Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
PEPTIDES COMPRISING A T-CELL EPITOPE SPECIFIC TO COLLAGEN II
The present invention provides new peptides derived from collagen CII, methods
for their
s preparation and their use in medical therapy, particularly in the treatment
of rheumatoid
arthritis and related autoimmune conditions
Collagen molecules are some of the main structural proteins of connective
tissue. They
consist of three polypepti.de chains forming an extended triple helical
structure with a
~o unique X-Y-Gly repetitive amino acid sequence. The extracellular matrix of
cartilage is
unique in containing coll~agens mainly of type II, but also of types IX and
XI. CII has
recently been cloned fronn the mouse and its entire sequence of 1419 amino
acids
determined (Metsarantam et al., 1991, J Biol Chem, 266: 16862-9).
a .Type IIrollagen is thouglht to be "hidden " from cells of the immune system
as it is only
found in avascular tissues, such as the cartilage, and the vitreous body of
the eye. The
immune system is therefore not completely tolerant of its own type II collagen
As a
sequestered protein, CII has the ability, when injected in Freund's complete
adjuvant, to
induce tissue-specific autoimmune disease. The disease that it induces is
termed collagen
2o induced arthritis (CIA). ('_IA is normally induced by injection of foreign
CII, which
provokes the production of T cells and antibodies able to recognise the
corresponding self
protein.
Susceptibility to CIA in mice is limited to MHC types I-Aq and I-Ar. The
binding motifs
2s of these molecules are similar to those of human DR4 and DR 1. Therefore it
is believed
that CII is an autoantigen in rheumatoid arthritis(RA).
No causal therapy is currently available for RA. Existing treatment methods
e.g.
application of corticosteroids, are unspecific, as they suppress the immune
response in
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
2
general. Current research suggest an important role for autoreactive T cells
in the
pathogenesis of RA which has lead to the concept that tolerization of these
pathogenic T
cells by nasal/oral administration of immunogenic peptides might be of
therapeutic
potential.
Miyahara, H. et al ( Immunology 1995 86 110-1 IS) describe the use of a
fragment of type
II collagen in the suppression of arthritis in mice. The fragment used (CII
607-621 ) does
however not contain a binding motif to any of the HLA-DR molecules associated
with RA
in man and would therefore not be effective for use as a toleragen in human RA
therapy.
io
WO 96/20950 purports to describe type II collagen peptides capable of binding
to the
human HLA DRB 1 MHC protein which it is suggested would be of use in RA
therapy.
However these peptides which are present in the 273-404 region of collagen CII
protein
show only weak activity in relevant assays for putative toleragens for RA .
The peptides of
is the present invention are of a significantly greater therapeutic efficacy.
The present invention provides peptides which have been found to be
particularly effective
in inducing immune tolerance to collagen CII derived T-cell epitopes. These
peptides may
be used in the treatment of autoimmune conditions such as rheumatoid
arthritis.
zo
There is therefore provided according to the present invention, an isolated
peptide having,
or comprising, an amino acid sequence of formula (I):
A1-Xaa-GIy-A4-AS-Gly-A~-Xaa-Gly (I)
2s wherein;
Ai represents an amino acid residue with an aromatic or aliphatic side chain,
A4 represents an asparagine or arginine residue or an amino acid residue with
an aromatic
or aliphatic side chain,
AS represents any naturally occurring amino acid,
so A~ represents an amino acid with a negatively charged residue,
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98I00128
3
Xaa represents any amino acid residue, and
Gly represents a glycine residue.
Peptide of the present invention are preferably 9, 10 , 11, 12, 13, 14 or 15
amino acid
s peptides; and are more preferably 9 amino acid peptides.
In peptides of the present invention the following independent preferences
apply;
-A i is F, I, L, A or P and is most preferably F
~o -A4 is F, I) L, A, R, N or P and is most preferably F
-ASisKorR
-A~ is E , D, Q, P or N and is most preferably Q.
Preferred peptides accordiing to the present invention include those
comprising, or having,
is one of the following sequences:
ESG SPG ENG, PPG AL~G QPG, ARG NDG QPG, QPG AKG DQG, APG AKG EAG,
PTG VTG PKG, AQG SP;G EPG, RVG PPG ANG, PAG ASG NPG, ANG NPG PAG,
TDG IPG AKG, DPG LQG PAG, SAG APG IAG, APG EKG EPG, IAG APG FPG, PQG
2o LAG QRG, FPG PRG PPG, PKG ANG DPG, APG ASG DRG, LPG ARG LTG, DAG
PQG KVG, ALG APG Al'G, PAG ANG EKG, KQG DRG EAG, or ARG APG EPG (SEQ
IDs Nos 1 to 25 respective;ly); and more preferably either RVG PPG ANG (SEQ
1T7 No 8)
or ANG NPG PAG (SEQ ID No 10).
2s Prefer ed examples of peptides according to the invention also include
those comprising, or
having, one of the following amino acid sequences:
VKG ESG SPG ENG SPCA; FAG PPG ADG QPG AKG; AAG ARG NDG QPG PAG;
ADG QPG AKG DQG EAG; APG AKG EAG PTG ARG; PQG PTG VTG PKG ARG;
3o PEG AQG SRG EPG NPCi; AAG RVG PPG ANG NPG; PAG ASG NPG TDG IPG; PPG
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
4
ANG NPG PAG PPG; NPG TDG IPG AKG SAG; RAG DPG LQG PAG APG; AKG
SAG APG IAG APG; PAG APG EKG EPG DDG; APG IAG APG FPG PRG; PPG PQG
LAG QRG IVG; APG FPG PRG PPG PQG; LAG PKG ANG DPG RPG; KQG APG ASG
DRG PPG; EPG LPG ARG LTG RPG; RPG DAG PQG KVG PSG; ETG ALG APG APG
PPG; PPG PAG ANG EKG EVG; PTG KQG DRG EAG AQG; or STG ARG APG EPG
ETG (SEQ IDs Nos 26 to 52 respectively); more preferably AAG RVG PPG ANG NPG
(SEQ ID No 33), and most preferably PPG ANG NPG PAG PPG (SEQ ID No 35).
Preferably the peptide according to the invention comprises a 9 to 15 amino
acid sequence
~o present unintemzpted in the 701-721 region of collagen IL
The invention also relates to a peptide comprising a T-cell epitope specific
to collagen II
which peptide comprises at least nine amino acids having the same sequence as
and
selected consecutively from the 110-239, 338-379 and 587-895 portions of the
sequence of
i s collagen II wherein each amino acid is optionally replaced by a
functionally equivalent
amino acid and wherein the peptide is of formula I as defined above.
According to the invention there is further provided a pharmaceutical
composition
comprising a peptide according to the invention in association with a
pharmaceutically
zo acceptable carrier or diluent. The compositions are preferably for use in
providing
tolerance against an autoimmune condition such as rheumatoid arthritis and
relapsing
polychondritis.
The invention further provides the use of a peptide according to the invention
or of a
is composition according to the invention in the manufacture of a medicament
for use in the
treatment of an autoimmune condition.
According to the invention there is also provided a method of treating a human
or animal
suffering from an autoimmune condition such as rheumatoid arthritis and
relapsing
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
S
polychondritis, which method comprises supplying the human or animal with a
therapeutically effective: amount of the peptide or of the composition.
The peptides according to the invention may be prepared using methods known to
the
s skilled man. For examF~le by using the standard solid phase sequential
coupling technique
utilising an automatic pE;ptide synthesiser (see for example: Jones, J. The
Chemical
Synthesis of Peptides, p:p 132-156) first edition, Oxford University Press,
1991 and R.
Epton (ed) Innovation and Perspectives in Solid Phase Peptide Synthesis, SPCC
(UK), Ltd,
1990). The preparation starts from the C-terminal amino acid which can be
obtained
io grafted to a methylbenzhydrylamine, benzhydrylamine or chloromethylated
resin or a
suitable solid support. The other amino acids are grafted step by step, after
having
protected the side chains thereof. In this coupling method the alpha-amino
groups of the
amino acids are protected with F-moc or t-Boc methodology. Protective groups
for the side
chains of amino acids are well known in the art. The whole protected peptide
is released
~s from the chloromethylat~ed resin by ammoniolysis to obtain the protected
amide, or from
the methylbenzhydrylamine or benzhydrylamine resins by acidolysis.
Peptides according to thf; invention may also be prepared using solution
methods, by either
stepwise or fragment condensations (see for example: Jones, J. The Chemical
Synthesis of
2o Peptides, pp 115-131, first edition, Oxford University Press, 1991). An
appropriately alpha
aminoprotected amino acid is coupled to an appropriately alpha carboxyl
protected amino
acid {such protection may not be required depending on the coupling method
chosen) using
diimides, symmetrical or unsymmetrical anhydrides, or other coupling reagents
or
techniques known to those skilled in the art. These techniques may be either
chemical or
is enzymatic. The alpha amino acid an/or alpha carboxyl protecting groups are
removed and
the next suitably protected amino acid or block of amino acids are coupled to
extend the
growing peptide. Variou<,~ combinations of protecting groups and of chemical
and/or
enzymatic techniques and assembly strategies can be used in each synthesis.
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
6
The peptides according to the invention are defined as comprising a T-cell
epitope. In
other words they are capable of activating T-cells. There are several known
techniques for
determining binding strength . Preferably a peptide according to invention can
activate the
T-cells with a binding strength of at least 2 in an IFN=y release assay and/or
with a
stimulation index of at least 3. The IFN-'y release assay can be carried out
using methods
known in the art or by using the methodology described in the Examples herein.
Similarly
the stimulation index can be determined using known proliferation assays, for
example
those described in "Analysis of type II collagen reactive T cells in the
mouse" Andersson
and Holmdahl, Eur J Immunol 20:1061-1066, 1990, preferably the assay is
carried out
~o using the methodology used in the Examples herein.
The peptides of the invention are of use in therapy without modification but
alternatively
the peptides may be modified, for example they may be conjugated, e.g. bound
covalently,
to delivery systems, for example to mucosal binding structures which include
the cholera ~i
~s toxin which assists absorption in the intestine.
The peptides of the present invention may be used in the treatment, prophlaxis
or diagnosis
of autoimmune conditions and the terms 'therapy' and 'treatment' as used
herein should be
taken also to include prophylaxis and diagnosis.
A peptide of the present invention is to be taken to be an isolated peptide in
the sense that
peptides of the present invention do not include peptides present in an
organism. Peptides
of the present invention may be either isolated from a naturally or
recombinantly produced
peptide or protein or may be chemically synthesised as herein described.
zs
The compounds may be administered at a dosage from about 10 p,g to 10 mg per
day either
as a single dose or in divided doses 2 to 4 times per day. Thus unit doses
comprise from 2.5
p.g to 10 mg of a compound according to the invention. The compounds may be
administered intranasally in the form of solutions, suspensions, HFA aerosols
and dry
3o powder formulations, e.g. Turbuhale ~ formulations; or systemically, e.g.
by oral
CA 02278638 1999-07-19
WO 98133811 PCT/SE98/00128
7
administration in the form of tablets) pills, capsules, syrups, powders or
granules, or by
parenteral administration in the form of sterile parenteral solutions or
suspensions) or by
rectal administration in the form of suppositories.
The compounds of the invention may be administered on their own or as a
pharmaceutical
composition comprising the compound of the invention in combination with a
pharmaceutically acceptable diluent, adjuvant or carrier. Particularly
preferred are
compositions not containing material capable of causing an adverse, e.g. an
allergic,
reaction.
~o
Dry powder formulations and pressurized HFA aerosols of the compounds of the
invention
may be administered by nasal inhalation. For inhalation the compound is
desirably finely
divided. The finely divided compound preferably has a mass median diameter of
less than
~.m, and may be suspended in a propellant mixture with the assistance of a
dispersant,
~s such as a Cg-CZp fatty acid or salt thereof, (e.g. oleic acid), a bile
salt, a phospholipid, an
alkyl saccharide, a perfluorinated or polyethoxylated surfactant, or other
pharmaceutically
acceptable dispersant.
The compounds of the invention may also be administered by means of a dry
powder
zo inhaler. The inhaler may ibe a single or a mufti dose inhaler, and may be a
breath actuated
dry powder inhaler.
One possibility is to mix the finely divided compound with a carrier
substance, e.g. a
mono-, di- or polysaccharide, a sugar alcohol or another polyols. Suitable
carriers are
zs sugars) e.g. lactose, glucose, raffinose, melezitose, lactitol, maltitol,
trehalose, sucrose,
mannitol; and starch. Alternatively the finely divided compound may be coated
by another
substance. The powder mixture may also be dispensed into hard gelatine
capsules, each
containing the desired dose of the active compound.
3o The pharmaceutical composition comprising the compound of the invention may
conveniently be tablets, pills, capsules, syrups) powders or granules for oral
administration;
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98I00128
8
sterile parenteral solutions or suspensions for parenteral administration or
suppositories for
rectal administration.
For oral administration the active compound may be admixed with an adjuvant or
a carrier,
s e.g. lactose, saccharose, sorbitol, mannitol, starches such as potato
starch, corn starch or
amylopectin, cellulose derivatives) a binder such as gelatine or
polyvinylpyrrolidone, and a
lubricant such as magnesium stearate, calcium stearate, polyethylene glycol,
waxes,
paraffin, and the like, and then compressed into tablets. If coated tablets
are required, the
cores, prepared as described above, may be coated with a concentrated sugar
solution
~o which may contain e.g. gum arabic, gelatine, talcum, titanium dioxide, and
the like.
Alternatively, the tablet may be coated with a suitable polymer dissolved in a
readily
volatile organic solvent.
For the preparation of soft gelatine capsules, the compound may be admixed
with e.g. a
is vegetable oil or polyethylene glycol. Hard gelatine capsules may contain
granules of the
compound using either the above mentioned excipients for tablets, e.g.
lactose, saccharose,
sorbitol , mannitol, starches, cellulose derivatives or gelatine. Also liquid
or semisolid
formulations of the drug may be filled into hard gelatine capsules.
2o Liquid preparations for oral application may be in the form of syrups or
suspensions, for
example solutions containing the compound) the balance being sugar and a
mixture of
ethanol, water, glycerol and propylene glycol. Optionally such liquid
preparations may
contain colouring agents, flavouring agents, saccharine and
carboxymethylcellulose as a
thickening agent or other excipients known to those skilled in art.
The invention is now illustrated by the following Examples which should not be
interpreted as limiting the present invention.
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
9
Example 1
The CII peptide chains listed in Table 1 each consisting of 15 amino acids
were synthesised
using an SMPS 350 automated synthesiser (Zinsser, Frankfurt/Main, Germany)
using
known Fmoc chemistry (;>ee Atherton and Sheppard) Solid Phase Peptide
Synthesis - A
s Practical Approach. IRL 1?ress) Oxford). Each peptide was then acetylated at
the N-
terminus and amidated at the C- terminus. The quality of the peptides was
assessed by
HPLC and mass spectroscopy of a sample of the peptides confirmed the expected
molecular weight.
Table 1
0
Pe tideAmino acid residues Pe tide Amino acid residues
110-124VKG ESG SPG ENG SPG 635-649 FAG PPG ADG PG AKG
140-154AAG ARG NDG PG PAG 641-655 ADG QPG AKG D G EAG
161-175GPG FPG APG AKG EAG 665-679 PSG APG P G PTG VTG
I70-184APG AKG EAG PTG ARG 677-691 P G PTG VTG PKG ARG
I85-199PEG A G SRG EPG NPG 701-715 AAG RVG PPG ANG NPG
203-217PAG ASG NPG TDG IPG 707-721 PPG ANG NPG PAG PPG
209-223NPG TDG IPG AKG SAG 740-754 RAG DPG LQG PAG APG
218-232AKG SAG APG IAG APG 749-763 PAG APG EKG EPG DDG
224-238APG IAG APG FPG PRG 770-784 PPG P G LAG QRG IVG
230-244APG FPG 1PRG PPG P 779-793 PG IVG LPG PG ERG
G
338-352LAG PKG ANG DPG RPG 806-820 K G APG ASG DRG PPG
353-367EPG LPG ARG LTG RPG 821-835 PVG PPG LTG PAG EPG
365-379RPG DAG P G KVG PSG 857-871 ETG ALG APG APG PPG
593-607PPG PAG ANG EKG EVG 881-895 PTG K G DRG EAG A G
614-628STG ARG .APG EPG ETG
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
IO
The portions of the amino acid which are shown in bold were found to be the
core
sequences, i.e. the parts of the amino acids which were bound most strongly,
by comparing
the binding strengths of structurally closely related peptides.
Example 2
Mouse CII was extracted from xiphisterna by pepsin digestion using known
techniques and
then further purified by salt precipitation. Rat CII was purified from the
Swarm
chondrosarcoma again using known techniques. The collagens were dissolved in
0.1 M
acetic acid. Collagen for use in restimulating primed lymph node cells was
denatured by
io incubating at 56°C for 30 minutes.
Example 3
To test proliferative responses and cytokine release in drained lymph node
cells, male
(B10.Q X DBA/1) F1 mice, 7-10 weeks of age, were immunised in each hind foot
pad with
is 50 p.g mouse CiI or synthetic peptide emulsified in CFA (containing H37Ra,
Difco,
Detroit, MI). Arthritis was induced in the same mice when they were 7 to 10
weeks old, by
immunising the mice at the base of the tail with 100p.g of rat CII emulsified
CFA as
prepared in Example 2.
2o After 5 weeks, mice were boosted with 50~tg of CII emulsified in a 1:1
ratio by weight with
IFA (DIFCO, Detroit) MI). Lymph node cells drained from severely arthritic
joints from
these mice were then removed and pooled to test reactivity to the panel of
mouse CII
peptides prepared in Examples 1 in a proliferation assay in the following way.
2s Cytokine release was assayed from 50 ~.1 supernatants of primary cultures
of mouse CII
primed lymph node cells were restimulated in vitro with the panel of CII
peptides prepared
according to Example 1 using IFN y and IL-4 minikits (Endogen) Cambridge, MA).
The results are shown in Table 2.
CA 02278638 1999-07-19
WO 98/33811 PCTISE98/00128
11
Table 2
Pe tideSI IFN-y Release Pe tideSI IFN-y Release
(nglml) (n /ml)
110-1242.2 2.2 635-6492.7 0.4
140-1542.1 1.5 641-6552.0 0.9
161-1753.1 4.2 665-6793.5 1.5
170-184 1.5 677-6912.2 2.9
185-1994.1 1.9 701-71518.0 2.5
203-2173.6 6.5 707-72119.4 5,g
209-2235.2 740-7542.8 2.4
218-2323.9 2.3 749-7634.8 1.0
224-2385.0 4.4 770-7842.7 1.4
230-2443.9 5.8 779-7933.9
338-3524.4 2.0 806-8202.3
353-3673.1 1.5 821-835 l,g
365-3792.8 857-8712.4 0.3
-
1593-6072.9 0.7 881-8952.6 0.6
'~ 614-6282.7 2.3
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98/00128
12
Example 4
Lymph node cells from mice primed with mouse CII were depleted of either T
cells or B
cells by passage through a magnetically activated cell sorter (MACS) using
super-
paramagnetic microbeads conjugated with monoclonal rat anti-mouse L3T4 (CD4)
antibodies or rat anti-mouse B220 (CD45R) (both from Miltenyi Biotec GmbH)
Bergisch
Gladbach, Germany) as recommended by the manufacturer except that PBS
containing 2%
FCS, S mM EDTA, 50 ~.M 2-ME and 10 mM HEPES was substituted for PBSBSA buffer.
The fractions were analysed on a FACScan flow cytometer (Becton Dickinson)
using
FITC-conjugated anti-mouse-CD3-E for staining T-cells and FTTC-conjugated anti-
mouse-
~o x-light-chain for B cells (Pharmigen, San Diego, CA). The cells were washed
three times
with DMEM medium without serum after separation and then put into
proliferation assays
using the method described in Example 3. Enriched CD4+ T cells were mixed
together
with spleen cells from spleens from syngeneic mice as antigen presenting cells
in a ratio of
1:2 by weight. AP cells are used in the form of a single cell suspension from
spleens
is treated with 0.84% NH4C1 at pH 7.4 to lyse red blood cells.
Table 3
CeII T a CPM x 10 3
Unfractionated 13.6
CD4+ Enriched 21.4
CD4+ De leted 0.3
I CD45R+ De leted 12.5
I CD45R+ Enriched 0.4
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98N10128
13
. SEQUENCE LISTING
SEQUENCE ID No. 1: ESG SPG ENG
s SEQUENCE ID No. 2: PPG ADG QPG
SEQUENCE ll~ No. 3: ARG NDG QPG
SEQUENCE ID No. 4: QPG AKG DQG
SEQUENCE ID No. 5: APG AKG EAG
SEQUENCE 1D No. 6: PTG VTG PKG
io SEQUENCE ID No. 7: .AQG SRG EPG
SEQUENCE ID No. 8: 1(tVG PPG ANG
SEQUENCE ID No. 9:1?AG ASG NPG
SEQUENCE ID No. 10: ANG NPG PAG
SEQUENCE ID No. 11: TDG IPG AKG
is SEQUENCE 1D No. 12: DPG LQG PAG
SEQUENCE 1D No. 13: SAG APG IAG
SEQUENCE 1D No. 14: APG EKG EPG
SEQUENCE ID No. 15: IAG APG FPG
SEQUENCE ID No. 16: PQG LAG QRG
zo SEQUENCE ID No. 17: FPG PRG PPG
SEQUENCE ID No. 18: PKG ANG DPG
SEQUENCE ID No. 19: APG ASG DRG
SEQUENCE m No. 20: :LPG ARG LTG
SEQUENCE ID No. 21: :DAG PQG KVG
zs SEQUENCE ID No. 22: ,ALG APG APG
SEQUENCE ID No. 23: PAG ANG EKG
SEQUENCE ID No. 24: I~QG DRG EAG
SEQUENCE ID No. 25: ARG APG EPG
SEQUENCE ID No. 26: VKG ESG SPG ENG SPG
3o SEQUENCE 117 No. 27: FAG PPG ADG QPG AKG
SEQUENCE B~ No. 28: AAG ARG NDG QPG PAG
CA 02278638 1999-07-19
WO 98/33811 PCT/SE98100128
14
SEQUENCE ID No. 29: ADG QPG AKG DQG EAG
SEQUENCE ID No. 30: APG AKG EAG PTG ARG
SEQUENCE ID No. 31: PQG PTG VTG PKG ARG
SEQUENCE ID No. 32: PEG AQG SRG EPG NPG
s SEQUENCE ID No. 33: AAG RVG PPG ANG NPG
SEQUENCE ID No. 34: PAG ASG NPG TDG IPG
SEQUENCE ID No. 35: PPG ANG NPG PAG PPG
SEQUENCE ID No. 36: NPG TDG IPG AKG SAG
SEQUENCE ID No. 37: RAG DPG LQG PAG APG
~o SEQUENCE ID No. 38: AKG SAG APG IAG APG
SEQUENCE ID No. 39: PAG APG EKG EPG DDG
SEQUENCE ID No. 40: APG IAG APG FPG PRG
SEQUENCE ID No. 41: PPG PQG LAG QRG IVG
SEQUENCE ID No. 42: APG FPG PRG PPG PQG
is SEQUENCE ID No. 43: LAG PKG ANG DPG RPG
SEQUENCE ID No. 44: KQG APG ASG DRG PPG
SEQUENCE ID No. 45: EPG LPG ARG LTG RPG
SEQUENCE ID No. 46: RPG DAG PQG KVG PSG
SEQUENCE ID No. 47: EXG ALG APG APG PPG
2o SEQUENCE ID No. 48: PPG PAG ANG EKG EVG
SEQUENCE ID No. 49: PTG KQG DRG EAG AQG
SEQUENCE 117 No. 50: STG ARG APG EPG ETG
SEQUENCE ID No. S 1: AAG RVG PPG ANG NPG
SEQUENCE ID No. 52: PPG ANG NPG PAG PPG
