U.S. patent application number 14/877679 was filed with the patent office on 2016-04-07 for conjugates comprising mannan and myelin basic protein (mbp).
This patent application is currently assigned to VIANEX S.A. The applicant listed for this patent is Vianex S.A. Invention is credited to Vasso Apostolopoulos, Maria Katsara, John Matsoukas, Lesley Probert, Theodoros Tselios, Vivian Tseveleki.
Application Number | 20160095935 14/877679 |
Document ID | / |
Family ID | 40785428 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160095935 |
Kind Code |
A1 |
Matsoukas; John ; et
al. |
April 7, 2016 |
CONJUGATES COMPRISING MANNAN AND MYELIN BASIC PROTEIN (MBP)
Abstract
A first aspect of the invention relates to a conjugate including
mannan and at least one epitope comprising a peptide fragment of a
protein selected from myelin basic protein (MBP), myelin
oligodentrocyte glycoprotein (MOG) and proteolipid protein (PLP),
said peptide fragment being in linear or cyclic form; and wherein
said epitope is linked to mannan via a [(Lys-Gly).sub.n] bridge,
where n is an integer from 1 to 10. Further aspects of the
invention relate to pharmaceutical compositions comprising said
conjugates, and their use in the preparation of a medicament for
treating an immune disorder.
Inventors: |
Matsoukas; John; (Patras,
GR) ; Tselios; Theodoros; (Mesologgi, GR) ;
Apostolopoulos; Vasso; (St. Albans, AU) ; Tseveleki;
Vivian; (Drosia, GR) ; Katsara; Maria;
(Kalamata, GR) ; Probert; Lesley; (Kifisia,
GR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vianex S.A |
Nea Erythrea Attikis |
|
GR |
|
|
Assignee: |
VIANEX S.A
Nea Erythrea Attikis
GR
|
Family ID: |
40785428 |
Appl. No.: |
14/877679 |
Filed: |
October 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12864019 |
Oct 28, 2010 |
9180174 |
|
|
PCT/IB09/00382 |
Jan 22, 2009 |
|
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14877679 |
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Current U.S.
Class: |
514/17.9 ;
514/21.1; 514/21.4; 530/317; 530/326 |
Current CPC
Class: |
C07K 14/4713 20130101;
A01K 2227/105 20130101; A61K 39/0008 20130101; A61K 2039/627
20130101; A01K 2267/0325 20130101; A61K 47/64 20170801; C07K 7/08
20130101; A01K 2207/10 20130101; C07K 7/64 20130101; A61P 37/06
20180101; A61K 2039/6087 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 7/64 20060101 C07K007/64; C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2008 |
GB |
0801424.3 |
Feb 8, 2008 |
GB |
0802405.1 |
Feb 29, 2008 |
GR |
20080100151 |
Claims
1. A conjugate comprising: (i) mannan; and (ii) at least one
epitope comprising a peptide fragment of myelin basic protein
(MBP), said peptide fragment being in linear or cyclic form;
wherein said epitope is linked to mannan via a [(Lys-Gly).sub.n]
bridge, where n is an integer from 1 to 10.
2. (canceled)
3. A conjugate according to claim 1, wherein the epitope comprises
a peptide selected from MBP.sub.83-99, MBP.sub.82-101,
[Ala.sup.86]MBP.sub.83-89, [Ala.sup.88]MBP.sub.83-89
[Tyr.sup.89]MBP.sub.83-89 and MBP.sub.110-118, and variants
thereof, in linear or cyclic form.
4. A conjugate according to claim 1, wherein the epitope comprises
the peptide MBP.sub.83-99, in linear or cyclic form.
5. A conjugate according to claim 1, wherein the epitope
corresponds to the peptide of SEQ ID NO. 1, or a variant thereof:
TABLE-US-00016 [SEQ ID NO. 1]
H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-
Thr-Pro-Arg-Thr-Pro-OH
in linear or cyclic form.
6.-14. (canceled)
15. A conjugate according to claim 1, wherein the mannan is reduced
mannan.
16. A conjugate according to claim 1, wherein the mannan is
oxidised mannan.
17. A conjugate according to claim 1, wherein two or more epitopes,
or cyclic counterparts thereof, are linked to mannan.
18. A conjugate according to claim 17 wherein the epitopes (or
cyclic counterparts thereof) are different.
19. A mixture comprising two or more conjugates according to claim
1.
20. A mixture according to claim 19 wherein the conjugates are
different.
21. A pharmaceutical preparation comprising a conjugate according
to claim 1 and a pharmaceutically acceptable carrier, diluent or
excipient.
22.-26. (canceled)
27. A method of treating an immune disorder, said method comprising
administering to a subject a conjugate according to claim 1.
28. A method according to claim 27 wherein the immune disorder is
an autoimmune disease.
29. A method according to claim 27 wherein the immune disorder is
multiple sclerosis (MS).
30. A method according to claim 27 wherein the immune disorder is
experimental autoimmune encephalomyelitis (EAE).
31. A method of immunizing a subject against an immune disorder,
said method comprising administering to a subject a conjugate
according to claim 1.
32. A method according to claim 27 wherein the conjugate is
administered in an amount sufficient to cause suppression of
chronic experimental autoimmune encephalomyelitis (EAE).
33. (canceled)
34. A process for preparing a conjugate according to claim 1, said
process comprising the steps of: (i) reacting an epitope comprising
a peptide fragment of myelin basic protein (MBP), said peptide
fragment being in linear or cyclic form, with a peptide bridge
[(Lys-Gly).sub.n], wherein n is an integer from 1 to 10; (ii)
reacting the product formed in step (i) with oxidized mannan; and
(iii) optionally reducing the product formed in step (ii) to form a
reduced mannan conjugate.
35. A vaccine comprising a conjugate according to claim 1.
36. (canceled)
37. A conjugate according to claim 1 further comprising: an epitope
comprising a peptide fragment of myelin oligodentrocyte
glycoprotein (MOG); and an epitope comprising a peptide fragment of
proteolipid protein (PLP); each peptide fragment being in linear or
cyclic form; wherein each epitope is linked to mannan via a
[(Lys-Gly).sub.n] bridge, where n is an integer from 1 to 10.
38. A conjugate according to claim 1 further comprising one of (i)
epitope comprising a peptide fragment of myelin oligodentrocyte
glycoprotein (MOG); and (ii) an epitope comprising a peptide
fragment of proteolipid protein (PLP).
39. A conjugate according to claim 5 wherein the epitope is linked
to mannan via a [(Lys-Gly).sub.5] bridge.
40. A conjugate according to claim 1 wherein the peptide has the
amino acid sequence of formula (I), TABLE-US-00017
ENPVVHFFK.sup.91NIVTP.sup.96RTP (I)
wherein at least one of K.sup.91 and P.sup.96 is substituted by a
natural or unnatural amino acid, wherein said peptide is in linear
or cyclic form.
41. A conjugate according to claim 40 wherein K.sup.91 is
substituted by an amino acid selected from A, R, E, F and Y.
42. A conjugate according to claim 40 wherein P.sup.96 is
substituted by the amino acid A.
43. A conjugate according to claim 40 wherein the peptide is
selected from: TABLE-US-00018 [R.sup.91, A.sup.96]MBP.sub.83-99
ENPVVHFFRNIVTARTP; [A.sup.91, A.sup.96]MBP.sub.83-99
ENPVVHFFANIVTARTP; [E.sup.91]MBP.sub.83-99 ENPVVHFFENIVTPRTP;
[F.sup.91]MBP.sub.83-99 ENPVVHFFFNIVTPRTP; and
[Y.sup.91]MBP.sub.83-99 ENPVVHFFYNIVTPRTP.
44. A conjugate according to claim 1 wherein the peptide is
selected from the following: TABLE-US-00019 peptide analogues
sequence cyclo(83-99)MBP.sub.83-99 SEQ ID NO. 1 cyclo(83-99)E N P V
V H F F K N I V T P R T P cyclo(83-99)[A.sup.91]MBP.sub.83-99 SEQ
ID NO. 18 cyclo(83-99)E N P V V H F F A N I V T P R T P
cyclo(83-99)[R.sup.91]MBP.sub.83-99 SEQ ID NO. 19 cyclo(83-99)E N P
V V H F F R N I V T P R T P cyclo(83-99)[F.sup.91]MBP.sub.83-99 SEQ
ID NO. 15 cyclo(83-99)E N P V V H F F F N I V T P R T P
cyclo(83-99)[Y.sup.91]MBP.sub.83-99 SEQ ID NO. 16 cyclo(83-99)E N P
V V H F F Y N I V T P R T P cyclo(83-99)[E.sup.91]MBP.sub.83-99 SEQ
ID NO. 14 cyclo(83-99)E N P V V H F F E N I V T P R T P
cyclo(83-99)[A.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 13
cyclo(83-99)E N P V V H F F A N I V T A R T P
cyclo(83-99)[R.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 12
cyclo(83-99)E N P V V H F F R N I V T A R T P
cyclo(83-99)[F.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 20
cyclo(83-99)E N P V V H F F F N I V T A R T P
cyclo(83-99)[Y.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 21
cyclo(83-99)E N P V V H F F Y N I V T A R T P
cyclo(83-99)[S.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 22
cyclo(83-99)E N P V V H F F S N I V T A R T P.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
Ser. No. 12/864,019, filed Oct. 28, 2010, which is a .sctn.371
national phase application of International Application No.
PCT/IB09/00382, filed Jan. 22, 2009, which claims priority to
United Kingdom Application Nos. 0801424.3, filed Jan. 25, 2008 and
0802405.1, filed Feb. 8, 2008 and to Greece Application No.
20080100151, filed Feb. 29, 2008, contents of which are
incorporated by reference herein in their entirety.
[0002] The present invention relates to conjugates of myelin
antigens that are useful candidates for the immunotherapy of
multiple sclerosis (MS).
BACKGROUND TO THE INVENTION
[0003] Multiple sclerosis (MS) is a chronic disease of the central
nervous system (CNS) characterized by local T cell and macrophage
infiltrates, demyelination and loss of neurologic function
[Steinman, 1996; Martin, et al., 1992; Mantzourani et al., 2005].
MS is an autoimmune disease triggered by CNS-specific CD4.sup.+ T
lymphocytes. Candidate autoantigens include constituents of the
myelin sheath, such as myelin basic protein (MBP), proteolipid
protein (PLP) and myelin oligodendrocyte glycoprotein (MOG).
[0004] An association between major histocompability complex (MHC)
class II alleles and disease has been observed in MS patients, in
particular HLA-DR1, HLA-DR2 and HLA-DR4. Although the pathology of
MS remains unclear, there is evidence that T cells recognizing
encephalitogenic epitopes of myelin, such as MBP, play a pathogenic
role in the induction of MS. Studies have shown that T cell
responses in patients are associated with the recognition of the
81-105 region of MBP (QDENPVVHFFKNIVTPRTPPPSQGK; SEQ ID NO. 4), and
with highest affinity and binding to HLA-DR2 for the peptide
epitope MBP.sub.83-99 (ENPVVHFFKNIVTPRTP; SEQ ID NO. 1). T cell
recognition of this region of MBP has also been shown in healthy
individuals, although at relatively low precursor frequencies. The
binding of MBP.sub.83-99 to HLA-DR2 is via hydrophobic V.sup.87 and
F.sup.90 residues, whilst, H.sup.88, F.sup.89, and K.sup.91 are TCR
contact residues [Mantzourani et al., 2005].
[0005] The pathogenic role of autoimmune T cells recognizing
encephalitogenic epitopes of MBP has also been noted in
experimental autoimmune encephalomyelitis (EAE), one of the
best-studied experimental animal models of MS. EAE represents an
invaluable in vivo system for the evaluation of therapeutic
approaches. EAE is induced in susceptible animals by immunodominant
epitopes of the myelin sheath. Similar clinical and
histopathological features to MS can be induced in susceptible
mouse strains by immunization of myelin components. EAE is mediated
by CD4.sup.+ T cells of the Th1 phenotype (IFN-.gamma.). Like MS,
EAE susceptibility is dependent on the MHC background of the mouse
and different peptides are immunogenic and induce EAE in different
strains. Myelin Oligodendrocyte Glycoprotein (MOG) residue 35-55
induces chronic (non-relapsing) EAE in C57BL/6 mice, guinea pig MBP
residue 74-85 induces acute (relapsing-remitting) EAE in Lewis rats
and proteolipid protein (PLP) residue 139-151 induces acute EAE in
SJL/J mice [Zamvil et al., 1990].
[0006] The SJL/J mouse strain (H-2.sup.s haplotype) is commonly
used for EAE since numerous histopathological, clinical and
immunological features resemble that of human MS compared to other
mouse or rat strains. In the SJL/J mouse strain, residues from the
encephalitogenic epitope MBP.sub.81-100 have been shown to bind
with high affinity. In fact, the minimum epitope required for
binding is MBP.sub.83-99. Furthermore, in SJL/J mice, CD4 T cell
responses to PLP residues 139-151 (PLP.sub.139-151: sequence
HSLGKWLGHPDKF; SEQ ID NO. 3) is qualitatively different from
responses to encephalitogenic of MBP.sub.83-99 in that the PLP
peptide-specific clones are heterogeneous. As a first step toward
understanding the cellular and molecular basis and the biologic
relevance of this heterogeneity, studies have determined whether
multiple overlapping epitopes within the PLP.sub.139-151 are
responsible for the diversity. Initial studies demonstrated that
the panel of T cell clones reacted with overlapping PLP peptides
only when the peptides contained residue 144, thereby suggesting
that this is an important site for the activation of all of the
clones [Kuchroo, et al., 1992]. Furthermore, W.sup.144 is the
dominant TCR contact residue, as substitution at position 144 with,
alanine (A), or other hydrophobic residues, such as phenylalanine
(F), abolishes the in vitro stimulatory activity of the peptide and
such analogues do not induce EAE [Kuchroo, et al., 1992]. The
single TCR antagonist peptide analogue (L.sup.144/R.sup.147), in
which both of the major TCR contact residues are substituted,
showed the maximum antagonist activity (L.sup.144/R.sup.147), in
vitro and also gave the best inhibition of EAE [Kuchroo, et al.,
1994].
[0007] In the C57BL/6 mouse strain, residues 35-55 from MOG protein
have been found to be encephalitogenic [McFarlin, et al., 1973].
Disease is elicited by immunization with MOG.sub.35-55, resulting
in a CD4.sup.+ T helper-1 (Th1)-cell response that attacks the
myelinated areas of CNS [Zamvil and Steinman, 1990]. T cells
supported by monocytes and activated microglial cells mediate
inflammation and demyelination. B cells and antibodies are not
critical for EAE induction in mice, although antibodies that bind
to epitopes of MOG.sub.35-55 enhance demyelination in some models
[Linington, et al., 1988]. In Lewis rats, epitope MBP.sub.74-85 has
been identified as immunodominant for EAE induction. Moreover,
peptide analogues based on the human MBP.sub.83-99 epitope have
been found to suppress the EAE symptoms induced from the
encephalitogenic MBP.sub.74-85 epitope. [Mendel et al., 1995;
Tselios et al., 1999; Tselios et al., 2000a; Tselios et al.,
2000b].
[0008] In the light of the above, the peptides MOG.sub.35-55,
PLP.sub.139-151, MBP.sub.74-85 and MBP.sub.83-99 and their head to
tail cyclic counterparts clearly represent a promising starting
point for the design of altered peptide ligands and peptide
analogues, which could be used to alter T cell responses in these
animal models and thus lead to new therapeutic approaches against
MS and other autoimmune diseases. Moreover, epitopes of MBP, PLP
and MOG from multiple sclerosis patients, MBP.sub.82-100,
[Ala.sup.86]MBP.sub.83-99, [Ala.sup.88]MBP.sub.83-99,
[Tyr.sup.89]MBP.sub.83-99, MBP.sub.110-118, MOG.sub.97-108,
PLP.sub.97-117, PLP.sub.185-206, PLP.sub.40-60, PLP.sub.190-209,
PLP.sub.184-199, PLP.sub.80-88, PLP.sub.30-49, PLP.sub.180-199 and
the like are recognized/presented by T cells/B cells from the
peripheral blood of MS patients or are able to induce
peptide-specific T cells responses in individuals [Greer, et al.,
1997, Singh, et al., 2004, Greer, et al., 2004, Tsuchida, et al.,
1994, Trotter, et al., 1998].
Current Peptide Therapies for MS
[0009] Current peptide therapies of MS include treatment with
interferons (interferon beta-1.alpha. and interferon beta-1.beta.)
and glatiramer acetate (copolymer-1) which is a synthetic protein
comprised of the major amino acids Glu, Gln, Lys, Arg of MBP. These
immunomodulators have been approved by the FDA for patients with
relapsing-remitting MS. Interferons given by subcutaneous injection
reduce the frequency, severity and duration of exacerbation, but
their impact on preventing long term disability has not yet been
established. In addition, side effects are common and consist of
reactions at the injection site, fever, myalgia and flu-like
syndrome. So far the reported benefits from the use of interferons
and copolymers are marginal and the need for improved therapeutics
is imperative.
[0010] Another approach under clinical investigation for autoimmune
suppression is the oral administration of autoantigens. Orally
administered antigens have been shown to suppress autoimmunity in
animal models, including EAE, collagen and adjuvant-induced
arthritis, uveitis and diabetes in the non-obese diabetic mouse.
Low doses of oral antigen induce antigen-specific regulatory
T-cells which act by releasing inhibitory cytokines such as
TGF-.beta., IL-4, and IL-10 at the target organ. Thus, one can
suppress inflammation at a target organ by orally administering an
antigen derived from the site of inflammation, even if it is not
the target of the autoimmune response. Initial human trials of
orally administered antigen have shown positive findings in
patients with MS and rheumatoid arthritis. A double-blind,
placebo-controlled, phase III multi-centre trial of oral myelin in
relapsing-remitting MS patients is in progress, as are phase II
clinical trials investigating the oral administration of type II
collagen in rheumatoid arthritis, S-antigen in uveitis and insulin
in type I diabetes. This promising method allows for oral
administration which is advantageous over previous treatments with
interferons and copolymer-1. However, issues relating to the
peptidic nature and cost of the administered substance renders the
non-peptide mimetic approach, even in its infancy, an attractive
goal to pursue. In this regard, our cyclic epitopes, which are more
stable than their linear epitope counterparts, offer this
advantageous property.
[0011] The present invention seeks to provide a new approach
towards the therapeutic management of MS. More specifically, the
invention focuses on the design and use of peptide analogues of
disease-associated myelin epitopes to induce peripheral T-cell
tolerance.
STATEMENT OF INVENTION
[0012] The present invention provides an immunotherapeutic approach
in which immunodominant/antigenic peptide analogues of myelin basic
protein (MBP), myelin oligodentrocyte glycoprotein (MOG) or
proteolipid protein (PLP) are conjugated (each one or as cluster)
to oxidized or reduced mannan via a [Lys-Gly].sub.n bridge for the
treatment of multiple sclerosis (MS).
[0013] A first aspect of the invention relates to a conjugate
comprising: [0014] (i) mannan; and [0015] (ii) at least one epitope
comprising a peptide fragment of a protein selected from myelin
basic protein (MBP), myelin oligodentrocyte glycoprotein (MOG) and
proteolipid protein (PLP), said peptide fragment being in linear or
cyclic form; wherein said epitope is linked to mannan via a
[(Lys-Gly).sub.n] bridge, where n is an integer from 1 to 10.
[0016] Glycosylation is a universal characteristic of proteins in
nature, which determines their physicochemical and biological
properties. Design and synthesis of glycopeptides is a topic of
intense research in the last years, since the glyco-part improves
the pharmacokinetic characteristics, enhances or alters the
biologic activity and can be used as a tool to study the biologic
functions.
[0017] Without wishing to be bound by theory, it is believed that
conjugating the immunodominant epitopes of MBP, PLP and MOG (linear
or cyclic) to mannan actively inhibits or prevents disease through
the activation of antigen-specific regulatory T cells or by
inducing T cell tolerance to self antigens.
[0018] A second aspect of the invention relates to a mixture
comprising two or more conjugates as defined above.
[0019] A third aspect of the invention relates to a pharmaceutical
preparation comprising a conjugate or mixture as defined above, and
a pharmaceutically acceptable carrier, diluent or excipient.
[0020] A fourth aspect of the invention relates to a conjugate or
mixture as defined above for use in medicine.
[0021] A fifth aspect of the invention relates to the use of a
conjugate or mixture as defined above in the preparation of a
medicament for treating MS and other immune disorders.
[0022] A sixth aspect of the invention relates to a method of
treating an immune disorder, said method comprising administering
to a subject a conjugate or a mixture as defined above.
[0023] A seventh aspect of the invention relates to a method of
immunizing a subject against an immune disorder, said method
comprising administering to a subject a conjugate or a mixture as
defined above.
[0024] An eighth aspect of the invention relates to the use of a
conjugate as defined above, in an assay for elucidating agents
capable of regulating experimental autoimmune encephalomyelitis
(EAE) or regulating multiple sclerosis.
[0025] A ninth aspect of the invention relates to a process for
preparing a conjugate as defined above, said process comprising the
steps of: [0026] (i) reacting an epitope comprising a peptide
fragment of a protein selected from myelin basic protein (MBP),
myelin oligodentrocyte glycoprotein (MOG) and proteolipid protein
(PLP), said peptide fragment being in linear or cyclic form, with a
peptide bridge [(Lys-Gly).sub.n], wherein n is an integer from 1 to
10; [0027] (ii) reacting the product formed in step (i) with
oxidized mannan; and [0028] (iii) optionally reducing the product
formed in step (ii) to form a reduced mannan conjugate.
[0029] A tenth aspect of the invention relates to a vaccine
comprising a conjugate or mixture as defined above.
[0030] An eleventh aspect of the invention relates to a conjugate
or a mixture as defined above for the treatment of MS and other
immune disorders.
DETAILED DESCRIPTION
[0031] The present invention relates to potent peptides (linear or
cyclic) of immunodominant epitopes of myelin sheath, MBP, PLP or
MOG which are conjugated with oxidized or reduced mannan via a
(Lys-Gly).sub.n spacer. The conjugates are useful in the treatment
of EAE and thus have implications in the treatment of MS. For the
first time, epitopes of myelin proteins conjugated to
oxidized/reduced mannan have been synthesized, via a
(Lys-Gly).sub.5 spacer and shown to completely prevent and protect
animals from EAE symptoms without the use of an adjuvant.
Preferably, the conjugates of the invention are administrated to
animals diluted in buffer solution (pH 6.0-9.0). Evidence suggests
that conjugating these disease-associated epitopes to oxidized or
reduced mannan via a (Lys-Gly).sub.n linker induces reduced levels
of proliferative CD4.sup.+ T cells and cytokines.
[0032] Experiments have shown that active immunization of animals
with oxidized/reduced mannan conjugated peptides confers protection
against EAE (the most widely used animal model for human MS). This
protection is evident in two different species, using three
different models of EAE. One model represents the relapse-remitting
form of the disease (MOG.sub.35-55 in C57BL/6 mice) and the others
represent acute monophasic disease followed by complete remission
(MPB.sub.72-85 in Lewis rats or PLP.sub.139-151 in SJL/J mice). The
results indicate that mannan peptide conjugates could potentially
be of significant therapeutic value in the treatment of MS.
Conjugate
[0033] One aspect of the invention relates to a conjugate
comprising: [0034] (i) mannan; and [0035] (ii) at least one
epitope, or cyclic counterpart thereof, of a protein selected from
myelin basic protein (MBP), myelin oligodentrocyte glycoprotein
(MOG) and proteolipid protein (PLP); wherein said epitope is linked
to mannan via a [(Lys-Gly).sub.n] bridge, where n is an integer
from 1 to 10.
[0036] More particularly, the invention relates to a conjugate
comprising: [0037] (i) mannan; and [0038] (ii) at least one epitope
comprising a peptide fragment of a protein selected from myelin
basic protein (MBP), myelin oligodentrocyte glycoprotein (MOG) and
proteolipid protein (PLP), said peptide fragment being in linear or
cyclic form; wherein said epitope is linked to mannan via a
[(Lys-Gly).sub.n] bridge, where n is an integer from 1 to 10.
[0039] As used herein, the term "epitope" refers to a molecular
region on the surface of an antigen that is capable of eliciting an
immune response and of combining with the specific antibody
produced by such a response.
[0040] In the context of the present invention, the term "peptide
fragment" refers to an amino acid sequence (or variant thereof)
derived from a full length protein. Preferably, the peptide
fragment has one or more amino acid residues deleted from the full
length protein.
[0041] In the context of the present invention, these epitopes are
typically derived from, or constitute, specific amino acid
fragments of the protein sequences of myelin basic protein (MBP),
myelin oligodentrocyte glycoprotein (MOG) and/or proteolipid
protein (PLP). The epitopes may be linear or cyclic.
[0042] In one preferred embodiment, the epitope comprises a peptide
fragment of myelin basic protein (MBP).
[0043] More preferably, the epitope comprises a peptide selected
from MBP.sub.83-99, MBP.sub.82-101, [Ala.sup.86]MBP.sub.83-89,
[Ala.sup.8]MBP.sub.83-89 [Tyr.sup.89]MBP.sub.83-89 and
MBP.sub.110-118, and variants thereof, in linear or cyclic
form.
[0044] Even more preferably, the epitope is a peptide selected from
MBP.sub.83-99, MBP.sub.82-101, [Ala.sup.86]MBP.sub.83-89,
[Ala.sup.88]MBP.sub.83-89, [Tyr.sup.8]MBP.sub.83-89 and
MBP.sub.110-118, and variants thereof, in linear or cyclic
form.
[0045] In one preferred embodiment, the epitope comprises the
peptide MBP.sub.83-99, or a variant thereof. Even more preferably,
the epitope is MBP.sub.83-99, or a variant thereof in linear or
cyclic form.
[0046] In one particularly preferred embodiment, the epitope
corresponds to the peptide sequence SEQ ID NO. 1, or a variant
thereof:
TABLE-US-00001 [SEQ ID NO. 1]
H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-
Thr-Pro-Arg-Thr-Pro-OH
in linear or cyclic form. Preferably, the peptide is in linear
form.
[0047] In one preferred embodiment, the peptide comprises the amino
acid sequence of formula (I),
TABLE-US-00002 ENPVVHFFK.sup.91NIVTP.sup.96RTP (I; SEQ ID NO.
5)
wherein at least one of K.sup.91 and P.sup.96 is substituted by a
natural or unnatural amino acid, wherein said peptide is in linear
or cyclic form.
[0048] In another preferred embodiment of the invention, the
peptide comprises the amino acid sequence of formula (Ia),
TABLE-US-00003 ENPVVHFFK.sup.91NIVTP.sup.96RTP (Ia; SEQ ID NO.
5)
wherein each of K.sup.91 and P.sup.96 is substituted by a natural
or unnatural amino acid.
[0049] In another preferred embodiment, the peptide consists of the
amino acid sequence of formula (Ia), wherein each of K.sup.91 and
P.sup.96 is substituted by a natural or unnatural amino acid.
[0050] In one preferred embodiment, K.sup.91 is substituted by a
natural amino acid.
[0051] In one preferred embodiment, K.sup.91 is substituted by an
amino acid selected from A, R, E, F and Y.
[0052] In a more preferred embodiment, K.sup.91 is substituted by
an amino acid selected from A, E and Y.
[0053] In one highly preferred embodiment, K.sup.91 is substituted
by the amino acid Y.
[0054] In one preferred embodiment, P.sup.96 is substituted by a
natural amino acid.
[0055] In one particularly preferred embodiment, P.sup.96 is
substituted by the amino acid A.
[0056] In one especially preferred embodiment, K.sup.91 is
substituted by an amino acid selected from A, R, E, F and Y and
P.sup.96 is substituted by the amino acid A.
[0057] In one preferred embodiment, K.sup.91 is substituted by the
amino acid R and P.sup.96 is substituted by the amino acid A.
[0058] In another preferred embodiment, K.sup.91 is substituted by
the amino acid A and P.sup.96 is substituted by the amino acid
A.
[0059] In one particularly preferred embodiment, the peptide of the
invention is selected from the following sequences:
TABLE-US-00004 [R.sup.91, A.sup.96]MBP.sub.83-99 ENPVVHFFRNIVTARTP
(SEQ ID NO. 12) [A.sup.91, A.sup.96]MBP.sub.83-99 ENPVVHFFANIVTARTP
(SEQ ID NO. 13)
[A.sup.91,A.sup.96]MBP.sub.83-99 is particularly preferred.
[0060] In another preferred embodiment, the peptide comprises the
amino acid sequence of formula (Ib),
TABLE-US-00005 ENPVVHFFK.sup.91NIVTP.sup.96RTP (Ib; SEQ ID NO.
14)
wherein at least one of K.sup.91 and P.sup.96 is substituted by an
amino acid selected from R, E, F and Y.
[0061] In another preferred embodiment, the peptide consists of the
amino acid sequence of formula (Ib), wherein at least one of
K.sup.91 and P.sup.96 is substituted by an amino acid selected from
R, E, F and Y.
[0062] In one highly preferred embodiment, K.sup.91 is substituted
by an amino acid selected from R, E, F and Y.
[0063] More preferably still, the peptide of formula (Ib) is
selected from the following:
TABLE-US-00006 [E.sup.91]MBP.sub.83-99 ENPVVHFFENIVTPRTP (SEQ ID
NO. 15) [F.sup.91]MBP.sub.83-99 ENPVVHFFFNIVTPRTP (SEQ ID NO. 16)
[Y.sup.91]MBP.sub.83-99 ENPVVHFFYNIVTPRTP (SEQ ID NO. 17)
[0064] Peptide [Y.sup.91]MBP.sub.83-99 is particularly preferred,
linear or cyclic.
[0065] Preferably, the peptide of formula (Ia) or (Ib) is a linear
peptide
[0066] In another preferred embodiment of the invention, the
peptide is a cyclic peptide comprising the amino acid sequence of
formula (Ic),
TABLE-US-00007 ENPVVHFFK.sup.91NIVTP.sup.96RTP (Ic; SEQ ID NO.
5)
or a variant thereof wherein one or two amino acids are substituted
by a natural or unnatural amino acid.
[0067] Preferably, the peptide consists of the amino acid sequence
of formula (Ic) cyclised head to tail.
[0068] Preferably, at least one of K.sup.91 and P.sup.96 is
substituted by a natural or unnatural amino acid.
[0069] In one preferred embodiment, one or two amino acids are
substituted by an amino acid selected from A, R, E, F, S and Y.
[0070] In one preferred embodiment, K.sup.91 is substituted by an
amino acid selected from A, R, E, F and Y.
[0071] In one preferred embodiment, P.sup.96 is substituted by a
natural amino acid.
[0072] More preferably, P.sup.96 is substituted by the amino acid
A.
[0073] In one particularly preferred embodiment, K.sup.91 is
substituted by an amino acid selected from A, R, F, S and Y and
P.sup.96 is substituted by the amino acid A.
[0074] More preferably still, the K.sup.91 residue is substituted
by the amino acid A. Thus, in one particularly preferred
embodiment, the cyclic analogue is
cyclo(83-99)[A.sup.91]MBP.sub.83-99, with alanine substitution at
position 91 and head-tail cyclization between residues 83-99.
[0075] In one particularly preferred embodiment, the cyclic peptide
is selected from the following:
TABLE-US-00008 peptide analogues sequence cyclo(83-99)MBP.sub.83-99
SEQ ID NO. 1 cyclo(83-99)E N P V V H F F K N I V T P R T P
cyclo(83-99)[A.sup.91]MBP.sub.83-99 SEQ ID NO. 18 cyclo(83-99)E N P
V V H F F A N I V T P R T P cyclo(83-99)[R.sup.91]MBP.sub.83-99 SEQ
ID NO. 19 cyclo(83-99)E N P V V H F F R N I V T P R T P
cyclo(83-99)[F.sup.91]MBP.sub.83-99 SEQ ID NO. 15 cyclo(83-99)E N P
V V H F F F N I V T P R T P cyclo(83-99)[Y.sup.91]MBP.sub.83-99 SEQ
ID NO. 16 cyclo(83-99)E N P V V H F F Y N I V T P R T P
cyclo(83-99)[E.sup.91]MBP.sub.83-99 SEQ ID NO. 14 cyclo(83-99)E N P
V V H F F E N I V T P R T P cyclo(83-99)[A.sup.91,
A.sup.96]MBP.sub.83-99 SEQ ID NO. 13 cyclo(83-99)E N P V V H F F A
N I V T A R T P cyclo(83-99)[R.sup.91, A.sup.96]MBP.sub.83-99 SEQ
ID NO. 12 cyclo(83-99)E N P V V H F F R N I V T A R T P
cyclo(83-99)[F.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 20
cyclo(83-99)E N P V V H F F F N I V T A R T P
cyclo(83-99)[Y.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 21
cyclo(83-99)E N P V V H F F Y N I V T A R T P
cyclo(83-99)[S.sup.91, A.sup.96]MBP.sub.83-99 SEQ ID NO. 22
cyclo(83-99)E N P V V H F F S N I V T A R T P
[0076] In the above nomenclature, (83-99) denotes the site of
cyclisation. For example, amino acid residue 83 is joined to
residue 99; i.e. the peptide is cyclised "head-to-tail".
Cyclo(83-99)[Y.sup.91]MBP.sub.83-99 is particularly preferred.
[0077] In another preferred embodiment of the invention, the
peptide comprises the amino acid sequence of formula (IIa),
TABLE-US-00009 VHFFK.sup.91NIVTP.sup.96RTP (IIa; SEQ ID NO. 23)
in linear or cyclic form.
[0078] In another preferred embodiment of the invention, the
peptide comprises the amino acid sequence of formula (IIa),
TABLE-US-00010 VHFFK.sup.91NIVTP.sup.96RTP (IIa; SEQ ID NO. 24)
wherein K.sup.91 is substituted by the amino acid A and P.sup.96 is
substituted by the amino acid A, wherein said peptide is in linear
form.
[0079] In one highly preferred embodiment, the peptide of formula
(IIa) consists of the sequence VHFFANIVTARTP (SEQ ID NO. 24).
[0080] In yet another preferred embodiment of the invention, the
peptide comprises the amino acid sequence of formula (IIb),
TABLE-US-00011 VHFFK.sup.91NIVTP.sup.96RTP (IIb; SEQ ID NO. 24)
wherein K.sup.91 is substituted by the amino acid A and P.sup.96 is
substituted by the amino acid A, wherein said peptide is in cyclic
form.
[0081] In one highly preferred embodiment, the peptide of formula
(IIb) consists of the sequence VHFFANIVTARTP (SEQ ID NO. 24)
cyclised head to tail, i.e. cyclization between residues 87-99.
[0082] In another preferred embodiment of the invention, the
epitope comprises a peptide fragment of myelin oligodentrocyte
glycoprotein (MOG) in linear or cyclic form.
[0083] Preferably, the epitope comprises a peptide selected from
MOG.sub.35-55 and MOG.sub.97-108, and variants thereof, in linear
or cyclic form.
[0084] More preferably, the epitope is a peptide selected from
MOG.sub.35-55 and MOG.sub.97-108, and variants thereof, in linear
or cyclic form.
[0085] In one preferred embodiment, the epitope comprises the
peptide MOG.sub.35-55, or a variant thereof. More preferably, the
epitope is MOG.sub.35-55, or a variant thereof, in linear or cyclic
form.
[0086] In one particularly preferred embodiment, the epitope
corresponds to the peptide sequence SEQ ID NO. 2, or a variant
thereof:
TABLE-US-00012 [SEQ ID NO. 2]
H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-
Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys-OH
in linear or cyclic form. Preferably, the peptide is in linear
form.
[0087] In another preferred embodiment of the invention, the
epitope comprises a peptide fragment of proteolipid protein (PLP)
in linear or cyclic form.
[0088] Preferably, the epitope comprises a peptide selected from
PLP.sub.97-117, PLP.sub.185-206, PLP.sub.40-60, PLP.sub.139-151,
PLP.sub.190-209, PLP.sub.184-199, PLP.sub.80-88, PLP.sub.30-49,
PLP.sub.180-199, and variants thereof, in linear or cyclic
form.
[0089] More preferably, the epitope is a peptide selected from
PLP.sub.97-117, PLP.sub.185-206, PLP.sub.40-60, PLP.sub.139-151,
PLP.sub.190-209, PLP.sub.184-199, PLP.sub.80-88, PLP.sub.30-49,
PLP.sub.180-199, and variants thereof, in linear or cyclic
form.
[0090] In one preferred embodiment, the epitope comprises the
peptide PLP.sub.139-151, or a variant thereof. Even more
preferably, the epitope is PLP.sub.139-151, or a variant thereof,
in linear or cyclic form.
[0091] In one particularly preferred embodiment, the epitope
corresponds to the peptide sequence SEQ ID NO. 3, or a variant
thereof:
TABLE-US-00013 [SEQ ID NO. 3]
H-His-Ser-Leu-Gly-Lys-Trp-Leu-Gly-His-Pro-Asp-Lys- Phe-OH
in linear or cyclic form. Preferably, the peptide is in linear
form.
[0092] As used herein, the term "variant" includes any variation
wherein; (a) one or more amino acid residues are replaced by a
naturally or non-naturally occurring amino acid residue (b) the
order of two or more amino acid residues is reversed, (c) both (a)
and (b) are present together, (d) a spacer group is present between
any two amino acid residues, (e) one or more amino acid residues
are in peptoid form, (f) the (N--C--C) backbone of one or more
amino acid residues of the peptide has been modified, or any of
(a)-(f) in combination. Preferably, the variants arise from one of
(a), (b) or (c).
[0093] More preferably, one or two amino acids residues are
substituted by one or more other amino acid residues. Even more
preferably, one amino acid residue is substituted by another amino
acid residue. Preferably, the substitution is homologous.
[0094] Homologous substitution (substitution and replacement are
both used herein to mean the interchange of an existing amino acid
residue, with an alternative residue) may occur i.e. like-for-like
substitution such as basic for basic, acidic for acidic, polar for
polar etc. Non-homologous substitution may also occur i.e. from one
class of residue to another or alternatively involving the
inclusion of unnatural amino acids such as ornithine (hereinafter
referred to as Z), diaminobutyric acid ornithine (hereinafter
referred to as B), norleucine ornithine (hereinafter referred to as
0), pyridylalanine, thienylalanine, naphthylalanine and
phenylglycine, a more detailed list of which appears below. Within
each peptide carrier moiety more than one amino acid residue may be
modified at a time.
[0095] As used herein, amino acids are classified according to the
following classes;
basic; H, K, R acidic; D, E non-polar; A, F, G, I, L, M, P, V, W
polar; C, N, Q, S, T, Y, (using the internationally accepted single
letter amino acid notation) and homologous and non-homologous
substitution is defined using these classes. Thus, homologous
substitution is used to refer to substitution from within the same
class, whereas non-homologous substitution refers to substitution
from a different class or by an unnatural amino acid.
[0096] Suitable spacer groups that may be inserted between any two
amino acid residues of the carrier moiety include alkyl groups such
as methyl, ethyl or propyl groups in addition to amino acid spacers
such as glycine or .beta.-alanine residues. A further form of
variation, type (e), involving the presence of one or more amino
acid residues in peptoid form, will be well understood by those
skilled in the art. For the avoidance of doubt, "the peptoid form"
is used to refer to variant amino acid residues wherein the
.alpha.-carbon substituent group is on the residue's nitrogen atom
rather than the .alpha.-carbon. Processes for preparing peptides in
the peptoid form are known in the art, for example Simon R J et
al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends
Biotechnol. (1995) 13(4), 132-134. Type (f) modification may occur
by methods such as those described in International Application
PCT/GB99/01855.
[0097] Within the definition of formula (I) it has been
demonstrated that it is preferable for amino acid variation,
preferably of type (a) or (b), to occur independently at any
position. As mentioned above more than one homologous or
non-homologous substitution may occur simultaneously. Further
variation may occur by virtue of reversing the sequence of a number
of amino acid residues within a sequence.
[0098] In one embodiment the replacement amino acid residue is
selected from the residues of alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine.
[0099] The replacement amino acid residue may additionally be
selected from unnatural amino acids. Non-natural amino acid
derivatives that may be used in the context of the present
invention include alpha* and alpha-disubstituted* amino acids,
N-alkyl amino acids*, lactic acid*, halide derivatives of natural
amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*,
p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*,
.beta.-alanine*, L-.alpha.-amino butyric acid*, L-.gamma.-amino
butyric acid*, L-.alpha.-amino isobutyric acid*, L-.epsilon.-amino
caproic acid.sup.#, 7-amino heptanoic acid*, L-methionine
sulfone.sup.#*, L-norleucine*, L-norvaline*,
p-nitro-L-phenylalanine*, L-hydroxyproline.sup.#, L-thioproline*,
methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*,
pentamethyl-Phe*, L-Phe (4-amino).sup.#, L-Tyr (methyl)*, L-Phe
(4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl
acid)*, L-diaminopropionic acid.sup.# and L-Phe (4-benzyl)*. The
notation * has been utilised for the purpose of the discussion
above, to indicate the hydrophobic nature of the derivative whereas
# has been utilised to indicate the hydrophilic nature of the
derivative, #* indicates amphipathic characteristics.
[0100] In one particularly preferred embodiment, one amino acid of
the peptide sequence is substituted by an alanine residue. Even
more preferably, each amino acid residue in turn is substituted by
an alanine residue in accordance with routine "alanine
scanning".
[0101] The peptide of the present invention may comprise amino
acids in the L or D form, i.e. one or more residues, preferably all
the residues may be in the L or D form.
[0102] The conjugate of the invention is linked to mannan via a
[(Lys-Gly).sub.n] bridge, where n is an integer from 1 to 10.
Preferably, n is an integer from 1 to 5, and even more preferably,
n is 5. In one preferred embodiment, n is from 6 to 10, more
preferably, from 7 to 10. In another preferred embodiment, n is
from 1 to 4, more preferably, from 1 to 3.
[0103] Conjugation occurs via Schiff base formation between the
free amino groups of the lysine and oxidized mannan. Reduced mannan
conjugates may be prepared by adding a reducing agent (e.g. sodium
borohydride) to the oxidized mannan conjugates.
[0104] Mannan (poly-mannose) conjugated to MUC1 FP or peptides
(MUC1, an antigen found on adenocarcinoma cells) in the oxidized
(comprising aldehydes) or in the reduced form (aldehydes reduced to
alcohols) generates differential immune responses. Mannan has been
used as a successful carrier to target peptides to the mannose
receptor, which is predominantly found on macrophage and dendritic
cells (DCs). Upon binding, MHC class I or MHC class II presentation
of peptides is generated, stimulating either CTL/Ab or Th1/Th2
immune responses. Th1 cytokines released after therapeutic
administration are associated with exacerbation of MS. However, Th2
cytokines (such as IL-4 and IL-10) have anti-inflammatory
properties and down-regulate Th1 responses. Mannose-antigen leads
to 100-10,000 fold enhanced potency to stimulate MHC Class II
presentation to T cell [Apostolopoulos, et al., 2000a;
Apostolopoulos, et al., 2000b]. Mannan has been investigated
extensively for its ability to generate responses in several model
systems. Its adjuvant function has been shown to stem from its
ability to target the mannose receptor on antigen presenting cells.
Mice (inbred or MUC1 transgenic) immunized with mannan-MUC1 protein
are protected against a MUC1 expressing tumor challenge, as well as
reversing established tumors in mice [Apostolopoulos, et al., 1996;
Acres, et al., 2000]. Similar results were observed in MUC1
transgenic mice. Either a Th1 response (IL-2, IFN-.gamma., IL-12,
TNF-.alpha. and IgG2a antibodies) or Th2 response (no IFN-.gamma.
or IL-12, but significant amounts of IL-4, IL-10 and TGF-.beta. and
IgG1 antibodies) is generated depending on the mode of conjugation,
and whether the mannan is in an oxidized or reduced state [Lees, et
al., 1999; Lees, et al., 2000a; Lees, et al., 2000b]. Other
cytokines, IL-5, IL-6, IL-13, IL-15, and IL-18 have also been
measured with either oxidized or reduced mannan immunogens. In
addition to Th1/Th2 type responses to MUC1 in mice, similar
responses have been demonstrated in humans and monkeys [Vaughan, et
al., 1999; Vaughan, et al., 2000] with MUC1 protein and to an
Anaplasma marginale MSP-1 peptide in cows [Davis, et al., 2002].
The use of reduced or oxidized mannan conjugated with
MBP.sub.83-99, PLP.sub.139-151 or MOG.sub.35-55 epitopes, and in
particular, the use of reduced mannan to further divert immune
responses to Th2 when conjugated to MBP peptides, constitutes a
promising strategy for the immunotherapy of MS.
[0105] In one preferred embodiment of the invention, the mannan is
reduced mannan. In another preferred embodiment of the invention,
the mannan is oxidised mannan.
[0106] In one especially preferred embodiment, more than one
epitope is linked to mannan, i.e. multiple epitopes are attached to
a single mannan residue. For this embodiment, the epitopes may be
the same or different.
[0107] Thus, in one particularly preferred embodiment, the
conjugate comprises a mixture (or "cocktail") of more than one of
said epitopes conjugated to a single mannan residue via a
[(Lys-Gly).sub.n] bridge, where n is an integer from 1 to 10, i.e.
the mannan may be conjugated to a plurality of epitopes. As above,
the mannan may be oxidised or reduced.
[0108] In one highly preferred embodiment, the invention relates to
a conjugate comprising: [0109] (i) mannan; and [0110] (ii)(a) an
epitope comprising a peptide fragment of myelin basic protein
(MBP); and [0111] (ii)(b) an epitope comprising a peptide fragment
of myelin oligodentrocyte glycoprotein (MOG); and [0112] (ii)(c) an
epitope comprising a peptide fragment of proteolipid protein (PLP);
each peptide fragment being in linear or cyclic form; wherein each
epitope is linked to mannan via a [(Lys-Gly).sub.n] bridge, where n
is an integer from 1 to 10.
[0113] Another embodiment of the invention relates to a mixture
comprising two or more conjugates as defined above. For this
embodiment, the two or more conjugates may be the same or
different. Moreover, each conjugate may itself comprise one or more
epitopes, which may be the same or different.
Therapeutic Applications
[0114] Another aspect of the invention relates to a conjugate as
described above for use in medicine.
[0115] Yet another aspect relates to the use of a conjugate of the
invention in the preparation of a medicament for treating an immune
disorder.
[0116] In one preferred embodiment, the immune disorder is an
autoimmune disease.
[0117] In one particularly preferred embodiment, the disorder is
multiple sclerosis (MS).
[0118] MS is a serious autoimmune disease in which the destruction
of myelin sheath and loss of neurologic function takes place
[Steinman, 1996]. The linear peptides MBP.sub.83-99:
H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-OH
(SEQ ID NO. 9), PLP.sub.139-151:
H-His-Ser-Leu-Gly-Lys-Trp-Leu-Gly-His-Pro-Asp-Lys-Phe-OH (SEQ ID
NO. 10) and MOG.sub.35-55:
H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro[Ser]-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Ty-
r-Arg-Asn-Gly-Lys-OH (SEQ ID NO. 11) were found to induce EAE in
animal models. However, their conjugation to reduced or oxidized
mannan completely prevented the induction of EAE.
[0119] In another preferred embodiment, the disorder is
experimental autoimmune encephalomyelitis (EAE).
[0120] In one preferred embodiment, the EAE is
MBP.sub.74-85-induced EAE.
[0121] In one preferred embodiment, the EAE is
MBP.sub.83-89-induced EAE.
[0122] In another preferred embodiment, the EAE is
MOG.sub.35-55-induced EAE.
[0123] In yet another preferred embodiment, the EAE is
PLP.sub.131-139-induced EAE.
[0124] A further aspect of the invention relates to a method of
treating an immune disorder, said method comprising administering
to a subject a conjugate as defined above.
[0125] Preferably, the immune disorder is an autoimmune disease.
More preferably, the disorder is multiple sclerosis (MS) or
experimental autoimmune encephalomyelitis (EAE).
[0126] Yet another aspect of the invention relates to a method of
immunizing a subject against an immune disorder, said method
comprising administering to a subject a conjugate as defined
above.
[0127] Preferably, the peptide or conjugate is administered in an
amount sufficient to cause suppression of chronic experimental
autoimmune encephalomyelitis (EAE).
[0128] Another aspect of the invention relates to a conjugate or
mixture according the invention for the treatment of an immune
disorder.
Pharmaceutical Composition
[0129] Another aspect relates to a pharmaceutical composition
comprising a peptide or a conjugate according to the invention,
admixed with a pharmaceutically acceptable diluent, excipient or
carrier.
[0130] Even though the conjugates of the present invention
(including their pharmaceutically acceptable salts, esters and
pharmaceutically acceptable solvates) can be administered alone,
they will generally be administered in admixture with a
pharmaceutical carrier, excipient or diluent, particularly for
human therapy. The pharmaceutical compositions may be for human or
animal usage in human and veterinary medicine.
[0131] Examples of such suitable excipients for the various
different forms of pharmaceutical compositions described herein may
be found in the "Handbook of Pharmaceutical Excipients, 2.sup.nd
Edition, (1994), Edited by A Wade and P J Weller.
[0132] Acceptable carriers or diluents for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985).
[0133] Examples of suitable carriers include lactose, starch,
glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol
and the like. Examples of suitable diluents include ethanol,
glycerol and water.
[0134] The choice of pharmaceutical carrier, excipient or diluent
can be selected with regard to the intended route of administration
and standard pharmaceutical practice. The pharmaceutical
compositions may comprise as, or in addition to, the carrier,
excipient or diluent any suitable binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
[0135] Examples of suitable binders include starch, gelatin,
natural sugars such as glucose, anhydrous lactose, free-flow
lactose, beta-lactose, corn sweeteners, natural and synthetic gums,
such as acacia, tragacanth or sodium alginate, carboxymethyl
cellulose and polyethylene glycol.
[0136] Examples of suitable lubricants include sodium oleate,
sodium stearate, magnesium stearate, sodium benzoate, sodium
acetate, sodium chloride and the like.
[0137] Preservatives, stabilizers, dyes and even flavoring agents
may be provided in the pharmaceutical composition. Examples of
preservatives include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
Vaccines
[0138] Another aspect of the invention relates to a vaccine or
immunogenic composition comprising a conjugate as defined
above.
[0139] The immunogenic compositions of the invention are preferably
adjuvanted. Adjuvants used in the present invention are those which
are physiologically acceptable to humans, these include, but are
not limited to an aluminium salt such as aluminium hydroxide gel
(alum) or aluminium phosphate, but may also be a salt of calcium,
iron or zinc, oil/surfactant based emulsion adjuvants such as
Montanide.TM. in which different surfactants (especially mannityl
oleate) are combined with a mineral oil, squalene-containing
emulsions such as MF59.TM., monophosphoryl lipid A, or Neisseriae
mutant lipopolysaccharide, an insoluble suspension of acylated
tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes.
[0140] Preferably the adjuvant is administered at the same time as
of the invention and in preferred embodiments are formulated
together.
[0141] The vaccine composition of the present invention preferably
is sterile. Furthermore, the composition may contain components
that preserve against infestation with, and growth of,
micro-organisms.
[0142] It is preferred that the vaccine composition is manufactured
in the form of a sterile aqueous liquid which is ready for
immediate administration.
[0143] In a preferred embodiment of the invention, the inventive
vaccine composition may be formulated in dosage unit form as
heretofore described to facilitate administration and ensure
uniformity of dosage. Formulation may be effected using available
techniques, such as those applicable to preparations of
emulsions.
Salts/Esters
[0144] The peptides/conjugates of the invention can be present as
salts or esters, in particular pharmaceutically acceptable salts or
esters.
[0145] Pharmaceutically acceptable salts of the peptides/conjugates
of the invention include suitable acid addition or base salts
thereof. A review of suitable pharmaceutical salts may be found in
Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for
example with strong inorganic acids such as mineral acids, e.g.
sulphuric acid, phosphoric acid or hydrohalic acids; with strong
organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4
carbon atoms which are unsubstituted or substituted (e.g., by
halogen), such as acetic acid; with saturated or unsaturated
dicarboxylic acids, for example oxalic, malonic, succinic, maleic,
fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids,
for example ascorbic, glycolic, lactic, malic, tartaric or citric
acid; with aminoacids, for example aspartic or glutamic acid; with
benzoic acid; or with organic sulfonic acids, such as
(C.sub.1-C.sub.4)-alkyl- or aryl-sulfonic acids which are
unsubstituted or substituted (for example, by a halogen) such as
methane- or p-toluene sulfonic acid.
[0146] Esters are formed either using organic acids or
alcohols/hydroxides, depending on the functional group being
esterified. Organic acids include carboxylic acids, such as
alkanecarboxylic acids of 1 to 12 carbon atoms which are
unsubstituted or substituted (e.g., by halogen), such as acetic
acid; with saturated or unsaturated dicarboxylic acid, for example
oxalic, malonic, succinic, maleic, fumaric, phthalic or
tetraphthalic; with hydroxycarboxylic acids, for example ascorbic,
glycolic, lactic, malic, tartaric or citric acid; with aminoacids,
for example aspartic or glutamic acid; with benzoic acid; or with
organic sulfonic acids, such as (C.sub.1-C.sub.4)-alkyl- or
aryl-sulfonic acids which are unsubstituted or substituted (for
example, by a halogen) such as methane- or p-toluene sulfonic acid.
Suitable hydroxides include inorganic hydroxides, such as sodium
hydroxide, potassium hydroxide, calcium hydroxide, aluminium
hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms
which may be unsubstituted or substituted, e.g. by a halogen).
Enantiomers/Tautomers
[0147] In all aspects of the present invention previously
discussed, the invention includes, where appropriate all
enantiomers and tautomers of the peptides/conjugates. The man
skilled in the art will recognise compounds that possess an optical
properties (one or more chiral carbon atoms) or tautomeric
characteristics. The corresponding enantiomers and/or tautomers may
be isolated/prepared by methods known in the art.
Stereo and Geometric Isomers
[0148] Some of the peptides/conjugates of the invention may exist
as stereoisomers and/or geometric isomers--e.g. they may possess
one or more asymmetric and/or geometric centres and so may exist in
two or more stereoisomeric and/or geometric forms. The present
invention contemplates the use of all the individual stereoisomers
and geometric isomers, and mixtures thereof. The terms used in the
claims encompass these forms, provided said forms retain the
appropriate functional activity (though not necessarily to the same
degree).
[0149] The present invention also includes all suitable isotopic
variations of the peptides/conjugates or pharmaceutically
acceptable salts thereof. An isotopic variation is defined as one
in which at least one atom is replaced by an atom having the same
atomic number but an atomic mass different from the atomic mass
usually found in nature. Examples of isotopes that can be
incorporated into the peptides/conjugates and pharmaceutically
acceptable salts thereof include isotopes of hydrogen, carbon,
nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such
as .sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.17O,
.sup.18O, .sup.31P, .sup.32P, .sup.35S, .sup.18F and .sup.36Cl,
respectively. Certain isotopic variations of the
peptides/conjugates and pharmaceutically acceptable salts thereof,
for example, those in which a radioactive isotope such as .sup.3H
or .sup.14C is incorporated, are useful in drug and/or substrate
tissue distribution studies. Tritiated, i.e., .sup.3H, and
carbon-14, i.e., .sup.14C, isotopes are particularly preferred for
their ease of preparation and detectability. Further, substitution
with isotopes such as deuterium, i.e., .sup.2H, may afford certain
therapeutic advantages resulting from greater metabolic stability,
for example, increased in vivo half-life or reduced dosage
requirements and hence may be preferred in some circumstances.
Isotopic variations of the peptides/conjugates of the present
invention and pharmaceutically acceptable salts thereof of this
invention can generally be prepared by conventional procedures
using appropriate isotopic variations of suitable reagents.
Solvates
[0150] The present invention also includes the use of solvate forms
of the peptides/conjugates of the present invention. The terms used
in the claims encompass these forms.
Polymorphs
[0151] The invention furthermore relates to the peptides/conjugates
of the present invention in their various crystalline forms,
polymorphic forms and (an)hydrous forms. It is well established
within the pharmaceutical industry that chemical compounds may be
isolated in any of such forms by slightly varying the method of
purification and or isolation form the solvents used in the
synthetic preparation of such compounds.
Prodrugs
[0152] The invention further includes the peptides/conjugates of
the present invention in prodrug form. Such prodrugs are generally
peptides/conjugates wherein one or more appropriate groups have
been modified such that the modification may be reversed upon
administration to a human or mammalian subject. Such reversion is
usually performed by an enzyme naturally present in such subject,
though it is possible for a second agent to be administered
together with such a prodrug in order to perform the reversion in
vivo. Examples of such modifications include ester (for example,
any of those described above), wherein the reversion may be carried
out be an esterase etc. Other such systems will be well known to
those skilled in the art.
Administration
[0153] The pharmaceutical compositions of the present invention may
be adapted for oral, rectal, vaginal, parenteral, intramuscular,
intraperitoneal, intraarterial, intrathecal, intrabronchial,
subcutaneous, intradermal, intravenous, nasal, buccal or sublingual
routes of administration.
[0154] For oral administration, particular use is made of
compressed tablets, pills, tablets, gellules, drops, and
capsules.
[0155] Other forms of administration comprise solutions or
emulsions which may be injected intravenously, intraarterially,
intrathecally, subcutaneously, intradermally, intraperitoneally or
intramuscularly, and which are prepared from sterile or
sterilisable solutions. The pharmaceutical compositions of the
present invention may also be in form of suppositories, pessaries,
suspensions, emulsions, lotions, ointments, creams, gels, sprays,
solutions or dusting powders.
[0156] An alternative means of transdermal administration is by use
of a skin patch. For example, the active ingredient can be
incorporated into a cream consisting of an aqueous emulsion of
polyethylene glycols or liquid paraffin. The active ingredient can
also be incorporated, at a concentration of between 1 and 10% by
weight, into an ointment consisting of a white wax or white soft
paraffin base together with such stabilisers and preservatives as
may be required.
[0157] Compositions may be formulated in unit dosage form, i.e., in
the form of discrete portions containing a unit dose, or a multiple
or sub-unit of a unit dose.
Dosage
[0158] A person of ordinary skill in the art can easily determine
an appropriate dose of one of the instant compositions to
administer to a subject without undue experimentation. Typically, a
physician will determine the actual dosage which will be most
suitable for an individual patient and it will depend on a variety
of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the individual undergoing
therapy. The dosages disclosed herein are exemplary of the average
case. There can of course be individual instances where higher or
lower dosage ranges are merited, and such are within the scope of
this invention.
Combinations
[0159] In a particularly preferred embodiment, one or more
peptides/conjugates of the invention are administered in
combination with one or more other therapeutically active agents,
for example, existing drugs available on the market. In such cases,
the compounds of the invention may be administered consecutively,
simultaneously or sequentially with the one or more other
agents.
[0160] Combination therapy is desirable in order to avoid an
overlap of major toxicities, mechanism of action and resistance
mechanism(s). Furthermore, it is also desirable to administer most
drugs at their maximum tolerated doses with minimum time intervals
between such doses. The major advantages of combining
chemotherapeutic drugs are that it may promote additive or possible
synergistic effects through biochemical interactions and also may
decrease the emergence of resistance in early tumor cells which
would have been otherwise responsive to initial chemotherapy with a
single agent.
Assay
[0161] A further aspect of the invention relates to a conjugate as
defined above in an assay for elucidating agents capable of
regulating experimental autoimmune encephalomyelitis (EAE) or
regulating multiple sclerosis.
[0162] The present invention is further described by way of the
following non-limiting examples, and with reference to the
following figures wherein:
[0163] FIG. 1 shows the mean clinical score of groups of mice which
received prophylactic vaccination with oxidized/reduced
mannan-MOG.sub.35-55 peptide conjugates prior to MOG.sub.35-55-EAE
induction (n=6 for all groups);
[0164] FIG. 2 shows the mean body weight changes in groups of mice
that had received prophylactic vaccination with oxidized/reduced
mannan-MOG.sub.35-55 peptide conjugates prior to MOG.sub.35-55-EAE
induction.
[0165] FIG. 3 shows the mean clinical scores of mice that received
vaccination with MOG.sub.35-55 peptide (black squares, n=6) prior
to MOG.sub.35-55-EAE induction. Non-vaccinated animals were used as
controls (n=3, white squares).
[0166] FIG. 4 shows the mean body weight changes in mice that
received vaccination with MOG.sub.35-55 peptide (black squares,
n=6) prior to MOG.sub.35-55-EAE induction. Non-vaccinated animals
were used as controls (n=3, white squares).
[0167] FIG. 5 shows hematoxylin-eosin and luxol fast blue staining
of spinal cord sections from the indicated groups of mice to
observe inflammatory cell infiltration and demyelination
respectively.
[0168] FIG. 6 shows the results for individual mice of each of the
experimental groups for which histological analysis was performed
following MOG.sub.35-55-EAE induction. Spinal cord infiltration and
demyelination in addition to brain infiltration and demyelination
are shown. (Abbreviations: ubi=ubiquitous, cer=cerebellum,
men=meninges, vac=vacuolation). Inflammation in the spinal cord was
determined by absolute true quantification; the numbers mean
inflammatory infiltrates/mm.sup.2 of tissue. In the brain a
semiquantitative scoring was used according to which 0.5=single
perivascular infiltrates; 1=multiple inflammatory infiltrates.
Demyelination was scored as follows: 0.5: single perivascular
sleeves of demyelination, 1: ubiquitous perivascular or subpial
demyelination, 2: confluent demyelinated plaques, 3: profound focal
demyelination, involving about 1/2 of the spinal cord white matter
at least in one spinal cord segment, 4: extensive demyelination,
for instance complete demyelination of spinal cord white matter at
least in one segment of the spinal cord. Controls 1, 2, 3 are the
same as PBS-control, because the experiment was carried out
twice.
[0169] FIG. 7 shows the proliferation of splenocytes isolated 25
days post-MOG.sub.35-55-EAE induction from mice immunized with
oxidized/reduced mannan-MOG.sub.35-55, oxidized/reduced mannan
alone, or PBS as control. Splenocytes were ex vivo stimulated with
increasing concentrations of MOG.sub.35-55 peptide. (PBS control,
closed circles, n=4; oxidized mannan MOG.sub.35-55, closed squares,
n=6; reduced mannan MOG.sub.35-55, open squares, n=6; oxidized
mannan, closed triangles, n=5; reduced mannan, open triangles,
n=4).
[0170] FIG. 8 shows the proliferation of splenocytes isolated 25
days post-MOG.sub.35-55-EAE induction from mice that were immunized
with MOG.sub.35-55 peptide alone, and stimulated ex vivo with
increasing concentrations of MOG.sub.35-55 peptide. (PBS control,
open circles, n=3; MOG.sub.35-55 peptide, closed circles, n=6).
[0171] FIG. 9 shows cytokine production measured in culture
supernatants of splenocytes that were stimulated for 48 hours with
10 .mu.g/ml MOG.sub.35-55 peptide. Splenocytes were isolated 25
days post MOG.sub.35-55-EAE induction. Statistical significance
after pair-wise comparisons (using T-Test and Mann Whitney rank sum
test) of each experimental group with the non-vaccinated control
(PBS) group is shown. PBS1, PBS2 are controls.
[0172] FIG. 10 shows the mean clinical score of groups of mice
which received prophylactic vaccination with oxidized/reduced
mannan-PLP.sub.139-151 peptide conjugates prior to
PLP.sub.139-151-EAE induction; control mice were non immunized (n=5
for all groups). Mice immunized with oxidized mannan alone, reduced
mannan alone, or control mice were culled on day 14 for ethical
reasons. The vaccinated group with PLP.sub.139-151-oxidized mannan
has the same clinical score as the control. It is completely
protected.
[0173] FIG. 11 shows the average clinical score Lewis rats that had
received oxidized/reduced mannan-MBP.sub.83-99 or oxidized/reduced
mannan alone injections, prior to MBP.sub.74-85 EAE induction.
Naive mice (non immunized) were used as controls (n=5 for all
groups).
[0174] FIG. 12 shows the mean body weight changes in Lewis rats
that were immunized with oxidized/reduced mannan-MBP.sub.83-99 or
oxidized/reduced mannan alone, or non immunized (controls), after
MBP.sub.74-85 EAE induction (n=5 for all groups).
EXAMPLES
Synthesis and Purification of Linear Myelin Epitopes:
MBP.sub.83-99, PLP.sub.139-151 and MOG.sub.35-55 with a
[(Lys-Gly).sub.5] Bridge
General Procedure
[0175] In general, peptides were synthesized by Fmoc/tBu
methodology using 2-chlorotrityl chloride (CLTR-Cl) resin (0.7 mmol
Cl.sup.-/g) and N.sup.a-Fmoc
(9-fluorenylmethyloxycarboxyl)-protected amino acids [Tselios et
al., 1999; Tselios et al., 2000a; Tselios et al., 2000b; Tselios et
al., 2002; Matsoukas et al., 2005; Mantzourani et al., 2006a;
Mantzourani et al., 2006b; Mantzourani et al., 2007]. In
particular, the linear protected peptides: MBP.sub.83-99:
H-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Glu(tB-
u)-Asn(Trt)-Pro-Val-Val-His(Trt)-Phe-Phe-Lys(Boc)-Asn(Trt)-Ile-Val-Thr(tBu-
)-Pro-Arg(Pdf)-Thr(tBu)-Pro-OH (SEQ ID NO. 9), PLP.sub.139-151:
H-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-His(Tr-
t)-Ser(tBu)-Leu-Gly-Lys(Boc)-Trp-Leu-Gly-His(Trt)-Pro-Asp-Lys(Boc)-Phe-OH
(SEQ ID NO. 10) and MOG.sub.35-55:
H-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Lys(Boc)-Gly-Met-Gl-
u(tBu)-Val-Gly-Trp-Tyr(tBu)-Arg(Pbf)-Pro(Ser)-Pro-Phe-Ser(tBu)-Arg(Pbf)-Va-
l-Val-His(Trt)-Leu-Tyr(tBu)- Arg(Pbf)-Asn(Trt)-Gly-Lys(Boc)-OH (SEQ
ID NO. 11) were synthesized step by step on the solid polymer
(CLTR-Cl) using N,N'-diisopropyl-carbodiimide and
1-hydroxybenzotriazol as coupling reagent in DMF. The Kaiser test
and thin layer chromatography (TLC) in n-butanol/acetic acid/water
(4:1:1) (BAW) as elutant verified the completeness of each
coupling. The protected, on the resin, peptides were treated with
the splitting mixture DCM/HFIP (7/3) for 6 h at room temperature to
remove peptide from the resin. The mixture was filtered off and the
resin was washed twice with the splitting mixture and with DCM. The
solvent was removed on a rotary evaporator and the obtained oily
product was precipitated from cold and dry diethyl ether as a white
solid. The protected linear peptides were treated with the
deprotection mixture DCM/TFA/ethanedithiol/anisole (32/65/2/1) for
6 h at room temperature. The resulting solution was concentrated
under vacuum to a small volume (0.5 ml). The final crude products
were further purified using semi-preparative Reverse Phase-High
Performance Liquid Chromatography (RP-HPLC). The purity of peptides
was determined using RP-HPLC, and the identification was achieved
by ESI-MS and Amino Acid analysis.
Solid-Phase Peptide Synthesis of Linear and Cyclic Analogues
[0176] Peptides (Table 1) were prepared on 2-chlorotrityl chloride
resin (CTLR-Cl) using Fmoc/tBu methodology. The cyclization was
achieved with TBTU/HOAt and 2,4,6-collidine as base, as previously
described. Preparative HPLC for peptide analogues were performed
using a Lichrosorb RP-18 reversed phase semipreparative column with
7 nm packing material. The peptides were >95% pure as analysed
by mass spectrometry.
TABLE-US-00014 TABLE 1 MBP.sub.83-99 peptide analogues used in this
study Sequence Peptide analogues P1 (SEQ ID NO. 1) E N P V V H F F
K N I V T P R T P MBP.sub.83-99 P2 (SEQ ID NO. 1) cyclo(83-99)E N P
V V H F F K N I V T P R T P cyclo(83-99)MBP.sub.83-99 P3 (SEQ ID
NO. 18) E N P V V H F F A N I V T P R T P [A.sup.91]MBP.sub.83-99
P4 (SEQ ID NO. 18) cyclo(83-99)E N P V V H F F A N I V T P R T P
cyclo(83-99)[A.sup.91]MBP.sub.83-99 P5 (SEQ ID NO. 15) E N P V V H
F F E N I V T P R T P [E.sup.91]MBP.sub.83-99 P6 (SEQ ID NO. 16) E
N P V V H F F F N I V T P R T P [F.sup.91]MBP.sub.83-99 P7 (SEQ ID
NO. 12) E N P V V H F F R N I V T A R T P [R.sup.91,
A.sup.91]MBP.sub.83-99 P8 (SEQ ID NO. 17) E N P V V H F F Y N I V T
P R T P [Y.sup.91]MBP.sub.83-99 P9 (SEQ ID NO. 13) E N P V V H F F
A N I V T A R T P [A.sup.91, A.sup.96]MBP.sub.83-99
Examples of Cyclization
[0177] Cyclization of linear MBP.sub.83-99 protected analogue was
achieved using O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole, 2,4,6
collidine in DMF solution, allowing fast reactions and high yield
cyclization products. Recent results indicate that HOAt, a
4-nitrogen containing variant, is a very effective coupling
additive, more efficient than HOBt for solution or solid phase
synthesis leading in high yield products.
Synthesis of Human MBP Cyclic Peptides
TABLE-US-00015 [0178] (SEQ ID NO. 12) Cyclo(83-99)[Arg.sup.91,
Ala.sup.96]MBP.sub.83-99 (Glu-Asn-Pro-
Val-Va1.sup.87-His-Phe-Phe-Arg.sup.91-Asn-Ile-Val-Thr-Ala.sup.96-
Arg-Thr-Pro.sup.99) (SEQ ID NO. 25) Cyclo(91-99)[Ala.sup.96]
MBP.sub.83-99 (Glu-Asn-Pro-Val-
Val.sup.87-His-Phe-Phe-Lys.sup.91-Asn-Ile-Val-Thr-A1a.sup.96-Arg-
Thr-Pro.sup.99)
[0179] Human MBP cyclic analogues were prepared on 2-chlorotrityl
chloride resin using Fmoc/tBu methodology. The peptide synthesis
was achieved using DIC/HOBt in DMF and the N.sup..alpha.--NH.sub.2
of amino acids was protected with the Fmoc group. The side chain of
peptides was protected as following: Trt for His, Pbf for Arg, tBu
for Ser, Thr, Asp, Glu, Boc for Lys, as regarding the cyclic
analogue cyclo(91-99)[Ala.sup.96] MBP.sub.83-99 (by
N.epsilon.-NH.sub.2 of Lys and C-terminous) Mtt protected group was
used, because it can easily be removed using mixture
HFIP(1,1,1,3,3,3 hexafluoro-2-propanol)/DCM (2/8), which cleaves
peptides from the resin. Otherwise, the side chain of Lys of the
cyclo(87-99)[Arg.sup.91, Ala.sup.96]MBP.sub.87-99 was protected
with Boc group. The final protected linear peptides on resin were
dried in vacuum and then treated with the splitting mixture
DCM/HFIP (8/2) for 7 h at room temperature to release the peptide
from the resin and deprotect Lys from Mtt of the
cyclo(91-99)[Ala.sup.96] MBP.sub.83-99. Each one of the linear
protected peptide was dissolved in DMF and collidine/HOAt was
added. This mixture was added dropwise in a solution of TBTU in DMF
for 8 hours. The cyclization was determined by TLC and analytical
reversed phase HPLC (RP-HPLC). The solvent was removed under
reduced pressure affording a light yellow oily residue. The cyclic
protected peptide (purity .gtoreq.90%) was precipitated from
H.sub.2O and dried in vacuum for 16 h. The cyclic protected peptide
was treated with 65% TFA in DCM and 3% ethanodithiol as scavanger
for 4 hours at room temperature. The resulting solution was
concentrated to a small volume and the final free peptide was
precipitated as a light yellow amorphous solid added diethylether
(purity .gtoreq.80%). Peptide purity was assessed by analytical
HPLC reruns, thin layer chromatography (TLC) and mass spectrometry
(ESIMS).
Solid-Phase Peptide Synthesis of Linear Analogs
[0180] Peptides MBP.sub.87-99 (VHFFKNIVTPRTP; SEQ ID NO. 23),
[R.sup.91, A.sup.96]MBP.sub.87-99 (VHFFRNIVTARTP; SEQ ID NO. 26)
and [A.sup.91, A.sup.96]MBP.sub.87-99 (VHFFANIVTARTP; SEQ ID NO.
24) were prepared on 2-chlorotrityl chloride resin (CLTR-Cl) using
Fmoc/tBu methodology.sup.22, 48-51. Preparative HPLC for
MBP.sub.87-99, [R.sup.91, A.sup.96]MBP.sub.87-99 and [A.sup.91,
A.sup.96]MBP.sub.87-99 peptide analogs were performed using a
Lichrosorb RP-18 reversed phase semipreparative column with 7 .mu.m
packing material. The peptides were >95% pure as analyzed by
analytical RP-HPLC and ESI-MS.
References for Solid-Phase Peptide Synthesis of Linear Analogs
[0181] 22. Matsoukas, J.; Apostolopoulos, V.; Kalbacher, H.;
Papini, A. M.; Tselios, T.; Chatzantoni, K.; Biagioli, T.; Lolli,
F.; Deraos, S.; Papathanassopoulos, P.; Troganis, A.; Mantzourani,
E.; Mavromoustakos, T.; Mouzaki, A. Design and synthesis of a novel
potent myelin basic protein epitope 87-99 cyclic analogue: enhanced
stability and biological properties of mimics render them a
potentially new class of immunomodulators. J Med Chem 2005, 48,
1470-80. [0182] 48. Tselios, T.; Apostolopoulos, V.; Daliani, I.;
Deraos, S.; Grdadolnik, S; Mavromoustakos, T.; Melachrinou, M.;
Thymianou, S.; Probert, L.; Mouzaki, A.; Matsoukas, J. Antagonistic
effects of human cyclic MBP(87-99) altered peptide ligands in
experimental allergic encephalomyelitis and human T-cell
proliferation. J Med Chem 2002, 45, 275-83. [0183] 49. Tselios, T.;
Daliani, I.; Deraos, S.; Thymianou, S.; Matsoukas, E.; Troganis,
A.; Gerothanassis, I.; Mouzaki, A.; Mavromoustakos, T.; Probert,
L.; Matsoukas, J. Treatment of experimental allergic
encephalomyelitis (EAE) by a rationally designed cyclic analogue of
myelin basic protein (MBP) epitope 72-85. Bioorg Med Chem Lett
2000, 10, 2713-7. [0184] 50. Tselios, T.; Daliani, I.; Probert, L.;
Deraos, S.; Matsoukas, E.; Roy, S.; Pires, J.; Moore, G.;
Matsoukas, J. Treatment of experimental allergic encephalomyelitis
(EAE) induced by guinea pig myelin basic protein epitope 72-85 with
a human MBP(87-99) analogue and effects of cyclic peptides. Bioorg
Med Chem 2000, 8, 1903-9. [0185] 51. Tselios, T.; Probert, L.;
Daliani, I.; Matsoukas, E.; Troganis, A.; Gerothanassis, I. P.;
Mavromoustakos, T.; Moore, G. J.; Matsoukas, J. M. Design and
synthesis of a potent cyclic analogue of the myelin basic protein
epitope MBP72-85: importance of the Ala81 carboxyl group and of a
cyclic conformation for induction of experimental allergic
encephalomyelitis. J Med Chem 1999, 42, 1170-7.
Solid-Phase Peptide Synthesis of Cyclic Analogs
[0186] Peptides MBP.sub.87-99 (VHFFKNIVTPRTP; SEQ ID NO. 23) and
cyclic double mutant peptide with Ala mutations at positions 91 and
96, cyclo(87-99)[A.sup.91, A.sup.96]MBP.sub.87-99 (cyclo,
head-to-tail, VHFFANIVTARTP; SEQ ID NO. 24) were prepared on
2-chlorotrityl chloride resin (CLTR-Cl) using Fmoc/tBu methodology.
Head-to-tail cyclization of MBP.sub.87-99[A.sup.91, A.sup.96]
peptide was achieved using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate
(TBTU) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine in DMF
solution, allowing fast reactions and high yield cyclization
products. Results indicate that HOAt, a 4-nitrogen containing
variant, is a very effective coupling additive, more efficient than
HOBt for solution or solid phase synthesis.sup.14-17. Preparative
HPLC for peptide analogs were performed using a Lichrosorb RP-18
reversed phase semi-preparative column with 7 .mu.m packing
material. The peptides were >95% pure as analyzed by mass
spectrometry (ESI-MS).
References for Solid-Phase Peptide Synthesis of Cyclic Analogs
[0187] 14. Tselios, T.; Apostolopoulos, V.; Daliani, I.; Deraos,
S.; Grdadolnik, S.; Mavromoustakos, T.; Melachrinou, M.; Thymianou,
S.; Probert, L.; Mouzaki, A.; Matsoukas, J. Antagonistic effects of
human cyclic MBP(87-99) altered peptide ligands in experimental
allergic encephalomyelitis and human T-cell proliferation. J Med
Chem 2002, 45, 275-83. [0188] 15. Tselios, T.; Daliani, I.; Deraos,
S.; Thymianou, S.; Matsoukas, E.; Troganis, A.; Gerothanassis, I.;
Mouzaki, A.; Mavromoustakos, T.; Probert, L.; Matsoukas, J.
Treatment of experimental allergic encephalomyelitis (EAE) by a
rationally designed cyclic analogue of myelin basic protein (MBP)
epitope 72-85. Bioorg Med Chem Lett 2000, 10, 2713-7. [0189] 16.
Tselios, T.; Daliani, I.; Probert, L.; Deraos, S.; Matsoukas, E.;
Roy, S.; Pires, J.; Moore, G.; Matsoukas, J. Treatment of
experimental allergic encephalomyelitis (EAE) induced by guinea pig
myelin basic protein epitope 72-85 with a human MBP(87-99) analogue
and effects of cyclic peptides. Bioorg Med Chem 2000, 8, 1903-9.
[0190] 17. Tselios, T.; Probert, L.; Daliani, I.; Matsoukas, E.;
Troganis, A.; Gerothanassis, I. P.; Mavromoustakos, T.; Moore, G.
J.; Matsoukas, J. M. Design and synthesis of a potent cyclic
analogue of the myelin basic protein epitope MBP72-85: importance
of the Ala81 carboxyl group and of a cyclic conformation for
induction of experimental allergic encephalomyelitis. J Med Chem
1999, 42, 1170-7.
Conjugation of Peptides to Mannan
[0191] The peptide-mannan binding was achieved following a protocol
earlier described [Apostolopoulos et al., 1996; Tselios et al.,
2005]. Briefly, 14 mg mannan (poly-mannose from Saccharomyces
cerevisiae, Sigma-Aldrich Ltd, Athens, Greece) was dissolved in 1
ml phosphate buffer, pH 6.0, and oxidized to polyaldehyde by
treating with sodium periodate. The mixture was passed through a
PD-10 column (Sephadex G-25 M column, Pharmacia Biotech. Sweden)
equilibrated with 0.1 M bicarbonate buffer pH 9.0 and the mannan
fraction collected. Oxidized mannan (7.0 mg/ml) was eluted with 2
ml of pH 9.0 phosphate buffer, to which 1 mg peptides containing
[(Lys-Gly).sub.5] bridge were added and allowed to react overnight
at room temperature in the dark. Conjugation occurs via Schiff base
formation between free amino groups of Lys and oxidized mannan.
Reduced mannan-peptide complexes were prepared by adding 1 mg
sodium borohydride to each mixture for 6-8 h at room temperature in
the dark. The cyclic peptide-mannan binding was achieved through a
Lys residue following same protocol as above.
Mannan Conjugated Peptide Vaccinations
[0192] Groups of female animals (age range 6-10 week old C57BL/6 or
SJL/J mice and 12-15 week old Lewis rats) were immunized
intra-dermally with 100 .mu.l solution containing 30 .mu.g peptide
(MOG.sub.35-55) or 50 .mu.g PLP.sub.139-151 peptide for mice and 30
.mu.g MBP.sub.83-99 for Lewis rats) conjugated to 70 .mu.g oxidized
or reduced mannan diluted in bi-carbonate buffer, pH 9.0. As
controls, age matched groups of animals were vaccinated with
oxidized or reduced mannan alone, saline or 30-50 .mu.g of peptide
alone. Three consecutive vaccinations were performed spaced 15 days
apart. Fifteen days after the last vaccination EAE was induced.
Induction-Inhibition and Assessment of MOG.sub.35-55-EAE in C57BL/6
Mice
[0193] EAE was induced in C57BL/6 mice by subcutaneous (s.c) tail
base injection of 50 .mu.g of MOG.sub.35-55 emulsified in Complete
Freund's Adjuvant (CFA) supplemented with 400 .mu.g of H37Ra M.
tuberculosis (Difco). Mice also received an intraperitoneal (i.p)
injection of 200 ng of pertussis toxin (Sigma-Aldrich, Greece) on
days 0 and 2. Mice were assessed daily for clinical signs according
to the following scale: 0, normal; 1, limp tail; 2, hind limb
weakness; 3, hind limb paralysis; 4, forelimb paralysis; and 5,
moribund or death (0.5 gradations represent intermediate scores).
Weights of mice were also monitored daily. Mice were allowed free
access to food and water throughout the experiment.
Induction-Inhibition and Assessment of PLP.sub.139-151-EAE in SJL/J
Mice
[0194] EAE was induced in SJL/J mice using a standard protocol. 150
.mu.g of PLP.sub.139-151 was dissolved in PBS and emulsified in an
equal volume of CFA (containing 1 mg/ml of heat killed
Mycobacterium Tuberculosis HR37a, Sigma). On Day 0 (day of disease
induction), one s.c injection was given by injecting 150 .mu.g
PLP.sub.139-151/200 .mu.l per mouse in both of the flanks. Each
mouse also received 400 ng pertussis toxin (Sigma, P2980) (i.p)
dissolved in 200 .mu.l PBS. On day 2, mice were received a booster
of pertussis toxin (200 ng pertussis toxin dissolved in 200 .mu.l
PBS per mouse) Animals were daily evaluated for clinical signs of
disease, starting from day 1 post immunisation, using a 6-grade
clinical scale: 0, normal animal; 0.5, weight loss; 1, inability to
elevate the tail above the horizontal level, tail weakness; 2, tail
paralysis; 3, tail paralysis/hind limb paresis; 4, hind limb
paralysis/forelimb weakness; 5, quadriplegia/moribund; 6, death
from EAE. Moreover, animals were daily weighted throughout the
entire experimental period.
Induction and Assessment of MBP.sub.74-85-EAE in Lewis Rats
[0195] EAE was induced in female Lewis rats by s.c. injection in
hind foot pads of 30 .mu.g MPB.sub.74-85 peptide emulsified in CFA
supplemented with 400 .mu.g of H37Ra M. tuberculosis (Difco). Rats
were assessed daily for clinical signs according to the following
scale: 0, normal; 1, tail paralysis; 2, hind limb weakness; 3, hind
limb paralysis; 4, forelimb paralysis; and 5, moribund or dead (0.5
gradations represent intermediate scores). Weights of the animals
were also daily monitored. Rats were allowed free access to food
and water throughout the experiment.
T Cell Priming and Proliferation Assays
[0196] Isolated splenocytes were cultured for 72 h in 96-well
plates in RPMI 1640 (Invitrogen Life Technologies, Gaithersburg,
Md.) containing 10% heat-inactivated FCS, 50 .mu.M 2-ME, and
increasing concentrations of MOG.sub.35-55 peptide. Cells were
stimulated in triplicate at 2.times.10.sup.6 cells/ml in
round-bottom, 96-well plates (Costar). Cells were pulsed with 1
.mu.Ci/5.times.10.sup.5 cells [.sup.3H]-thymidine in triplicate
(Amersham Radiochemicals) for the last 16 h of culture.
[.sup.3H]-thymidine incorporation was measured by liquid
scintillation counting (Wallac, Turku, Finland). Results are
expressed as the stimulation index (ratio between radioactivity
counts of cells cultured in presence of peptide and cells cultured
with media alone).
Measurement of Cytokines
[0197] The mouse Th1/Th2 cytokine cytometric bead array kit (BD
Biosciences) was used to measure cytokine levels in culture
supernatants according to the manufacturer's instructions. The
cytokines measured with this kit were: IL-2, IL-4, IL-5,
IFN-.gamma. and TNF-.alpha.. In addition a mouse IL-17 ELISA set
(R&D Systems, Germany) was used to measure cytokine secretion
from splenocyte cell culture supernatants according to
manufacturer's instructions. The sensitivity of the assay for
different cytokines was as follows: IL-2, IL-4 and IL-5=5.0 pg/ml;
IFN-.gamma.=2.5 pg/ml; TNF-.alpha.=6.3 pg/ml.
Histopathological Analysis
[0198] Mice were transcardially perfused with ice-cold 4%
paraformaldehyde in PBS under deep anesthesia. CNS tissues were
postfixed in the same fixative for 3 h at 4.degree. C. and
processed for standard histopathological analysis. Inflammation was
visualized by staining with H&E, whereas demyelination was
demonstrated by a Luxol Fast Blue/periodic acid-chiff stain.
Quantification of inflammation and demyelination was done in a
blinded manner. Inflammation in the spinal cord was determined by
absolute true quantification; the numbers mean inflammatory
infiltrates/mm.sup.2 of tissue. In the brain a semiquantitative
scoring was used according to which 0.5 means single perivascular
infiltrates; 1 means multiple inflammatory infiltrates.
Demyelination was scored as follows: 0.5: single perivascular
sleeves of demyelination, 1: ubiquitous perivascular or subpial
demyelination, 2: confluent demyelinated plaques, 3: profound focal
demyelination, involving about 1/2 of the spinal cord white matter
at least in one spinal cord segment, 4: extensive demyelination,
for instance complete demyelination of spinal cord white matter at
least in one segment of the spinal cord.
Results and Discussion
[0199] Vaccination with Oxidized/Reduced Mannan-MOG.sub.35-55
Peptide Conjugates Protect C57BL/6 Mice from MOG.sub.35-55-EAE
[0200] Triplicate vaccination of C57BL/6 mice with oxidized/reduced
mannan-MOG.sub.35-55 peptide conjugates allowed their significant
protection from MOG.sub.35-55-EAE development that was induced 15
days following the last vaccination (FIG. 1). MOG.sub.35-55 peptide
conjugated to oxidized mannan showed the greatest degree of
protection as judged from the lowering of clinical score (FIG. 1)
and the absence of wasting symptoms usually accompanying disease
onset and progression as daily weight-measurement of experimental
animals showed (FIG. 2). MOG.sub.35-55 peptide conjugated to
reduced mannan showed significant inhibition from disease
manifestation as well. In order to exclude the possibility that
there was an induction of tolerance to peptide due to repeated
stimulation with it, we performed an equivalent experiment in which
mice received triplicate vaccinations with unconjugated
MOG.sub.35-55 peptide spaced 15 days apart before MOG.sub.35-55-EAE
induction. Results showed that mice receiving three prior
challenges with peptide alone were still susceptible to MOG-EAE and
showed clinical signs of disease but characteristically: 1) EAE
initiation was delayed in MOG.sub.35-55 vaccinated animals by 3
days, 2) EAE was significantly lower in MOG.sub.35-55 vaccinated
animals compared to non vaccinated controls at initial stages of
experimental disease and 3) disease was exacerbated in
MOG.sub.35-55 vaccinated animals after day 20 of disease
progression (FIGS. 3 and 4).
Absence of Inflammatory Infiltrates and Demyelinating Lesions in
Mice Immunized with Oxidized/Reduced Mannan-MOG.sub.35-55
Conjugates Prior to EAE Induction
[0201] Animals were sacrificed 24 days following EAE induction.
Spinal cords and brains were examined for the presence of
inflammation and demyelination. Evaluation of the extent of
lymphocyte infiltration and demyelination (FIG. 5) shows mice that
had not received any vaccination had substantial amount of
mononuclear cells infiltrating the spinal cord accompanied by
extensive demyelination. In contrast, oxidized mannan MOG.sub.35-55
vaccinated mice showed little inflammatory infiltration and no
demyelinating lesions in the spinal cord. Intermediate protective
effects were seen in reduced mannan MOG.sub.35-55 vaccinated mice,
MOG.sub.35-55 peptide and mannan alone were used as controls.
[0202] In a detailed examination of the demyelination and
inflammation scores for each individual mouse (FIG. 6) the marked
reduction in the inflammatory and demyelination index for the
oxidized mannan MOG.sub.35-55 treated mice is observed for both
brain and spinal cord. This analysis provides strong
pathophysiological evidence in support of a therapeutic nature of
the vaccination with the MOG.sub.35-55 mannan conjugates.
[0203] Decreased Proliferative T Cell Responses in Mice Immunized
with Oxidized/Reduced Mannan MOG.sub.35-55 Peptide Conjugates
Following MOG.sub.35-55-EAE Induction
[0204] Splenocytes were isolated from animals on day 25 post
MOG.sub.35-55-EAE induction. Single cell suspensions were ex vivo
stimulated in triplicate with increasing concentrations of
MOG.sub.35-55 peptide. Proliferation was assessed by
[.sup.3H]-thymidine incorporation. Significant reduced
proliferation was noted in spleens from mice that were
pre-immunized with oxidized/reduced mannan-MOG.sub.35-55 peptide
conjugates (FIG. 7), compared to mice that had received oxidized
mannan alone, reduced mannan alone or control (PBS), prior to
MOG.sub.35-55-EAE induction (FIG. 7). At 100 .mu.g/ml MOG.sub.35-55
recall peptide, T cell proliferation peaked with a stimulation
index of 7 compared to a stimulation index of 3 (in mannan-peptide
immunized group) (FIG. 7). Likewise, immunization with
MOG.sub.35-55 peptide alone or control mice (PBS) did not show a
reduction in T cell proliferative responses (FIG. 8). The results
indicate that the presence of oxidized or reduced mannan in the
peptide conjugates is able to tolerize (downregulate) T cell
responses to self peptides.
Cytokine Release from Spleens of Vaccinated Mice
[0205] Splenocytes were isolated from C57BL/6 mice on day 25 post
MOG.sub.35-55-EAE induction. Single cell suspensions were
stimulated ex vivo in triplicate wells with 10 .mu.g/ml
MOG.sub.35-55 peptide. Supernatants were collected and subjected to
a bead-based flow cytometric assay for measurement of Th1/Th2
cytokines. Results shown are for IFN-.gamma., TNF-.alpha. and IL-2.
Since IL-17 production has also been implicated in the effector
phase of EAE and has recently been shown to play important role in
disease development and progression [Sutton, et al., 2006;
Komiyama, et al., 2006], we also measured IL-17 production using a
commercially available ELISA kit. Overall protective vaccination
with reduced or oxidized mannan-MOG.sub.35-55 peptide conjugates
induced reduction in the levels of Th1 cytokines and in the amount
of IL-17 secreted by the MOG.sub.35-55 specific T cells. The
results that reached a level of significance with p values <0.05
are highlighted in FIG. 9.
Vaccination with Oxidized/Reduced Mannan-PLP.sub.139-151 Peptide
Conjugates Protect SJL/J Mice Against PLP.sub.139-151-EAE
[0206] Female mice were injected with oxidized/reduced mannan alone
or oxidized/reduced mannan-PLP.sub.139-151 conjugates, with three
consecutive immunizations. On day 24 mice were challenge with EAE,
in order to test the protective efficacy of oxidized/reduced mannan
conjugated peptides. Mice vaccinated with reduced mannan alone or
control mice (non immunized) induced severe EAE clinical signs with
clinical score up to, 4, Mice immunized with oxidized mannan
induced less severe EAE with the peak clinical score of 2.5 (FIG.
10). Interestingly, both of the groups of mice immunized with
oxidized or reduced mannan-PLP.sub.139-151 conjugates did not
induce EAE (clinical score 0.5, weight loss), thus indicating that
the use of mannan conjugates with the encepahalitogenic
PLP.sub.139-151 peptide could protect mice from inducing EAE (FIG.
10).
Vaccination with Oxidized/Reduced Mannan-MBP.sub.83-99 Peptide
Conjugates Inhibits MBP.sub.74-85-EAE in Lewis Rats
[0207] Lewis rats were used as an alternative species to test the
therapeutic efficacy of oxidized/reduced mannan conjugated peptides
in the treatment of EAE. For this series of experiments Lewis rats
received three consecutive vaccinations with mannan in reduced or
oxidized form conjugated to MPB.sub.83-99 peptide spaced 15 days
apart, including the necessary control groups that received mannan
alone in the reduced or oxidized form. Induction of EAE in Lewis
rats was done using the MBP.sub.74-85 peptide. Clinical scoring
(FIG. 11) of the experimental animals and weight monitoring (FIG.
12) showed similar protective findings in that, vaccination with
oxidized/reduced mannan MBP.sub.83-99 conjugates protected Lewis
rats from EAE, delaying disease onset by 5 days and lowering the
mean clinical severity of disease.
[0208] Various modifications and variations of the described
aspects of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
of carrying out the invention which are obvious to those skilled in
the relevant fields are intended to be within the scope of the
following claims.
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Sequence CWU 1
1
26117PRTHomo sapiensMISC_FEATUREPeptide can be linear or cyclic
1Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr 1
5 10 15 Pro 221PRTArtificial SequenceSynthetic peptide 2Met Glu Val
Gly Trp Tyr Arg Xaa Pro Phe Ser Arg Val Val His Leu 1 5 10 15 Tyr
Arg Asn Gly Lys 20 313PRTArtificial SequenceSynthetic peptide 3His
Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe 1 5 10 425PRTHomo
sapiens 4Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val
Thr Pro 1 5 10 15 Arg Thr Pro Pro Pro Ser Gln Gly Lys 20 25
517PRTArtificial SequenceSynthetic peptide 5Glu Asn Pro Val Val His
Phe Phe Xaa Asn Ile Val Thr Xaa Arg Thr 1 5 10 15 Pro 613PRTHomo
sapiens 6His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe 1 5 10
717PRTArtificial SequenceSynthetic peptide 7Glu Asn Pro Val Val His
Phe Phe Xaa Asn Ile Val Thr Xaa Arg Thr 1 5 10 15 Pro
813PRTArtificial SequenceSynthetic peptide 8Val His Phe Phe Xaa Asn
Ile Val Thr Xaa Arg Thr Pro 1 5 10 927PRTArtificial
SequenceSynthetic peptide 9Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly
Glu Asn Pro Val Val His 1 5 10 15 Phe Phe Lys Asn Ile Val Thr Pro
Arg Thr Pro 20 25 1023PRTArtificial SequenceSynthetic peptide 10Lys
Gly Lys Gly Lys Gly Lys Gly Lys Gly His Ser Leu Gly Lys Trp 1 5 10
15 Leu Gly His Pro Asp Lys Phe 20 1131PRTArtificial
SequenceSynthetic peptide 11Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly
Met Glu Val Gly Trp Tyr 1 5 10 15 Arg Xaa Pro Phe Ser Arg Val Val
His Leu Tyr Arg Asn Gly Lys 20 25 30 1217PRTArtificial
SequenceSynthetic peptide; [R91,A96]MBP83-99 12Glu Asn Pro Val Val
His Phe Phe Arg Asn Ile Val Thr Ala Arg Thr 1 5 10 15 Pro
1317PRTArtificial SequenceSynthetic peptide; [A91,A96]MBP83-99
13Glu Asn Pro Val Val His Phe Phe Ala Asn Ile Val Thr Ala Arg Thr 1
5 10 15 Pro 1417PRTArtificial SequenceSynthetic peptide 14Glu Asn
Pro Val Val His Phe Phe Xaa Asn Ile Val Thr Xaa Arg Thr 1 5 10 15
Pro 1517PRTArtificial SequenceSynthetic peptide; [E91]MBP83-99
15Glu Asn Pro Val Val His Phe Phe Glu Asn Ile Val Thr Pro Arg Thr 1
5 10 15 Pro 1617PRTArtificial SequenceSynthetic peptide;
[F91]MBP83-99 16Glu Asn Pro Val Val His Phe Phe Phe Asn Ile Val Thr
Pro Arg Thr 1 5 10 15 Pro 1717PRTArtificial SequenceSynthetic
peptide; [Y91]MBP83-99 17Glu Asn Pro Val Val His Phe Phe Tyr Asn
Ile Val Thr Pro Arg Thr 1 5 10 15 Pro 1817PRTArtificial
SequenceSynthetic peptide; [A91]MBP83-99 18Glu Asn Pro Val Val His
Phe Phe Ala Asn Ile Val Thr Pro Arg Thr 1 5 10 15 Pro
1917PRTArtificial SequenceSynthetic peptide;
cyclo(83-99)[R91]MBP83-99 19Glu Asn Pro Val Val His Phe Phe Arg Asn
Ile Val Thr Pro Arg Thr 1 5 10 15 Pro 2017PRTArtificial
SequenceSynthetic peptide; cyclo(83-99)[F91, A96]MBP83-99 20Glu Asn
Pro Val Val His Phe Phe Phe Asn Ile Val Thr Ala Arg Thr 1 5 10 15
Pro 2117PRTArtificial SequenceSynthetic peptide; cyclo(83-99)[Y91,
A96]MBP83-99 21Glu Asn Pro Val Val His Phe Phe Tyr Asn Ile Val Thr
Ala Arg Thr 1 5 10 15 Pro 2217PRTArtificial SequenceSynthetic
peptide; cyclo(83-99)[S91, A96]MBP83-99 22Glu Asn Pro Val Val His
Phe Phe Ser Asn Ile Val Thr Ala Arg Thr 1 5 10 15 Pro
2313PRTArtificial SequenceSynthetic peptide 23Val His Phe Phe Lys
Asn Ile Val Thr Pro Arg Thr Pro 1 5 10 2413PRTArtificial
SequenceSynthetic peptide 24Val His Phe Phe Ala Asn Ile Val Thr Ala
Arg Thr Pro 1 5 10 2517PRTArtificial SequenceSynthetic peptide;
Cyclo(91-99)[Ala96] MBP83-99 25Glu Asn Pro Val Val His Phe Phe Lys
Asn Ile Val Thr Ala Arg Thr 1 5 10 15 Pro 2613PRTArtificial
SequenceSynthetic peptide; [R91, A96]MBP87-99 26Val His Phe Phe Arg
Asn Ile Val Thr Ala Arg Thr Pro 1 5 10
* * * * *