were maintained at 37.degree.
C. in 5% humidified CO.sub.2 for 20 to 24 hours, after which seven
80 .mu.l two-fold serial dilutions were made for determination of
IL-2 titers. Four thousand HT-2 cells were added to each
supernatant dilution, and after 16 to 20 hours HT-2 cell viability
was evaluated by visual inspection. IL-2 titers were determined by
the reciprocal of the highest two-fold serial dilution maintaining
90% viability of the HT-2 cells. Results are presented as units of
IL-2 per ml of undiluted supernatant as described by Kapper et al.
Kappler, J., J. White, D. Wegman, E. Mustain, and P. Marrack, 1982,
Antigen Presentation by Ia.sup.+B Cell Hybridomas to H-2 Restricted
T Cell Hybridomas. Proc. Natl. Acad. Sci. USA 79:3604. The
disclosure of this article is incorporated by reference.
[0024] T cell stimulation assays were performed in 96 well plates
and the degree of stimulation was quantitated by measurements of
.gamma.-IFN production. Myers, L. K., K. Terato, J. M. Seyer, J. M.
Stuart, and A. H. Kang, 1992, Characterization of a Tolerogenic T
Cell Epitope of Type II Collagen and its Relevance to
Collagen-Induced Arthritis. J. Immunol. 149:1439. The disclosure of
this article is incorporated by reference. Spleens and lymph nodes
from mice immunized with CII 14 to 21 days prior were individually
minced into single cell suspensions in Hank's Balanced Salt
Solution (BSS) and washed 3 times. 5.times.10.sup.5 cells were
cultured with 100 .mu.m of antigen (synthetic peptides, collagen,
or PPD) in 0.3 ml of Dulbecco's Modified Eagle Medium (Gibco, Gand
Island, N.Y.) supplemented with 5% fetal bovine serum (Hyclone
Laboratories, Logan, Utah). Supernatants were collected from 72 to
120 hours later and either 8 analyzed for .gamma.-IFN production
immediately or stored at -70.degree. C. prior to analysis.
Quantitative measurement of murine gamma interferon was done using
a solid-phase enzyme-linked immunosorbent assay (Amgen Biologicals,
Thousand Oaks, Calif.). Supernatant samples and standards were
incubated in microliter plates coated with a monoclonal antibody
recognizing murine .gamma.-interferon. Plates were washed and
incubated with a pre-formed detector complex consisting of a
biotinylated second monoclonal antibody to .gamma.-interferon and
an anti-biotin-alkaline phosphatase conjugate. The absorbance was
measured at 405 nm with a spectrophotometer, and a standard curve
was obtained by plotting the absorbance versus the corresponding
concentration of the standards. Units of .gamma.-interferon were
calculated based on NIH standard number Gg02-901-533. Each sample
was tested in duplicate wells.
[0025] As shown in FIG. 1, substitution of alanine for proline at
position 248, and hydroxyproline for leucine at position 249 had
almost no effect on T cell stimulation compared to the response of
T cells to the wild type peptide, CII 245-270. However, when the
substitution at residue 248 was combined with an alanine for
proline substitution at residues 251 and 269, the ability of the T
cells to respond to this peptide was greatly reduced (25% of the
wild type peptide response), yet still above background levels. The
measure of a material immunogenic response may vary in particular
circumstances or for particular individuals. Generally, however, to
provoke a material immunogenic response if more than about 5 units
of interferon are measured by the foregoing test. In contrast, the
CII-primed T cells did not respond to the analog peptides
containing substitutions at residues 260, 261 and 263, and residues
266, 267, and 269 (FIG. 1).
6 1
[0026] All of these substitutions are based on type I collagen
sequences, and are non-conservative substitutions, with the
exception of the conservative substitution of aspartic acid for the
glutamic acid at residue 266. These data indicated, among other
things, that the amino acid(s) at positions 260-270 are important
for I-A.sup.q-restricted presentation of the CII 245-270 peptide to
T cells.
EXAMPLE II
[0027] The inability of some analog peptides to stimulate T cells
likely occurs either because of disruption of peptide binding to
the I-A.sup.q molecule or the inability of the T cell receptor to
recognize the peptide. In order to determine whether the analog
peptides could bind to the class II molecule, competitive antigen
presentation assays were performed. Antigen presenting cells (APC)
were pulsed with various molar ratios of CII 245-270 and an analog
peptide, and tested for their ability to stimulate CII 245-270
specific T cell hybridomas. When APC were competitively pulsed with
the CII 245-270[s260, 261, 263] and CII 245-270 at molar ratios of
6:1 or greater, respectively, their ability to stimulate the T-cell
hybridomas was greatly reduced (Table II).
7TABLE II Competitive inhibition of antigen binding to 1-A.sctn. on
the surfacE on antigen presenting cells. IL-2 U/ml Molar Ratio
(Competitor: T-cell CII 245-270).sctn. Hybrid Competitor Peptide
13:1 6.5:1 3.2:1 1.6:1 0:1 qCII85.33 CII 245-270 --* 40 320 640 640
[s260, 261, 263] CII245-270 640 640 640 640 640 [s266, 267, 269]
2qCII92.33 CII 245-270 -- 20 80 160 160 [s260, 261, 263] CII
245-270 160 160 160 160 160 [s266, 267, 269] qCII98.10 CII 245-270
-- 40 80 160 160 [s260, 261, 263,] CII 245-270 160 160 160 160 160
[s266, 267, 269] .sctn.DBA/1 spleen cells were pulsed with various
molar ratios of analog peptide and CII 245-270, washed, and tested
for their ability to present antigen to a set of CII 245-270
specific T-cell hybridomas. Production of IL-2 by the T-cell
hybridomas was determined by the ability of culture supernatants to
support the growth of IL-2 dependent cell line, HT-2. *A "--"
indicates less than 20 U/ml of IL-2 detected.
[0028] These data indicate that the analog peptide designated CII
245-270 [s260, 261, 263] is capable of binding to I-A.sup.q. The
amino acid substitutions in this peptide are believed to disrupt
the ability of the T cell receptor to recognize the peptide. In
contrast, the CII 245-270[s266, 267, 269] analog peptide did not
compete for the presentation of the wild type peptide to the T-cell
hybridomas.
EXAMPLE III
[0029] The same two analog peptides used in Example II were tested
for their ability to inhibit the presentation of antigen to
CII-primed, bulk T cells. Similar results were observed to those
noted in Example II. In these experiments analog peptides were
co-cultured with either CII 245-270 or native CII at various ratios
with primed T cells from CII-immunized DBA/1 mice. As was observed
with the T-cell hybridomas, the addition of peptide CII
245-270[s260, 261, 263] to the T cell cultures significantly
decreased responses to both CII 245-270 and CII in a dose dependent
manner while CII 245-270[s266, 267, 269] had no significant effect
(FIG. 2).
8 2
[0030] The molar ratios required for inhibition were similar for
the competitive presentation of the wild type peptide, and
significantly higher molar ratios were required for the inhibition
of the presentation of CII. This may reflect variation in the
antigen processing required, or the differing numbers of class II
binding determinants within the CII molecule and CII 245-270.
EXAMPLE IV
[0031] Since the analog peptide CII 245-270[260, 261, 263]
inhibited the presentation of antigen in vitro, it was tested for
its ability to inhibit the induction of experimental arthritis in
vivo with DBA/1 mice. Arthritis induced in mice is considered a
model for human rheumatoid arthritis. Anderson, Banerjee, Luthra
and David, 1991, Role of Mls-1 Locus and Cloned Deletion of T Cells
in Susceptibility to Collagen-Induced Arthritis in Mice, J. Imm.
Vol. 147, 1189-1193.
[0032] DBA/1 mice, obtained from Jackson Laboratories (Bar Harbor,
Me.), were maintained in groups of six in polycarbonate cages and
fed standard rodent chow (Ralston Purina Co, St. Louis, Mo.) and
water ad libitum. The environment was specific pathogen-free and
sentinel mice were tested routinely for mouse hepatitis and Sendai
viruses. Neonatal mice were obtained by breeding mice from Jackson
Laboratories in our facility. Mice were immunized at 8-12 weeks of
age. Stuart, J. M., A. S. Townes, and A. H. Kang, 1982. DBA/1 mice
were immunized with either CII, CII plus CII 245-270, or CII plus
CII 245-270[s260, 261, 263] at various molar ratios and were
observed for the development of arthritis.
[0033] Mice were bled at four weeks after immunization and the
serum was tested for antibodies reactive with type II collagen by
enzyme-linked immunoassay (ELISA) described in Stuart, J. M., A. S.
Townes, and A. H. Kang. 1982. Nature and Specificity of the Immune
Response to Collagen in Type II Collagen-Induced Arthritis in Mice,
J. Clin. Invest. 69:673. The disclosure of this article is
incorporated by reference An anti-CII serum standard was used in
each assay. A standard curve was derived by computer analysis using
a 4 parameter logistic curve. Results are reported as units of
activity, derived by comparison of test sera with the curve derived
from the anti-CII standard which was arbitrarily defined as having
50 units of activity. Sera from mice were tested individually, and
means were calculated for each experimental group.
[0034] Chick CH II obtained as described above, was dissolved in
0.01 N acetic acid and emulsified with an equal volume of complete
Freund's adjuvant (CFA). In some experiments, a synthetic peptide
was added to the emulsion in varying concentrations. That is, in
coimmunization experiments synthetic peptide was added to the same
emulsion as the native CII peptide. The resulting emulsion was
injected intradermally into the base of the tail. Each mouse
received a total volume of 0.005 ml containing 100 .mu.g of MTb and
100.mu. of antigen.
[0035] To measure the incidence of arthritis in immunized mice,
individuals examined and scored each of the forepaws and hind paws
on a scale of 0-4 as described in Wooley, P. H., H. W. Luthra, J.
M. Stuart and C. S. David. 1981. "Type 11 Collagen-induced
Arthritis in Mice". I. Major Histocompatibility Complex (I region)
Linkage and Antibody Correlates. J. Exp. Med. 154:688. This article
is incorporated herein by reference. There were two separate
examiners, one of whom was unaware of the identity of the treatment
groups. Each mouse was scored three times a week beginning three
weeks post immunization and continuing through 8 weeks post
immunization. The incidence of arthritis (number of animals with
one or more arthritic limbs) was reported at 6 weeks post
immunization, the time point at which the control group reached its
peak of disease. The incidence of arthritis in various groups of
mice was compared using Fisher's Exact Test. Student's T test was
used to compare means of antibody responses to CII.
[0036] DBA/1 mice co-immunized with CII 245-270[s260, 261, 263]
demonstrated a dose-dependent decrease in the incidence of
arthritis and number of arthritic limbs (Table III).
9TABLE III Suppression of Arthritis by Simultaneous Immunization
with CII and an Analog Peptide Molar Ratio Number of Number of
Peptide.sctn. (CII:Peptide) Arthritic Mice Arthritic Limbs CII
245-270 110 10/12 (83%) 24/48 (50%) [s260-261, 263] 1:160 4/6 (67%)
10/24 (42%) 1:320 6/12 (50%) 11/48 (23%)** 1:480 0/10 (0%)* 0/40
(0%)** CII 245-270 1:480 4/6 (67%) 8/24 (33%) .sctn.DBA/1 mice were
immunized intradermally with a single emulsion containing either
CII 245-270[s260, 261, 263] and CII, or wild type CII 245-270 and
CII in complete Freund's adjuvant. *p .ltoreq. .002 using Fisher's
Exact Test. **p .ltoreq. .005 using Fisher's Exact Test
[0037] When molar ratios of native CII to CII 245-270[s260, 261,
263] of 1:480, respectively, were co-injected, arthritis did not
develop. Simultaneous immunization with CII plus CII 245-270 did
not alter the incidence of disease.
[0038] In order to assess the effects of co-immunizing mice with
both CII 245-270[s260, 261, 263] and CII, the mean antibody titers
to native CII were measured for each immunization group in Table
III, four weeks after immunization (Table IV).
10TABLE IV Measurement of anti-CII response in DBA/1 mice
co-immunized with analog peptides abd CII. Molar Ratio Antibodies
Peptide (CII:peptide) to CII.sctn. CII 245-270[s260, 261, 263]
(1:480) 17 .+-. 3** CII 245-270[s260, 261, 263] (1:320) 34 .+-. 22*
CII 245-270[s260, 261, 263] (1:160) 53 .+-. 25 CII 245-270 (1:480)
54 .+-. 25 None (1:0) 60 .+-. 20 .sctn.Serum was collected from
mice co-immunized with the various peptides and CII, and antibody
titers to type II collagen were tested using an ELISA. *p .ltoreq.
.05 using Student's T test. **p .ltoreq. .005 using Student's T
test.
[0039] Concordant with a decrease in the incidence and severity of
arthritis, antibody production to native CII was also significantly
decreased. These data indicate that peptide CII 245-270[s260, 261,
263] significantly down regulated the immune responses to CII in
vivo as well as in vitro.
EXAMPLE V
[0040] The foregoing data support the hypothesis that analog
peptide CII 245-270[s260, 261, 263] competes for binding to
I-A.sup.q. Further tests showed that the induction of T cell
tolerance to CII 245-270 is not a likely explanation for the test
results. Synthetic peptides were solubilized directly in phosphate
buffered saline (PBS) at a concentration of 1 mg/ml. Neonatal mice
were tolerized using a protocol described by Gammon et al..sup.21
in which antigen emulsified with incomplete Freund's adjuvant was
injected intraperitoneally. Gammon, Dunn, Shastri, Oki, Wilbur,
Sercarz, 1986, Neonatal T Cell Tolerance to Minimal Immunogenic
Peptides is Caused by Clonal Inactivation, Nature (Lond) 319:413.
The disclosure of this article is incorporated herein by reference.
Each mouse received 100 .mu.m of antigen in 0.1 ml of emulsion
within 24 hours of birth. When they reached eight weeks of age,
mice were immunized with CII and observed for arthritis as
described above.
[0041] CII 245-270[s260, 261, 263] was administered to neonatal
mice prior to immunization with CII, in order to induce tolerance
and evaluate the effects on arthritis. While peptide CII 245-270
was an effective tolerogen, capable of inhibiting the subsequent
induction of arthritis and also depressing the resulting mean
antibody titers to CII, the analog was ineffective as a CII
tolergen. It had no significant effect on either the development of
arthritis or the development of antibodies to CII (Table V).
11TABLE V Inability of analog peptide to induce neonatal tolerance.
Number of Antigen.sctn. Arthritic Mice Antibodies to CII No Antigen
16/18 (89%) 63.5 .+-. 25 CII 245-270[s260, 261, 263] 5/5 (100%)
55.6 .+-. 19 CII 245-270 6/20 (30%) 18.5 .+-. 8* .sctn.Neonatal
mice were injected intrapentoneally with 100 .mu.g of antigen
emulsified in incomplete Freund's adjuvant within 24 hours of
birth. At eight weeks of age the mice were challenged with CII in
CFA and observed for the development of arthritis. Serum was
collected from the mice four weeks after immunization and mean
antibody titers to CII were evaluated by an ELISA.
[0042] In vivo administration of a synthetic peptide, an analog of
an antigenic determinant of type If collagen, successfully
inhibited the development of collagen-induced arthritis. The
simultaneous immunization of this analog peptide with CII not only
reduced the incidence and severity of arthritis, but also
significantly decreased the humoral immune response to collagen. In
addition, the direct binding of the peptide to I-A.sup.q is
currently believed to result in competitive inhibition of the T
cell responses to CII. In this manner, peptides of applicants block
formation of trimolecular complexes of antoimmune antigenic
peptide, MHC and T cell receptors without provoking a material
immunogenic response.
[0043] The data shown in Table II indicates that inhibition of CII
245-270-specific T cell responses occurs by competitive inhibition
induced by direct binding of inhibitor to I-A.sup.q. APC's
prepulsed with competitor and antigen, then washed before culturing
with antigen-specific T cell hybridomas, were ineffective at
presentation of antigen. Since .alpha.1(II) is 40 times the size of
CII 245-270 and likely contains a number of T cell antigenic sites,
the greater molar ratio of the inhibitor peptide required to
prevent T cell responses to .alpha.1(II) than to inhibit responses
to CII 245-270 (FIG. 2B) indicates a mechanism in which the
inhibitor binds to a site common to multiple antigenic peptides
which are recognized by I-A.sup.q-restricted T cells. Guery and
coworkers, Guery, J. C., A. Sette, J. Leighton, A. Dragomir, and L.
Adorini, 1992, Selective Immunosuppression by Administration of
Major Histocompatibility Complex (MHC) class II-binding peptides.
I. Evidence for In Vivo MCH Blockade Preventing T Cell Activation.
J. Exp. Med. 175:1345, recently demonstrated that such a
competition for class II binding may also occur in vivo. The
disclosure of the foregoing article is incorporated herein by
reference.
[0044] A toxic effect of the tested analog peptide is not likely,
as T-cell hybrids specific for lysozyme in the context of
1-A.sup.k, were not inhibited by CII 245-270[s260, 261, 263] when
this peptide was added to cultures containing APC's and the HEL
antigen. More specifically, the cells responded and were not
killed. Data using peptide as neonatal tolerogens (Table V) also
indicate that the analog peptide CII 245-270[s260, 261, 263] is a
very poor tolerogen. These data make the induction of
antigen-specific tolerance unlikely, as a regulatory mechanism.
[0045] Administration of peptides of applicant's invention may
occur through familiar techniques. In humans, the most likely
routes are subcutaneous injection or oral administration. If
subcutaneous injection is used, the peptide would be dissolved and
injected with a pharmaceutically acceptable saline solution.
[0046] The foregoing disclosure illustrates currently preferred
embodiments of applicants invention. It will be understood by those
of ordinary skill in the art that modifications of the disclosed
invention may be made without departing from the invention.
Sequence CWU 1
1
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