U.S. patent application number 14/616320 was filed with the patent office on 2015-08-27 for treatment.
The applicant listed for this patent is Queen Mary & Westfield College. Invention is credited to Fulvio D'Acquisto, Roderick John Flower, Mauro Perretti.
Application Number | 20150239965 14/616320 |
Document ID | / |
Family ID | 40262550 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150239965 |
Kind Code |
A1 |
Perretti; Mauro ; et
al. |
August 27, 2015 |
Treatment
Abstract
The present invention provides a specific binding molecule which
binds to Annexin-1 (Anx-A1) for use in the treatment of T
cell-mediated disease.
Inventors: |
Perretti; Mauro; (London,
GB) ; D'Acquisto; Fulvio; (London, GB) ;
Flower; Roderick John; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Queen Mary & Westfield College |
London |
|
GB |
|
|
Family ID: |
40262550 |
Appl. No.: |
14/616320 |
Filed: |
February 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13131927 |
Oct 5, 2011 |
8980255 |
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PCT/GB2009/002810 |
Dec 2, 2009 |
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14616320 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/139.1 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 37/08 20180101; A61P 31/18 20180101; A61P 1/04 20180101; A61P
37/00 20180101; A61P 25/00 20180101; A61P 3/10 20180101; A61P 17/06
20180101; A61P 43/00 20180101; A61P 5/14 20180101; A61P 27/02
20180101; A61P 19/02 20180101; A61P 37/06 20180101; A61P 5/38
20180101; A61K 2039/505 20130101; A61P 9/10 20180101; A61P 21/00
20180101; A61P 29/00 20180101; C07K 16/18 20130101; A61P 1/00
20180101; A61P 17/00 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 2, 2008 |
GB |
0822011.3 |
Claims
1-12. (canceled)
13. A method of treating a T cell-mediated disease in an animal
comprising administering to the animal in need thereof a
therapeutic amount of an antibody, or fragment thereof, that binds
to Annexin-1 (Anx-A1).
14. The method of claim 13 wherein the antibody binds to an
N-terminal peptide of Anx-A1 of at least 50 amino acid
residues.
15. The method of claim 13 wherein the antibody binds to the
N-terminal peptide Ac.2-26 of Anx-A1.
16. The method of claim 13 wherein the antibody binds to a fragment
of at least 6 amino acids of the N-terminal peptide Ac.2-26 of
Anx-A1.
17. The method of claim 13 wherein the antibody is a monoclonal
antibody.
18. The method of claim 13 wherein the antibody fragment is chosen
from Fab fragment, F(ab').sub.2 fragment, Fv fragment, and single
chain Fv (scFv) molecule.
19. The method of claim 13 wherein the antibody is a chimeric
antibody.
20. The method of claim 13 wherein the antibody is a humanized
antibody.
21. The method of claim 13 wherein the T cell-mediated disease is
chosen from graft-versus-host disease, graft rejection,
atherosclerosis, HIV, AIDS, psoriasis, and an autoimmune
disease.
22. The method of claim 13 wherein the T cell-mediated disease is
chosen from atherosclerosis, HIV, AIDS, and psoriasis.
23. The method of claim 21 wherein the autoimmune disease is chosen
from rheumatoid arthritis, multiple sclerosis, systemic lupus
erythematosus, Addison's disease, Grave's disease, scleroderma,
polymyositis, diabetes mellitus, autoimmune uveoretinitis,
ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease,
and autoimmune thyroiditis.
24. The method of claim 21 wherein the autoimmune disease is chosen
from rheumatoid arthritis, multiple sclerosis, and systemic lupus
erythematosus.
Description
[0001] The present invention relates to methods for treating T
cell-mediated disease by modulating the activity of Annexin-1.
[0002] Autoimmune diseases are chronic disabling pathologies caused
by malfunction of the immune system. In most cases they are
initiated by an uncontrolled T cell response to autoantigens
presented in the context of MHC molecules of antigen presenting
cells (APCs). Several factors have been described as being involved
in the pathogenesis of autoimmune diseases including environmental,
genetic and viral factors, with one overarching feature: the
hyperresponsivity of T cells.
[0003] Glucocorticoids (GCs) are often used for the therapy of a
variety of chronic autoimmune diseases because of their ability to
simultaneously block both the innate and adaptive immune response.
Studies over the last 10 years or so by the present inventors and
other research groups have shown that some of the inflammatory
effects of GCs on the innate immune response are mediated by a
protein called Annexin-1 (Anx-A1). This protein has been proven to
exert a homeostatic control over a number of cell types including
neutrophils, macrophages and endothelial cells. However, one aspect
that has always been neglected is the role of Anx-A1 in the
adaptive immune response. This is surprising considering that
Anx-A1 has been proposed as one of the second messengers of the
pharmacological effects of GCs.
[0004] The present inventors have previously shown that Anx-A1
plays a homeostatic role in T cells by modulating the strength of T
cell receptor (TCR) signaling (D'Acquisto et al., Blood 109:
1095-1102, 2007).
[0005] Furthermore, the inventors have shown that high levels of
Anx-A1 lower the threshold of T cell activation and favour the
differentiation into Th1 cells, whereas Anx-A1 deficient mice show
impaired T cell activation and increased differentiation into Th2
cells (D'Acquisto et al., Eur. J. Immunol. 37: 3131-3142,
2007).
[0006] WO 2005/027965 describes the discovery of a mechanism by
which apoptotic neutrophils deliver anti-inflammatory signals to
dendritic cells and identifies an antibody that interferes with
this process. In particular, WO 2005/027965 describes the
identification of Anx-1 as a signalling molecule which is said to
be expressed by apoptotic neutrophils to inhibit the activation and
maturation of dendritic cells. WO 2005/027965 proposes that an
antibody termed DAC5 (Detector of Apoptotic Cells Nr. 5) recognizes
and blocks the anti-inflammatory effects of Anx-1 presented on the
surface of apoptotic neutrophils upon phagocytosis by dendritic
cells. WO 2005/027965 thus refers to the possibility of treatment
of various diseases by targeting such apoptotic cells and deleting
them by causing an inflammatory response, but does not discuss a
role for Anx-1 in T cell activation.
[0007] WO 2005/027965 claims that annexins are expressed on cells
that are undergoing apoptosis (see for example page 8, lines 6-7
and 29-30) and that these annexins are presented on the surface of
such cells (see for example page 6, lines 10-11 and page 8, lines
16-17). However, two separate studies (Maderna et al., J Immunol.,
174: 3727-3733, 2005; Scannell et al., J Immunol., 178: 4595-4605,
2007) have shown that apoptotic cells, including neutrophils,
release annexin-1, rather than expressing the protein and
presenting it on the cell surface. Since annexin-1 is released from
the cell, it cannot be claimed that DAC5 would identify only
apoptotic cells expressing the protein on the surface, as the
antibody would also identify released annexin-1.
[0008] Furthermore, WO 2005/027965 claims that co-incubation of
apoptotic neutrophils expressing annexin-1 on their cell membrane
with dendritic cells activated with LPS causes inhibition of
TNF-.alpha. secretion and upregulation of the activation markers
CD83, CD86 and HLA-DR, and that addition of DAC5 to this culture
reverses the inhibitory effects of the annexin-1 expressing
apoptotic neutrophils (page 5, line 31 to page 6, line 8). Data
from the present inventors (Huggins et al., FASEB J. 2008, in
press) demonstrates that dendritic cells release Anx-1 upon
stimulation with LPS and thus the DAC5 described in WO 2005/027965
would bind the Anx-1 externalized on the neutrophils as well as the
annexin-1 released by dendritic cells. Furthermore, the present
inventors have found that the absence of annexin-1 in dendritic
cells causes an increased expression of maturation/activation
markers and production of inflammatory cytokines such as TNF-cc and
IL-113 and IL-12. Therefore, the antibody DACS described in WO
2005/027965 should affect the maturation and activation of
dendritic cells and thus the subsequent modulation of the immune
response.
[0009] In support of this, the present inventors have shown that
co-culturing Anx-A1.sup.-/- dendritic cells with nave T cells
within a mixed lymphocyte reaction (MLR) showed a significantly
reduced ability to induce either T cell proliferation or IL-2 and
IFN-.gamma. production. Thus, agents blocking Anx-A1 function in
dendritic cells should reduce their capacity to stimulate a robust
T cell mediated immune response. The antibodies referred to in WO
2005/027965 would therefore not be suitable for treating the
diseases referred to in that patent application.
[0010] The present invention provides the use of specific binding
molecules which bind to Annexin-1 (Anx-A1) in the treatment of T
cell-mediated disease.
[0011] According to a first aspect of the invention there is
therefore provided a specific binding molecule which binds to
Annexin-1 (Anx-A1) for use in the treatment of T cell-mediated
disease.
[0012] The present inventors have previously shown that Anx-A1
modulates the strength of T cell receptor (TCR) signaling and that
high levels of Anx-A1 lower the threshold of T cell activation and
favour differentiation into Th1 cells. The inventors have now
identified the annexin pathway, and the ensuing signal, as a target
for blockade in order to treat T cell-mediated diseases. Such
diseases include those in which there is aberrant T cell
activation, for example many autoimmune diseases, and those in
which it is desirable to skew differentiation of T cells in favour
of Th1 rather than Th2 cells.
[0013] The present invention utilises a specific binding molecule
which binds to Annexin-1 (Anx-A1).
[0014] Annexins are a group of calcium- and phospholipid-binding
cellular proteins and are also known as lipocortins. The annexin
family has 13 members, including Annexin A1, Annexin A2 and Annexin
A5. Annexin-A1 is also known as Annexin-1 and is referred to herein
as "Anx-A1". Annexin-1 (Anx-A1) is a 37-kDa protein and was
originally described as a mediator of the actions of
glucocorticoids. Over the last few years evidence has shown than
Anx-A1 plays a homeostatic role in the adaptive immune system, in
particular T cells, by modulating the strength of T cell receptor
(TCR) signalling. Anx-A1 acts as an endogenous down-regulator of
inflammation in cells of the innate immune system in vivo. FIG. 1A
is a ribbon diagram showing the three-dimensional structure of
Anx-A1.
[0015] There are eight human nucleotide sequences which encode
Anx-A1. Of these, only four are translated and thus there are four
isoforms of Anx-A1, designated ANXA1-002, ANXA1-003, ANXA1-004 and
ANXA1-006. These sequences are available from the Ensembl website
(www.ensembl.org) and are designated OTTHUMT00000052664
(ANXA1-002), OTTHUMT00000052665 (ANXA1-003), OTTHUMT00000052666
(ANXA1-004) and OTTHUMT00000052668 (ANXA1-006). The amino acid and
nucleotide sequences of one isoform of human Annexin-1 (Anx-A1),
ANXA1-003, are shown in FIG. 2a. The amino acid sequences of
isoforms ANXA1-002, ANXA1-004 and ANXA1-006 are shown in FIGS. 2b,
2c and 2d respectively. As can be seen from FIG. 2, isoforms
ANXA1-002, ANXA1-004 and ANXA1-006 are either short splice variants
of ANXA1-003 or variants of ANXA1-003 with a small number of amino
acid changes.
[0016] A number of studies have shown that an N-terminal peptide of
Anx-A1 named Ac.2-26 acts as a bioactive surrogate of the whole
protein (see e.g. Lim et al., Proc Natl Acad Sci U S A95, 14535-9,
1998).
[0017] FIG. 1B is a schematic representation of the annexin repeats
and the location of this bioactive sequence. Peptide Ac.2-26 is an
acetylated peptide having the sequence of amino acid residues 2-26
of the full-length amino acid sequence of Anx-A1 shown in FIG. 2.
The sequence of peptide Ac.2-26 is shown in FIG. 1C and is as
follows: [0018] CH.sub.3CO-AMVSEFLKQAWFIENEEQEYVQTVK
[0019] Anx-A1 and its N-terminal derived bioactive peptides mediate
their biological effects through members of the formyl peptide
receptor (FPR) family. Anx-A1 exerts its counterregulatory actions
on neutrophil extravasation and innate immunity by direct binding
and activation of one member of this family, formyl peptide
receptor like-1 (FPRL-1). The present inventors have previously
found that stimulation of T cells in the presence of hrAnx-A1
increases T cell activation via stimulation of FPRL-1 (D'Acquisto
et al., Blood 109: 1095-1102, 2007).
[0020] The present invention utilises a specific binding molecule
which binds to Annexin-1 (Anx-A1). The Anx-A1 to which the specific
binding molecule binds is typically human Anx-A1 having the
polypeptide sequence shown in FIG. 2a, or a variant or fragment
thereof, such as one of the isoforms of human Anx-A1 having the
polypeptide sequence shown in FIG. 2b, FIG. 2c or FIG. 2d. The
fragment of human Anx-A1 to which the specific binding molecule
binds is typically the polypeptide having the sequence shown in
FIG. 1C. The Anx-A1 to which the specific binding molecule binds is
typically encoded by the nucleotide sequence shown in FIG. 2a.
[0021] As used herein the term "variant" relates to proteins which
have a similar amino acid sequence and/or which retain the same
function. For instance, the term "variant" encompasses proteins or
polypeptides which include one or more amino acid additions,
deletions, substitutions or the like. Amino acid substitutions are
typically conservative substitutions, i.e. the substitution of an
amino acid with another with generally similar properties, such
that the overall functioning is likely not to be seriously
affected.
[0022] Thus the amino acids glycine, alanine, valine, leucine and
isoleucine can often be substituted for one another (amino acids
having aliphatic side chains). Of these possible substitutions it
is preferred that glycine and alanine are used to substitute for
one another (since they have relatively short side chains) and that
valine, leucine and isoleucine are used to substitute for one
another (since they have larger aliphatic side chains which are
hydrophobic). Other amino acids which can often be substituted for
one another include: phenylalanine, tyrosine and tryptophan (amino
acids having aromatic side chains); lysine, arginine and histidine
(amino acids having basic side chains); aspartate and glutamate
(amino acids having acidic side chains); asparagine and glutamine
(amino acids having amide side chains); and cysteine and methionine
(amino acids having sulphur containing side chains).
[0023] Using the three letter and one letter codes the amino acids
may be referred to as follows: glycine (G or Gly), alanine (A or
Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile),
proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr),
tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg),
histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or
Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine (C or
Cys), methionine (M or Met), serine (S or Ser) and Threonine (T or
Thr). Where a residue may be aspartic acid or asparagine, the
symbols Asx or B may be used. Where a residue may be glutamic acid
or glutamine, the symbols Glx or Z may be used. References to
aspartic acid include aspartate, and references to glutamic acid
include glutamate, unless the context specifies otherwise.
[0024] Amino acid deletions or insertions may also be made relative
to the amino acid sequence of the protein referred to above. Thus,
for example, amino acids which do not have a substantial effect on
the activity of the polypeptide, or at least which do not eliminate
such activity, may be deleted. Such deletions can be advantageous
since the overall length and the molecular weight of a polypeptide
can be reduced whilst still retaining activity. This can enable the
amount of polypeptide required for a particular purpose to be
reduced--for example, dosage levels can be reduced.
[0025] Amino acid insertions relative to the sequence of the fusion
protein above can also be made. This may be done to alter the
properties of a substance (e.g. to assist in identification,
purification or expression).
[0026] Amino acid changes relative to the sequence given above can
be made using any suitable technique e.g. by using site-directed
mutagenesis or solid state synthesis.
[0027] It should be appreciated that amino acid substitutions or
insertions within the scope of the present invention can be made
using naturally occurring or non-naturally occurring amino acids.
Whether or not natural or synthetic amino acids are used, it is
preferred that only L-amino acids are present.
[0028] One can use a program such as the CLUSTAL program to compare
amino acid sequences. This program compares amino acid sequences
and finds the optimal alignment by inserting spaces in either
sequence as appropriate. It is possible to calculate amino acid
identity or similarity (identity plus conservation of amino acid
type) for an optimal alignment. A program like BLASTx will align
the longest stretch of similar sequences and assign a value to the
fit. It is thus possible to obtain a comparison where several
regions of similarity are found, each having a different score.
Both types of identity analysis are contemplated in the present
invention.
[0029] Variants of the proteins and polypeptides described herein
should retain the function of the original protein or polypeptide.
Alternatively or in addition to retaining the function of the
original protein or polypeptide, variants of the proteins and
polypeptides described herein typically have at least 60% identity
(as discussed above) with the proteins or polypeptides described
herein, in particular the polypeptide sequences shown in FIG. 1C or
FIG. 2. Typically, variants for use in the invention have at least
70%, at least 80%, at least 90%, at least 95%, at least 97% or at
least 99% identity to the proteins or polypeptides described
herein, in particular the polypeptide sequences shown in FIG. 1C or
FIG. 2.
[0030] The percent identity of two amino acid sequences or of two
nucleic acid sequences is determined by aligning the sequences for
optimal comparison purposes (e.g., gaps can be introduced in the
first sequence for best alignment with the sequence) and comparing
the amino acid residues or nucleotides at corresponding positions.
The "best alignment" is an alignment of two sequences which results
in the highest percent identity. The percent identity is determined
by the number of identical amino acid residues or nucleotides in
the sequences being compared (i.e., % identity =number of identical
positions/total number of positions x 100).
[0031] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm known to those
of skill in the art. An example of a mathematical algorithm for
comparing two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877.
The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.
Biol. 215:403-410 have incorporated such an algorithm. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to nucleic acid molecules of the invention. BLAST protein searches
can be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilised as described in Altschul et al. (1997)
Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be
used to perform an iterated search which detects distant
relationships between molecules (Id.). When utilising BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov. Another example of a mathematical
algorithm utilised for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0)
which is part of the CGC sequence alignment software package has
incorporated such an algorithm. Other algorithms for sequence
analysis known in the art include ADVANCE and ADAM as described in
Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and
FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
85:2444-8. Within FASTA, ktup is a control option that sets the
sensitivity and speed of the search.
[0032] In an alternative approach, the variants can be fusion
proteins, incorporating moieties which render purification easier,
for example by effectively tagging the desired protein or
polypeptide. It may be necessary to remove the "tag" or it may be
the case that the fusion protein itself retains sufficient
functionality to be useful.
[0033] A "specific binding molecule which binds to Anx-A1" as used
herein is a molecule which binds with greater affinity to Anx-A1
than to other molecules, i.e. which binds specifically to Anx-A1.
Specific binding molecules which bind to Anx-A1 include anti-Anx-A1
antibodies and aptamers. The anti-Anx-A1 antibodies for use in the
present invention function by blocking the activation of T cells
and thus, when administered, can be used in the treatment of T
cell-mediated diseases, which are typically caused by aberrant T
cell activation.
[0034] Anti-Anx-A1 antibodies can be raised, for example, against
human Anx-A1 having the amino acid sequence set out in FIG. 2,
typically the amino acid sequence set out in FIG. 2a.
Alternatively, anti-Anx-A1 antibodies can be directed to a
particular epitope or epitopes of human Anx-A1 having the amino
acid sequence set out in FIG. 2, typically the amino acid sequence
set out in FIG. 2a. For example, anti-Anx-A1 antibodies can be
directed against an N-terminal fragment of Anx-A1, for example an
N-terminal fragment of at least 188, 100, 50 or 25 amino acid
residues from the N-terminus of the amino acid sequence set out in
FIG. 2a. Typically, the anti-Anx-A1 antibody for use in the
invention is an antibody against the N-terminal fragment of Anx-A1
termed Ac2-26 and which has the sequence shown in FIG. 1C, or
against a fragment of at least 6 amino acids thereof Specific
binding molecules which bind to Anx-A1 therefore include
anti-Anx-A1 antibodies which are antibodies against the Anx-A1
fragment Ac2-26 having the sequence shown in FIG. 1C or a fragment
of at least 6 amino acids thereof In this embodiment, the
anti-Anx-A1 antibody is raised against a fragment of the sequence
shown in FIG. 1C which is antigenic and capable of stimulating the
production of antibodies which, when administered, can be used in
the treatment of T cell-mediated diseases, which are typically
caused by aberrant T cell activation.
[0035] As stated above, an active subfragment of the specified
sequence may be used as defined. Active subfragments may consist of
or include a fragment of at least 6 continuous amino acid residues
(a hexapeptide) of the N-terminal fragment of Anx-A1 termed Ac2-26
having the sequence set out in FIG. 1C, including one or more
of:
TABLE-US-00001 AMVSEF MVSEFL VSEFLK SEFLKQ EFLKQA FLKQAW LKQAWF
KQAWFI QAWFIE AWFIEN WFIENE FIENEE IENEEQ ENEEQE NEEQEY EEQEYV
EQEYVQ QEYVQT EYVQTV YVQTVK
[0036] Active subfragments may consist of or include a fragment of
more than 6 continuous amino acid residues of the N-terminal
fragment of Anx-A1 termed Ac2-26 having the sequence set out in
FIG. 1C, for example a fragment of at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17, at least 18, at least 19, at
leat 20, at least 21, at least 22, at least 23 or at least 24 amino
acids of the sequence set out in FIG. 1C.
[0037] Anti-Anx-A1 antibodies include monoclonal and polyclonal
antibodies. Typically, the anti-Anx-A1 antibody is a monoclonal
antibody. The anti-Anx-A1 antibody can be a commercially available
antibody, for example a rabbit polyclonal or mouse monoclonal
antibody. Typically, the anti-Anx-A1 antibody is humanised, as
described in detail below.
[0038] Monoclonal antibodies can be produced from hybridomas. These
are typically formed by fusing myeloma cells and spleen cells which
produce the desired antibody in order to form an immortal cell
line. The well-known Kohler & Milstein technique (Nature
256:495-497 (1975)) or subsequent variations upon this technique
can be used to produce a monoclonal antibody for use in accordance
with the invention.
[0039] Polyclonal antibodies can be raised by stimulating their
production in a suitable animal host (e.g. a mouse, rat, guinea
pig, rabbit, sheep, goat or monkey) by injection of Anx-A1, or a
variant or fragment thereof, into the animal. If desired, an
adjuvant may be administered together with the Anx-A1 protein.
Well-known adjuvants include Freund's adjuvant (complete and
incomplete) and aluminium hydroxide. The antibodies can then be
purified by virtue of their binding to Anx-A1.
[0040] Techniques for producing monoclonal and polyclonal
antibodies that bind to a particular polypeptide/protein are now
well developed in the art and are discussed in standard immunology
textbooks, for example in Roitt et al, Immunology second edition
(1989), Churchill Livingstone, London.
[0041] In addition to whole antibodies, the present invention
includes derivatives thereof which are capable of binding to Anx-A1
as described herein. Thus the present invention includes antibody
fragments and synthetic constructs. Examples of antibody fragments
and synthetic constructs are given by Dougall et al in Trends
Biotechnol., 12: 372-379 (1994).
[0042] Antibody fragments include, for example, Fab, F(ab').sub.2
and Fv fragments. Fab fragments are discussed in Roitt et al
[supra]. Fv fragments can be modified to produce a synthetic
construct known as a single chain Fv (scFv) molecule. This includes
a peptide linker covalently joining variable heavy chain (V.sub.H)
and variable light chain (V.sub.L) regions, which contributes to
the stability of the molecule. The linker may comprise from 1 to 20
amino acids, such as for example 1, 2, 3 or 4 amino acids, 5, 10 or
15 amino acids, or other intermediate numbers in the range 1 to 20
as convenient. The peptide linker may be formed from any generally
convenient amino acid residues, such as glycine and/or serine. One
example of a suitable linker is Gly.sub.4Ser. Multimers of such
linkers may be used, such as for example a dimer, a trimer, a
tetramer or a pentamer, e.g. (Gly.sub.4Ser).sub.2,
(Gly.sub.4Ser).sub.3, (Gly.sub.4Ser).sub.4 or (Gly.sub.4Ser).sub.5.
However, in other embodiments no peptide linker is present and the
V.sub.L domain is linked to the V.sub.H domain by a peptide
bond.
[0043] The specific binding molecule may be an analogue of a
single-chain variable fragment (scFv). For example, the scFv may be
linked to other specific binding molecules (for example other
scFvs, Fab antibody fragments and chimeric IgG antibodies (e.g.
with human frameworks)). The scFv may be linked to other scFvs so
as to form a multimer which is a multi-specific binding protein,
for example a dimer, a trimer or a tetramer. Bi-specific scFv's are
sometimes referred to as diabodies, tri-specific as triabodies and
tetra-specific as tetrabodies.
[0044] An scFv can be prepared by any suitable technique using
standard chemical or molecular biology techniques. In one
embodiment of the invention, the monoclonal antibody analogues can
be prepared as scFv's from a nave human antibody phage display
library (McCafferty et al., Nature 348, 552-554 (1990); and as
described in WO 92/01047).
[0045] Other synthetic constructs that can be used include
Complementarity Determining Region (CDR) peptides. These are
synthetic peptides comprising antigen-binding determinants Peptide
mimetics can also be used. These molecules are usually
conformationally restricted organic rings that mimic the structure
of a CDR loop and that include antigen-interactive side chains.
[0046] Synthetic constructs include chimeric molecules. Thus,
humanised antibodies or derivatives thereof are within the scope of
the antibodies for use in the present invention. Methods for
humanising antibodies are well known in the art. The antibody can
be humanised by modifying the amino acid sequence of the antibody.
An example of a humanised antibody is an antibody having human
framework regions, but rodent (for example murine) hypervariable
regions. Ways of producing chimeric antibodies are discussed for
example by Morrison et al in PNAS, 81: 6851-6855 (1984) and by
Takeda et al in Nature, 314: 452-454 (1985). Humanisation can be
performed, for example, as described by Jones et al in Nature, 321:
522-525 (1986); Verhoeyen et al in Science, 239: 1534-1536;
Riechmann et al in Nature 332: 323-327, 1988. Methods to reduce the
immunogenicity of the specific binding molecules of the invention
may include CDR grafting on to a suitable antibody framework
scaffold or variable surface residue remodelling, e.g. by
site-directed mutagenesis or other commonly used molecular
biological techniques (Roguska et al Protein Eng. 9 895-904
(1996)).
[0047] Other methods applicable include the identification of
potential T-cell epitopes within the molecule, and the subsequent
removal of these e.g. by site-directed mutagenesis
(de-immunisation). Humanisation of the CDR regions or of the
surrounding framework sequence may be carried out as desired.
[0048] Synthetic constructs also include molecules comprising an
additional moiety that provides the molecule with some desirable
property in addition to antigen binding. For example the moiety may
be a label (e.g. a fluorescent or radioactive label).
Alternatively, it may be a pharmaceutically active agent.
[0049] The present invention relates to the use of a specific
binding molecule which binds to Anx-A1 for the treatment of T
cell-mediated disease.
[0050] The present invention can be used to treat a wide range of
diseases which are mediated by T cells. In the present context, "T
cell-mediated disease" means any disease or condition in which T
cells play a role in pathogenesis or development of the disease or
condition. T cell-mediated diseases are typically caused by
aberrant T cell activation. Accordingly, such diseases can be
treated by preventing the activation of T cells by blocking the
activity of Anx-A1. Typically, the T cell-mediated diseases treated
in the present invention are diseases in which Th1 cells play a
role.
[0051] T cell-mediated diseases include but are not limited to
graft-versus-host disease, graft rejection, atherosclerosis, HIV
and/or AIDS, psoriasis and some autoimmune diseases. Autoimmune
diseases which can be treated according to the present invention
include rheumatoid arthritis (RA), multiple sclerosis (MS),
systemic lupus erythematosus (SLE), Addison's disease, Grave's
disease, scleroderma, polymyositis, some forms of diabetes mellitus
(for example juvenile onset diabetes), autoimmune uveoretinitis,
ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease
and autoimmune thyroiditis. The T cell-mediated disease is
typically rheumatoid arthritis, multiple sclerosis, systemic lupus
erythematosus or atherosclerosis.
[0052] The T cell-mediated disease is typically rheumatoid
arthritis. In rheumatoid arthritis (RA), it is thought that T cells
recognise and interact with antigen presenting cells in the
synovium. Once activated, these cells produce cytokines and
effector molecules; this sequential, expanded production of
cytokines constitutes the "cytokine cascade" that results in the
activation of macrophages and induction of the inflammatory
process, culminating in degradation and resorption of cartilage and
bone. Over time, bone erosion, destruction of cartilage, and
complete loss of joint integrity can occur. Eventually, multiple
organ systems may be affected.
[0053] In another embodiment, the T cell-mediated disease is
atherosclerosis. Inflammation plays a key role in coronary artery
disease and other manifestations of atherosclerosis. Immune cells
dominate early atherosclerotic lesions, their effector molecules
accelerate progression of the lesions, and activation of
inflammation can elicit acute coronary syndromes. Adaptive immunity
is highly involved in atherogenesis since it has been shown to
interact with metabolic risk factors to initiate, propagate, and
activate lesions in the arterial tree.
[0054] Two mouse models with features of hypercholesterolemia and
rapid development of atherosclerosis, the ApoE.sup.-/- and the
low-density lipoprotein receptor-knockout mouse (LDLR.sup.-/-), are
useful in the study of atherosclerosis as they mimic the cellular
composition of human lesions, particularly in content of T
lymphocytes. Lymphocyte recruitment is increased in the arteries of
the atherosclerotic-prone ApoE.sup.-/- mice even well before the
onset of the pathology.
[0055] The presence of T-lymphocytes has functional consequences as
their complete absence reduces lesion formation during moderate
hypercholesterolemia. CD4+ IFN-.gamma.-secreting type-1 helper
(Th1) cells are the predominant type of T cell found in plaques,
and these T cells exert pro-atherogenic and plaque-destabilising
effects.
[0056] The inventors have now found that Anx-A1 is expressed in
both human and murine atherosclerotic plaques and that there is a
correlation between Anx-A1 expression and MS in a mouse model.
[0057] In another embodiment, the T cell-mediated disease is
systemic lupus erythematosus (SLE). The inventors have now found
that Anx-A1 mRNA and protein are expressed at higher levels in T
cells from SLE patients than in T cells from healthy
volunteers.
[0058] In relation to the ability of Anx-A1 to favour
differentiation of Th1 cells, the present invention can also be
used, for example, to limit uncontrolled protective cellular (Th1)
responses against intracellular pathogens and to treat
extracellular infection (Th2 response) by suppressing Th1
differentiation and favouring Th2 differentiation.
[0059] The specific binding molecule which binds to Anx-A1 is
typically formulated for use with a pharmaceutically acceptable
carrier, excipient, vehicle, adjuvant and/or diluent. The present
invention thus encompasses a pharmaceutical composition comprising
a specific binding molecule which binds to Annexin-1 (Anx-A1) for
use in the treatment of T cell-mediated disease. The pharmaceutical
composition comprises a specific binding molecule which binds to
Annexin-1 (Anx-A1) and a pharmaceutically acceptable carrier,
excipient, vehicle, adjuvant and/or diluent. Such compositions may
be prepared by any method known in the art of pharmacy, for example
by admixing the active ingredient with the carrier, excipient,
vehicle, adjuvant and/or diluent under sterile conditions.
[0060] Suitable carriers, vehicles, adjuvants and/or diluents are
well known in the art and include saline, phosphate buffered saline
(PBS), carboxymethylcellulose (CMC), methylcellulose,
hydroxypropylmethylcellulose (HPMC), dextrose, liposomes, polyvinyl
alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose, sucrose
(and other sugars), magnesium carbonate, gelatin, oil, alcohol,
detergents, emulsifiers or water (preferably sterile). The specific
binding molecule which binds to Anx-A1 can be formulated as a
liquid formulation, which will generally consist of a suspension or
solution of the specific binding molecule which binds to Anx-A1 in
a suitable aqueous or non-aqueous liquid carrier or carriers, for
example water, ethanol, glycerine, polyethylene glycol (PEG) or an
oil.
[0061] Typically, when the specific binding molecule which binds to
Anx-A1 is an antibody, the antibody is PEGylated, i.e. covalently
attached to a polyethylene glycol. Typically, this has the effect
of reducing the immunogenicity and increasing the half-life of said
antibody.
[0062] The specific binding molecule which binds to Anx-A1 can be
administered alone or together with another agent.
[0063] The specific binding molecule which binds to Anx-A1 for use
in the present invention is typically administered to a subject in
a therapeutically effective amount. Such an amount is an amount
effective to ameliorate, eliminate or prevent one or more symptoms
of T cell-mediated disease. Preferably, the subject to be treated
is a human. However, the present invention is equally applicable to
human or veterinary medicine. For example, the present invention
may find use in treating companion animals, such as dogs and cats,
or working animals, such as race horses.
[0064] The specific binding molecule which binds to Anx-A1 can be
administered to the subject by any suitable means. The specific
binding molecule which binds to Anx-A1 can be administered
systemically, in particular intra-articularly, intra-arterially,
intraperitoneally (i.p.), intravenously or intramuscularly.
However, the specific binding molecule which binds to Anx-A1 can
also be administered by other enteral or parenteral routes such as
by subcutaneous, intradermal, topical (including buccal, sublingual
or transdermal), oral (including buccal or sublingual), nasal,
vaginal, anal, pulmonary or other appropriate administration
routes.
[0065] Pharmaceutical compositions adapted for oral administration
may be presented as discrete units such as capsules or tablets; as
powders or granules; as solutions, syrups or suspensions (in
aqueous or non-aqueous liquids; or as edible foams or whips; or as
emulsions). Suitable excipients for tablets or hard gelatine
capsules include lactose, maize starch or derivatives thereof,
stearic acid or salts thereof Suitable excipients for use with soft
gelatine capsules include for example vegetable oils, waxes, fats,
semi-solid, or liquid polyols etc. For the preparation of solutions
and syrups, excipients which may be used include for example water,
polyols and sugars. For the preparation of suspensions, oils (e.g.
vegetable oils) may be used to provide oil-in-water or water in oil
suspensions.
[0066] Pharmaceutical compositions adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active ingredient may
be delivered from the patch by iontophoresis as generally described
in Pharmaceutical Research, 3(6):318 (1986).
[0067] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils. For infections of the eye or other external tissues, for
example mouth and skin, the compositions are preferably applied as
a topical ointment or cream. When formulated in an ointment, the
active ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base. Pharmaceutical compositions adapted for topical
administration to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent. Pharmaceutical compositions adapted
for topical administration in the mouth include lozenges, pastilles
and mouth washes.
[0068] Pharmaceutical compositions adapted for rectal
administration may be presented as suppositories or enemas.
[0069] Pharmaceutical compositions adapted for nasal administration
wherein the carrier is a solid include a coarse powder having a
particle size for example in the range 20 to 500 microns which is
administered in the manner in which snuff is taken, i.e. by rapid
inhalation through the nasal passage from a container of the powder
held close up to the nose. Suitable compositions wherein the
carrier is a liquid, for administration as a nasal spray or as
nasal drops, include aqueous or oil solutions of the active
ingredient.
[0070] Pharmaceutical compositions adapted for administration by
inhalation include fine particle dusts or mists which may be
generated by means of various types of metered dose pressurised
aerosols, nebulizers or insufflators.
[0071] Pharmaceutical compositions adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations.
[0072] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solution which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation substantially isotonic
with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents. Excipients which may be used for injectable
solutions include water, alcohols, polyols, glycerine and vegetable
oils, for example. The compositions may be presented in unit-dose
or multi-dose containers, for example sealed ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carried, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets.
[0073] The pharmaceutical compositions may contain preserving
agents, solubilising agents, stabilising agents, wetting agents,
emulsifiers, sweeteners, colourants, odourants, salts, buffers,
coating agents or antioxidants. They may also contain
therapeutically active agents in addition to the specific binding
molecule which binds to Anx-A1.
[0074] The dose of the specific binding molecule which binds to
Anx-A1 to be administered may be determined according to various
parameters, especially according to the specific binding molecule
which binds to Anx-A1 used; the age, weight and condition of the
patient to be treated; the route of administration; and the
required regimen. A physician will be able to determine the
required route of administration and dosage for a particular
patient.
[0075] This dosage may be repeated as often as appropriate. If side
effects develop the amount and/or frequency of the dosage can be
reduced, in accordance with normal clinical practice.
[0076] For administration to mammals, and particularly humans, it
is expected that the daily dosage of the active agent will be from
1 .mu.g/kg to 10mg/kg body weight, typically around 10 .mu.g/kg to
1 mg/kg body weight. The physician in any event will determine the
actual dosage which will be most suitable for an individual which
will be dependant on factors including the age, weight, sex and
response of the individual. The above dosages are exemplary of the
average case. There can, of course, be instances where higher or
lower dosages are merited, and such are within the scope of this
invention
[0077] In a second aspect of the invention, there is provided the
use of a specific binding molecule which binds to Anx-A1 in the
manufacture of a medicament for the treatment of T cell-mediated
disease.
[0078] In a third aspect of the invention, there is provided a
method for the treatment of T cell-mediated disease comprising
administering to a subject in need thereof a therapeutic amount of
a specific binding molecule which binds to Anx-A1. As stated above,
the method of treatment may of a human or an animal subject and the
invention extends equally to methods of treatment for use in human
and/or veterinary medicine.
[0079] Preferred features for the second and third aspects of the
invention are as for the first aspect mutatis mutandis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] The invention will now be further described by way of
reference to the following Examples and Figures which are provided
for the purposes of illustration only and are not to be construed
as limiting on the invention. Reference is made to a number of
Figures, in which:
[0081] FIG. 1A is a ribbon diagram of annexin-1 structure showing
the four annexin repeats and the N-terminal domain. FIG. 1B is a
schematic representation of the annexin repeats and the location of
the bioactive sequence, Annexin-1 peptide Ac.2-26. FIG. 1C shows
the amino acid sequence of peptide Ac.2-26, which is an acetylated
N-terminal peptide fragment of Anx-A1.
[0082] FIG. 2a shows (i) the amino acid sequence and (ii) the
nucleotide sequence of human Annexin-1 (Anx-A1), isoform ANXA1-003.
FIG. 2b shows the amino acid sequence of human Annexin-1 (Anx-A1),
isoform ANXA1-002.
[0083] FIG. 2c shows the amino acid sequence of human Annexin-1
(Anx-A1), isoform ANXA1-004. FIG. 2d shows the amino acid sequence
of human Annexin-1 (Anx-A1), isoform ANXA1-006.
[0084] FIGS. 3A-3D show the effect of human recombinant Annexin-1
(hrAnx-A1) on T cell activation. Pre-treatment of murine nave CD4+
primary cells with hrAnx-A1 followed by activation with different
concentrations of anti-CD3/CD28 augmented cell proliferation (FIG.
3A), IL-2 production (FIG. 3B) and cell surface expression of CD25
and CD69 (FIGS. 3C and 3D).
[0085] FIGS. 4A-4D show that endogenous Anx-A1 modulates T cell
proliferation. Stimulation of Anx-A1.sup.+/+ or Anx-A1.sup.-/- T
cells with anti-CD3, anti-CD3/CD28 or PMA/Ionomycin showed a
decrease rate of .sup.3H-thymidine incorporation (FIGS. 4A, 4B and
4C, respectively) and IL-2 production (FIG. 4D) in the Anx-A1
deficient T cells compared to control unstimulated T cells.
[0086] FIGS. 5A and 5B show activation of Activator Protein-1
(AP-1), Nuclear Factor-.kappa.B (NF-.kappa.B) and Nuclear Factor of
Activated T cells (NFAT) in the presence or absence of Anx-A1 (FIG.
5A), and a comparison of the activation of AP-1, NF-.kappa.B and
NFAT in Anx-A1.sup.-/+ and Anx.sup.-/-T cells (FIG. 5B).
[0087] FIG. 6A shows FACS analysis of FPRL-1 expression in T cells
stimulated with anti-CD3/CD28 (5.0 .mu.g/ml) for the indicated
times. FIG. 6B shows the cellular localization of Anx-A1 in T cells
before (Control) or after stimulation with anti-CD3/CD28 (5.0
.mu.g/ml). FIG. 6C is a schematic representation of the role of the
Anx-A1/FPRL-1 system in T cells.
[0088] FIGS. 7A and 7B show that exogenous and endogenous Anx-A1
modulates Th1/Th2 differentiation. FIG. 7A shows the results when
nave lymph node T cells were differentiated in vitro in Th1 (black
bars) or Th2 (white bars) conditions in presence or absence of
hrAnx-A1 and then restimulated with platebound anti-CD3 to measure
Th1 or Th2 cytokine production. FIG. 7B shows the results when nave
lymph node T cells from Anx-A1.sup.+/+ or Anx-A1.sup.-/- mice were
differentiated in vitro in Th1 (first and second column graphs from
the left) or Th2 (third and fourth column graphs from the left)
conditions and then restimulated with platebound anti-CD3 to
measure Th1 or Th2 cytokine production.
[0089] FIG. 8A and 8B paw volume (FIG. 8A) and clinical score (FIG.
8B) of DBA mice treated with PBS or hrAnx-A1 for 12 days during the
immunization phase of the collagen-induced arthritis (CIA) model.
FIG. 8C is an analysis of Anx-A1 expression in CD4+ cells of
healthy control volunteers (HC) or rheumatoid arthritis (RA)
patients. FIG. 8D shows an immunohistochemical analysis of Anx-A1
expression in synovial tissue from RA patients.
[0090] FIG. 9 shows the effects of full length hrAnx-A1 and the
N-terminal peptide Ac 2-26 on T cell activation.
[0091] FIGS. 10A and 10B show the expression of Anx-A1 in human
atherosclerotic plaques Immunohistochemical analysis of Anx-A1
expression with mouse monoclonal anti-human Anx-A1 antibody 1B
(FIG. 10A) or with nonimmune IgG (FIG. 10B) in carotid
atherosclerotic plaques removed from patients during carotid
endarteretomy surgery. Photographs are from a single patient and
representative of six different patients with similar
conditions.
[0092] FIGS. 11A-11C show expression of Anx-A1 in murine
atherosclerotic plaques. FIG. 11 shows immunofluorescence
visualization of Anx-A1 in ApoE.sup.-/- mice aortic sinus (FIG. 11A
and FIG. 11B) and brachiocefalic artery (FIG. 11C). Sections were
stained with Dapi to locate nuclei. Results illustrated are from a
single experiment and are representative of three separate
experiments. Original magnification: .times.200 (FIG. 11A and FIG.
11B), .times.400 (FIG. 11C).
[0093] FIG. 12 shows expression of Annexin-1 in Systemic Lupus
Erythematosus (SLE) patients. RT-PCR (upper panel) and Western Blot
(lower panel) analysis of Anx-A1 expression in T cells from healthy
(Control) or Systemic Lupus Erythematosus (SLE) patients. The
numbers in the figure indicate the volume (p1) of cDNA or the
amount (pg) of proteins obtained from the same number
(2.times.10.sup.6) of T cells collected from healthy (Control) or
Systemic Lupus Erythematosus (SLE) patients.
[0094] FIG. 13 shows inhibition of activation of the T cell
receptor (TCR), measured in terms of interleukin-2 (IL-2)
production, in human peripheral T cells from one donor incubated
with a neutralising monoclonal antibody raised against human
recombinant annexin-1 (anti-AnxA1 mAblA).
[0095] FIG. 14 shows inhibition of activation of the T cell
receptor (TCR), measured in terms of interleukin-2 (IL-2)
production, in human peripheral T cells from a different donor
incubated with a neutralising monoclonal antibody raised against
human recombinant annexin-1 (anti-AnxA1 mAblA).
[0096] FIGS. 15A and 15B show spinal cord sections from C57BL/6
mice immunized with MOG.sub.35-55 and CFA and from which spinal
cords removed at day 12 (score 0), day 18 (score 2) and day 20
(score 4). The sections were stained with hematoxylin and eosin
(H&E, FIG. 15A) or anti-AnxA1 (FIG. 15B). For each staining,
the right panels (20.times.) show a higher magnification of an area
of the left panels (4X). Results representative of 3
experiments.
[0097] FIG. 16 shows spinal cord sections from C57BL/6 mice
immunized with MOG.sub.35-55 and CFA and from which spinal cords
removed at day 20 (score 4). The sections were stained with
anti-AnxA1 and anti-CD3 (A) or anti-F4/80 (B). The right panels
show an overlay of the two single stainings on the right. Results
representative of 3 experiments.
[0098] FIG. 17 shows the results from a study in which C57BL/6 mice
were immunized with MOG.sub.35-55 and CFA and monitored daily for
signs and symptoms of EAE (A) or weight gain/loss (B) for 23 days.
Results are means.+-.SEM (n=10/group). ** p<0.01, representative
of 3 experiments.
[0099] FIG. 18 shows the incorporation of .sup.3H-Thymidine (A) and
the production of IL-2 (B) of lymph node cells obtained from
AnxA1.sup.+/+ and AnxA1.sup.-/- mice immunized with MOG.sub.35-55
and CFA and sacrificed after 14 days. Cells were stimulated with
MOG.sub.35-55 for 48 hours and pulsed with 1 .mu.Ci
.sup.3H-Thymidine for 12 hours. Cell supernatants were used to
measure IL-2 production. Results are means.+-.SEM (n=4/group). *
p<0.05, ** p<0.01, representative of 3 experiments.
[0100] FIG. 19 shows the total cell number of spleen (A) and lymph
node (B) cells obtained from AnxA1.sup.+/+ and AnxA1.sup.-/- mice
immunized with MOG.sub.35-55 and CFA and sacrificed after 14 days.
C and D show the cytofluorimetric analysis of lymph node cells with
anti-CD4 FITC and anti-CD8 PE. Results are means.+-.SEM
(n=10/group). ** p<0.01, representative of 3 experiments.
[0101] FIG. 20 shows levels of (A) IFN-.gamma., (B) IL-2, (C)
TNF-.alpha. and (D) IL-17 in the cell supernatants of lymph node
cells obtained from AnxA1.sup.+/+ and AnxA1.sup.-/- mice immunized
with MOG.sub.35-55 and CFA and sacrificed after 14 days. Cells were
stimulated with the indicated concentration of MOG.sub.35-55 for 4
days and the supernatants used for cytokine ELISA. Results are
means.+-.SEM (n=4/group). * p<0.05, ** p<0.01, representative
of 3 experiments.
[0102] FIG. 21 shows haematoxylin-eosin staining of spinal cord
sections obtained from AnxA1.sup.+/+ (A) and AnxA1.sup.-/- (B) mice
immunized with MOG.sub.35-55 and CFA and sacrificed after 22 days.
For each staining, the right panels (20X) show a higher
magnification of an area of the left panels (4X). Consecutive
sections were stained with anti-CD3 (C) or anti-F4/80 (D). Pictures
are representative of three separate experiments with similar
results.
[0103] FIG. 22 shows FACS analysis of CD3 (A) and F4/80 (B)
positive mononuclear cells recovered by Percoll gradient of spinal
cord homogenates obtained from AnxA1.sup.+/+ and AnxA1.sup.-/- mice
immunized with MOG.sub.35-55 and CFA and sacrificed after 14 days.
The dot plots and histograms are from a single mouse and
representative of 2 experiments with n=4 mice. The numbers in the
histograms indicate the percentage of CD3.sup.+ and F4/80.sup.+
cells.
EXAMPLES 1 TO 10
[0104] Materials and Methods
[0105] Reagents
[0106] Anti-mouse CD3 (clone 145-2C11), anti-mouse CD28 (clone
37.51), anti-human CD3 (clone OKT3), anti-human CD28 (clone
CD28.2), PE-conjugated anti-CD69 (clone H1.2F3), FITC-conjugated
anti-CD25 (clone PC61.5), murine IL-2, IL-4, IFN-.gamma., IL-12,
antiIL-4 (clone 11B11), and antiIFN-.sub.7 (clone XMG1.2) were
purchased from eBioscience (Wembley, United Kingdom).
Endotoxin-free human recombinant Anx-A1 (hrAnx-A1) was prepared as
described. In some experiments, we used denatured hrAnx-A1
(heat-inactivated at 95.degree. C. for 5 minutes) as positive
control. Unless otherwise specified, all the other reagents were
from Sigma-Aldrich (St Louis, Mo.).
[0107] Mice
[0108] BALB/C, C57/BL6 and DBA/1 male mice were obtained from
Charles River Laboratories (Wilmington, Mass.). Annexin 1 null mice
on BALB/C were generated in our lab and bred in pathogen-free
conditions in our animal facilities. All mice used in these studies
were between 6 and 8 weeks old. Animal work was performed according
to United Kingdom Home Office regulations (Guidance on the
Operation of Animals, Scientific Procedures Act 1986) and along the
directives of the European Union.
[0109] Isolation of cells from Patients
[0110] Peripheral blood mononuclear cells (PBMCs) were prepared
from peripheral blood using Ficoll density centrifugation
(Ficoll-Paque Plus; Amersham Biosciences, Freiburg, Germany).
CD4.sup.+ cells were selected from peripheral blood using positive
selection. Briefly, peripheral blood was subjected to Ficoll
density centrifugation (Ficoll-Paque Plus; Amersham Biosciences).
Adherent cells were removed from the mononuclear cells by adherence
to serum-coated plastic. Nonadherent cells were incubated with
mouse anti-human CD4 antibody (RFT4), washed in buffer
(phosphate-buffered saline [PBS], 0.5% bovine serum albumin [BSA],
2 mM EDTA pH 7.2) and incubated with goat antimouse antibody
conjugated to a magnetic bead (Miltenyi Biotec, Auburn, Calif.).
Cells were run through a MACS column (Miltenyi Biotec) and
CD4.sup.+ cells were collected. The purity of the cells was
assessed by flow cytometry. The median percentage of CD3.sup.+
CD4.sup.+ cells following the 10 depletions was 98% (range,
97%-99.3%). Remaining cells were resuspended in lysis buffer
(Ambion, Huntingdon, United Kingdom).
[0111] Cell Culture
[0112] Primary murine T cells were prepared from lymph nodes by
negative selection. Briefly, axillary, inguinal, and mesenteric
lymph nodes were teased apart to make a single cell suspension,
then washed and layered over Ficoll. The buffy coat was washed 2
times and then incubated with the antibody mix and the magnetic
beads following the manufacturer's instructions (mouse T-cell
negative isolation kit; Dynal, Bromborough, United Kingdom). In
some experiments, cells were further purified to obtain naive
CD62L.sup.+ CD4.sup.+ T cells by using the Miltenyi Biotec
CD62L.sup.+ CD4.sup.+ T-cell isolation kit. Th0 conditions were
created by T cells for 4 days in 6-well plates precoated with
anti-CD3 (5 ng/mL) and anti-CD28 (5 ng/mL) in complete RPMI medium
(10% fetal calf serum [FCS], 2 mM L-glutamine, and 100 units
mL.sup.-1 gentamycin) containing murine IL-2 (20 U/mL). Th1
conditions were created with murine IL-12 (3.4 ng/mL; eBioscience),
IL-2 (20 U/mL; eBioscience), and anti-IL4 (clone 11B11; 2 ng/mL).
Th2 conditions were created with IL-4 (3000 U/mL; Peprotech, Rocky
Hill, N.J.), IL-2 (20 U/mL), and antiIFN-.sub.7 (clone XMG1.2; 2
ng/mL). Jurkat cells were obtained from ATCC (Manassas, Va.) and
were cultured in complete RPMI medium.
[0113] Flow Cytometric Analysis
[0114] Purified lymph node T cells were pretreated with human
recombinant Anx-A1 for 2 hours at 37.degree. C. in Eppendorf tubes
and then stimulated with plate-bound anti-CD3 and anti-CD28 as
indicated in the figures. After 16 hours, the cells were stained
with PE-conjugated anti-CD69 (clone H1.2F3) and FITC-conjugated
anti-CD25 (clone PC61.5) diluted in FACS buffer (PBS containing 1%
FCS and 0.02% NaN.sub.2). Intact cells were gated by using forward
and side scatter and were analyzed with the CellQuest program
(Becton Dickinson, Franklin Lakes, N.J.) on a FACScan flow
cytometer. To analyze FPRL-1 expression, human peripheral blood T
cells were stimulated with plate-bound anti-CD3 and anti-CD28 for
different times and thereafter stained with mouse anti-human FPRL-1
(clone 6C7-3-A; 5 ug/mL), followed by FITC-conjugated antibody.
[0115] Cell Proliferation Assay
[0116] Purified lymph node T cells (105 cells/mL) were incubated
with medium alone or with different concentrations of hrAnx-A1 for
2 hours at 37.degree. C. in Eppendorf tubes. Thereafter, aliquots
of 200-0_, cell suspension were stimulated by plate-bound anti-CD3
and anti-CD28 for 24 hours in 96-well plates. After 18 hours,
cultures were pulsed for 8 hours with 1 uCi (3.7 x 10.sup.4 Bg)
[.sup.3H]-thymidine (Amersham Pharmacia Biotech, Piscataway, N.J.)
and incorporated radioactivity was measured by automated
scintillation counter (Packard Instruments, Pangbourne, United
Kingdom).
[0117] Electromobility Shift Assays
[0118] Nuclear extracts were harvested from 3.times.10.sup.6 to
5.times.10.sup.6 cells according to previously described protocols.
Nuclear extracts (3 .mu.g to 5 .mu.g) were incubated with 1 .mu.g
(for NFAT) or 2 .mu.g (for NF-M3 and AP-1) of poly (dI:dC) in 20
.mu.L, binding buffer with .sup.32P end-labeled, double-stranded
oligonucleotide probes (5.times.10.sup.5 cpm), and fractionated on
a 6% polyacrylamide gel (29:1 cross-linking ratio) in 0.5% TBE for
2.5 hours at 150 V. The NF-.kappa.B and AP-1 binding buffer
(10.times.) contained 100 mM Tris-HCl, (pH 7.5), 500 mM NaCl, 10 mM
EDTA, 50% glycerol, 10 mg/mL albumin, 30 mM GTP, 10 mM DTT. The
NFAT binding buffer (10.times.) contained 100 mM Hepes (pH 7.9),
500 mM KCl, 1 mM EDTA, 1 mM EGTA, 50% glycerol, 5 mg/mL albumin, 1%
Nonidet P-40, 10 mM DTT. The NF-M3 and AP-1 double-stranded
oligonucleotide probes were from Promega and the NFAT was from
Santa Cruz Biotechnology (Santa Cruz, Calif.).
[0119] Western Blotting Analysis
[0120] Lymph node T cells were incubated as indicated in the
figures. After incubation at 37.degree. C. for various time
periods, cells were lysed in ice-cold lysis buffer (1% NP-40, 20 mM
Tris pH 7.5, 150 mM NaCl, 1 mM MgCl.sub.2, 1 mM EGTA, 0.5 mM PMSF,
1 .mu.M aprotinin, 1 .mu.M leupeptin, 1 .mu.M pepstatin, 50 mM NaF,
10 mM Na.sub.4P.sub.2O.sub.7, and 1 mM NaVO.sub.4, 1 mM
B-glycerophosphate). The cell lysates were centrifuged at 13/226g
(13 000) rpm for 5 minutes at 4.degree. C. and the supernatants
were collected and subjected to electrophoresis on SDS-10%
polyacrylamide gel. After transfer, the membranes were incubated
overnight with antibodies diluted in Tris-buffered saline solution
containing Tween-20 (TTBS: 0.13 M NaCl; 2.68 mM KCl; 0.019 M
Tris-HCl; 0.001% vol/vol Tween-20; pH 7.4) with 5% nonfat dry milk
at 4.degree. C. For the experiments with anti-pERK1/2 and anti-Akt,
the TTBS buffer was supplemented with 50 mM NaF and bovine serum
albumin (5%) was used instead of milk. For each condition, extract
equivalents obtained from the same number of cells were used
Immunoblotting and visualization of proteins by enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech) were performed
according to the manufacturer's instructions. To obtain cytosolic
and membrane fractions, cells were first collected and washed in
ice-cold PBS and then centrifuged briefly for 2 minutes at 300 g.
The resultant cell pellet was lysed in lysis buffer (20 mM
Tris-HCl, pH 7.5; protease inhibitors as listed in the lysis
buffer) and passed through a 25-gauge needle at least 5 times to
ensure sufficient lysis. The suspension was then centrifuged for 2
minutes at 300 g, the supernatant collected, and centrifuged again
for 45 minutes at 800g (4.degree. C.). At this stage the
supernatant (cytosolic fraction) was collected and the pellet
(membrane fraction) resuspended in lysis buffer containing 1%
(vol/vol) Triton X-100. All fractions were kept on ice throughout
the experiments.
[0121] Cytokine ELISA
[0122] For Th1/Th2 cytokine production analysis, Th0/Th1/Th2 cells
(10.sup.6/mL) obtained after 4-day differentiation in skewing
conditions and 1 day of resting in complete RPMI medium, were
stimulated with plate-bound anti-CD3 (5 .mu.g/mL) for 8 h in
24-well plates. Culture supernatants were collected and analyzed
for IFN-.gamma., IL-2, IL-4 and IL-10 content by using Th1/Th2
panel ELISA kit (eBioscience). The IL-13 ELISA kit was also
purchased from eBioscience.
Example 1
Effect of Human Recombinant Annexin-1 (hrAnx-A1) on T Cell
Activation
[0123] Murine naive lymph nodes T cells were stimulated with 5.0
(v), 2.5 (G) and 1.25 (T) .mu.g/ml of anti-CD3/CD28 in the absence
of or in the presence of different concentrations of hrAnx-A1 for
24 hrs and were then pulsed with .sup.3H-thymidine to measure
proliferation. The results are shown in FIG. 3A.
[0124] FIG. 3B shows IL-2 production from primary murine nave lymph
node T cells stimulated with anti-CD3/CD28 (1.25 .mu.g/ml) in the
absence of or in the presence of different concentrations of
hrAnx-A1 for 24 hrs.
[0125] Murine nave lymph node T cells were stimulated with
anti-CD3/CD28 at a concentration of 1.25 .mu.g/ml (left column),
2.5 .mu.g/ml (middle column) and 5.0 .mu.g/ml (right column), in
the absence of (upper panels) or in the presence of (lower panels)
hrAnx-A1 (600nM) for 12 hrs and then analyzed for CD25 and CD69
expression by FACS. The results are shown in FIG. 3C.
[0126] In FIG. 3D, murine nave lymph node T cells were stimulated
with the indicated concentration of anti-CD3/CD28 in the presence
of 150 (.times.), 300 (.OMEGA.) and 600 (.mu.) nM of hrAnx-A1 for
12 hours and then analysed for CD25 (left graph) and CD69 (right
graph) expression by FACS.
[0127] In all of the experiments values are mean.+-.S.E. of n=3-4
mice. *P<0.05; **P<0.01.
[0128] The results show that pre-treatment of murine naive CD4+
primary cells with hrAnx-A1 followed by activation with different
concentrations of anti-CD3/CD28 augmented cell proliferation (FIG.
3A), IL-2 production (FIG. 3B) and cell surface expression of CD25
and CD69 (FIGS. 3C and D).
Example 2
Endogenous Anx-A1 Modulates T Cell Proliferation
[0129] FIG. 4 shows that: (A) anti-CD3 (5.0 mg/ml) (B)
anti-CD3/CD28 (5.0 mg/ml) or (C) PMA (20 ng/ml) and Ionomycin (2
ng/ml) induced proliferation of wild type and Anx-A1 deficient T
cells, expressed as a percentage of .sup.3H-thymidine incorporation
compared to control unstimulated T cells. In some experiments,
cells were also activated in presence of mouse recombinant IL-2 (20
ng/ml). Values are mean.+-.S.E. of n=4-5 t t P<0.01 vs IL-2
stimulated Anx-A1.sup.+/+ cells; **P<0.01 vs anti-CD3 or
anti-CD3/CD28 or PMA/Ionomycin stimulated Anx-A1.sup.+/+ cells;
.sctn..sctn.P<0.01 vs anti-CD3 or anti-CD3/CD28 or PMA/Ionomycin
stimulated Anx-A1.sup.-/- cells.
[0130] FIG. 4D shows IL-2 production from nave lymph node T cells
stimulated with anti-CD3, anti-CD3/CD28 (5.0 mg/ml) or PMA (20
ng/ml) and Ionomycin (2 ng/ml) for 24 h. Values are mean.+-.S.E. of
n=4-5 mice. **P<0.01.
[0131] The results show that stimulation of Anx-A1.sup.+/+ or
Anx-A1.sup.-/-T cells with anti-CD3, anti-CD3/CD28 or PMA/ionomycin
showed a decrease rate of .sup.3H-thymidine incorporation (FIGS.
4A, 4B and 4C, respectively) and IL-2 production (FIG. 4D) in the
Anx-A1 deficient T cells compared to control unstimulated T
cells.
Example 3
Activation of AP-1, NF-.kappa.B and NFAT in Presence or Absence of
Anx-A1
[0132] Investigations were carried out into how exogenous and
endogenous Anx-A1 modulates T cell activation. The three major
transcriptional activators of T cells, namely Activator Protein-1
(AP-1), Nuclear Factor-.kappa.B (NF-.kappa.B) and Nuclear Factor of
Activated T cells (NFAT) were analysed in cells stimulated in
presence of hrAnx-A1.
[0133] FIG. 5A is an Electrophoretic Mobility Shift Assay for AP-1,
NF-.kappa.B and NFAT activation in T cells stimulated with
anti-CD3/CD28 (1.25 .mu.g/ml) in presence or absence of the
indicated concentration of hrAnx-A1 . FIG. 5B shows a comparison of
the activation of AP-1, NF-.kappa.B and NFAT in Anx-A1.sup.+/+ and
Anx.sup.-/-T cells stimulated with anti-CD3/CD28 (5.0
.mu.g/ml).
[0134] The results demonstrated increased activation of all three
transcription factors (FIG. 5A). Conversely, Anx-A1.sup.-/- T cells
showed a decreased activation of these transcription factors
compared to their control littermates (FIG. 5B).
Example 4
Externalization of FPRL-1 and Anx-A1 in T Cells
[0135] We investigated whether T cells express the receptor for
Anx-A1, the Formyl Peptide Receptor Like-1 (FPRL-1). FACS staining
of unstimulated human Peripheral Blood T (PBT) cells with specific
monoclonal anti-FPRL-1 antibody demonstrated no receptor
expression. However, stimulation with anti-CD3/CD28 induced the
externalization of FPRL-1 within 1 hour followed by a stable steady
state expression on the cell surface (FIG. 6A). Interestingly, a
similar pattern was observed for Anx-A1. Thus, analysis of Anx-A1
distribution in human PBT demonstrated that the protein is evenly
distributed between the cytosol and membrane. However, when cells
were stimulated with anti-CD3/CD28, accumulation of Anx-A1 at the
membrane was observed.
[0136] The protein is then exported to the outer side of the
membrane and released into the extracellular milieu. Consistent
with this model, when we immunoprecipitated Anx-A1 from the culture
supernatant of human PBT stimulated with anti-CD3/CD28, we observed
an increased release of Anx-A1 compared to control unstimulated
cells (FIG. 6B). Collectively, these observations demonstrate that
signalling through the TCR increases Anx-A1 release, concomitant
with the upregulation of its receptors.
[0137] In physiological conditions, the Anx-A1/FPRL-1 integrates
with the TCR to modulate the strength of TCR signalling. However,
in pathological conditions, such as in RA or systemic lupus
erythematosus (unpublished data), where the protein is expressed at
higher levels this could lead to increased T cell activation due to
lower threshold of TCR signalling (FIG. 6C).
Example 5
Exogenous and Endogenous Anx-A1 modulates Th1/Th2
Differentiation
[0138] Recent studies have postulated that the strength of TCR
signalling influences T cell lineage commitment to Th1 or Th2
effector cells. Given the increased or decreased TCR signalling in
T cells treated with hrAnx-A1 (FIGS. 3 and 5A) or Anx-A1.sup.-/-
cells (FIGS. 4 and 5B), we then sought to determine whether
different levels of Anx-A1 would influence T cell differentiation
into Th1 or Th2 cells.
[0139] Naive lymph node T cells were differentiated in vitro in Th1
(black bars) or Th2 (white bars) conditions in presence or absence
of hrAnx-A1 (600 nM) and then restimulated with platebound anti-CD3
(5.0 mg/ml) for 8 h to measure Th1 or Th2 cytokine production. The
results are shown in FIG. 7A. Values are mean.+-.S.E. of n=4-5
mice. **P<0.01
[0140] As shown in FIG. 7A, differentiation of nave T cells
(CD441o, CD62Lhi) in Th1 (anti-CD3/CD28, IL2, IL12 and anti-IL4) or
Th2 (anti-CD3/CD28, IL2, IL4 and anti-IFN.gamma.) conditions in the
presence of hrAnx-A1 increased IL2 and IFN.gamma. production with a
concomitant decrease of IL4 and IL10 release upon anti-CD3
re-stimulation.
[0141] Naive lymph node T cells from Anx-A1.sup.+/+ or
Anx-A1.sup.-/- mice were differentiated in vitro in Th1 (first and
second graphs from the left) or Th2 (third and fourth graphs from
the left) conditions and then restimulated with platebound anti-CD3
(5.0 mg/ml) for 8 h to measure Th1 or Th2 cytokine production. The
results are shown in FIG. 7B. Values are mean.+-.S.E. of n=4-5
mice. **P<0.01
[0142] As shown in FIG. 7B, similar findings were also obtained
with respect to the endogenous protein: analysis of Th1/Th2
cytokine production in differentiated Th1/Th2 cells from
Anx-A1.sup.+/+ or Anx-A1.sup.4- mice yielded higher levels of IL2
and IFN.gamma. wild-type mice compared with knockout mice, with
opposite profiles for IL4 and IL13 production.
Example 6
Anx-A1 and Rheumatoid Arthritis
[0143] To prove that hrAnx-A1 increased T cell activation in vivo,
we used a mouse model of chronic autoimmune disease, the
collagen-induced arthritis (CIA) model in DBA mice. Mice were
injected with hrAnx-A1 daily for 12 days after immunization with
collagen (time during which nave cells differentiate in Th effector
cells) and thereafter the progression of the disease upon antigen
challenge was analyzed. FIG. 8 shows paw volume (FIG. 8A) and
clinical score (FIG. 8B) of the mice treated with PBS (100 .mu.l)
or hrAnx-A1 (1 pg s.c. twice a day). Synchronization of disease
onset was obtained by boosting with collagen on day 21, and
clinical signs were evident from day 22 (day 1 of the onset of the
diseases). Values are mean.+-.S.E. of n=6-8 mice. Groups were
compared using the Mann-Whitney test. *P<0.01
[0144] As can be seen from FIGS. 8A and 8B, treatment of mice with
hrAnx-A1 exacerbated the signs and symptoms of arthritis compared
to mice treated with PBS vehicle, confirming that high levels of
Anx-A1 influence T cell activation and differentiation and that
these effects influence the disease development in a mouse model of
RA.
[0145] To investigate the clinical relevance of these studies
Anx-A1 expression was analyzed in CD4+ peripheral T cells and
synovial CD3+ cells from RA patients. FIG. 8C shows the results.
The median values are indicated by horizontal lines and p values of
the Mann-Whitney test are shown.*P<0.01. As shown in FIG. 8C, RA
CD4+ cells express high levels of Anx-A1 mRNA and protein (data not
shown) compared with cells from healthy control volunteers
(HC).
[0146] Fluorescence immunohistochemistry was also carried out using
green and red fluorescent tagged secondary antiserum, as shown in
each panel of FIG. 8D. This immunohistochemical analysis of Anx-A1
expression in the synovial tissue of RA patients revealed a high
degree of colocalization with CD3+ cells. Therefore, considering
that CD4 cells from RA patients express higher levels of Anx-A1, it
can be concluded that the dysregulated expression of this protein
might contribute to the development of this disease.
Example 7
Effects of Full Length hrAnx-A1 and the N-Terminal Peptide Ac 2-26
on T Cell Activation
[0147] The effects of an N-terminal peptide of hrAnx-A1 (peptide Ac
2-26) and of full length hrAnx-A1 on T cell activation were
investigated. IL-2 production from murine nave lymph node T cells
was stimulated with 0.6, 1.25 or 2.5 .mu.g/ml of anti-CD3/CD28 in
the presence or absence of full length hrAnx-A1 (300nM) or the
Anx-A1 derived N-terminal peptide Ac.2-26 (100pM) for 24 hrs.
[0148] It was found that the N-terminal peptide Ac.2-26 retains
most of the biological activity of the full-length protein, i.e.
increased IL-2 production (FIG. 9) and T cell proliferation (data
not shown).
Example 8
Anx-A1 and Atherosclerosis
[0149] To investigate if Anx-A1 is expressed in human
atherosclerotic plaques, sections of carotid atherosclerotic
plaques removed from patients during carotid endarteretomy surgery
were stained with a mouse monoclonal anti human Anx-A1 antibody
(mAb 1B). The production of this antibody is described in Pepinsky
et al FEBS Letters 261: 247-252, 1990. Briefly, BALB/c mice were
immunized with an intraperitoneal injection of annexin-1 (referred
to as lipocortin-1 in Pepinsky et al) in complete Freund's
adjuvant. The animals were boosted on days 14 and 28 with annexin-1
in incomplete Freund's adjuvant. After 6 weeks, test bleeds were
taken and screened for antibodies that blocked annexin-1 activity.
Spleen cells from mice whose antisera displayed anti-annexin
activity were fused with SP3.times.Ag8 cells for hybridoma
production. Hybridoma culture supernatants were assayed for
antibodies that could precipitate radiolabeled annexin-1, and
hybridomas producing antibodies that precipitated over 50% of the
input counts were subcloned by limiting dilution. The most
promising lines were grown as ascites in pristane-primed mice and
the monoclonal antibodies were affinity-purified on protein A
sepharose, using the Pierce binding and elution buffer systems.
[0150] As shown in FIG. 10, a compact and clear staining for Anx-A1
could be observed within the plaque confirming that the
inflammatory infiltrate within these tissues expresses high levels
of Anx-A1.
[0151] Similar analysis was also carried out in ApoE.sup.-/- mice.
Localization of Anx-A1 in the aortic sinus and the brachiocefalic
artery (BCA) of 10 month old ApoE.sup.-/- mice were performed by
confocal microscopy to determine the expression and spatial
distribution of Anx-A1. Non atherosclerotic arterial tissue lacked
immunoreactive Anx-A1 (data not shown). In contrast,
atherosclerotic plaque from both aortic sinus and BCA stain
strongly for Anx-A1 (FIG. 11).
[0152] A clear immunoreactivity for Anx-A1 was detected in
proximity of the fibrous cap in both aortic sinus (FIG. 11A) and
BCA (FIG. 11B) and in proximity of the necrotic core of the plaque
in the aortic sinus (FIG. 11B). These results demonstrate that
Anx-A1 is expressed in both human and murine atherosclerotic
plaques and suggest that its expression could potentially influence
plaque stability.
Example 9
Anx-A1 and Systemic Lupus Erythematosus (SLE)
[0153] Clinical studies on the biological functions of Annexin-1
have associated the presence of autoantibodies against this protein
with the development of autoimmune diseases including systemic
lupus erythemathosus (SLE), rheumatoid arthritis and inflammatory
bowel disease. In light of these findings we hypothesized that the
generation of these autoantibodies might be due to an uncontrolled
expression of Annexin-1 in these patients. To verify this
hypothesis, the expression level of Annexin-1 in T cells collected
from healthy volunteers and SLE patients was analyzed. Annexin-1
mRNA and protein were expressed at a much more marked level in the
SLE T cells (FIG. 12). Thus, these results support the hypothesis
that increased Annexin-1 expression in SLE T cells, and therefore
in T cells from patients with other autoimmune pathologies, might
be responsible for the increased levels of Th1 cytokines described
in these pathologies, thereby representing a risk factor for the
development of autoimmune diseases.
Example 10
Inhibition of T Cell Activation by Anti-Anx-A1 Antibodies
[0154] Purified human peripheral blood T cells were incubated with
a mixture of anti-CD3 and anti-CD28 antibodies (5 mg/ml) to
activate the T cell receptor (TCR): this occurred as demonstrated
in FIG. 13 by the remarkable production of interleukin-2 (IL-2), a
cytokine central to T cell activation and differentiation.
[0155] Cells were then incubated with different concentrations
(1.0, 0.1, 0.01 and 0.001 micrograms/ml) of a neutralising mouse
monoclonal antibody raised against human recombinant annexin 1 (mAb
1A). The production of this antibody is described in Pepinsky et al
FEBS Letters 261: 247-252, 1990 and in Example 8.
[0156] Treatment with mAblA produced a concentration dependent
inhibition of IL-2 production (FIG. 13) and cell proliferation
(data not shown). IgG was used as a control at concentrations of
1.0, 0.1, 0.01 and 0.001 micrograms/ml and was without efficacy at
all concentrations. The results showed that blockade of annexin-1
effects seemed to be more effective at lower concentrations of the
specific monoclonal antibody mAblA.
[0157] In all cases, data are mean.+-.SE of triplicate
measurements. *P<0.01.
[0158] Purified human peripheral blood T cells from a different
donor were then incubated with a mixture of anti-CD3 and anti-CD28
antibodies at a different concentration (1 mg/ml) to activate the T
cell receptor (TCR). Again, this occurred as demonstrated in FIG.
14 by the production of interleukin-2 (IL-2), but at a lower level
due to the lower concentration of anti-CD3 and anti-CD28 antibodies
used.
[0159] Again, cells were then incubated with different
concentrations (1.0, 0.1, 0.01 and 0.001 micrograms/ml) of the
neutralising mouse monoclonal antibody raised against human
recombinant annexin 1 (mAb 1A). Treatment with mAblA produced a
concentration dependent inhibition of IL-2 production (FIG. 14).
IgG was used as a control at a concentration of 1.0 micrograms/ml
and was without efficacy. Again, the results showed that blockade
of annexin-1 effects seemed to be more effective at lower
concentrations of the specific monoclonal antibody mAb 1A, except
with a concentration of 1.0 micrograms/ml of mAblA.
[0160] In all cases, data are mean.+-.SE of triplicate
measurements. *P<0.01.
[0161] These experiments demonstrate that endogenous annexin 1
promotes T cell activation in the presence of a specific stimulus.
In addition, blockade of the annexin 1 pathway attenuated T cell
activation by up to 50%. The fact that inhibition reached a maximum
of around 50% suggests that "normal and housekeeping" immunity
would not be affected by treatment of T cell-mediated diseases
using a molecule which specifically binds to Anx-A1, as
claimed.
Example 11
Anx-A1 and Multiple Sclerosis (MS)
[0162] Materials and Methods
[0163] Reagents
[0164] The Myelin Oligodendrocyte Glycoprotein peptide
(MOG).sub.33-55 (MEVGWYRSPFSRVVHLYRNGK) was synthesized and
purified by Cambridge Research Biochemicals (Billingham, UK).
Complete Freund's adjuvant containing Mycobacterium tuberculosis
H37a was purchased from Difco while Bordetella pertussis toxin was
from Sigma-Aldrich Co (Poole, UK). Unless otherwise specified, all
the other reagents were from Sigma-Aldrich Co.
[0165] Mice
[0166] Male AnxA1 null mice were as previously described (Hannon et
al., Faseb J, 17: 253-255, 2003; Roviezzo et al., J. Physiol
Pharmacol 53: 541-553, 2002) (9-11 week old) and were backcrossed
on a C57BL/6 background for >10 generations and bred at
[0167] B&K animal care facilities (Hull, UK). Age and
gender-matched control C57BL/6 mice were used as control for all
experiments. Animals were kept under standard conditions and
maintained in a 12h/12h light/dark cycle at 22 .+-.1.degree. C. in
accordance with United Kingdom Home Office regulations (Animal Act
1986) and of the European Union directives.
[0168] Induction of EAE
[0169] Mice were immunized subcutaneously on day 0 with 300 .mu.l
of emulsion consisting of 300 pg of MOG.sub.35-55 in PBS combined
with an equal volume of CFA containing 300 .mu.g heat-killed M.
tuberculosis H37Ra. The emulsion was injected in both flanks and
followed by an intraperitoneal injection of B. pertussis toxin (500
ng/100 .mu.l) in 100 .mu.l of saline on days 0 and 2. Mice were
observed daily for signs of EAE and weight loss. Disease severity
was scored on a 6-point scale: 0=no disease; 1=partial flaccid
tail; 2=complete flaccid tail; 3=hind limb hypotonia; 4=partial
hind limb paralysis; 5=complete hind limb paralysis; 6=moribund or
dead animal
[0170] Cell Proliferation Assay
[0171] Lymph node cells (10.sup.5 cells/200 .mu.l) obtained from
mice immunized with MOG.sub.33-55 for 14 days were stimulated with
MOG.sub.33-55 (50-100 .mu.g/200 .mu.l) for 48 h in 96 well plates.
During the last 12 h, cultures were pulsed with 1 .mu.Ci of
[.sup.3H]-thymidine (Amersham Pharmacia Biotech, Buckinghamshire,
UK) and incorporated radioactivity was measured by automated
scintillation counter (Packard Instrument Company, Inc., Ill.,
US).
[0172] Cytokine ELISA
[0173] Lymph node cells (10.sup.6 cells/ml) obtained from mice
immunized with MOG.sub.33-55 for 14 days were stimulated with
MOG.sub.33-55 (100 .mu.g/ml) for 4 days. Cell supernatants were
collected and analyzed for IFN-.gamma., IL-2, IL-17A and
TNF-.alpha. content using ELISA kits (eBioscience, Dorset, UK)
according to manufacturer's instructions.
[0174] Isolation of Inflammatory Cell from the Spinal Cord
[0175] Mice were killed using CO.sub.2. The spinal cords were
expelled from the spinal column with PBS by hydrostatic pressure
using a syringe attached to a 21-gauge needle.
[0176] Tissues were cut in small pieces and passed through cell
strainer (70 nm; BD Falcon) using the plunger of a sterile 1 ml
syringe. The single cell suspension was centrifuged for 10 min at
390.times.g, resuspended in 20 ml of PBS containing 30% of Percoll
(Sigma) and overlayed onto 10 ml of PBS containing 70% Percoll.
After centrifugation at 390xg for 20 min, the mononuclear cells
were removed from the interphase, washed, and resuspended in FACS
buffer (PBS containing 1% FCS and 0.02% NaN.sub.2) for further
analysis.
[0177] Flow Cytometry
[0178] Cell samples from Percoll-purified spinal cord tissues or
Ficoll-purified lymph nodes were resuspended in FACS buffer
containing CD16/CD32 FcyIIR blocking antibody (clone 93;
eBioscience) for 30 min at 4.degree. C. Thereafter, cell
suspensions were labelled with the FITC-conjugated anti-CD3 (1:100;
clone 145 2C11) or anti-F4/80 (1:100; clone BMT) while lymph node
cells were stained with anti-CD4-FITC (1:500; clone L3T4) and
anti-CD8 (1:1000; clone Ly-2) for 30 min at 4.degree. C., prior to
analysis by FACS calibur using CellQuest software (Becton
Dickinson). At least 10.sup.4 cells were analyzed per sample, and
determination of positive and negative populations was performed
based on the staining attained with irrelevant IgG isotypes.
[0179] Histology
[0180] Spinal cord tissues were dissected and fixed in 4% neutral
buffered formalin for 48 hrs and then incubated with decalcifying
solution containing EDTA (0.1 mM in PBS) for 14 days prior to
paraffin embedding. Histological evaluation was performed on
paraffin-embedded sections sampled at various time points depending
on disease severity. Spinal cord sections (5 .mu.m) were
deparaffinized with xylene and stained with haematoxylin and eosin
(H&E) to assess inflammation. The staining for AnxA1 was
performed on frozen sections using anti-AnxA1 (dilution 1:500;
Zymed, Invitrogen) and anti-rabbit Ig horseradish peroxidase
(HRP)-conjugated antibodies (dilution 1:500; Dako). Double staining
for AnxA1 and CD3 or F4/80 was carried out as previously described
using FITC-conjugated anti-CD3 (1:100; clone 145 2C11) or
anti-F4/80 (1:100; clone BMT). Sections were also counterstained
with haematoxylin.
[0181] In all cases, a minimum >3 sections per animal were
evaluated. Phase-contrast digital images were taken using the Image
Pro image analysis software package.
[0182] Statistical Analysis
[0183] Prism software (GraphPad software) was used to run all the
tests. Statistical evaluations of cell frequency, proliferation and
cytokine production were performed using two-tailed, unpaired
Student's t tests. ANOVA were applied to analyze the EAE clinical
grading. A p value of <0.05 was considered to be statistically
significant. P-values lower than 0.05 were considered significant.
Data are presented as mean.+-.S.E.M of n samples per group.
[0184] Results
[0185] AnxA1 expression correlates with the severity of
experimental autoimmune encephalomyelitis (EAE)
[0186] The correlation between AnxA1 levels in the spinal cord
content and extent of infiltrating mononuclear cells in the CNS
were assessed in a mouse model of MS induced by immunization with
MOG.sub.35-55, EAE. MOG.sub.35-55-induced EAE is a model for
autoimmune demyelination of the CNS and has been widely used to
investigate pathogenic mechanisms responsible for the development
of MS. To this aim, spinal cords and brains of wild type mice
immunized with MOG.sub.35-55 peptide at different stages of the
diseases i.e. at day 12 (score 0), day 18 (score 2) and day 20
(score 4) were collected and immunohistochemistry for AnxA1 was
performed side by side with hematoxylin and eosin staining
[0187] As shown in FIG. 15, spinal cord tissues collected during
the induction phase of mice with no signs of disease showed a faint
staining for AnxA1 (score 0, FIGS. 15A and B, respectively).
However, with the onset of clinical signs and the appearance of
inflammatory infiltrates in the CNS, discrete patches of AnxA1
immunostaining were observed all around the meninges (score 2, FIG.
15A and B, respectively). As the disease progressed, an increase in
number of AnxA1-positive cellular infiltrate patches was observed
(score 4, FIG. 15A and B, respectively), suggesting that the
infiltration of inflammatory cells expressing high levels of AnxA1
is correlated with the severity of the disease.
[0188] To identify the cellular sources of AnxA1 immunoreactivity
in the spinal cord, double immunofluorescence staining of the
sections was performed with anti-AnxA1 and either anti-CD3 (marker
for T cells) or anti-F4/80 (marker for macrophages). A large number
of infiltrated T cells and macrophages was detected in the spinal
cord sections of mice at the peak of EAE (FIG. 16A and B, middle
panels, respectively). However, AnxA1 staining in the same sections
showed a partial co-localization with both T cells and macrophages
without particular preference for one or the other cell types (FIG.
16A and B, right panels, respectively).
[0189] AnxA1.sup.-/- Mice Develop an Impaired EAE
[0190] Since AnxA1 expression was upregulated at the peak of EAE,
the role of this protein on the development of EAE was
investigated. AnxA1.sup.+/+ and AnxA1.sup.-/- mice were immunized
s.c. with MOG.sub.35-55 peptide in CFA on day 0, and then injected
i.v. with B. pertussis toxin on both day 0 and day 2. Both
AnxA1.sup.+/+ and AnxA1.sup.-/- mice started to develop EAE from
day 12 after immunization, reaching peak disease around day 20.
However, AnxA1.sup.-/- mice had reduced levels of disease compared
to AnxA1.sup.+/+ (FIG. 17A). Interestingly, this was evident and
significant only at the later stage of the disease i.e. from day 18
to 23 and onwards.
[0191] Studies on animal models of EAE have demonstrated that the
acute phase of the disease coincides with weight loss, probably due
to anorexia and deficient fluid uptake. Weight measurement of
immunized mice correlated with the severity of the clinical score
and showed a reduced weight loss from day 18 onwards--in the
AnxA1.sup.-/- mice compared to AnxA1.sup.+/+ controls (FIG. 17B).
Further comparison of development of EAE in AnxA1.sup.+/+ and
AnxA1.sup.-/- mice showed a decrease in both the mortality rate and
maximum disease score, without differences in the incidence rate or
disease onset (Table 1).
TABLE-US-00002 TABLE 1 Clinical parameters of MOG.sub.35-55-induced
EAE in AnxA1.sup.+/+ and AnxA1.sup.-/- mice (mean .+-. SEM, n =
10/group) Onset day Max. score Mice Incidence.sup..sctn. Mortality
(mean .+-. SEM) (mean .+-. SEM) AnxA1.sup.+/+ 100% 33.3% 16.4 .+-.
2.3 5.7 .+-. 0.2 (10/10) (3/10) AnxA1.sup.-/- 100% 0% 15.9 .+-. 1.3
4.3 .+-. 0.1** (10/10) (0/10)** **p < 0.01, representative of 3
experiments .sup..sctn.EAE clinical score equal or greater than
1.
[0192] In vitro Recall Response to MOG.sub.35-55 in AnxA1.sup.-/-
Mice
[0193] T cells play a key role in the development of EAE and
AnxA1.sup.-/- T cells have an impaired capacity to respond to
anti-CD3/CD28 stimulation. In light of these findings, it was
investigated whether the decreased development of EAE in
AnxA1.sup.-/- mice was associated with a lower response to
antigen-stimulation. Lymph node cells from AnxA1.sup.+/+ and
Am(A1.sup.-/- mice, collected 14 days after immunization, were
stimulated in vitro with MOG.sub.35-55. ArD(A1.sup.-/- lymph node
cells showed a decreased rate of proliferation and produced lower
levels of IL-2 when stimulated with MOG.sub.35-55 compared to
wild-type mice (FIGS. 18A and B, respectively). Similar results
were obtained with splenocytes (data not shown).
[0194] These results on cell proliferation were mirrored in the
number of cells recovered from the spleen and the draining lymph
nodes of the immunized mice. The total cell count of
Ficoll-purified spleen and lymph node mononuclear cells from the
same animals, revealed a significant decrease in AnxA1.sup.-/- mice
compared to controls (FIG. 19A and B, respectively), with no
measurable changes in the percentages of CD4 or CD8 positive cells
(FIG. 19C and D, respectively).
[0195] Reduced MOG.sub.35-55-Specific Th1 and Th17 Cytokine
Responses in AnxA1.sup.-/- Mice
[0196] Studies using draining lymph node cells from MOG.sub.35-55
immunized C57/BL6 mice showed significant changes in Th1 and Th17
cytokine production. Analysis of cytokine production from
AnxA1.sup.-/- lymph node cells upon re-challenge with MOG.sub.35-55
for 96 h showed a decreased production of Th1 cytokines
IFN-.gamma., IL-2, and TNF-.alpha. compared to wild type cells
(FIG. 20A-C). Similarly, measurement of Th17 signature product
IL-17, revealed decreased levels of this cytokine in AnxA1.sup.-/-
compared to wild type (FIG. 20D).
[0197] T cell infiltration in the nervous system of AnxA1.sup.-/-
mice during EAE The reduced signs of EAE in AnxA1.sup.-/- mice from
day 18 onwards, prompted us to investigate whether there could be a
neuro-pathological correlate. The spinal cords of AnxA1.sup.+/+ and
AnxA1.sup.-/- treated mice, collected at day 18 or 22, were
analyzed for histological evidence of inflammation. It was found
that there were reduced numbers of immune cell infiltrates detected
in AnxA1.sup.-/- mice compared to AnxA1.sup.+/+ animals. (FIG. 21A
and B).
[0198] The reduced histological signs of inflammation in
AnxA1.sup.-/- mice were associated with a reduced number of CD3 and
F4/80 positive cells infiltrating the CNS (FIG. 21C and D,
respectively). These qualitative analyses were confirmed by FACS
measuring the percentages of CD3 and F4/80 positive leucocytes
isolated from day 18 spinal cord tissues. Consistent with the
immunohistochemistry results, Anx1.sup.-/- mice had about 60 and
80% less T cells and macrophages, respectively, compared to
AnxA1.sup.+/+ mice (FIG. 22A and B, respectively).
[0199] The results show that there is a remarkable accumulation of
Annexin-1 expressing cells in the spinal cord of mice at the peak
of the disease. There is therefore a correlation between Annexin-1
expression and the development of EAE.
[0200] In addition, the results show that Annexin-1 deficient mice
develop less severe EAE. Ablating Annexin-1 expression therefore
limits the development of EAE, a mouse model for MS.
Sequence CWU 1
1
27125PRTHomo sapiensACETYLATION(1)..(1) 1Ala Met Val Ser Glu Phe
Leu Lys Gln Ala Trp Phe Ile Glu Asn Glu 1 5 10 15 Glu Gln Glu Tyr
Val Gln Thr Val Lys 20 25 26PRTHomo sapiens 2Ala Met Val Ser Glu
Phe 1 5 36PRTHomo sapiens 3Met Val Ser Glu Phe Leu 1 5 46PRTHomo
sapiens 4Val Ser Glu Phe Leu Lys 1 5 56PRTHomo sapiens 5Ser Glu Phe
Leu Lys Gln 1 5 66PRTHomo sapiens 6Glu Phe Leu Lys Gln Ala 1 5
76PRTHomo sapiens 7Phe Leu Lys Gln Ala Trp 1 5 86PRTHomo sapiens
8Leu Lys Gln Ala Trp Phe 1 5 96PRTHomo sapiens 9Lys Gln Ala Trp Phe
Ile 1 5 106PRTHomo sapiens 10Gln Ala Trp Phe Ile Glu 1 5 116PRTHomo
sapiens 11Ala Trp Phe Ile Glu Asn 1 5 126PRTHomo sapiens 12Trp Phe
Ile Glu Asn Glu 1 5 136PRTHomo sapiens 13Phe Ile Glu Asn Glu Glu 1
5 146PRTHomo sapiens 14Ile Glu Asn Glu Glu Gln 1 5 156PRTHomo
sapiens 15Glu Asn Glu Glu Gln Glu 1 5 166PRTHomo sapiens 16Asn Glu
Glu Gln Glu Tyr 1 5 176PRTHomo sapiens 17Glu Glu Gln Glu Tyr Val 1
5 186PRTHomo sapiens 18Glu Gln Glu Tyr Val Gln 1 5 196PRTHomo
sapiens 19Gln Glu Tyr Val Gln Thr 1 5 206PRTHomo sapiens 20Glu Tyr
Val Gln Thr Val 1 5 216PRTHomo sapiens 21Tyr Val Gln Thr Val Lys 1
5 2221PRTArtificialMyelin Oligodendrocyte Glycoprotein peptide
(MOG)33-55 22Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val
Val His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20 23346PRTHomo sapiens
23Met Ala Met Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn 1
5 10 15 Glu Glu Gln Glu Tyr Val Gln Thr Val Lys Ser Ser Lys Gly Gly
Pro 20 25 30 Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser
Ser Asp Val 35 40 45 Ala Ala Leu His Lys Ala Ile Met Val Lys Gly
Val Asp Glu Ala Thr 50 55 60 Ile Ile Asp Ile Leu Thr Lys Arg Asn
Asn Ala Gln Arg Gln Gln Ile 65 70 75 80 Lys Ala Ala Tyr Leu Gln Glu
Thr Gly Lys Pro Leu Asp Glu Thr Leu 85 90 95 Lys Lys Ala Leu Thr
Gly His Leu Glu Glu Val Val Leu Ala Leu Leu 100 105 110 Lys Thr Pro
Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys 115 120 125 Gly
Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg 130 135
140 Thr Asn Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu
145 150 155 160 Lys Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser
Gly Asp Phe 165 170 175 Arg Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp
Arg Ser Glu Asp Phe 180 185 190 Gly Val Asn Glu Asp Leu Ala Asp Ser
Asp Ala Arg Ala Leu Tyr Glu 195 200 205 Ala Gly Glu Arg Arg Lys Gly
Thr Asp Val Asn Val Phe Asn Thr Ile 210 215 220 Leu Thr Thr Arg Ser
Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys Tyr 225 230 235 240 Thr Lys
Tyr Ser Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu 245 250 255
Lys Gly Asp Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr 260
265 270 Ser Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys
Gly 275 280 285 Val Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val
Ser Arg Ser 290 295 300 Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr
Gln Lys Met Tyr Gly 305 310 315 320 Ile Ser Leu Cys Gln Ala Ile Leu
Asp Glu Thr Lys Gly Asp Tyr Glu 325 330 335 Lys Ile Leu Val Ala Leu
Cys Gly Gly Asn 340 345 241041DNAHomo sapiens 24atggcaatgg
tatcagaatt cctcaagcag gcctggttta ttgaaaatga agagcaggaa 60tatgttcaaa
ctgtgaagtc atccaaaggt ggtcccggat cagcggtgag cccctatcct
120accttcaatc catcctcgga tgtcgctgcc ttgcataagg ccataatggt
taaaggtgtg 180gatgaagcaa ccatcattga cattctaact aagcgaaaca
atgcacagcg tcaacagatc 240aaagcagcat atctccagga aacaggaaag
cccctggatg aaacactgaa gaaagccctt 300acaggtcacc ttgaggaggt
tgttttggct ctgctaaaaa ctccagcgca atttgatgct 360gatgaacttc
gtgctgccat gaagggcctt ggaactgatg aagatactct aattgagatt
420ttggcatcaa gaactaacaa agaaatcaga gacattaaca gggtctacag
agaggaactg 480aagagagatc tggccaaaga cataacctca gacacatctg
gagattttcg gaacgctttg 540ctttctcttg ctaagggtga ccgatctgag
gactttggtg tgaatgaaga cttggctgat 600tcagatgcca gggccttgta
tgaagcagga gaaaggagaa aggggacaga cgtaaacgtg 660ttcaatacca
tccttaccac cagaagctat ccacaacttc gcagagtgtt tcagaaatac
720accaagtaca gtaagcatga catgaacaaa gttctggacc tggagttgaa
aggtgacatt 780gagaaatgcc tcacagctat cgtgaagtgc gccacaagca
aaccagcttt ctttgcagag 840aagcttcatc aagccatgaa aggtgttgga
actcgccata aggcattgat caggattatg 900gtttcccgtt ctgaaattga
catgaatgat atcaaagcat tctatcagaa gatgtatggt 960atctcccttt
gccaagccat cctggatgaa accaaaggag attatgagaa aatcctggtg
1020gctctttgtg gaggaaacta a 104125346PRTHomo sapiens 25Met Ala Met
Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn 1 5 10 15 Glu
Glu Gln Glu Tyr Val Gln Thr Val Lys Ser Ser Lys Gly Gly Pro 20 25
30 Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val
35 40 45 Ala Ala Leu His Lys Ala Ile Met Val Lys Gly Val Asp Glu
Ala Thr 50 55 60 Ile Ile Asp Ile Leu Thr Lys Arg Asn Asn Ala Gln
Arg Gln Gln Ile 65 70 75 80 Lys Ala Ala Tyr Leu Gln Glu Thr Gly Lys
Pro Leu Asp Glu Thr Leu 85 90 95 Lys Lys Ala Leu Thr Gly His Leu
Glu Glu Val Val Leu Ala Leu Leu 100 105 110 Lys Thr Pro Ala Gln Phe
Asp Ala Asp Glu Leu Arg Ala Ala Met Lys 115 120 125 Gly Leu Gly Thr
Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg 130 135 140 Thr Asn
Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu 145 150 155
160 Lys Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe
165 170 175 Arg Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp Arg Ser Glu
Asp Phe 180 185 190 Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg
Ala Leu Tyr Glu 195 200 205 Ala Gly Glu Arg Arg Lys Gly Thr Asp Val
Asn Val Phe Asn Thr Ile 210 215 220 Leu Thr Thr Arg Ser Tyr Pro Gln
Leu Arg Arg Val Phe Gln Lys Tyr 225 230 235 240 Thr Lys Tyr Ser Lys
His Asp Met Asn Lys Val Leu Asp Leu Glu Leu 245 250 255 Lys Gly Asp
Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr 260 265 270 Ser
Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys Gly 275 280
285 Val Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser
290 295 300 Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met
Tyr Gly 305 310 315 320 Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr
Lys Gly Asp Tyr Glu 325 330 335 Lys Ile Leu Val Ala Leu Cys Gly Gly
Asn 340 345 26204PRTHomo sapiens 26Met Asn Leu Ile Leu Arg Tyr Thr
Phe Ser Lys Met Ala Met Val Ser 1 5 10 15 Glu Phe Leu Lys Gln Ala
Trp Phe Ile Glu Asn Glu Glu Gln Glu Tyr 20 25 30 Val Gln Thr Val
Lys Ser Ser Lys Gly Gly Pro Gly Ser Ala Val Ser 35 40 45 Pro Tyr
Pro Thr Phe Asn Pro Ser Ser Asp Val Ala Ala Leu His Lys 50 55 60
Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr Ile Ile Asp Ile Leu 65
70 75 80 Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile Lys Ala Ala
Tyr Leu 85 90 95 Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu Lys
Lys Ala Leu Thr 100 105 110 Gly His Leu Glu Glu Val Val Leu Ala Leu
Leu Lys Thr Pro Ala Gln 115 120 125 Phe Asp Ala Asp Glu Leu Arg Ala
Ala Met Lys Gly Leu Gly Thr Asp 130 135 140 Glu Asp Thr Leu Ile Glu
Ile Leu Ala Ser Arg Thr Asn Lys Glu Ile 145 150 155 160 Arg Asp Ile
Asn Arg Val Tyr Arg Glu Glu Leu Lys Arg Asp Leu Ala 165 170 175 Lys
Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe Arg Asn Ala Leu Leu 180 185
190 Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe Gly 195 200
27115PRTHomo sapiens 27Met Ala Met Val Ser Glu Phe Leu Lys Gln Ala
Trp Phe Ile Glu Asn 1 5 10 15 Glu Glu Gln Glu Tyr Val Gln Thr Val
Lys Ser Ser Lys Gly Gly Pro 20 25 30 Gly Ser Ala Val Ser Pro Tyr
Pro Thr Phe Asn Pro Ser Ser Asp Val 35 40 45 Ala Ala Leu His Lys
Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr 50 55 60 Ile Ile Asp
Ile Leu Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile 65 70 75 80 Lys
Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu 85 90
95 Lys Lys Ala Leu Thr Gly His Leu Glu Glu Val Val Leu Ala Leu Leu
100 105 110 Lys Thr Pro 115
* * * * *
References