U.S. patent application number 12/092724 was filed with the patent office on 2009-09-10 for compound comprising an autoantigenic peptide and a carrier with a mhc binding motif.
This patent application is currently assigned to Ahlstrom Corporation. Invention is credited to Balik Dzhambazov, Rikard Holmdahl, Jan Kihlberg, Mikael Vestberg.
Application Number | 20090227516 12/092724 |
Document ID | / |
Family ID | 38048898 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090227516 |
Kind Code |
A1 |
Holmdahl; Rikard ; et
al. |
September 10, 2009 |
COMPOUND COMPRISING AN AUTOANTIGENIC PEPTIDE AND A CARRIER WITH A
MHC BINDING MOTIF
Abstract
The invention relates a compound comprising (a) a peptide and
(b) a carrier, wherein said peptide having at least the motif
X-X-X-X-X-X-X, wherein at least one amino acid residue X is
glycosylated, said peptide being linked to the peptide binding
protein and said carrier comprises at least a MHC binding motif
being linked to said peptide as well as pharmaceutical compositions
comprising said compound and the use of said compound or
pharmaceutical composition for the treatment of a disease, such as
an inflammatory joint disease. The subject matter of the
application is exemplified with peptides derived from type II
collagen such as peptides having at least the sequence AGFKGEA, or
IAGFKGEQPKG, or the peptide AAAKAAA. Preferably a hydroxylysine in
the peptides are glycosylated.
Inventors: |
Holmdahl; Rikard; (Lund,
SE) ; Kihlberg; Jan; (Pixbo, SE) ; Dzhambazov;
Balik; (Plovdiv, BG) ; Vestberg; Mikael;
(Sodra Sandby, SE) |
Correspondence
Address: |
MANELLI DENISON & SELTER
2000 M STREET NW SUITE 700
WASHINGTON
DC
20036-3307
US
|
Assignee: |
Ahlstrom Corporation
Helsinki
FI
Centre NAtional De La Recherche Scientifique
Paris
FR
Univertise Claude Bernard
Vileurbanne
FR
|
Family ID: |
38048898 |
Appl. No.: |
12/092724 |
Filed: |
November 15, 2006 |
PCT Filed: |
November 15, 2006 |
PCT NO: |
PCT/SE06/01290 |
371 Date: |
May 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60758481 |
Jan 12, 2006 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
530/328; 530/329 |
Current CPC
Class: |
A61K 47/6425 20170801;
C07K 16/18 20130101; C07K 2319/33 20130101; A61P 19/02 20180101;
A61K 2039/6056 20130101; C07K 14/705 20130101; A61K 2039/605
20130101; A61K 47/6811 20170801; A61K 47/6843 20170801; C07K
14/4713 20130101; A61K 39/0008 20130101; C07K 7/08 20130101 |
Class at
Publication: |
514/15 ; 530/329;
530/328; 514/16 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 17/00 20060101 C07K017/00; A61P 19/02 20060101
A61P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2005 |
SE |
0502530-9 |
Claims
1. A compound comprising (a) a peptide and (b) a carrier, wherein
a. said peptide having at least the motif X-X-X-X-X-X-X, wherein at
least one amino acid residue X is glycosylated, said peptide being
linked to the peptide binding protein and b. said carrier comprises
at least a MHC binding motif being linked to said peptide.
2. The compound according to claim 1, wherein at least one amino
acid residue X of said peptide (a) is O-linked glycosylated.
3. The compound according to claim 2, wherein the glycosylated
amino acid reside X of said peptide (a) is hydroxylysine.
4. The compound according to claim 3, wherein said peptide (a)
comprises 4 to 50 amino acid residues.
5. The compound according to claim 4, wherein said peptide (a)
comprises X-F-K-X-X-X-X-X-X-X, wherein K is hydroxylysine.
6. The compound according to claim 5, wherein said peptide (a)
comprises X-F-K-X-E-X-X-X-X-X, wherein K is hydroxylysine.
7. The compound according to claim 6, wherein at least one amino
acid residue has been substituted.
8. The compound according to claim 1, wherein said carrier
comprises the variable region of an MHC class II molecule selected
from the group consisting of DRB 1*0401/DRA, DRBI*0402/DRA,
DRBI*0403/DRA, DRBI*0404/DRA, DRBI*0405/DRA, DRB I*0406/DRA,
DRBI*0407/DRA, DRBI*0408/DRA, DRBI*0409/DRA, DRBI*0410/DRA,
DRBI*0101/DRA, DRBI*0102/DRA and DRBI*1001/DRA, DRBI*1002/DRA.
9. The compound according to claim 8, wherein at least one amino
acid residue of the variable region has been substituted.
10. The compound according to claim 8, wherein said variable region
comprises a peptide having 80% identity to the polypeptide sequence
in its full length shown in SEQ ID NO 1 and a second polypeptide
having 80% identity in its full length to the polypeptide sequence
shown in SEQ ID NO 2.
11. The compound according to claim 10, wherein said variable
region comprises a peptide having 90% identity to the polypeptide
sequence in its full length shown in SEQ ID NO 1 and a second
polypeptide having 90% identity in its full length to the
polypeptide sequence shown in SEQ ID NO 2.
12. The compound according to claim 11, wherein said variable
region comprises a peptide having 95% identity to the polypeptide
sequence in its full length shown in SEQ ID NO 1 and a second
polypeptide having 95% identity in its full length to the
polypeptide sequence shown in SEQ ID NO 2.
13. The compound according to claim 1, wherein said peptide (a) is
selected from the group consisting of A-G-F-K-G-E-A and
A-A-A-K-A-A-A.
14. The compound according to claim 1, wherein said peptide (a) is
at least IAGFKGEQGPKG.
15. The compound according to claim 1, wherein said peptide (a) is
covalently bound to said carrier (b).
16. The compound according to claim 1, wherein said peptide (a) is
hydroxylated.
17. The compound according to claim 1, wherein said carrier
comprises a constant region being linked to said MHC binding
motif.
18. The compound according to claim 17, wherein said constant
region being selected from the group consisting of the constant
region of an MHC class II molecule or an immunoglobulin.
19. The compound according to claim 1, wherein said constant region
comprises a peptide having 80% identity to the polypeptide sequence
in its full length shown in SEQ ID NO 3 and a second polypeptide
having 80% identity in its full length to the polypeptide sequence
shown in SEQ ID NO 4.
20. The compound according to claim 19, wherein said constant
region comprises a peptide having 90% identity to the polypeptide
sequence in its full length shown in SEQ ID NO 3 and a second
polypeptide having 90% identity in its full length to the
polypeptide sequence shown in SEQ ID NO 4.
21. The compound according to claim 20, wherein said constant
region comprises a peptide having 95% identity to the polypeptide
sequence in its full length shown in SEQ ID NO 3 and a second
polypeptide having 95% identity in its full length to the
polypeptide sequence shown in SEQ ID NO 4.
22. The compound according to claim 1, wherein said constant region
of said carrier comprises at least 25% of the polypeptide sequence
shown in SEQ ID NO 3 and 4.
23. The compound according to claim 22, wherein said constant
region of said carrier comprises at least 30% of the polypeptide
sequences shown in SEQ ID NO 3 and 4.
24. A pharmaceutical composition comprising a compound comprising a
peptide having at least the motif X-X-X-X-X-X-X, wherein at least
one amino acid residue X is glycosylated, wherein said peptide
being linked to the peptide binding protein, and a carrier
comprising at least a MHC binding motif being linked to said
peptide; and a pharmaceutically acceptable carrier, excipient or
diluter.
25. A method of using a pharmaceutical composition for the
treatment of a disease or disorder wherein the pharmaceutical
composition comprising a compound comprising a peptide having at
least the motif X-X-X-X-X-X-X, wherein at least one amino acid
residue X is glycosylated, wherein said peptide being linked to the
peptide binding protein, and a carrier comprising at least a MHC
binding motif being linked to said peptide; and a pharmaceutically
acceptable carrier, excipient or diluter.
26. Method according to claim 25 for the treatment of a disease or
disorder selected from the group consisting of arthritis, reumatoid
arthritis, ankylosing spondylitis, psoriasis arthritis,
osteoarthritis, relapsing polychondritis and Menieres disease.
Description
FIELD OF INVENTION
[0001] The invention relates a compound comprising (a) a peptide
and (b) a carrier, wherein said peptide having at least the motif
X-X-X-X-X-X-X, wherein at least one amino acid residue X is
glycosylated, said peptide being linked to the peptide binding
protein and said carrier comprises at least a MHC binding motif
being linked to said peptide as well as pharmaceutical compositions
comprising said compound and the use of said compound or
pharmaceutical composition for the treatment of a disease, such as
an inflammatory joint disease.
BACKGROUND OF INVENTION
[0002] There is an increasing population of humans suffering from
different kinds of inflammatory joint diseases. Diseases, which
sometimes are impossible to cure, where the treatment is lifelong
and where the symptoms often become worse during the years. So far
the focus of the treatment has been on trying to find compounds,
which reduce the symptoms but not cure the disease or make the
disease decline.
[0003] One example of such a disease is rheumatoid arthritis (RA),
which is characterized by chronic inflammation of the articular
synovial tissues initiated by leukocyte infiltration (mainly
neutrophils, macrophages and T cells) and secretion of inflammatory
cytokines (TNF-alpha, IFN-gamma, IL-1, IL-6), chemokines and
destructive enzymes such as matrix metalloproteases. Activation of
T cells is believed to be an important pathogenic factor in the
disease although its exact role and potential as a therapeutic
target has not yet been identified. The abnormal activation of T
cells do, however, most likely occur years before the clinical
diagnosis of the disease as T cell dependent IgG antibodies
specific for immunoglobulin Fc (i.e rheumatoid factors) and
citrullinated protein epitopes are highly predictive for disease
(1, 2). Importantly, the risk for developing arthritis is
dramatically increased in individuals who have both such antibodies
and express certain MHC class II molecules, that share a specific
peptide pocket, the so called MHC shared epitope (3, 4). The MHC
class II region is also the strongest known genetic factor
associated with RA. Taken together, these findings argue for a
pathogenic role of MHC class II restricted autoreactive T cells. It
has however been difficult to identify a single specificity of such
T cells although T cell reactivity to several autoantigens, such as
BiP, RA33 and GPI and also joint specific antigens such as type II
collagen (CII), have been reported (5-8).
[0004] Since there is no way to cure inflammatory joint diseases
today there is a need for developing a way to cure the disease.
SUMMARY OF THE INVENTION
[0005] The object of the present invention is to solve the above,
discussed problem in connection with inflammatory joint
diseases.
[0006] It has surprisingly been found that by the use of a compound
comprising (a) a peptide and (b) a carrier, wherein said peptide
having at least the motif X-X-X-X-X-X-X, wherein at least one amino
acid residue X is glycosylated, said peptide being linked to the
peptide binding protein and said carrier comprises at least a MHC
binding motif being linked to said peptide it is possible for the
first time to reduce and/or eliminate an inflammatory joint
disease. Thereby a mammal suffering from such a disease will bee
cured or at least the disease will be reduced.
[0007] Additionally the invention relates to a pharmaceutical
composition comprising said compound as well as the use of said
compound for the treatment of an inflammatory disease or disorder,
such as arthritis, rheumatoid arthritis, ankylosing spondylitis,
psoriasis arthritis, osteoarthritis, relapsing polychondritis and
Menieres disease
[0008] In a specific embodiment, said compound is used to vaccinate
a mammal and thereby cure or prevent an inflammatory disease or
disorder.
[0009] Further advantages and objects with the present invention
will be described in more detail, inter alia with reference to the
accompanying drawings.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows peptide/A.sup.q complexes activate
antigen-specific T-cell hybridomas.
(A) HCQ.3 hybridoma, specific for GalOK264 CII1259-273 epitope; (B)
HCQ.4 hybridoma, specific for non-modified (1264) CII259-273
epitope.
[0011] FIG. 2 shows a criss-cross test of T-cell hybridoma
specificities for peptide/A.sup.q complexes. (A) HCQ.3 hybridoma,
specific for GalOK264 CII259-273 epitope; (B) HCQ.4 hybridoma,
specific for non-modified (1264) CII259-273 epitope.
[0012] FIG. 3 shows that GalOK264/Aq complexes suppress development
of CIA. (A) Incidence of arthritis (percent of affected mice); (B)
Mean clinical score of arthritis severity including both arthritic
and healthy mice; (C) Anti-CII IgG serum levels. All data represent
mean.+-.SE of 10 mice per group. *, p<0.05; **, p<0.01 and
***, p<0.001.
[0013] FIG. 4 shows that GaIOK264/A.sup.q complexes reduce
arthritis progression in chronic stage. (A) Mean arthritis score
for 202 days of the chronic mice chosen for treatment; (B) Mean
clinical score of GalOK264/A.sup.q treated mice after
reimmunisation; (C) Anti-CII IgG serum levels.
[0014] FIG. 5 shows transfer of T cells from GalOK264/A.sup.q
treated mice provided protection against CA development. (A) Mean
clinical score of arthritis after T-cell transfer; (B) Anti-CII IgG
serum levels.
[0015] FIG. 6 shows that GalOK264/A.sup.q treatment blocked
arthritis progression in 5H2q/r F1 mice.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0016] In the context of the present application and invention the
following definitions apply:
[0017] The term "MHC class II molecule" is intended to mean a
protein consisting of an alpha and a beta chain, coded from two
distinct genes. This protein is normally cell surface bound on
so-called antigen presenting cells (APC) and serve as a receptor
for peptides. The peptide is bound to a specific site, the peptide
binding site, in the MHC molecule and the resulting protein
structure (the peptide bound to the two chain MHC class II
molecule) is recognized by the T cell receptor. This interaction is
the crucial molecular and antigen specific event in the immune
response and has been well described.
The term "MHC binding motif" is intended to mean the amino acids of
the variable region, (i.e. polymorphic) of an MHC class II
molecule, which can contact and bind the peptide of the invention
in the peptide-binding site. The variable region of an MHC class II
molecule being defined as the first amino acid residues 1-90 of the
alpha and the beta chain in the form seen on the cell surface.
[0018] The term "variable region on the MHC molecule" is intended
to mean the first domain of both alpha and beta chains each
encompassing amino acids 1-90.
[0019] The term "constant region on a MHC or Ig molecule" is
intended to mean the part of the MHC class II molecule that is not
part of the variable domains/regions.
[0020] The term "carrier" is intended to mean a compound, such as a
protein, which can bind to the peptide of the invention and present
that particular peptide to specific T cell receptors. T cell
receptors (TCR), which normally are bound to T cells normally
recognising type II collagen, a joint cartilage protein.
[0021] The term "peptide" is intended to mean a sequence of amino
acid residues having from six to 50 amino acid residues.
[0022] The term "polypeptide" is intended to mean a sequence of
amino acid residues having more 51 or more amino acid residues.
[0023] In the present context, amino acid names and atom names are
used as defined the Protein DataBank (PDB) (www.pdb.or), which is
based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism
for Amino Acids and Pep-tides (residue names, atom names etc.), Eur
J Biochem., 138, 9-37 (1984) together with their corrections in Eur
J Biochem., 152, 1 (1985). The term "amino acid" is intended to
indicate an amino acid from the group consisting of alanine (Ala or
A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid
(Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine
(His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu
or L), methionine (Met or M), asparagine (Asn or N), proline (Pro
or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or
S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W)
and tyrosine (Tyr or Y), or derivatives thereof.
DESCRIPTION
Compound
[0024] The invention relates to a compound, comprising a peptide
and a carrier.
[0025] Said peptide having at least the motif X-X-X-X-X-X-X,
wherein at least one amino acid residue X is glycosylated, such as
an O-linked galactose. Other examples of the glycosylated
structures are N-acetyl galactosamine, glucose, N-acetyl
glucosamine, glucose, mannose, fucose, as well as their mono- and
dideoxygenated, mono- and difluorogenated, and C-glycoside
derivatives. Said peptide being linked to the carrier and the
linking between said peptide and said peptide binding protein may
be covalent or they may be bound in another way as long as they can
bind and remain bound to each other. The glycosylated amino acid
residue X of said peptide may be hydroxylysine or a variant
thereof. A variant of the lysine side chain could be any structure
that is acceptable for recognition by a T cell receptor, such as
hydroxynorvaline. Said peptide may comprise from 4 to 50 amino acid
residues, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 amino acid residues. Said peptide may comprise at
least one of the following amino acid sequences
X-F-K-X-X-X-X-X-X-X, X-F-K-X-E-X-X-X-X-X, A-G-F-I-G-E-A or
A-A-A-K-A-A-A wherein K may be hydroxylysine. Additionally, said
peptide also have a MHC class II binding motif, i.e., have the
ability to bind to such a molecule. A MHC class II binding motif is
capable of binding to a three-dimensional structure formed by the
alpha and the beta chains of the first domain of the MHC class II
molecule. The amino acids of critical importance for this peptide
binding structure are both located in a beta pleated sheath forming
the bottom of the peptide binding structure (alpha 1-49, beta 1-49)
and two alpha helices (alpha50-80, beta 50-90) forming the sides of
these cleft-forming structure.
[0026] Peptides forming specific motifs unique for each MHC class
II allele binds to this peptide binding structure. MHC class II
alleles associated with rheumatoid arthritis forms peptide binding
structure with many similarities encompassing them to bind peptides
with certain binding motifs. One example of a peptide, according to
the invention, is derived from collagen II, positions 260-273
(IAGFKGEQGPKGEP) which binds the DRB1*0401/DRA, the DRB1*1001/DRA
and the DRB1*0101/DRA molecules. However, the peptide may be
synthetic or semisynthetic or derived from other proteins as long
as the peptide is identical or have similar structure as the
peptide above. The MHC binding positions for binding to the
DRB1*0401/DRA molecule has been hypothesised to be F263 and E266 in
this peptide and the TCR binding positions K264, Q267 and K270.
These TCR contacting positions could be of different importance for
different TCR, i.e., they could be substituted into other amino
acid residues.
[0027] Additionally, the above, mentioned peptide (IAGFKGEQGPKGEP)
may have one or more modifications such as hydroxylation,
galactosylation or galactoglucosylation. Examples of positions,
which could be modified, are positions K264 and K270 as well as
deamidation of position Q267. Accordingly one or more of the amino
acid residues may be substituted with another amino acid residue as
long as it have the capability to be part of the MHC binding motif
and function as a composition which can be used to prevent/reduce
or treat a mammal suffering from a joint inflammatory disease or
disorder or induce an immune response and thereby function as a
vaccine. Examples of mammals includes human, dog, pig, sheep, cat,
camel and horse.
[0028] The carrier of the compound to which the peptide is linked
comprises at least the variable region of the MHC class II
molecule, being defined as the first domains of the alpha and the
beta chain, i.e., the first amino acid residues 1-90 of the alpha
and the beta chain and a constant region. The alpha and the beta
chains being linked to each other, such as by a leucine zipper
domain.
The carrier comprises at least at least a MHC binding motif being
linked to said peptide. However, one or more amino acid residues
may be substituted as long as the carrier can bind the peptide and
induce an immune response in a mammal. The variable part may be
modified by up to 30 amino acid residues, such as 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 amino acid residues. Examples of
variable regions are those, which originate form MHC class II
molecules DRB1*0401/DRA, DRB1*0402/DRA, DRB1*0403/DRA,
DRB1*0404/DRA, DRB1*0405/DRA, DRB1*0406/DRA, DRB1*0407/DRA,
DRB1*0408/DRA, DRB1*0409/DRA, DRB1*0410/DRA, DRB1*0101/DRA,
DRB1*0102/DRA, DRB1*1001/DRA, DRB1*1002/DRA. Example sequences of
one of the sequences to be used are depicted; SEQ NO 1, DRA V
domain and SEQ NO 2, DRB1*0401 V domain.
[0029] According to one embodiment the compound comprises the above
identified peptide and the carrier comprises a variable and a
constant region, wherein said variable region comprises a peptide
having 80% identity to the polypeptide sequence in its full length
shown in SEQ ID NO 1 and a second polypeptide having 80% identity
in its full length to the polypeptide sequence shown in SEQ ID NO
2, such as 85, 90, 95 or 100% identity in its full length to one or
both of the polypeptides shown in SEQ ID NO 1 and 2.
[0030] The carrier may also contain one or more constant regions,
such as the constant regions of the MHC class II molecule or
regions of an immunoglobulin such as the constant regions of IgG.
For example, the class II first domain (i.e the variable or
polymorphic domain) can be covalently linked to the IgG structure
changing the first V domains of the VH and the VL chains. The
constant and the variable regions may be covalently linked to each
other. Said constant region may be selected from the group
consisting of the constant region of an MHC class II molecule or an
immunoglobulin. Example of a constant region being one comprising a
peptide having 80% identity to the polypeptide sequence in its full
length shown in SEQ ID NO 3 and a second polypeptide having 80%
identity in its full length to the polypeptide sequence shown in
SEQ ID NO 4, such as having 90% or 95% identity to the polypeptide
sequence in its full length shown in SEQ ID NO 3 and a second
polypeptide having 90% or 95% identity in its full length to the
polypeptide sequence shown in SEQ ID NO 4 or being identical to the
polypeptide sequence shown in SEQ ID NO 3 and 4.
[0031] In another embodiment, the constant region of the carrier
may be a polypeptide sequence which is at least 25% of the
polypeptide sequences SEQ ID NO 3 and 4, i.e., the constant region
of the MHC class II, such as 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100%. Additionally there may be one or more substitutions of the
polypeptides as long as the polypeptide sequences are linked
together to form a carrier as defined above. The polypeptide
sequences may be covalently linked to each other through a leucine
zipper domain or part of a leucine zipper domain.
TABLE-US-00001 SEQ ID NO1: DR alpha V domain:
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGR
FASFEAQGALANIAVDKANLEIMTKRSNYT SEQ ID NO2: The DR beta V domain
(from DRB1*0401) GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAV
TELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFT SEQ ID NO3: DR alpha
constant domain to be used:
PITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT
GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEF DAPSPLPETTEN SEQ
ID NO4: DR beta (from DRB1*0401) constant domain to be used:
VQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKT
GVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRA RSESAQS
A Pharmaceutical Composition
[0032] According to another aspect the invention relates to a
pharmaceutical composition comprising above defined compound and a
pharmaceutically acceptable carrier, excipient or diluter.
[0033] Pharmaceutical formulations of the compound of the invention
are typically administered in a composition that includes one or
more pharmaceutically acceptable carriers and diluters. Such
pharmaceutical compositions may be prepared in a manner known in
the art that is sufficiently storage stable and suitable for
administration to humans and animals. The pharmaceutical
composition may be lyophilised.
[0034] "Pharmaceutically acceptable" means a carrier, diluent or
excipient that at the dosage and concentrations employed does not
cause any unwanted effects in the patients to whom it is
administered. Such pharmaceutically acceptable carriers or
excipients are well-known in the art (see Remington's
Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack
Publishing Company (1990) and handbook of Pharmaceutical
Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press
(2000).
[0035] The pharmaceutical composition may be admixed with adjuvants
such as lactose, sucrose, starch powder, cellulose esters of
alkanoic acids, stearic acid, talc, magnesium stearate, magnesium
oxide, sodium and calcium salts of phosphoric and sulphuric acids,
acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or
polyvinyl alcohol, and tableted or encapsulated for conventional
administration. Alternatively, they may be dissolved in saline,
water, polyethylene glycol, propylene glycol, ethanol, oils (such
as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth
gum, and/or various buffers. Other adjuvants and modes of
administration are well known in the pharmaceutical art. The
carrier or diluent may include time delay material, such as
glyceryl monostearate or glyceryl distearate alone or with a wax,
or other materials well known in the art.
[0036] The pharmaceutical compositions may be subjected to
conventional pharmaceutical operations such as sterilisation and/or
may contain conventional adjuvants such as preservatives,
stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.,
e.g., as disclosed elsewhere herein.
[0037] The pharmaceutical composition according to the invention
may be administered locally or systemically such as topically,
intravenously, orally, parenterally or as implants and even rectal
use is possible. Suitable solid or liquid pharmaceutal preparation
forms are, for example granules, powders, tablets, coated tablets,
(micro) capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampule form and
also preparations with protracted release of active compounds, in
the preparation of excipients, diluents, adjuvants or carriers are
customarily used as described above.
[0038] The pharmaceutical composition will be administered to a
patient in a pharmaceutically effective dose. By "pharmaceutically
effective dose" is meant a dose that is sufficient to produce the
desired effects in relation to the condition for which it is
administered. The exact dose is dependent on the activity of the
compound, manner of administration, nature and severity of the
disorder, age and body weight of the patient and different doses
may be needed. The administration of the dose can be carried out
both by single administration in the form of an individual dose
unit or else several smaller dose units and also by multiple
administration of subdivided doses at specific intervals, e.g.
vaccination.
[0039] The pharmaceutical composition of the invention may be
administered alone or in combination with other therapeutic agents.
These agents may be incorporated as part of the same pharmaceutical
composition or may be administered separately.
[0040] The "patient" for the purposes of the present invention
includes both humans and other mammals. Thus the methods are
applicable to both human therapy and veterinary applications.
[0041] Pharmaceutical formulations of the nucleotide sequence
molecule or polypeptide of the invention are typically administered
in a composition that includes one or more pharmaceutically
acceptable carriers or excipients. Such pharmaceutical compositions
may be prepared in a manner known in the art that is sufficiently
storage stable and suitable for administration to humans and
animals.
[0042] In a final aspect the above mentioned compound as well as
pharmaceutical composition may be used for the treatment of a
disease or a disorder such as an inflammatory joint disease threw
vaccination. Examples of diseases are arthritis, reumatoid
arthritis, anylosing spondylitis, psoriasis arthritis,
osteoarthritis, relapsing polychondritis and Menieres disease.
The vaccination may be performed by the use of any vaccination
system such as DNA vaccination in which DNA is used that is
translated into proteins in vivo corresponding to the above
described structures. The said DNA can be administered as pure DNA
or inserted into carrier structures.
[0043] Following examples are intended to illustrate, but not to
limit, the invention in any manner, shape, or form, either
explicitly or implicitly.
EXAMPLES
Example 1
Design of the MHC Class II A.sup.q Constructs
[0044] The cDNAs for Aalpha.sup.q and Abeta.sup.q were amplified
from a first strand cDNA reaction (first strand cDNA, Pharmacia,
Piscataway, N.J.). The cDNAs were further modified to include
cloning sites immediately upstream of the start codon, and the 3'
end from the transmembrane domain and downstream was replaced by an
inframe cloning site. Next, DNA for the leucine zipper (13) domain
from Jun including a 3' end coding for 6 histidines was cloned in
frame with the beta-chain cDNA. The DNA for the leucine zipper
domain from Fos was added to the alpha chain construct. The
resulting constructs were cloned separately into pMTAL (Invitrogen,
La Jolla, Calif.) or pRmHa-3 (14) to allow for heavy metal-induced
expression in insect cells. pMTAL contains the resistance gene for
hygromycin. Where pRmHa-3 was used a Copia promoter-driven
hygromycin gene was used as selection marker.
Example 2
Transfection, Expression and Purification of Soluble A.sup.q
[0045] The linearised A.sup.q alpha-chain and A.sup.q beta-chain
constructs were co-transfected at equimolar ratios into Drosophila
melanogaster SL2 cells (ATCC, CRL-1963) using calcium phosphate
transfection. Stable transfectants were derived by hygromycin
selection and kept under selection in Schneider's Drosophila medium
(Gibco.TM., Paisley, Scotland, UK) containing 100 .mu.g/ml of
hygromycin B (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Large-scale cell cultures were prepared in Fernbach bottles using a
magnetic stirrer. For expression of soluble A.sup.q, transfected
cells were grown in serum-free Insect express complete medium (PAA
Laboratories GmbH, Linz, Austria) at 25.degree. C., induced with
0.7 mM CuSO4 for three days, and the supernatants were clarified by
centrifugation and filtration. The SL2 cells produced .about.2-3 mg
of recombinant protein per liter of culture. The expressed soluble
A.sup.q molecules were purified from the clarified media using
Ni-NTA (Qiagen GmbH, Hilden, Germany) affinity chromatography and
the manufacturer's recommended protocol. The dialysed protein
fractions were examined by ELISA, SDS-PAGE and Western blot
analysis. Non-reducing SDS-PAGE analysis of NiNTA purified A.sup.q
on 4-20% gradient gel showed two bands with molecular weights of 29
and 33 kDa (approximately the predicted sizes of alpa and .beta.
beta chains), which demonstrates that the expressed proteins form
heterodimers consisting of alpha and beta chains. Positive
fractions were pooled, concentrated 5- to 10-fold by MICROSEP 30K
OMEGA (PALL, GelmanSciences, Ann Arbor, Mich.) or Amicon.RTM.
centrifugal filter devices (MILLIPORE Co, Billerica, Mass.) and
loaded with a peptide to form MHC-peptide complexes. All protein
concentrations were determined using a Dc protein assay (Bio-Rad
Laboratories, Hercules, Calif.).
Example 3
ELISA, SDS-PAGE and Western blot analyses of A.sup.q
[0046] The alpha- and beta-chains of the purified A.sup.q protein
were detected by sandwich ELISA, using Y3P mAb (specific for the
native alpha-chain) as capturing antibodies and biotinylated
7-16.17 (BD PharMingen, Los Angeles, Calif.) mAb (specific for the
beta-chain) as detecting antibodies. Flat-bottom 96-well plates
(Nunc, Roskilde, Denmark) were coated with 2.5 .mu.g/mL Y3P and
incubated overnight at 4.degree. C. The plates were then washed
with PBS, blocked with 1% BSA (Sigma, St Louis, Mo.) in PBS for 1
h, washed again, and incubated for 2 h with 50 .mu.L from the
protein fractions at room temperature. Plates were washed again,
followed by addition of 1 .mu.g/mL biotinylated 7-16.17 for 1 h.
After washing, the biotin-labeled antibody was detected by
europium-labeled streptavidin using the DELFIA system (Wallac,
Turku, Finland).
[0047] Protein purity was assessed by SDS-PAGE. Samples were
electrophoresed in 4-20% polyacrylamide gradient ready mini-gels
(Bio-Rad Laboratories, Hercules, Calif.) under denaturing and
non-reducing conditions and the gels were silver stained according
to the manufacturer's instructions. In parallel experiments, the
gels were electrotransferred onto nitrocellulose membranes (0.45
.mu.m). The membranes were blocked in 5% non-fat dry milk in PBS
for 1 h and blotted with different MHC class II specific antibodies
(M5/114, 7-16.17, 7-23.1, PCQ.6, 34-5-3, Y3P) at 4.degree. C.
overnight. After repeated washing, blots were incubated with
peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG (for
M5/114) antibodies (Jackson) for 1 h. Immunoblots were developed
using DAB (Vector Laboratories Inc., Burlingame, Calif.).
Example 4
Preparation of Peptide/A.sup.q Complexes
[0048] Empty soluble A.sup.q molecules were loaded with 5 to
50-fold molar excess of GalOK264 CII259-273, non-modified
CII259-273 or MOG79-90 peptides at 4.degree. C. for 72 h. GalOK264
CII259-273 is a peptide from type II collagen (CII) position
259-273 which has a lysine at position 264, which is hydroxylated
and galactosylated.
[0049] Non-modified is the same peptide but with a lysine without
modifications of its side chain. MOG=myelin oligodendrocytic
glycoprotein. MHC-peptide complexes were separated by
anion-exchange HPLC (Resource.TM. Q column) using an AKTA.TM.
explorer 100 Air system (Amersham Pharmacia Biotech AB, Uppsala,
Sweden) with UNICORN V4.00 software. Separations were done with a
loading solution of 10 mM Tris pH 8.5 (buffer A) and a gradient
elution up to 1 M NaCl (buffer B) in 10 mM Tris. The eluted protein
fractions were concentrated by ultrafiltration (MICROSEP 30K
OMEGA), dialyzed against PBS and examined by ELISA, SDS-PAGE and
T-cell hybridoma tests. MHC-peptide complexes were purified further
on a Superdex 200 gel filtration column (Amersham Pharmacia Biotech
AB, Uppsala, Sweden), concentrated again by Amicon.RTM. centrifugal
filter devices (MILLIPORE Co, Billerica, Mass.) and stored at
-20.degree. C. until used.
Example 5
Activation of T Cell Hybridomas
[0050] Peptide/A.sup.q complexes were diluted in sterile PBS and
coated onto plates by incubation at 4.degree. C. for overnight or
added directly in soluble form to the hybridomas. The coated plates
were then washed twice with sterile PBS to remove unbound protein
complexes, and 5.times.10.sup.4 T-hybridoma cells were added per
well in 200 .mu.L of DMEM supplemented with 5% FCS, 100 IU/mL
penicillin and 100 .mu.g/mL streptomycin. T-cell hybridoma HCQ.3
and HCQ.4, specific for GalOK264 and for non-modified CII259-273
(K264), respectively (12), have been used in FIG. 1. To block the
activation of the hybridomas, 5 ug/ml of 7-16.17 antibodies were
added to the immobilised complexes. In the criss-cross test (FIG.
2) five ug/ml of soluble peptide/Aq complexes were added directly
(without coating) to HCQ.3 and HCQ.4 hybridoma cells (5.times.104).
Medium alone (without antigen) was used as a negative control.
[0051] After 24 h, IL-2 in the culture supernatants was measured by
sandwich ELISA using DELFIA system (Wallac, Turku, Finland).
Recombinant mouse IL-2 served as a positive control and standard.
Data are represented as mean.+-.SE of triplicates
Example 6
Induction and Clinical Evaluation of Arthritis
Animals
[0052] Male B10.Q, (B10.QxB10.RIII)F1 or
B10.Q.times.(BALB/cxB10.Q)F2 mice, 8-10 weeks of age, were used in
the experiments. The founders of the B10.Q and B10.RIII mice were
originally provided by Dr. Jan Klein (Tubingen, Germany) and BALB/c
mice purchased from The Jackson Laboratory (Bar Harbor, Me.). The
mice were bred and used at the animal department of Medical
Inflammation Research (http://www.inflam.lu.se) and kept under
standardized conditions.
[0053] In FIG. 3 is used B10.Q mice (10 animals per group), which
were immunised with 100 ug rat CII in CFA on day 0 and boosted on
day 35 with 50 ug rat CII in IFA. On days 20 and 34 (arrows), mice
were treated by intravenous administration of purified peptide/Aq
complexes (100 ug in 200 ul PBS). Sample sera were collected at
days 35 and 70 after immunisation and incubated in serial dilutions
in rat CII-coated wells. Levels of IgG anti-CII antibodies were
measured by ELISA.
[0054] In FIG. 4 is used B10.Q(BALB/cxB10.Q)F1 mice were immunised
with 100 ug of rat CII emulsified in IFA on day 0 at the base of
the tail and boosted on day 35 with 50 ug of rat CII in IFA. The
mice were scored for a period of 202 days for arthritis
development. Mice, which developed chronic arthritis were selected
for the treatment experiment. All selected animals were reimmunised
on day 205 (day 0 of the reimmunisation) with 50 ug rat CII in IFA
and scored the next 75 days for clinical signs of arthritis. On
days 7, 11 and 28 after reimmunisation (arrows), mice were treated
by intravenous (100 ug in 200 ul PBS) administration of purified
GalOK264/A.sup.q complexes (10 mice in this group). PBS was
administrated (i.v.) as a control on the same days (7 mice in this
group). Sample sera were collected at days 0 and 75 after
reimmunisation and measured by ELISA. Data are represented as
mean.+-.SE.
[0055] In FIG. 5 three groups of donor B10.Q mice (5 mice in each
group) were injected i.v. with 200 ug GalOK264/A.sup.q in 100 ul
PBS, 200 ug MOG/A.sup.q in 100 ul PBS or 100 ul PBS alone. Five
days later, T cells were purified from each mouse individually by
negative selection and transferred i.v. (1.times.106 cells per
mouse) to the CII-immunised recipients (5 days after immunisation).
Sample sera were collected at days 35 and 70 after immunization
measured by ELISA. Results are expressed as the mean.+-.SE.
[0056] In FIG. 6 are (B10.QxB10.RIII)F1 mice immunised at day 0
with bovine CII. On days 20 and 34 (arrows), the mice were treated
by intravenous administration of purified peptide/A.sup.q complexes
(100 ug in 200 ul PBS (9 mice per group) (A). Sample sera were
collected at days 35 and 70 after immunisation and measured for
levels of anti-CII antibodies by ELISA (B). In another experiment,
(B10.QXB10.RIII)F1 mice (5 mice per group) were administrated once
with 200 ug in 200 ul PBS peptide/A.sup.q complexes at the day of
immunisation (day 0) and sera were collected at day 18 (C). All
data represent mean.+-.SE. *, p<0.05; **, p<0.01.
Antigens
[0057] Rat type II collagen (CII) was prepared from the Swarm
chondrosarcoma and bovine CII from joint cartilage, by limited
pepsin digestion, and further purified as previously described
(15). The CII peptides (non-modified CII259-273: GIAGFKGEQGPKGEP,
GalOK264 CII259-273: GIAGFK(Gal-Hyl)GEQGPKGEP, and the various
galactosylated peptides that were deoxygenated at OH groups on the
galactose (position 2, 3 and 4 respectively) were synthesized,
purified and characterized as previously described (9, 10, 11, 16,
17). The CII was dissolved in 0.1 M acetic acid. Mouse myelin
oligodendrocytic glycoprotein MOG79-90 peptide (GKVTLRIQNVRF) was
purchased from Schafer-N (Copenhagen, Denmark). All peptides were
dissolved in PBS. The collagen and peptides were stored at
4.degree. C. until used.
[0058] To induce CIA, each mouse was injected with 100 .mu.g of CII
(rat CII for B10.Q and bovine CII for (B10.QxB10.RIII)F1 mice),
emulsified 1:1 in complete Freund's adjuvant (CFA; Difco, Detroit,
Mich.) at the base of the tail in a total volume of 100 .mu.l.
Thirty-five days later, the mice were given a booster injection of
50 .mu.g of rat CII emulsified 1:1 in incomplete Freund's adjuvant
(IFA; Difco, Detroit, Mich.) in a total volume of 50 .mu.l.
Development of clinical arthritis was followed through visual
scoring of the animals based on the number of inflamed joints in
each paw, starting two weeks postimmunisation and continuing until
the end of the experiment. An extended scoring protocol (18)
ranging from 1-15 for each paw with a maximum score of 60 per mouse
was used. The mice were examined 2 to 4 times a week for at least
70 days after immunization.
[0059] The B10.Q(BALB/cxB10.Q)F2 mice were immunized with 100 .mu.g
of rat CII emulsified in IFA intradermally (i.d.) at the base of
the tail on day 0 and boosted on day 35 i.d. with 50 .mu.g of rat
CII in IFA. The mice were scored for a minimum period of 202 days
for arthritis development. Mice, which developed chronic arthritis
(mice with severe arthritis for a minimum period of 120 days were
considered as chronic) including the ones with clear relapses, were
selected for the treatment protocol.
Peptide/Aq Complex Treatment Protocols
[0060] Animals were treated by either intravenous (i.v.) or
intranasal (i.n.) administration of purified peptide/A.sup.q
complexes. In the intravenous treatment of CIA model, mice were
injected with GalOK264/Aq, deoxygenated GalOK264/Aq, K264/Aq or as
negative control MOG/Aq complex (100 .mu.g in 200 .mu.L PBS) on
days 20 and 34 postimmunisation (for the chronic model on days 7,
11 and 28 after reimmunisation). Control mice were injected
intravenously with 200 .mu.L PBS on the same days. In the
intranasal treatment experiments, mice were administrated with 10
.mu.g (in 20 .mu.L PBS) of peptide/A.sup.q complex on the days
mentioned above.
Example 6
Measurement of Serum Anti-CII Antibody Levels
[0061] Mice were bled at the time of boost immunization (day 35) as
well as at the termination of experiment (day 70) and sera were
analyzed for anti-CII IgG antibody levels by quantitative ELISA
(19). Briefly, 96-well ELISA plates (Nunc, Roskilde, Denmark) were
coated overnight at 4.degree. C. with 10 .mu.g/mL native rat CII in
PBS. The wells were washed three times with PBS-0.1% Tween 20 and
then 150 .mu.L of blocking buffer (5% BSA in PBS) was added to each
well and incubated for 1 h at room temperature. After washing, 50
.mu.L of samples in serial dilutions from 1/100 to 1/10.sup.5 were
added and incubated for 2 h at room temperature. After three
washes, peroxidase-conjugated goat anti-mouse IgG was added and
incubated at room temperature for 1 h. After extensive washing,
plates were developed using ABTS (Roche Diagnostics GmbH, Mannheim,
Germany) as substrate and the absorbance was then measured at 405
nm in a Spectra Max Plus reader (Goteborgs Termometerfabrik,
Goteborg, Sweden). A standard serum from arthritic and
non-immunised syngeneic mice was added to each plate in serial
dilutions as positive and negative controls, respectively.
TABLE-US-00002 TABLE 1 Peptide/A.sup.q complexes suppress
development of CIA in B10.Q mice. Mean anti-CII IgG Mean day Mean
peak (ug/mL) Treatment Incidence of onset of severity day 35 day 70
GalOK264/A.sup.q (i.v.) 0/10 (0%) N/A N/A 54.3 .+-. 10.5 36.9 .+-.
10.5 GalOK264/A.sup.q (i.n.) 1/10 (10%) 60 2 59.8 .+-. 15.4 38.8
.+-. 15.8 PBS (control) (i.v.) 8/10 (80%) 44 +/- 5 26.2 +/- 15.4
135.9 .+-. 30.9 96.6 .+-. 35.1
[0062] B10.Q mice (10 mice per group) were immunised with 100
microgrammes rat CII in CFA on day 0 and boosted on day 35 with 50
microgrammerat CII in IFA. On days 20 and 34, mice were treated by
intravenous (i.v.) (100 microgrammein 200 microlitres PBS) or
intranasal (i.n.) (10 microgrammein 20 microlitresPBS)
administration of purified GalOK264/Aq complex. PBS (200
microlitres) was administrated (i.v.) as a control on the same
days. Mice were monitored for clinical signs of arthritis for 70
days. Levels of IgG anti-CII were measured at days 35 and 70 by
ELISA. All values are shown as mean.+-.standard deviation. N/A
denotes not applicable.
Example 7
Histology
[0063] Hind paws were removed after ending the experiment, fixed in
4% neutral buffered formaldehyde overnight and then decalcified in
5% (w/v) EDTA at 4.degree. C. until the bones were pliable. Tissues
were then dehydrated in a gradient of alcohols, paraffin embedded,
sectioned at 5 .mu.m, mounted on glass slides, and stained with
hematoxylin and eosin (H&E). Serial H&E-stained sections
were analyzed microscopically for the degree of inflammation and
for cartilage and bone destruction. Analyses were performed in a
blinded fashion.
Example 8
T-Cell Transfer
[0064] For the T-cell transfer experiment, 15 B10.Q mice
(recipients) were immunized with CII/CFA (day 0) and boosted with
CII/IFA on day 35 using the standard immunization protocol. At the
same time (day 0), three groups (5 mice per each group) of other
B10.Q mice (donors) were injected i.v. with 200 .mu.g GalOK264/Aq
in 100 .mu.l PBS, 200 .mu.g MOG/Aq in 100 .mu.l PBS or 200 .mu.l
PBS alone, respectively. Five days later, erythrocyte free spleen
and lymph node cells from each mouse were passed through 40 .mu.m
nylon cell strainer (BD Biosciences Discovery Labware, Bedford,
Mass.) and then T cells were purified by negative selection using
antibodies against MRC class II+ (M5/114) and CD11b+ (M1/70)
expressing cells (BD Biosciences PharMingen, San Diego, Calif.) and
Dynabeads.RTM. (Dynal ASA, Oslo, Norway) followed by magnetic
sorting. The purity of the resulting T cells was measured by flow
cytometry and were found to be contaminated with <0.3% MLCII+
expressing cells. Purified T cells were analysed by FACS for
expression of CD25+, CD62L+, CD45RB+ and NK 1.1+ surface markers
but no differences between the individual mice or groups were
found. Purified T cells (1.times.106) from each individual donor
were resuspended in a final volume of 200 .mu.l sterile PBS and
transferred intravenously into recipient mice.
Statistics
[0065] Statistical difference in the incidence of disease between
groups of mice described in the examples was determined using Chi
Square test. To compare nonparametric data for statistical
significance, we applied the Mann-Whitney U or Kruskal Wallis test
on all clinical results and in vitro experiments using the
StatView.TM. programme (SAS, Institute Inc., USA).
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Sequence CWU 1
1
4180PRTHomo sapiens 1Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu
Phe Tyr Leu Asn Pro1 5 10 15Asp Gln Ser Gly Glu Phe Met Phe Asp Phe
Asp Gly Asp Glu Ile Phe20 25 30His Val Asp Met Ala Lys Lys Glu Thr
Val Trp Arg Leu Glu Glu Phe35 40 45Gly Arg Phe Ala Ser Phe Glu Ala
Gln Gly Ala Leu Ala Asn Ile Ala50 55 60Val Asp Lys Ala Asn Leu Glu
Ile Met Thr Lys Arg Ser Asn Tyr Thr65 70 75 80290PRTHomo sapiens
2Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His1 5
10 15Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
Tyr20 25 30His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu
Tyr Arg35 40 45Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
Asn Ser Gln50 55 60Lys Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp
Thr Tyr Cys Arg65 70 75 80His Asn Tyr Gly Val Gly Glu Ser Phe Thr85
903112PRTHomo sapiens 3Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val
Leu Thr Asn Ser Pro1 5 10 15Val Glu Leu Arg Glu Pro Asn Val Leu Ile
Cys Phe Ile Asp Lys Phe20 25 30Thr Pro Pro Val Val Asn Val Thr Trp
Leu Arg Asn Gly Lys Pro Val35 40 45Thr Thr Gly Val Ser Glu Thr Val
Phe Leu Pro Arg Glu Asp His Leu50 55 60Phe Arg Lys Phe His Tyr Leu
Pro Phe Leu Pro Ser Thr Glu Asp Val65 70 75 80Tyr Asp Cys Arg Val
Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys85 90 95His Trp Glu Phe
Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Asn100 105
1104107PRTHomo sapiens 4Val Gln Arg Arg Val Tyr Pro Glu Val Thr Val
Tyr Pro Ala Lys Thr1 5 10 15Gln Pro Leu Gln His His Asn Leu Leu Val
Cys Ser Val Asn Gly Phe20 25 30Tyr Pro Gly Ser Ile Glu Val Arg Trp
Phe Arg Asn Gly Gln Glu Glu35 40 45Lys Thr Gly Val Val Ser Thr Gly
Leu Ile Gln Asn Gly Asp Trp Thr50 55 60Phe Gln Thr Leu Val Met Leu
Glu Thr Val Pro Arg Ser Gly Glu Val65 70 75 80Tyr Thr Cys Gln Val
Glu His Pro Ser Leu Thr Ser Pro Leu Thr Val85 90 95Glu Trp Arg Ala
Arg Ser Glu Ser Ala Gln Ser100 105
* * * * *
References