Note: Descriptions are shown in the official language in which they were submitted.
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Modified peptides and peptidomimetics for use in immunotherapy
The present invention relates to modified peptides which are based on HC gp-39
(263-
275), pharmaceutical compositions comprising such peptides as well as the use
of these
s peptides for inducing tolerance induction in patients suffering from
autoimmune
diseases.
The immune system is established on a principle of discrimination between
foreign
antigens (non-self antigens) and autoantigens (self antigens, derived from the
individuals own body) achieved by a build-in tolerance against the
autoantigens.
i o The immune system protects individuals against foreign antigens and
responds to
exposure to a foreign antigen by activating specific cells such as T- and B
lymphocytes
and producing soluble factors like interleukins, antibodies and complement
factors. The
antigen to which the immune system responds is degraded by the antigen
presenting
cells (APCs) and a fragment of the antigen is expressed on the cell surface
associated
~s with a major histocompatibility complex (MHC) class II glycoprotein. The
MHC
glycoprotein-antigen-fragment complex is presented to a T cell which by virtue
of its T
cell receptor recognizes the antigen fragment conjointly with the MHC class II
protein
to which it is bound. The T cell becomes activated, i.e. proliferates and/or
produces
interleukins, resulting in the expansion of the activated lymphocytes directed
to the
2o antigen under attack (Grey et al., Sci. Am., 261:38-46. 1989).
Self antigens are also continuously processed and presented as antigen
fragments by the
MHC glycoproteins to T cells (Jardetsky et al., Nature 353:326-329, 1991).
Self
recognition thus is intrinsic to the immune system. Under normal circumstances
the
immune system is tolerant to self antigens and activation of the immune
response by
2s these self antigens is avoided.
When tolerance to self antigens is lost, the immune system becomes activated
against
one or more self antigens, resulting in the activation of autoreactive T cells
and the
production of autoantibodies. This phenomenon is referred to as autoimmunity.
As the
immune response in general is destructive, i.e. meant to destroy the invasive
foreign
3o antigen, autoimmune responses can cause destruction of the body's own
tissue.
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2
The contribution of T cells to autoimmune diseases has been established in
several
studies. In mice, experimental autoimmune encephalomyelitis (EAE) is mediated
by a
highly restricted group of T cells, linked by their specificity for a single
epitope of
myelin basic protein (MBP) complexed to an MHC class II molecule. In the Lewis
rat,
s a species with high susceptibility to various autoimmune diseases, disease
has been
shown to be mediated by T cells. In humans autoimmune diseases are also
thought to
be associated with the development of auto-aggressive T cells.
A destructive autoimmune response has been implicated in various diseases such
as
rheumatoid arthritis (RA), in which the integrity of articular cartilage is
destroyed by a
chronic inflammatory process resulting from the presence of large numbers of
activated
lymphocytes and MHC class II expressing cells. The mere presence of cartilage
appears
necessary for sustaining the local inflammatory response: it has been
suggested that
cartilage degradation is associated with the activity of cartilage-responsive
autoreactive
is T cells in RA (Sigall et al., Clin. Exp. Rheumat. 6:59, 1988; Glant et al.,
Biochem. Soc.
Traps. 18:796, 1990; Burmester et al., Rheumatoid arthritis Smolen, Kalden,
Maini
(Eds) Springer-Verlag Berlin Heidelberg, 1992). Furthermore, removal of
cartilage
from RA patients by surgery was shown to reduce the inflammatory process (R.S.
Laskin, J. Bone Joint Surgery (Am) 72:529, 1990). The cartilage proteins are
therefore
2o considered to be target autoantigens which are competent of stimulating T
cells.
Activation of these autoreactive T cells leads to development of autoimmune
disease.
However, the identification of the autoantigenic components that play a role
in the
onset of rheumatoid arthritis has so far remained elusive.
2s The inflammatory response resulting in the destruction of the cartilage can
be treated
by several drugs, such as for example steroid drugs. However, these drugs are
often
immunosuppressive drugs that are nonspecific and have toxic side effects. The
disadvantages of nonspecific immunosuppression makes this a highly
unfavourable
therapy.
3o The antigen-specific, nontoxic immunosuppression therapy provides a very
attractive
alternative for the nonspecific immunosuppression. This antigen-specific
therapy
involves the treatment of patients with the target autoantigen or with
synthetic T cell-
reactive peptides derived from the target autoantigen. These synthetic
peptides
correspond to T cell epitopes of the autoantigen and can be used to induce
specific T
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3
cell tolerance both to themselves and to the autoantigen. Desensitization or
immunological tolerance of the immune system is based on the long-observed
phenomenon that animals which have been fed or have inhaled an antigen or
epitope
are less capable of developing a systemic immune response towards said antigen
or
s epitope when said antigen or epitope is introduced via a systemic route.
Rheumatoid arthritis is an autoimmune disease that occurs more frequently in
HLA-
DR4-positive individuals. The disease association may indicate that DR4
molecules
present autoantigens to T-cells. The target of this autoimmune disease is the
joint
where the articular chondrocyte presents a unique cell type producing products
io organized in a matrix. It is thought that joint destruction as seen in RA
is mediated by
cartilage-specific, autoreactive T-cells. The cartilage-derived protein Human
Cartilage
gp-39 (HC gp-39) has recently been identified as a candidate autoantigen in
RA. A
dominant epitope of the HC gp-39 protein, the peptide covering the 263-275
sequence,
was preferentially recognized in RA patients suggesting that this epitope is a
target of
t s the autoimmune attack in rheumatoid arthritis. Eight out of 18 RA patients
responded
to this peptide and no responders were found in the healthy donor group
(Verheijden et
al., Arthtritis Rheum. 40:111 S, 1997). Thus, the data strongly suggest that
this peptide
or the HC gp-39 protein is a target for immune recognition in the joint.
The significance of HC gp-39 for arthritic disease was further demonstrated by
its
2o arthritogenicity in Balb/c mice. A single injection in the chest region
with ~g amounts
of protein mixed in IFA, induced a chronic joint inflammation reminiscent of
RA.
The response to the HC gp-39 peptide 263-275 was further examined by
generating a
set of DRB1*0401-restricted, peptide-specific T-T hybridomas from DRB1*0401-
transgenic mice following immunisation with HC gp-39. The fine specificity of
the
2s hybridomas specific for peptide 263-275 in the context of DR4 (DB 1 *0401 )
was
defined and compared. As a result 3 hybridomas differing in their recognition
of the
263-275 epitope presented by DRB 1 *0401 encoded molecules were identified.
(The
difference in epitope recognition between the three hybridomas used became
visible
when N- and C-terminal truncated peptides within the 263-275 sequence were
used for
3o stimulation of the different hybridomas). The SG11 hybridoma was found to
respond
optimally to the 265-275 sequence. In contrast, recognition by the 8B 12
hybridoma was
centered around sequence 264-274 whereas the 14611 hybridoma was optimally
responsive to 264-275.
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4
Tolerization of HC gp-39 (263-275)-reactive T-cells may be of benefit to RA
patients.
The present invention provides for modified peptide derivatives based on the
HC gp-39
(263-275) sequence which are superior in their capacity to induce an immune
response
and in their tolerizing capacity.
It was surprisingly found that specific peptide modifications based on HC gp-
39 (263
275) are agonistic for a set of T-cell hybridomas specific for HC gp-39 (263-
275)
peptide and superior in stimulation of two human T-cell clones generated
following
stimulation with peptides accommodating the 263-275 epitope sequence.
Moreover,
to these modified peptides showed a superior tolerizing capacity in vivo.
Thus according to one aspect of the invention there is provided a modified
peptide
derived from H-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (formula
I; SEQ ID NO:1 ) having general formula Q- A 1-A2-A3-A4-AS-A6-A7-A8-A9-A 10-
i s A 11-A 12-A 13- Z (formula II). In general formula II, A 1 through A 13
correspond with
the amino acids of formula I, Q corresponds with H and Z corresponds with OH.
The
modifications according to the present invention are selected from the group
consisting
of
a) substitution of 1-6, preferably 1-4 amino acids at A1 through A13 with non-
natural
2o amino acids or (3 amino acids
b) substitution of one or more amide bonds with reduced amide bonds or
ethylene
isosteres
c) substitutions at Q and/or Z.
The number of modifications to be selected from one or more of these groups
amounts
2s 1-6. In addition the amino acids may be substituted with other natural
amino acids
provided that the total number of modifications does not exceed the number of
6.
Modified peptides based on formula I (HC gp-39 (263-275)) may be stabilised by
C-
andor N- terminal modifications, which will decrease exopeptidase catalysed
3o hydrolysis. Such modifications may include N-terminal acylation, (e.g.
acetylation =
Ac-peptide), C-terminal amide introduction, (e.g. peptide-NHZ), combinations
of
acylation and amide introduction (e.g. Ac-peptide-NHZ) and e.g. introduction
of D-
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amino acids instead of L-amino acids. Other modifications are focused on the
prevention of hydrolysis by endopeptidases.
Table 1. Peptide linkages
Structure Name
Rs Peptide
\N~N NJ
II
H O R2 H O
R~ H R[3 ' Reduced peptide
w
~Nw/w
~
N
N
H R H
2
R~ H ~ Vinylogous peptides
sN / iN~Ni
~
I H
H
O RZ
R2 ~ Peptoid
N
N
wN~
IOI
R~ O R3
R~ HO O Rg N-hydroxy-peptide
w
~N
N
N II
OH O RZ OH O
R~ OI RZ H Oligocarbamates
Nw
~
~O
~
.H~O
~
H
O O
OII R1 H H Oligourea
\
~
~N
~N~
H
H
~
O R2
R_ 2 H R4 H Rs Hydrazinopeptides
~
HZN.N~ N~N ~
NN~
I
I
I
I
O
O
R
R5
R
1 3
0,o R2 0~o Oligosulphone
R~ O O R3
O\N R_ 1 0/N R2 Peptidosulphonamides
s
/j
/
~
H H
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6
R3 Ethylene isostere
w
~l
R2
Examples of these modifications are introduction of D-amino acids instead of L-
amino
acids, modified amino acids, cyclisation within the peptide, introduction of
modified
peptide bonds, e.g. reduced peptide bonds yr[CHZNH] and peptoids (N-alkylated
s glycine derivatives).
Other peptide analogues may be related to the peptides of formula I or general
formula
II but instead of the conventional -NH-C(O)- peptide bonds, the linkages shown
in
Table l, or any combination thereof may be used instead of the individual -NH-
C(O)
bonds. If the amino group at the N-terminus has been removed (e.g. A1
to desaminoarginine in formula II) Q in formula II corresponds to no atom.
Preferred peptides according to the invention are peptides wherein Q is H,
(C~_6)alkyl,
formyl, (C~_6)alkylcarbonyl, carboxy(Ci_6)alkyl, (C,_6)alkyloxycarbonyl, (C2_
6)alkenyloxycarbonyl, (C6_~4)aryl(C1_6)alkyl; (C6_14)aryl(C»)alkyloxycarbonyl,
CH3(OCH2CH2)~-OCHZ-C(O)- wherein n is 1-10, HOCHZ-(CHOH)m-CHZ- wherein m
is is 3-4; 1-methyl-pyridinium-3-carbonyl, 1-methyl-pyridinium-4-carbonyl or
Lys or Q is
absent if A1 is H2N-C(=NH)NH-(CHZ)~ C(O)-wherein n is 2-5;
Z is OR wherein R is H, (C~_6)alkyl, (C2_6)alkenyl, (C6_14)aryl(C»)alkyl,
(C6_,4)(C4_
,3)heteroaryl(C~_6)alkyl or NR1R2 wherein R, and R2 are independently selected
from H,
(C1_6)alkyl or (C6_i4)aryl(C1_6)alkyl;
2o and, optionally, Q and Z contain in addition together up to 10 amino acids
located next
to position A1 and/or A13. Substitution at A1 through A13 with one or more
other
natural amino acids preferably is performed at no more than four, more
preferably two
positions.
2s In the peptides according to the present invention the following
substitutions at general
formula II are to be preferred:
Q is H, (C1_6)alkyl, formyl, (C1_6)alkylcarbonyl, carboxy(C1_6)alkyl,
(C1_6)alkyloxy-
carbonyl, (CZ_6)alkenyloxycarbonyl, (C6_14)aryl(C~_6)alkyl; (C6_14)aryl(C1_
4)alkyloxycarbonyl, CH3(OCHZCH2)~-OCH2-C(O)- wherein n is 1-10, HOCHZ-
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(CHOH)m-CHZ- wherein m is 3-4; 1-methyl-pyridinium-3-carbonyl, 1-methyl-
pyridinium-4-carbonyl or Lys, or Q is absent if Al is HZN-C(=NH)NH-(CH2)~-C(O)-
wherein n is 2-5. More preferred are substitions wherein Q is H, (Ct_6)alkyl,
(C,_
6)alkylcarbonyl, carboxy(C,_6)alkyl, (C,_6)alkyloxycarbonyl, CH3(OCHZCHZ)~-
OCHZ-
s C(O)- wherein n is 1-10, HOCHZ-(CHOH)m-CHZ- wherein m is 3-4; 1-methyl-
pyridinium-3-carbonyl, 1-methyl-pyridinium-4-carbonyl or Lys, or Q is absent
if A1 is
HZN-C(=NH)NH-(CHZ)~-C(O)-wherein n is 2-5. Even more preferred are peptides
wherein is Q is H, methyl; acetyl; carboxymethylene, methoxycarbonyl;
CH3(OCHZCH2)3-OCHZ-C(O)-, D-1-glucityl, 1-methyl-pyridinium-3-carbonyl or 1-
to methyl-pyridinium-4-carbonyl, or Q is absent if A1 is HZN-C(=NH)NH-(CHZ)4-
C(O)-.
A 1 is L-Arg, D-Arg, L-Lys, D-Lys, L-Ala, D-Ala, HZN-C(=NH)NH-(CH2)~-C(O)-
wherein n is 2-5, H2N-(CH2)~-C(O)-, wherein n is 2-7, (R)-{-NH-CH[(CH2)~-NH-
C(=NH)-NH2]-CH2-C(O)-}, wherein n is 2-5 or (S)-{-NH-CH[(CH2)~ NH-C(=NH)-
NHz]-CH2-C(O)-}, wherein n is 2-5 or -N[(CHZ)"-NH-C(=NH)-NHz]CHzC(O)-,
t s wherein n is 2-5. Preferably A 1 is L-Arg, D-Arg, L-Ala, HZN-C(=NH)NH-
(CH2)~
C(O)-wherein n is 2-5, H2N-(CH2)~-C(O)-, wherein n is 2-7, (S)-{-NH-CH[(CH2)n-
NH-
C(=NH)-NHZ]-CHZ-C(O)-}, wherein n is 2-5 or -N[(CH2)~-NH-C(=NH)-
NH2]CHZC(O)-, wherein n is 2-5. More preferably A1 is L-Arg, D-Arg, L-Ala, HzN-
C(=NH)NH-(CHZ)4-C(O)-, H2N-(CH2)~-C(O)-, wherein n is 5-7, (S)-{-NH-CH[(CHZ)3-
2o NH-C(=NH)-NHZ]-CHZ-C(O)-} or -N[(CH2)3-NH-C(=NH)-NH2]CH2C(O)-.
A2 is L-Ser, D-Ser, L-hSer, D-hSer, L-Thr, D-Thr, L-Ala, D-Ala, Gly or -
N[(CH2)~-
OH]-CHZ-C(O)- wherein n is 2-5. Preferably A2 is L-Ser, L-Ala, D-Ala, Gly or -
N[(CHZ)n OH]-CH2-C(O)- wherein n is 2-5. More preferably A2 is L-Ser, L-Ala or
-
N[(CH2)2-OH]-CH2-C(O)-.
2s A3 is L-Phe, D-Phe, L-Phe(X), D-Phe(X) wherein X is independently selected
from
one or more of (Ct~)alkyl, hydroxy, halo, (Ct_6)alkylcarbonylamino, amino or
nitro, L-
Hfe, D-Hfe, L-Thi, D-Thi, L-Cha, D-Cha, L-Pal(3), D-Pal(3), L-1-Nal, D-1-Nal,
L-2-
Nal, D-2-Nal, L-Ser(Bzl), D-Ser(Bzl), (R)-{-NH-CH(CHZ-aryl)-CH2-} or (S)-{-NH-
CH(CHZ-aryl)-CH2-} or (R)-{-NH-CH(CH2-aryl)-CH2-} or (S)-{-NH-CH(CH2-aryl)-
3o CH2-}. Preferably A3 is L-Phe, D-Phe, L-Phe(X) or D-Phe(X) wherein X is
halo or
nitro, L-Hfe, L-Thi, L-Cha, L-Pal(3), L-1-Nal, L-2-Nal, L-Ser(Bzl) or (S)-{-NH
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CH(CHz-aryl)-CHZ-}. More preferably A3 is L-Phe, D-Phe, L-Phe(X) wherein X is
halo or nitro, L-Hfe, L-Thi, L-Cha, L-Pal(3), L-1-Nal, L-2-Nal or L-Ser(Bzl).
A4 is L-Thr, D-Thr- L-Ser-, D-Ser, L-hSer, D-hSer, L-Ala, D-Ala or Gly.
Preferably
A4 is L-Thr or L-Ala.
s AS is L-Leu, D-Leu, L-Ile, D-Ile, L-Val, D-Val-, L-Nva, D-Nva, L-Ala, D-Ala,
Gly,
(R)-{-NH-CH(CHZ-CH(CH3)2)-CHZ-}, or (S)-{-NH-CH(CHZ-CH(CH3)Z)-CHZ-}.
Preferably AS is L-Leu, L-Ala, or (S)-{-NH-CH(CHZ-CH(CH3)2)-CHZ-}.
A6 is L-Ala, D-Ala or Gly. Preferably A6 is L-Ala or Gly.
A7 is L-Ser, D-Ser, L-hSer, D-hSer, L-Thr, D-Thr, L-Ala, D-Ala or Gly.
Preferably A7
is L-Ser or L-Ala.
A8 is L-Ser, D-Ser, L-hSer, D-hSer, L-Thr, D-Thr, L-Ala, D-Ala or Gly.
Preferably A8
is L-Ser or L-Ala.
A9 is L-Glu, D-Glu, L-Asp, D-Asp, L-Ala, D-Ala or Gly. Preferably A9 is L-Glu
or L-
Ala.
is A10 is L-Thr, D-Thr, L-Ser, D-Ser, L-hSer, D-hSer, L-Ala, D-Ala or Gly.
Preferably
A 10 is L-Thr or L-Ala.
A11 is Gly, L-Ala, D-Ala or -NH-CH2-CHZ-. Preferably A11 is Gly, L-Ala or -NH-
CH2-CH2-.
A12 is L-Val, D-Val, L-Nva, D-Nva, L-Leu, D-Leu, L-Ile, D-Ile, (R)-{-NH-
2o CH[CH(CH3)2]-CHZ-}, (S)-{-NH-CH[CH(CH3)Z]-CHZ-}, (R)-{-NH-
CH[CHZCHZCH3]-CH2-}, (S)-{-NH-CH[CH2CH2CH3]-CH2-}, (R)-{-NH-
CH[CH2CH(CH3)2]-CH2-}, (S)-{-NH-CH[CH2CH(CH3)2]-CH2-}, (RR)-{-NH-
CH[CH2(CH(CH3)-CHZCH3]-CHZ-}, (RS)-{-NH-CH[CH2(CH(CH3)-CH2CH3]-CH2-},
(SR)-{-NH-CH[CH2(CH(CH3)-CHZCH3]-CH2-, or (SS)-{-NH-CH[CH2(CH(CH3)-
2s CHZCH3]-CHZ-}. Preferably A12 is L-Val or (S)-{-NH-CH[CH(CH3)2]-CH2-}.
A13 is Gly, L-Ala or D-Ala. Preferably A13 is Gly or L-Ala
Furthermore, in the peptides according to the invention Z is OR wherein R is
H, (C~_
6)alkyl, (C2_6)alkenyl, (C6_ia)aryl(C1~)alkyl, (C4_13)heteroaryl(C,_6)alkyl or
NR1R2
wherein Rl and R2 are independently selected from H, (C1_6)alkyl or
(C6_14)aryl(C~_
30 6)alkyl. Preferably Z is OR wherein R is H or NR1R2 wherein Rl and R2 are
independently selected from H or (C1_6)alkyl. More preferably Z is OH, NH2 or
NHEt.
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9
The peptides according to the invention optionally may be extended at the N
and C
terminal end, i.e. next to A 1 and /or A 13 with several amino acids.
Preferably they may
be extended with up to 10 amino acids. Thus, Q and Z may contain in addition
together
up to 10 amino acids located next to position A 1 and/or A 13.
s The peptides may differ from general formula I at several positions but
preferably they
are modified at 1-4 positions, more preferably at 2-3 positions.
As used herein the term (C ~ _6)alkyl means a branched or unbranched alkyl
group having
1-6 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, sec-
butyl, tert
i o butyl and hexyl. Most preferred are alkyl groups having 1-4 carbon atoms.
The term (C,~)alkyl means a branched or unbranched alkyl group having 1-4
carbon
atoms.
The term (C2_6)alkenyl means a branched or unbranched alkenyl group having 2-6
carbon atoms, such as ethenyl, 2-butenyl etc. (C,~)Alkenyl groups are
preferred, (C,_
is 3)alkenyl groups being the most preferred.
The term (C,_6)alkylcarbonyl means a branched or unbranched alkyl group having
I-6
carbon atoms, attached to a carbonyl group, for example an acetyl group. Most
preferred are alkyl groups having 1-4 carbon atoms.
The term carboxy-(C,_6)alkyl means a carboxy group attached to a branched or
2o unbranched alkyl group having 1-6 carbon atoms. Most preferred are alkyl
groups
having 1-4 carbon atoms.
The term (C i _6)alkyloxycarbonyl means a branched or unbranched alkyl group,
attached
to an oxycarbonyl group, for example a methoxycarbonyl-, or a tert-
butyloxycarbonyl-
(Boc-) group. Most preferred are alkyl groups having 1-4 carbon atoms.
2s The term (C2_6)alkenyloxycarbonyl means a branched or unbranched alkenyl
group
having 2-6 carbon atoms as defined previously, attached to an oxycarbonyl
group, for
example an allyloxycaxbonyl group. (C1~)Alkenyl groups are preferred,
(C1_3)alkenyl
groups being the most preferred.
The term (C1_6)(di)alkylamino means a (di)alkylamino group having I-6 carbon
atoms,
3o the alkyl moiety having the same meaning as previously defined. Preferred
are alkyl
groups having 1-4 carbon atoms.
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The term amino(C,_~)acyl means an acyl group having 1-6 carbon atoms,
functionalized
with an amino group. Preferred are acyl groups having 1-4 carbon atoms.
The term (C~_,4)aryl means an aromatic hydrocarbon group having 6-14 carbon
atoms,
such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, anthracyl, which may
optionally
s be substituted at the ortho and/or meta position with one or more
substituents such as -
but not limited to- hydroxy, halogen, nitro, cyano, amino((C,_6)acyl) or
(di)(C~_
~)alkylamino. the acyl and alkyl moiety having the same meaning as previously
defined.
(C6_,o)Aryl groups are preferred, phenyl being the most preferred.
The term (C,~_~3)heteroaryl(C1_6)alkyl means a substituted or unsubstituted
aromatic
~o group having 4-13 carbon atoms, preferably 4-9, at least including one
heteroatom
selected from N, O and/or S, connected to a branched or an unbranched alkyl
group
having 1-6 carbon atoms. The substituents on the heteroaryl group may be
selected
from the group of substituents listed for the aryl group. Nitrogen-containing
heteroaryl
groups may either be connected via a carbon or a nitrogen atom to the alkyl
group. Of
is the alkyl groups, groups having 1-4 carbon atoms are preferred.
The term (C, _6)alkyl(C6_, 4)aryl means means a branched or unbranched alkyl
group as
defined previously, attached to an aryl group as defined previously.
(C6_~o)Aryl groups
are preferred, phenyl being the most preferred. Of the alkyl groups, groups
having 1-4
carbon atoms are preferred.
2o The term (C6_ia)aryl(C~-6)alkyl means an arylalkyl group, wherein the alkyl
group is a
(C,_6)alkyl group and the aryl group is a (C6_,4)aryl as defined previously,
for example a
benzyl- (Bzl) or a triphenylmethyl- (Trt) group. (C6_,o)Aryl groups are
preferred, phenyl
being the most preferred. Of the alkyl groups, groups having 1-4 carbon atoms
are
preferred.
2s The term (C,_6)alkylcarbonylamino means an alkylcarbonylamino group, the
alkyl
group of which contains 1-6 carbon atoms and has the same meaning as
previously
defined. Alkyl groups having 1-4 carbon atoms are preferred.
The term (C6_,4)aryl(CI~)alkyloxycarbonyl means an (C6_14)aryl group connected
to an
alkyloxycarbonyl group, wherin the alkyl group is a (C »)alkyl group, and the
aryl
3o group is defined as previously, for example a benzyloxycarbonyl- (Z) or an
Fluorenyl-
methoxycarbonyl- (Fmoc) group. (C6_lo)Aryl groups are preferred, phenyl being
the
most preferred.
The term halo means F, Cl, Br or I.
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The naturally occurring amino acids are shown using their abbreviations (3-
letter code)
as follows: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid
(Asp), cysteine
(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),
serine (Ser),
isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine
(Phe),
s proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine
(Val). Of all
the amino acids the stereochemistry is defined as L-.
A non-natural amino acid is an, optionally Na-substituted, a-amino acid having
a
chemical structure not identical to those of the natural amino acids. Non-
natural amino
acids are e.g. Phe(X), with X is a substituent situated at the para position
of the phenyl
to ring of Phe, hSer (2-amino-4-hydroxybutanoic acid), norleucine (Nle, 2-
aminohexanoic
acid), norvaline (Nva, 2-aminopentanoic acid), L-Hfe (L-a-homophenylalanine),
D-Hfe
(D-a-homophenylalanine), L-Thi ((3-thienyl-L-alanine), D-Thi ((3-thienyl-D-
alanine),
L-Cha ((3-cyclohexyl-L-alanine), D-Cha ((3-cyclohexyl-D-alanine), L-Pal(3) ((3-
3-
pyridyl-L-alanine), D-Pal(3) ([i-3-pyridyl-D-alanine), L-1-Nal ((3-1-naphthyl-
L-
ts alanine), D-1-Nal ((3-1-naphthyl-D-alanine), L-2-Nal ((3-2-naphthyl-L-
alanine), D-2-
Nal ((3-2-naphthyl-D-alanine), L-Ser(Bzl) (O-benzyl-L-serine), D-Ser(Bzl) (O-
benzyl-
D-serine) and N-alkylglycine derivatives such as NVaI (N-isopropylglycine,
NArg (N-
(3-guanidinopropyl)glycine) and NhSer (N-(2-hydroxyethyl)glycine). Included
within
this group of amino acids are also the naturally occurring amino acids, the
2o stereochemistry of which is defined as D-.
It is to be understood that in a peptide incorporating a reduced amide bond
the original
carbonyl group of the amino acid has been replaced by a methylene group. In a
peptide
incorporating an ethylene isostere the original carboxamide function (-C(O)-NR-
) has
been replaced by an ethylene group (-CH=CR-).
2s The term yr[CH2NH] between two amino acid residues in a sequence means that
the
original amide bond (-C(O)-NH-) between those amino acid residues has been
replaced
by a reduced amide bond (-CH2NH-).
Several amino acids as indicated in formula I are preferred to be fixed at the
3o corresponding positions at general formula 2. Thus, a preferred embodiment
of the
invention is a modified peptide having general formula Q-Al-A2-A3-Thr-Leu-Ala-
Ser-
Ser-Glu-Thr-A11-A12-Gly-Z (formula III) wherein Q, Al, A2, A3, A11, A12 and Z
are
as defined previously. The most preferred substitutions in general formula III
are for
Al
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12
L-Arg, D-Arg, HZN-C(=NH)NI-I-(CHZ)4-C(O)-, HZN-(CHz)~-C(O)-, wherein n is 5-7
or
-N[(CHZ)3-NH-C(=NH)-NHZ]CHZC(O)-, for A2 L-Ser or -N[(CHZ)Z-OH]-CHZ-C(O)-
,and for A3 L-Phe, L-Phe(X) wherein X is halo, L-1-Nal, L-2-Nal, L-Ser(Bzl), L-
Thi,
L-Cha or L-Pal(3).
s More preferred are peptides according to general formula III wherein A 1 is
Arg, A3 is
Phe and A11 is Gly giving rise to general formula IV: Q-Arg-A2-Phe-Thr-Leu-Ala-
Ser-
Ser-Glu-Thr-Gly-A12-Gly-Z wherein the positions Q, A2, A12 and Z are as
defined
previously.
The most preferred peptides are selected from the group comprising
desaminoargininyl-
~o Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ, desaminoargininyl-Ser-
Phe-
Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, CH3-(OCHZCHZ)3-OCHZ-C(O)-Arg-
Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2, D-1-glucityl-Arg-Ser-Phe-
Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, CH30-C(O)-Arg-Ser-Phe-Thr-Leu-
Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, Ac-Arg-Ser-Phe-y~-[CHZNH]-Thr-Leu-Ala-
is Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2, Ac-Arg-Ser-Phe-Thr-Leu-~r-[CH2NH]-Ala-Ser-
Ser-Glu-Thr-Gly-Val-Gly-NHz, Ac-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-
Val-yr-[CH2NH]-Gly-NH2, Ac-Arg-N[(CH2)2-OH]-CH2-C(O)-Phe-Thr-Leu-Ala-Ser-
Ser-Glu-Thr-Gly- Val-Gly- NH2, Ac-Arg-N[(CH2)2-OH]-CHZ-C(O)-Phe-Thr-Leu-Ala-
Ser-Ser-Glu-Thr-Gly-Val-yl-[CHZNH]-Gly- NH2, H-Arg-Ser-Phe(Cl)-Thr-Leu-Ala-
2o Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, HZN-(CHZ)5-C(O)-Ser-Phe-Thr-Leu-Ala-Ser-Ser-
Glu-Thr-Gly-Val-Gly-OH, H2N-(CHZ)6-C(O)-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-
Gly-Val-Gly-OH, (N-methyl-nicotinoyl)+-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-
Gly-Val-Gly-OH.
2s A suitable methodology towards the synthesis of modified HC gp39 (263-275)
peptides
with N terminal modifications, as visualized in Formula V, commences with
derivatives of Formula VI. The modified peptides are synthesized via the
commonly
used Solid Phase Peptide Synthesis (SPPS) method (B. Merrifield, Solid Phase
Peptide
Synthesis, Peptides 1995, 93-169, Editor: B. Gutte, Academic, San Diego,
California,
3o USA; P. Lloyd-Williams, F. Albericio, E. Giralt, Tetrahedron 49: 11065-
11133, 1993).
Depending on the type of linker that is used, the peptide chain is connected
to the
support via either an ester (PAC linker) or an amide (PAL linker) linkage
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13
Rn1 Rn3
A N
C
Rn2
Formula V
Rn1 Rn3
A~ N linker r'"'
B
Rz
= polyethylene glycol (PEG)-
polystyrene (PS) solid support Formula VI
s After anchoring of the Fmoc-C-terminal amino acid to the solid support (m =
1, A-B =
Fmoc), using e.g. the coupling agents HATU (L. Carpino, A. El-Faham, C.A.
Minor, F.
Albericio, J. Chem. Soc., Chem. Comm. 201-203, 1994) or PyBOP (J. Coste, D. Le-
Nguyen, B. Castro, Tetrahedron Lett. 31:205-208, 1990) and DiPEA, the chain is
elongated (m = 2-12) by sequential acylation with the appropriately protected
Fmoc-
to amino acid derivatives followed by piperidine-mediated removal of the Fmoc
protective group (A-B = H) using an automated peptide synthesizer.
Alternatively,
pentafluorophenyl (Pfp) amino acid active esters (A. Dryland, R.C. Sheppard,
Tetrahedron 44: 859-876, 1988) may be used to effect the condensations.
Subsequently,
the N terminal amino acid B is introduced using the same protocol and the Fmoc-
group
1 s is removed. The thus obtained 13-meric peptide derivative (A = H, B = N
terminal
amino acid, m = 12) is then amenable to functionalization of the N terminus.
Introduction of an additional amide bond at the N terminus (A = alkylcarbonyl)
can be
accomplished by performing another HATU or PyBOP-mediated condensation with
the
desired acid (X-OH) or by coupling with an acyl chloride (X-Cl) in the
presence of
2o pyridine. The charged 1-methyl pyridinium-4-carbonyl unit or 1-methyl
pyridinium-3-
carbonyl unit may be introduced after cleavage (vide infra) from the resin (i.
a Formula
V, A = H, B = N terminal amino acid, Z = OR with R = H, alkyl or Z = NR1 R2
with R1,
R2 = H or alkyl) by DiPEA-mediated reaction of the free N terminus of the
completely
deprotected peptide with the N methyl (iso)nicotinium hydroxysuccinimide
active ester
2s (M.L. Tedjamulia, P.C. Srivastava, F.F. Knapp, J. Med. Chem., 28:1574-1580,
1985)
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14
in aqueous medium (cf. conversion of A = H to A = N-methyl(iso)nicotinium in
Formula V).
N-alkylation may be effected via reductive amination by treating the
immobilized
peptide with an appropriate aldehyde (Formula VI, conversion of A = H to A =
alkyl)
s in the presence of NaBH(OAc)3 in DMF/HOAc (99/1, v/v). Alternatively,
reaction of
the immobilized peptide (A = H) with an alkyl halide in the presence of DiPEA
also
gives access to N alkylated peptides (e.g. reaction with tert-butyl
bromoacetate). The
free NH2 (A = H in Formula VI) may also be functionalized with a carbamoyl
group
(e.g methoxycarbonyl) via reaction with the corresponding carbamoyl chloride
in
to CHZCIZ/DiPEA (conversion of A = H to A = alkyloxycarbonyl in Formula VI).
After
cleavage of the peptide from the solid support with concomitant removal of the
acid-
labile protective groups using TFA/Et3SiH/anisole/ROH (R = H, alkyl) the
peptides
with general Formula V (Z = OR) are purified by RP-HPLC. Alternatively, the C-
terminus may be equipped with an amide function (Z = NRiR2 with R1 and RZ = H
or
t s alkyl) during cleavage from the resin. In that case, a different linker
(PAL: B.
Merrifield, Peptides, 93-169, 1995) between the peptide chain (Formula VI) and
the
PEG-PS solid support is used. If the free amino group of the PAL linker is
alkylated
prior to attachment of the first (C-terminal) amino acid, C-terminal alkyl
amides will be
formed after cleavage from the polymer support.
zo Using the same Fmoc-SPPS strategy, peptides are accessible that contain
unnatural but
commercially available amino acids (e.g. D-amino acids or substituted
phenylalanine
derivatives). Apart from this, N-alkyl glycine derivatives (peptoid monomers,
R~1 =
amino acid side chain, R"2 = H in Formulas V and VI) are first synthesized in
solution
using literature procedures (J.A. Kruijtzer, L.J.F. Hofmeyer, W. Heerma, C.
Versluis,
2s R.M.J. Liskamp, Chem. Eur. J. 4:1570-1580, 1998). Also the modified N
terminal
amino acid (3-homo-L-arginine [B = NH-CH(CH2CH2CH2NH-CH(=NH)NH2)-CHZ-
C(O)] was prepared in solution prior to SPPS, according to known procedures
(H.M.M.
Bastiaans, A.E. Alewijnse, J.L. van der Baan, H.C.J. Ottenheijm, Tetrahedron
Lett.
35:7659-7660, 1994. After protection of the free NH2 group in the thus
obtained
3o monomeric amino acids with a Fmoc protective group, the compounds can be
incorporated in the elongating peptide (Formula VI) using the SPPS protocol.
The final class of modified peptides comprises peptides containing one or more
reduced amide bonds (R~3 = H2 in Formula V). These derivatives are accessible
(J.J.
Wen, A.R. Spatola, J. Pept. Res., 49:3-14, 1997) via a modified SPPS protocol
in
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which the free N-terminal amino acid of the growing chain ( 1 <m< 12 in
Formula VI) is
alkylated with the incoming amino acid aldehyde under reductive conditions
(NaBH3CN, DMF/HOAc, 99/1, v/v). The required N Fmoc protected amino acid
aldehydes are either commercially available or accessible by literature
methods (J.J.
s Wen, C.M. Crews, Tetrahedron: Asymmetry 9:1855-1858, 1998). The thus
elongated
chain (A-B = Fmoc, R~1 = H, Rn2 = amino acid side chain, R~3 = HZ) contains a
secondary amino function (R"+i ~ = H) which is subsequently protected with a
Boc
group. After removal of the Fmoc protective group the peptide chain may be
further
elongated using the SPPS protocol.
to
2
Rn+~
OH
Fmoc~
~ ~O
Rn+~ Formula VII
Alternatively, a dimeric structure with general Formula VII may be synthesized
in
solution prior to SPPS. Thus, the appropriate amino acid benzyl ester (H2N-
CH(Rn+12)-
ts C02Bz1) is reductively alkylated (NaBH3CN, DMF/HOAc, 99/1, v/v) with a Fmoc
protected amino acid aldehyde (Fmoc-NH-CH(R"2)-C(O)H) to give a dimeric
secondary amine. After protection of the amino function with a Boc group
(Rn+11 =
Boc) and subsequent hydrogenolysis of the benzyl ester a compound of Formula
VII is
formed, which can be incorporated into the growing chain using the SPPS
procedure.
The peptides according to the invention can be used as a therapeutical
substance. More
particularly, they can be used for the induction of specific T-cell tolerance
to an
autoantigen in patients who are suffering from autoimmune disease disorders,
more
specifically arthritis.
Modified peptides based on a MHC class II restricted T-cell epitope structure
with
enhanced stimulatory activity in vitro and an enhanced activity in vivo can be
selected
using known technologies.
in order to maintain the agonistic properties of a given T-cell epitope it is
regarded
3o essential not to interfere too much with either the residues involved in
binding to the
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16
relevant MHC molecule nor influence too much the residues involved in TCR
engagement of relevant T-cells. Thus, selection of agonistic modified peptides
would
involve:
1 ) definition of the affinity of a modified peptide for binding the relevant
MHC
s molecule and comparison with the affinity of the wild type, the non-
modified, peptide
epitope
2) definition of the stimulatory activity of a modified peptide and comparison
with the
activity of the wild type, non-modified, peptide, using an in vitro assay
(irradiated
antigen presenting cells co-incubated with peptide antigen and specific T-
cells).
t o Preferably a broad panel of epitope-specific, MHC class II restricted T-
cells, with
different TCR clonotypes, but reactive with the same epitope in the context of
the same
MHC class II molecule, should be evaluated. For this purpose, a panel of
specific T-cell
hybridomas or specific T-cell lines/clones can be employed. Selection of a
modified
epitope for human application will preferably require the use of human T-cell
is lines/clones to safeguard the relevance of the selected modified epitopes
for human T-
cell recognition.
3) definition of the activity of a modified peptide in vivo (optional). For
this purpose
different experimental set-ups may be used
a) a delayed type hypersensitivity test
zo b) an ex vivo T-cell activation assay following the administration of
antigen (with or
without adjuvant) in vivo
c) modulation of disease in experimental models of autoimmune disease by
administration of modified peptide antigen
Preferably compounds with enhanced agonistic activity in vitro, as compared to
the
2s wild type peptide or enhanced in vivo effects are to be selected.
Individual HC gp-39 derived peptides that are being recognised in mice are
expected to
downmodulate reactivity towards these peptides following nasal treatment. Such
reactivity can be measured by challenging the animal with the peptide in
question and
3o quantitating paw swelling as a result of a DTH response. Peptide
immunisations in
Balb/c mice result in immunological responses to the HC gp-39 peptide 263-275.
Thus,
mice immunised with HC gp-39 can be challenged with HC gp-39 263-275 in order
to
detect a DTH response. To delineate tolerogenicity of modified peptides in
vivo, mice
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17
can be treated per nostril with HC gp-39 263-275 or peptide derivatives in
various
concentrations. Modified peptide derivatives with a superior profile in terms
of
tolerance induction are expected to be active in this in vivo assay in lower
concentrations than the original peptide. To be able to quantitatively detect
effects of
s tolerance induction with the native peptide versus modified peptide
derivatives, various
application schemes and dosages can be tested. Finally, it can be investigated
whether
modified forms of HC gp-39 263-275 are more effective in downmodulating HC gp-
39
263-275 induced DTH responses in this model than the native 263-275 peptide.
to Tolerance can be attained by administering high or low doses of the
tolerogen or
peptides according to the invention. The amount of tolerogen or peptide will
depend on
the route of administration, the time of administration, the age of the
patient as well as
general health conditions and diet.
In general, a dosage of 0.01 to 1000 pg of peptide or protein per kg body
weight,
is preferably 0.05 to 500 fig, more preferably 0.1 to 100 ~g of peptide or
protein can be
used.
Another aspect of the invention resides in pharmaceutical compositions
comprising one
or more of the peptides according to the invention and a pharmaceutical
acceptable
carrier.
2o Pharmaceutical acceptable carriers are well known to those skilled in the
art and
include, for example, sterile salin, lactose, sucrose, calcium phosphate,
gelatin, dextrin,
agar, pectin, peanut oil, olive oil, sesame oil and water. Other carriers may
be, for
example MHC class II molecules, if desired embedded in liposomes.
In addition the pharmaceutical composition according to the invention may
comprise
2s one or more adjuvants. Suitable adjuvants include, amongst others,
aluminium
hydroxide, aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A,
muramyl dipeptide and saponins such as Quill A. The amount of adjuvant depends
on
the nature of the adjuvant itself.
Furthermore the pharmaceutical composition according to the invention may
comprise
30 one or more stabilizers such as, for example, carbohydrates including
sorbitol,
mannitol, starch, sucrosedextrin and glucose, proteins such as albumin or
casein, and
buffers like alkaline phosphates.
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18
Suitable administration routes are intramuscular injections, subcutaneous
injections,
intravenous injections or intraperitoneal injections, oral and intranasal
administration.
Oral and intranasal administration are preferred administration routes.
Especially,
modulator cells specific for the antigen could be generated by applying the
antigen via
s the mucosae, for instance the nasal mucosae. Mucosal administration of
antigens has
been shown to induce immunological tolerance to such antigens.
The peptides according to the invention are also very suitable for use in a
diagnostic
method to detect the presence of activated autoreactive T cells involved in
the chronic
inflammation of the articular cartilage.
to The diagnostic method according to the invention comprises the following
steps:
a) isolation of the peripheral blood mononuclear cells (PBMC) from a blood
sample
of an individual,
b) culture said PBMC under suitable conditions,
c) incubation of said PBMC culture in the presence of the autoantigen or one
or more
~ s peptides derived thereof according to the invention, and
d) detection of a response of T cells, for example a proliferative response,
indicating
the presence of activated autoreactive T cells in the individual.
In case of detection of a response by measuring the proliferative response of
the
autoreactive T cells, the incorporation of a radioisotope such as for example
3H-
zo thymidine is a measure for the proliferation. A response of the
autoreactive T cells
present in the PBMC can also be detected by measuring the cytokine release
with
cytokine-specific ELISA, or the cytotoxicity with S~Chromium release. Another
detection method is the measurement of expression of activation markers by
FACS
analysis, for example of Il-2R. A diagnostic composition comprising one or
more of the
2s peptides according to the invention and a suitable detecting agent thus
forms part of the
invention. Depending on the type of dection, the detection agent can be a
radioisotope,
an enzyme, or antibodies specific for cell surface or activation markers.
Also within the scope of the invention are test kits which comprise one or
more
peptides according to the invention. These test kits are suitable for use in a
diagnostic
3o method according to the invention.
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19
Thus, according to the present invention HC gp-39 derived modified peptides
can be
used to downmodulate autoimmune disease.
The following examples are illustrative for the invention and should in no way
be
interpreted as limiting the scope of the invention.
s
Legends to the figures
Figure 1
Proliferation of clone 235 following stimulation with lead peptide or selected
modified
peptides using irradiated, autologous PBMC as APCs was measured as described
in
to example 15. Peptides were tested for their stimulatory activity in
concentrations of 0,
0.4, 2, 10 and 50 p,g/ml. The response of the 235 clone following stimulation
with lead
peptide H-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (closed
circles), stimulation with Ac-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-
yr[CHZNH]-Gly-NH2 (closed squares), stimulation with Ac-Arg-NhSer-Phe-Thr-Leu-
is Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2 (open circles) or stimulation with Ac-
Arg-
NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-~r[CH2NH]-Gly-NHZ (open
squares) is shown.
Table 2
Hybridoma assay (first line t est) : + = compound stimulates all three
hybridomas in a
2o fashion comparable to or superior to the non-modified 263-275 peptide. +* =
agonist
activity demonstrated for 1 or 2 hybridomas but not for all three. Reactivity
of human
clones (proliferation of clone 235 and 243) in potency (stimulatory activity
of
analogue/stimulatory activity of lead peptide; e.g.HC gp-39 (263-275)). - =
potency <
0.6, + = potency 0.6 -12, ++ = potency > 12 - 100, +++ = potency > 100. Ac-Arg-
NhSer-
2s Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHz, Ac-Arg-Ser-Phe-Thr-Leu-Ala-
Ser-Ser-Glu-Thr-Gly-Val-yr[CHZNH]-Gly-NHZ, D-1-Glucityl-Arg-Ser-Phe-Thr-Leu-
Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, CH3(OCHZCHZ)3-OCH2C(O)-Arg-Ser-Phe-
Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2 and H-Arg-Ser-Phe(4C1)-Thr-Leu-
Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH were tested for their affinity to bind HLA-
3o DRB1*0401 and compared to the affinity of the lead-peptide (H-Arg-Ser-Phe-
Thr-Leu-
Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH). The most active compounds (Ac-Arg-NhSer-
Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHS and Ac-Arg-Ser-Phe-Thr-Leu-
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Ala-Ser-Ser-Glu-Thr-Gly-Val-y~[CHZNH]-Gly-NHz) showed a relative affinity for
binding to HLA-DRB1*0401 which was comparable to the affinity of the original
peptide.
s Examples
Example 1
H-Arg-Ser-Phe(4CI)-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (1)
The reaction vessel of the Millipore 9050 PepSynthesizer was charged with 0.5
g of
Fmoc-Gly-PAC-PEG-PS (commercially available at PerSeptive Biosystems, 0.20
io mmol/g) resin, pre-swollen in N-methyl-pyrrolidinone (NMP). Removal of the
Fmoc
group in each coupling cycle was effected with piperidine/DMF ( 1:4 v/v). The
coupling
efficiencies were determined by spectroscopic analysis of the Fmoc-cleavage
after each
elongation step. In each coupling step 4 equivalents of the appropriate acid-
labile side-
chain-protected Fmoc amino acid were used. The double syringe mode of the
is synthesizer was used in which one syrznge contains 0.50 M HATU in DMF p.a.
and the
other syringe contains 1.0 M DIPEA in DMF p.a. The main wash contained N-
methyl-
pyrrolidinone with 0.1% HOBt. The Analog Synthesis protocol was used. After
removal of the final Fmoc group the resin with the immobilized peptide was
taken out
of the reaction vessel and washed successively with DMF (20 mL), CH2C12 (20
mL),
2o diethylether (20 mL), CHZC12 (20 mL), diethylether (20 mL), CHZC12 (20 mL)
and
diethylether (20 mL). The immobilized peptide was dried in vacuo overnight.
The
peptide was then cleaved off with 10 mL of the mixture
TFA/(iPr)3SiH/anisole/H20
88/5/5/2 v/v/v/v for 3 hours. In this step all the acid-labile side-chain
protective groups
were also removed. After evaporation of the solvent the peptide was
precipitated with
2s 200 mL of diethyl ether. The ether layer was decanted and the peptide was
washed with
an additional amount (2 x 200 mL) of ether. The crude peptide was then dried
with a
stream of nitrogen and lyophilized. Purification of the peptide was carned out
by HPLC
chromatography on a PrepPak cartridge 40-100 mm Delta-PakTM C18 15 pm 100A
reverse phase column. The mobile phase consisted of a mixture of 20% of
phosphate
3o buffer pH 2.1 and a gradient of acetonitrile and water, as shown in the
analysis below.
The peptide was desalted on the HPLC, using 4°/0o aqueous acetic acid.
The purified
product was lyophilized.
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21
Mobile phase: A: 0.5 mol/L NaH2P04 + H~P04, pH=2.1
B: H20
C: CH3CN/H20 = 3/2 (v/v)
gradient: A: 20%; B: 80% ~ 20%; C: 0% ~ 60% in 40 min.
Yield: 68 mg; HPLC purity: 90.1%; MS: MW = 1346, this agrees with the
molecular
formula CSSHg9C1N,602,; amino acid analysis: all amino acids were found in the
required amounts; peptide content: 74.8%; ion chromatography: phosphate: 0.6%,
acetate: 0.6%, chloride: 3.4% (w/w).
to
Example 2
HZN-(CHZ)5-C(O)-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (2)
The peptide was synthesized using solid phase peptide chemistry, as reported
in the
synthesis of compound 1 (see above). In this case, commercially available Fmoc-
amino
t s acid pentafluorophenyl (Pfp) active esters were used instead of the free
Fmoc-amino
acids and HATU/DIPEA. The compound was prepared using 6-Fmoc-amino-hexanoic
acid as the N-terminal amino acid, obtained from 6-amino-hexanoic acid,
analogous to
the literature procedure (A. Marston, E. Hecker, Z. Naturforsch. B Anorg.
Chem. Org.
Chem., 38:1015-1021,1983). The support was Fmoc-Gly-PAC-PEG-PS (0.75 g, 0.170
2o mmol/g) and 3 equiv. Of the appropriate Pfp esters were used. For the
coupling of 6
Fmoc-amino-hexanoic acid PyBOP was applied as the coupling agent ( 199 mg).
Workup as reported in the standard procedure (example 1 ) gave 168 mg of crude
product. This was purified by HPLC (phosphate system pH=2.1, with CH3CN-H20
gradient). The product was desalted on the HPLC with 5
°/°° aqueous acetic acid and
2s freeze-dried to give 34 mg of the required peptide.
HPLC purity: 99.6%; MS: MW = 1268, this agrees with the molecular formula
CSSH89N13~21s amino acid analysis: all amino acids were found in the required
amounts; peptide content: 67.3%; ion chromatography: phosphate: 10% (w/w).
3o Example 3
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HZN-(CH2)~-C(O)-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (3)
Peptide 3 was prepared in an identical fashion as its N-terminal homolog 2
using 7-
Fmoc-amino-heptanoic acid (3a, prepared analogous to compound 2a: A. Marston,
E.
Hecker, Z. Naturforsch. B Anorg. Chem. Org. Chem., 38:1015-1021, 1983) as the
N-
s terminal amino acid. The support was Fmoc-Gly-PAC-PEG-PS (1.0 g, 0.17
mmol/g).
Workup, HPLC purification and desalting as reported in the standard procedure
(example 1 ) gave 45 mg of the required peptide.
HPLC purity: 95.0%; MS: MW = 1282, this agrees with the molecular formula
CS~HgiNi3O2,; amino acid analysis: all amino acids were found in the required
to amounts; peptide content: 87.4%; ion chromatography: acetate: 0.2% (w/w).
Example 4
(N-methyl-nicotinoyl)+-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-
OH (4)
t s Prior to the preparation of peptide 4, the starting material N-
succinimidyl ( 1-methyl-3-
pyridinio)formate iodide (4a) was synthesized via a literature procedure (M.L.
Tedjamulia, P.C. Srivastava, F.F. Knapp; J. Med. Chem. 28:1574-1580, 1985).
The
synthesis of compond 4 was carried out in solution. The peptide H-Arg-Ser-Phe-
Thr-
Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (4b, 26 mg, 0.02 mmol), prepared by
SPPS
2o methods according to example 1, was dissolved in DMF/H20 (1/99 v/v, 10 mL)
and
DIPEA/DMF ( 1 / 1, v/v) was added to give pH=9. Then N-succinimidyl ( 1-methyl-
3-
pyridinio)formate iodide (4a, 0.056 g. 0.15 mmol) was added in two portions.
The pH
was kept at pH=9 by adding a few drops of DIPEA/DMF ( 1 / 1, v/v). The mixture
was
stirred at room temperature for 4 hours and then diluted with 10 mL of H20 and
5 mL
2s of phosphate buffer pH=2.1. The product was purified immediately by HPLC
with the
phosphate buffer system as shown previously (example 1 ). Desalting with
5°/°° aqueous
acetic acid and lyophilisation provided 14 mg of the required peptide 4.
HPLC purity: 98.1%; MS: MW = 1430; amino acid analysis: all amino acids were
found in the required amounts; peptide content: 56.3%; ion chromatography:
chloride:
30 1.4%, phosphate: 1.0%, trifluoroacetate: 0.8%, acetate: 0.3% (w/w).
Example 5
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Desaminoargininyl-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (5)
Peptide 5 was synthesized according to the previously described procedure for
compound 1 using Fmoc-protected amino acids, HATU, DIPEA and 1.0 g of Fmoc-
Gly-PAC-PEG-PS-resin, support loading 0.17 mmol/g. In the final step desamino-
s Arg(Adoc)Z-OH (5a) was coupled to the immobilized peptide chain. Carboxylic
acid 5a
was prepared according to a known procedure (R. Presentini, G. Antoni, Int. J.
Pept.
Protein Res., 27:123-126, 1986). Workup and purification conditions were
identical to
those of peptide 1.
Yield: 58 mg; HPLC purity: 91.1%; MS: MW = 1296; amino acid analysis: all
amino
acids were found in the required amounts; peptide content: 76.2%; ion
chromatography: phosphate: 0.4%, trifluoroacetate: 0.6%, acetate: 0.2% (w/w).
Example 6
Desaminoargininyl-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2 (6)
is The assembly of peptide 6 was conducted in a fashion similar to that of
previously
described peptide 5, using PAL-PEG-PS resin (0.17 mmol/g) instead of PAC-PEG-
PS
as the solid support. In this case, the Fmoc group from commercially available
(PerSeptive Biosystems) Fmoc-PAL-PEG-PS resin was removed and the resulting H-
PAL-PEG-PS support was condensed with Fmoc-Gly-OH under the agency of
2o HATU/DIPEA. After elongation of the peptide chain and subsequent cleavage
from the
resin under the same conditions as described in example 1, the required
carboxamide
C-terminus was obtained. Workup and purification conditions were identical to
those
of peptide 1.
Yield: 43 mg; HPLC purity: 91.3%; MS: MW = 1295; amino acid analysis: all
amino
2s acids were found in the required amounts; peptide content: 76.5%; ion
chromatography: chloride: 0.5%, acetate: 4.0% (w/w).
Example 7
CH3(OCHzCH2)3-OCHZ-C(O)-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-
3o Val-Gly-NH2 (7)
For the synthesis of peptide 7 the starting material CH3(OCHZCH2)3-OCHZ-C02H
(7a)
was prepared first, according to a literature procedure (A.H. Haines, P.
Karntiang,
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Carbohydr. Res., 78:205-211, 1980). The synthesis of the protected and
immobilized
peptide H-Arg(Pmc)-Ser(tBu)-Phe-Thr(tBu)-Leu-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-
Thr(tBu)-Gly-Val-Gly-PAL-PEG-PS (7b) was conducted as shown in example 2 using
amino acid Pfp esters. The peptide on the resin (7b) was pre-swollen in NMP
and 142
s mg (0.64 mmol) of CH3(OCHZCHZ)3-OCHzCO2H (7a) was added, together with 169
mg (0.64 mmol) of the coupling agent TFFH (tetramethylfluoro-formamidinium
hexafluorophosphate). The combined reagents were circulated during 60 minutes
in de
Pepsynthesizer. Cleavage from the resin and workup were executed as described
in
example 5. The crude peptide was then purified by HPLC with the system and
solvents
to delineated in example 1. The product was desalted on the HPLC column using
2.5°/°°
of AcOH.
Yield: 120 mg; HPLC purity: 78%; MS: MW = 1515; ion chromatography: chloride:
0.1%, phosphate: 0.3%, trifluoroacetate: 4.0%, acetate: 0.3% (w/w).
t s Example 8
D-1-glucityl-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (8)
Prior to the assembly of N-terminally modified peptide 8, the peptide H-
Arg(Pmc)-
Ser(tBu)-Phe-Thr(tBu)-Leu-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-
PAC-PEG-PS (8a), having the same sequence as peptide 7b but differing in the
type of
20 linker (PAC instead of PAL), was prepared according to example 1. Reductive
amination was effected by overnight treatment of 6-O-trityl-a/(3-D-
glucopyranose (8b,
422 mg, 1.0 mmol, T. Utamura, K. Kuromatsu, K. Suwa, K. Koizumi, T. Shingu,
Tetsuro; Chem. Pharm. Bull. 34:2341-2353, 1986) with the immobilized peptide
8a
(500 mg, 0.2 mmol/g) in DMF/HOAc (99/ 1, v/v, 10 mL) using NaBH(Oac)3 (212 mg,
2s 1.0 mmol) as the reducing agent. Subsequent cleavage of the resulting fully
protected
derivative (6-O-trityl-D-1-glucityl)-Arg(Pmc)-Ser(tBu)-Phe-Thr(tBu)-Leu-Ala
Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-PAC-PEG-PS from the resin
using the conditions described in example 1, with concomitant removal of the
trityl
group and all amino-acid-protecting groups, furnished 38 mg of the target
peptide 8,
3o after purification by preparative HPLC and desalting with 5 %°
aqueous HOAc.
HPLC purity: 84.7%; MS: MW = 1475; amino acid analysis: all amino acids were
found in the required amounts; peptide content: 61.0%; ion chromatography:
chloride:
0.1 %, acetate: 1.7% (w/w).
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Example 9
Me0-C(O)-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH (9)
The synthesis of peptide 9 commenced by suspending the immobilized peptide H-
s Arg(Pmc)-Ser(tBu)-Phe-Thr(tBu)-Leu-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-
Gly-Val-Gly-PAC-PEG-PS (8a) in dioxane and cooling to 0 °C. To this
suspension,
100 ~1 of 4N aq NaOH and 100 p1 of methyl chloroformate were added. The
reaction
mixture was agitated for 16 h and subsequently, the resin was washed with
EtOH/HZO,
EtOH, CH2ClZ and ether. After drying in vacuo, the product was cleaved off the
resin
~o and purified as described in the previous peptide syntheses (example 1).
Finally, the
peptide was desalted on the HPLC using 5°/°° of aqueous
acetic acid and then
lyophilized to give peptide 9.
Yield: 11 mg; HPLC purity: 96.8%; MS: MW = 1368; amino acid analysis: all
amino
acids were found in the required amounts; peptide content: 60.5%; ion
is chromatography: chloride: 2.0%, phosphate: 0.2%, acetate: 0.4% (w/w).
Example 10
Ac-Arg-Ser-Phe-Thr-Leu-yr[CHZNH]-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2 (10)
Prior to the synthesis of peptide 10, the required amino acid aldehyde
building block
2o Fmoc-Leu-H (10a) was prepared via a known procedure (J.-P. Meyer, P. Davis,
K.B.
Lee, F. Porreca, H.I. Yamamura, V. Hruby, J. Med. Chem. 38:3462-3468, 1995).
Compound 10a was used without further purification. According to the method
described in example l, the resin was functionalized with an 8-amino acid
peptide
chain to give H-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-PAL-PEG-
2s PS (10b). The latter immobilized derivative (1 g, 0.2 mmol/g) was suspended
in S mL
of 1 % acetic acid in DMF. Two solutions were prepared, being 148 mg of Fmoc-
Leu-H
(10a) in 2.5 mL of DMF and 30 mg of NaCNBH3 in 2.5 mL of DMF. Both solutions
were combined and added to the suspension of peptide 10b. The mixture was
agitated
overnight at room temperature. Subsequently, the thus obtained intermediate
Fmoc-
3o Leu-yr[CHZNH]-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-PAL-PEG-
PS (10c) was protected at the newly introduced secondary amine function with
Boc20
and pyridine. The resin-bound peptide lOc was suspended in 10 mL of dry CH2C12
and
mg (0.16 mmol) of Boc20 and 13 ~.1 (0.16 mmol) of pyridine were added. The pH
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26
was kept at pH=8 with pyridine and the mixture was agitated overnight. Workup
involved washing of the resin with CHZC12, EtOH, CHzCIZ, ether and drying in
vacuo.
The synthesis was continued by SPPS using Fmoc amino acids and the HATU/DIPEA
protocol with NMP as the solvent (example 1 ). The last step involved coupling
with 4-
s nitrophenyl acetate to introduce the N-terminal acetyl group. After workup
as described
in example 1, the crude peptide was purified by HPLC, desalted with S
°/oo of acetic
acid and freeze-dried to give the target peptide 10.
Yield: 28 mg; HPLC purity: 76.3%; MS: MW = 1339; ion chromatography:
trifluoroacetate: 1.2%, acetate: 2.0% (w/w).
to
Example 11
Ac-Arg-Ser-Phe-y~[CH2NH]-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ (11)
The synthesis of 11 involved the reductive coupling of Fmoc-Phe-H (11a, J.-P.
Meyer,
P. Davis, K.B. Lee, F. Porreca, H.I. Yamamura, V. Hruby, J. Med. Chem.,
38:3462-
ts 3468, 1995) to the resin-bound protected peptide H-Thr(tBu)-Leu-Ala-
Ser(tBu)-
Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-PAL-PEG-PS (11b) obtained via the SPPS
protocol described in example 1. Peptide llb (1.0 g, 0.2 mmol/g) and aldehyde
lla
(200 mg) were suspended in 5 mL of 1% acetic acid/DMF and immediately 30 mg
(0.48 mmol) of NaCNBH3, dissolved in 5 mL of DMF, was added. The mixture was
2o stirred for 16 h, resulting in the formation of Fmoc-Phe-yr[CH2NH]-Thr(tBu)-
Leu-Ala-
Ser(tBu)-Ser(tBu)-Glu(OtBu)-Thr(tBu)-Gly-Val-Gly-PAL-PEG-PS. The peptide chain
was then elongated with the appropriate Fmoc-amino acids and N-terminal
acetylating
agent using the HATU/DIPEA SPPS protocol as described in example 8. Workup,
HPLC-purification and desalting were carried out as described in example 1.
2s Yield: 52 mg; HPLC purity: 97.9%; MS: MW = 1338; amino acid analysis: all
amino
acids were found in the required amounts; peptide content: 92.4%; ion
chromatography: acetate: 2.5% (w/w).
Example 12
3o Ac-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-yr[CHzNH]-Gly-NH2 (12)
For the synthesis of this compound, it was impossible to carry out the
reductive
alkylation with Fmoc-Val-H on the resin. Therefore, the dipeptide analogue
Fmoc-Val-
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27
y[CHZNH]-Gly-OH (12d) was prepared in solution prior to immobilization to the
resin.
Fmoc-Val-yr[CHzNH]-Gly-Obzl (12c)
s Fmoc-Val-H (12a, 3.16 g, 10 mmol, prepared according to T. Moriwake, S.-I.
Hamano,
S. Saito, S. Torii, S. Kashino, J. Org. Chem., 54:4114-4120, 1989) was
dissolved in
EtOH/HOAc (80 mL, 99/1, v/v) and HC1.H-Gly-Obzl (12b, 2.02 g, 10 mmol) was
added, followed by NaCNBH3 (0.94 g, 15 mmol). The reaction mixture was stirred
at
room temperature overnight. Subsequently, 5% aq. NaHC03 (20 mL) was added to
neutralize the reaction mixture. The mixture was then concentrated in vacuo
and the
residue was extracted with CHzCl2. The combined organic layers were washed
with
satd. Aq. NaCI, dried quickly over Na2S04, filtered and the solvent was
evaporated to
yield a yellow oil. After purification by silica gel chromatography (eluent: 0-
4%
methanol in CHZC12) compound 12c was isolated as a white solid. Yield: 1.85 g
(39 %).
is Analysis: TLC: (silica, CHZC12/MeOH 98/2) Rf = 0.45, MS: MW = 472.
Fmoc-Val-yr[CHZN(Boc)]-Gly-Obzl (12d)
Fmoc-Val-yr[CHZNH]-Gly-Obzl (12c, 0.910 g, 1.93 mmol), Boc20 (0.420 g, 1.93
mmol) and DIPEA (0.336 g, 1.93 mmol) were dissolved in dry CH2C12 (20 mL). The
2o pH was kept basic by addition of DIPEA and the mixture was stirred
overnight at room
temperature. The reaction mixture was then acidified by addition of 10% KHS04.
Water was added and the aqueous layer was extracted with CH2Cl2. The combined
organic layers were washed with aq. Satd. NaCI, dried quickly over MgSO.~ and
the
solvent was evaporated to yield 0.96 g (97%) of 12d. Analysis: TLC: (silica,
2s CH2Cl2/MeOH 98/2) Rf = 0.55; MS: MW=572.
Fmoc-Val-y[CHZN(Boc)]-Gly-OH (12e)
Fmoc-Val-yr[CH2N(Boc)]-Gly-Obzl (12d, 0.97 g, 1.70 mmol) was dissolved in a
mixture of MeOH/EtOAc (1/1, v/v, 100 mL) and hydrogenated at normal pressure
with
30 10% Pd/C for a time of 2 h. The palladium catalyst was filtered off and the
filtrate was
concentrated to afford carboxylic acid 12e as a slightly yellow oil. Yield:
0.661 g
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(81%). Analysis: TLC (silica, CHzCIz/MeOH/AcOH 90/9/1) Rf = 0.42; MS: MW =
482.
Using the Peptide synthesizer with HATU/DIPEA double syringe mode and with a
s double coupling with HATU/DIPEA, the compound Fmoc-Val-~r[CHZN(Boc)]-Gly-
OH (12e) (0.661 g, 1.37 mmol) was loaded onto PAL-PEG-PS resin ( 1.5 g, 0.1 S
mmol/g, 0.225 mmol). The substitution level was measured with the standard
Fmoc
cleavage procedure and was 0.13 mmol/g of loaded resin (yield: 87%). The
resulting
peptide H-Val-yr[CHZN(Boc)]-Gly-PAL-PEG-PS (12f7 was further elongated using
the
to HATU/DIPEA SPPS protocol (example 1) with double condensation steps of 60
min
for each Fmoc-amino acid. Similar to peptides 9 and 11 the N-terminal acetyl
group
was introduced using 4-nitrophenyl acetate. Workup, purification and desalting
were
carried out as described in example 1.
Yield: 17 mg; HPLC purity: 80.1%; MS: MW = 1338; amino acid analysis: all
amino
i s acids were found in the required amounts; peptide content: 63.7%; ion
chromatography: chloride: 1.0%, phosphate: 0.2%, acetate: 0.2% (w/w).
Example 13
Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ (13)
2o For the synthesis of peptide 13 the requisite peptoid monomer Fmoc-
NhSer(tBu)-OH
(13e) was prepared first.
Z-2-aminoethyl-tert butyl ether (13a)
3.25 g MgS04 (27 mmol) was suspended in 80 mL of CHZC12 (dry). Under N2 1.5 mL
2s of cone. H2S04 was added (procedure: S.W. Wright, D.L. Hageman, A.S.
Wright, L.
McClure, Tetrahedron Lett., 38:7345-7348, 1997). The mixture was stirred for
15 min
after which tent-BuOH (12.9 mL) and commercially available Z-2-aminoethanol
(5.28
g, 27 mmol), dissolved in CH2Cl2 (20 mL), were added. After stirring for 5
days, 200
mL of 5% aq. NaHC03 was added to the reaction mixture, which was stirred until
all
3o the MgS04 had dissolved. The layers were separated and the CH2C12 layer was
washed
with brine. The organic layer was dried over MgS04, filtered and the solvent
was
evaporated to yield 5.6 g of crude 13a. The product was purified by column
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chromatography (eluent: heptane/EtOAc 3:1 v/v). Yield 5.00 g (78%). ~ H NMR
(CDC13) 8: 1.1 S (s, 9H, tBu), 3.3-3.5 (dt, 4H, 2 x CHZ), S.1 (bs, 2H,
CHZBzI), 7.4 (m,
SH, Ar).
s 2-aminoethyl-tert-butyl ether.HCl (13b)
To a solution of 5.00 g of benzyl ester 13a in ethyl acetate (150 mL) 225 mg
of 10%
Pd/C was added and HZ was bubbled through for 2 hours. The catalyst was
filtered off
and 15 mL of 1 M aq. HC1 was added. The solvent was evaporated and a small
volume
of ether was added. The precipitated product 13b was filtered off and dried in
vacuo.
Yield: 2.35 g (77%). NMR (CDC13) 8: 1.20 (s, 9H, tBu), 3.15 (t, 2H, CHZ), 3.65
(t, 2H
CH2), 8.1-8.4 (bs, 2H, NHZ)
N-(2-tert-butoxyethyl)-glycine (H-NhSer(tBu)-OH) (13c)
To a solution of 13b (2.30 g, 15 mmol) in 25 mL of H20 was added 1.40 g (15.2
mmol)
is of glyoxylic acid.HzO. The pH was adjusted to pH=6 with 1.0 M aq NaOH. To
this
solution 230 mg of Pd/C was added and the reaction mixture was agitated at 45
psi HZ
pressure overnight. The catalyst was filtered off and washed with 5 mL H20.
The
filtrate containing 13c was used without further purification in the next
step.
2o Fmoc-NhSer(tBu)-OH (13d)
The reaction product 13c, still dissolved in H20, was brought to pH=9.5 with 1
N
NaOH. The basic solution was diluted with 25 mL of acetone and 5.40 g ( 16
mmol)
Fmoc-Osu, dissolved in 25 mL of acetone, was added dropwise. The pH was kept
at
pH=9.5 with 1 N NaOH. After stirring overnight, the reaction mixture was
2s concentrated to 150 mL and washed with 2 x 50 mL of ether/heptane (1/1,
v/v). The
H20 layer was acidified to pH = 2.5 with 20% citric acid and 3 x extracted
with 100
mL of ethyl acetate. The organic layers were combined and dried over NaZS04.
The
solvent was evaporated and the product was purified by column chromatography
(silica, CH2Clz/MeOH 5/1, v/v) and freeze-dried. Yield: 5.44 g (91 %). 1H NMR
30 (CDC13) 8: 1.20 (s, 9H, tBu), 3.2 (dt, 2H CH2), 3.6-3.7, (dt, 2H, CH2),
4.05 (s, 2H,
CH~C02H), 4.2 (b, 1H Fmoc), 4.4-4.6 (2H, 2 x d, Fmoc), 7.3-7.8 (m, 8H, ArH,
Fmoc).
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The synthesis of peptide 13 was carried out on the Pepsynthesizer using the
dual
syringe technique as described before (example 1 ). The support was Fmoc-PAL-
PEG-
PS, ( 1.0 g, 0.15 mmol/g) with NMP as the solvent. Double couplings (coupling
time 60
min) were used for all amino acids, including Fmoc-NhSer(tBu)-OH (13d). The N-
s terminal acetyl group was introduced using 4-nitrophenyl acetate. Workup and
cleaving
off the resin and protecting groups were conducted in the standard way
(example 1 ).
The crude peptide was purified by HPLC and desalted with
5°/°° of aqueous acetic acid.
Yield: 50 mg; HPLC purity: 98.6%; MS: MW = 1366; amino acid analysis: all
amino
acids were found in the required amounts; peptide content: 82.1%; ion
to chromatography: chloride: 0.3%, acetate: 1.3% (w/w).
Example 14
Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-yr [CH2NH]-Gly-NHZ
(14)
is The synthesis was carried out using the HATU/DIPEA protocol on the
Pepsynthesizer
(example 1). The previously described functionalized resin H-Val-yr[CH2N(Boc)]-
Gly-
PAL-PEG-PS (12f7 and the protected peptoid Fmoc-NhSer(tBu)-OH (13d) were used
as building blocks. As described before, the dual syringe technique and double
couplings of 60 min per coupling were used. Elongation of the peptide chain on
the
zo synthesizer was stopped before the coupling of Fmoc-NhSer(tBu)-OH (13d) and
this
amino acid was dissolved in DMSO with sonication prior to coupling to the
immobilized peptide chain (H-Phe-Thr(tBu)-Leu-Ala-Ser(tBu)-Ser(tBu)-Glu(OtBu)-
Thr(tBu)-Gly-Val-y[CH2NH]-Gly-PAL-PEG-PS). The synthesis was finished by
condensation of the remaining (Arg) amino acid and acetylation using 4-
nitrophenyl
zs acetate. Workup, purification and desalting of the peptide were standard,
as outlined in
example 1. Lyophilization furnished 47 mg of peptide 14.
HPLC purity: 72.9%; MS: MW = 1352; ion chromatography: trifluoroacetate: 5.5%
(w/w).
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31
Example 15
Pre-selection of agonist peptides using antigen-specific T-cell hybridomas
(first
line test).
To test the agonist activity of a modified peptide, 3 different, HC gp-39 (263-
275)-
s specific hybridoma cell lines were used (5G 11, 8B 12 and 14G 11 ). 5 x 104
hybridoma
cells and 2 x 105 irradiated (12000 RAD), EBV-transformed B cells carrying the
DRB 1 *0401 specificity were incubated in 150 ~1 volumes in wells of a round-
bottomed microtiter plate. Peptide antigen (HC gp-39 (263-275), and modified
peptides) was added in 50 ~1 volumes to duplicate wells. Forty eight hr later
100 p1 of
to the culture supernatant was assayed for antigen-specific IL-2 production
using a
sandwich ELISA with Pharmingen antibodies specific for mouse IL-2.
Selection of agonist peptides using antigen-specific T-cell clones (second
line test)
The 243 T-cell clone was isolated from a peptide-specific T-cell line obtained
from an
RA responder to peptide 263-275 (RA patient 243). The clones were obtained
is following four repetitive stimulations with HC gp-39 (263-275) peptide in
the presence
of DRB1*0401-matched PBMC. The H235 T-cell clone was isolated from a peptide-
stimulated T-cell line obtained from an HLA-DRB1*0401-positive donor. Upon 2
stimulations with peptide HC gp-39 (261-275) in the presence of DRB1 *0401-
matched
PBMC, clones were obtained by PHA cloning. Both clone 243 and 235 were found
to
2o be HLA-DRB 1 * 0401 restricted in the recognition of peptide antigen. Cells
were used
on day 10-14 after stimulation in each experiment.
Proliferative responses of clone 243 or clone 235 were measured by incubation
of 2 x
104 T cells and 105 DRB1*0401-matched (3,000 Rad irradiated) PBMC in 150 p.1
volumes of medium with 10% normal human pool serum (NHS, CLB, Amsterdam, The
2s Netherlands) in flat-bottomed microtiter plates. 50 p1 of antigen solution
(containing
the 263-275 sequence or modifications as indicated) was distributed in
triplicate wells.
3H-thymidine was added at day 2 or 3 of incubation. Cells were harvested on
glass fibre
filters and the incorporated radioactivity was measured.
Results
3o Most modified peptides as listed in Table 2 were able to stimulate all
three T-cell
hybridomas in a fashion comparable to the lead peptide H-Arg-Ser-Phe-Thr-Leu-
Ala-
Ser-Ser-Glu-Thr-Gly-Val-Gly-OH. Some peptides, however, did not stimulate all
three
hybridomas which exemplifies the difference in specificity of the hybridomas
used.
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
32
When these agonists were tested for their capacity to stimulate the two human
T-cell
clones, a clear difference in potency of the compounds tested became obvious
(Table
2). Most modified compounds induced a response of clone 235 and clone 243. One
compound (Ac-Narg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NH2) did
s not induce a proliferative response of either clone. Three compounds (H-
betahomoargininyl-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, Ac-Arg-
Ser-Phe-yr[CH2NH]-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ and Ac-Arg-Ser-
Phe-Thr-Leu-yr[CH2NH]-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ) were active on one
clone only (either clone 243 or 235). Three compounds (H-Arg-Ser-Phe(4Cl)-Thr-
Leu-
~o Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, H-D-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-
Thr-Gly-Val-Gly-OH and CH30C(O)-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-
Val-Gly-OH) induced a proliferative response in both clones which was in the
same
order of magnitude as the response induced by the lead peptide H-Arg-Ser-Phe-
Thr-
Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH. Seven compounds (Ac-Arg-Ser-Phe-Thr-
~s Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, CH3(OCH2CH2)3-OCH2C(O)-Arg-Ser-
Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-NHZ, D-1-glucityl-Arg-Ser-Phe-Thr-
Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, (N-methyl-nicotinoyl)+-Arg-Ser-Phe-Thr-
Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, Ac-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-
Glu-Thr-Gly-Val-yr[CH2NH]-Gly-NH2, Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-
2o Thr-Gly-Val-Gly-NHZ and Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-
Val-
~r[CH2NH]-Gly-NHZ) were superior in inducing a proliferative response of one
or both
clones. The most potent compounds identified being Ac-Arg-Ser-Phe-Thr-Leu-Ala-
Ser-Ser-Glu-Thr-Gly-Val-Gly-OH, Ac-Arg-Ser-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-
Gly-Val-yr[CHZNH]-Gly-NH2, Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-
2s Val-Gly-NHZ and Ac-Arg-NhSer-Phe-Thr-Leu-Ala-Ser-Ser-Glu-Thr-Gly-Val-
yr[CH2NH]-Gly-NHZ (Table 2 and Figure 1).
Example 16
Female Balb/c mice of approximately 8-10 weeks of age (Charles River Germany
or
Charles River France) were immunised on day 0 with 100 ~1 of antigen
preparation (50
3o Pg of HC gp-39 263-275) in Incomplete Freunds Adjuvant (IFA; Sigma
Chemicals, St.
Louis, USA). Antigen was given subcutaneously in two portions in the chest
region of
the mice. On day 7, mice were challenged with antigen preparation (HC gp-39
(263-
275)) diluted in 0.9% NaCI (NPBI, Emmer Compascuum, The Netherlands) in a
volume of 50 p.1 in 1 mg/ml alum (Pharmacy Donkers-Peterse, Oss, The
Netherlands)
CA 02386398 2002-04-05
WO 01/29081 PCT/TP00/10230
33
unilaterally in the footpad (left paw); the other (right) footpad was injected
with 50 p1
of alum solution in 0.9% NaCI as a control. Delayed type hypersensitivity
responses
(mean % specific swelling) were determined on day 8 by measuring the increase
in
footpad thickness of the left hind footpad compared to the right hind footpad
(swelling
s left (mm) - swelling right (mm) / swelling right (mm) x 100%), using a in-
house
designed micrometer.
Nasal application of antigen preparation (50, 10, 2 or 0.4 pg (or lower
concentrations))
of HC gp-39 (263-275) or of modified peptide derivatives was performed under
Isoflurane (Forene~, Abbott BV, Amstelveen, The Netherlands) anesthesia once
(day -
to S) before immunisation on day 0 with 100 ~1 of antigen preparation
containing 50 pg of
HC gp-39 263-275 in IFA. In these experiments, mice were immunised and
challenged
with HC gp-39 263-275 and DTH responses were determined as described above.
Using the above described assay system in which Balb/c mice immunised with HC
gp-
39 (263-275) in IFA responded to HC gp-39 (263-275), it became possible to
study the
is potential effects of tolerance induction by nasal application of HC gp-39
(263-275) in
comparison to those of modified peptide derivatives. Pre-treatment with HC gp-
39
(263-275) downmodulated the HC gp-39 (263-275) specific DTH reaction; this
effect
was dependent on the dose of peptide that was included in the pre-treatment
procedure.
Using a relatively high peptide concentration (SO ~g/mouse), nasal application
of one
2o dose of HC gp-39 (263-275) fully abrogated the DTH reaction whereas a dose
of 2
p.g/mouse was ineffective. Thus, a protocol was established to discriminate
between
effective (tolerogenic) and ineffective doses of peptide in a HC gp-39 (263-
275)
specific DTH assay system. Assuming that modified peptide derivatives based on
HC
gp-39 (263-275) may be active at lower concentrations than the original
peptide, such
2s peptides are expected to induce tolerance at relatively low peptide
concentrations.
Following this assumption, a series of modified peptides was tested in this
tolerance
induction protocol. In this experiment (in which a reliable HC gp-39 (263-275)
response was induced that could be downmodulated by pre-treatment with 50 but
not 2
p.g of HC gp-39 (263-275)) it was shown that specific modifications of the
peptide
3o were highly active in the induction of tolerance whereas others were not
(see Table 3).
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
34
..
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CA 02386398 2002-04-05
WO 01/29081 PCT/FP00/10230
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L c? oxxxx
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x > x ~ >, >, >, >,
Q?~~ ~c?c?c?V
xx z~c~ ~ N
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CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
SEQUENCE LISTING
<110> AKZO NOBEL N.V.
<120> Modified peptides and peptidomimetics for use in
immunotherapy
<130>
<140>
<141>
<160> 20
<170> PatentIn Ver. 2.1
<210> 1
<211> 13
<212> PRT
<213> Homo sapiens
<400> 1
Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 2
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at position 1 is desaminoargininyl; to the
C-terminal Gly NH2 is connected
<400> 2
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 3
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at position 1 is desaminoargininyl
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 3
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
2
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 4
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> At N-terminus connected to
CH3-(OCH2CH3)3-)CH2-C(0); at C-terminus connected
to NH2
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 4
Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 5
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> At N-terminus connected to D-1-glucityl
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 5
Arg Ser Phe Thr Leu Ala Ser Ser Glu Gly Thr Val Gly
1 5 10
<210> 6
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> At N-terminal position of peptide: CH30-C(0)
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<900> 6
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
3
Arg Ser Phe Thr Leu Ala Ser Ser Gly Thr Gly Val Gly
1 5 10
<210> 7
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> At N-terminus connected to Ac; At the C-terminus
NH2 is connected; Xaa at position 3 is
NH-CH(CH2Ph)-CH2
<400> 7
Arg Ser Xaa Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 8
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> At N-terminus connected to Ac; At the C-terminus
NH2 is connected; Xaa at position 5 is
NH-CH(CH2CH(CH3)2)-CH2
<400> 8
Arg Ser Phe Thr Xaa Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 9
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> At t'.2 N-terminus Ac is connected; At the
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
4
C-terminus NH2 is connected; Xaa at position 12 is
NH-CH(CH(CH3)2)-CH2
<900> 9
Arg Ser Phe Leu Thr Ala Ser Ser Glu Thr Gly Xaa Gly
1 5 10
<210> 10
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> At the N-terminus Ac is connected; At the
C-terminus NH2 is connected; Xaa at position 2 is
N[(CH2)2-OH]-CH2-C(O)
<400> 10
Arg Xaa Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 11
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> At the N-terminus Ac is connected; at the
C-terminus NH2 is connected; Xaa at position 2 is
N(CH2)2-OH]-CH2-C(O); Xaa at position 12 is
NH-CH(CH(CH3)2)-CH2
<400> 11
Arg Xaa Phe Thr Leu Ala Ser Ser Glu Thr Gly Xaa Gly
1 5 10
<210> 12
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
S
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> Xaa at position 3 is Phe(C1)
<400> 12
Arg Ser Xaa Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 13
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> Xaa at position 1 is H2N-(CH2)5-C(0)
<400> 13
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 14
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> Xaa at position 1 is H2N-(CH2)6-C(0)
<400> 14
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 15
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
6
<220>
<223> At the N-terminus is connected
(N-methyl-nicotinoyl)+
<900> 15
Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at position 3 is Phe(4Br)
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 16
Arg Ser Xaa Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 17
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at position 3 is cyclohexylalanine
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 17
Arg Ser Xaa Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 18
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at position 1 is H-betahomoargininyl
<220>
CA 02386398 2002-04-05
WO 01/29081 PCT/EP00/10230
7
<223> Description of Artificial Sequence: Synthetic
peptide
<900> 18
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
<210> 19
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> At the N-terminus Ac is connected; at the
C-terminus NH2 is connected; Xaa at position 11 is
NH-CH2-CH2
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 19
Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Xaa Val Gly
1 5 10
<210> 20
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> At the N-terminus Ac is connected; at the
C-terminus NH2 is connected; Xaa at position 1 is
N(CH2CH2CH2NH-C(=NH)-NH2)-CH2-CO
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 20
Xaa Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly Val Gly
1 5 10
