Note: Descriptions are shown in the official language in which they were submitted.
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60412-4434D1
Natural IgM Antibodies and Inhibitors Thereof
This application is a division of application 2,560,066 filed March 1, 2005.
1. Government support
This invention was made with government support under grant No. GM52585,
GM2489I, and 0M07560 from the National Institutes of Health. The government
has
certain rights in the invention. =
2. Cross-reference to related applications
This application claims the benefit of U.S. Provisional Application No.
60/588,648,
filed on July 16, 2004 and U.S. Provisional Application No. 60/549,123 filed
on March 1,
2004.
- 3. Background of the Invention
Nucleated cells are highly sensitive to hypoxia and even short periods of
ischemia in
multi-cellular organisms can have dramatic effects on cellular morphology,
gene .
transcription, and enzymatic processes. Mitochondria, as the major site of
oxygen
metabolism, are particularly sensitive to changes in oxygen levels and during
hypoxia
release reactive oxygen species that chemically modify intracellular
constituents such as
lipids and proteins. Clinically these effects manifest as an inflammatory
response in the
patient. Despite intensive investigations of cellular responses to hypoxia
little is known
regarding the initiation of acute inflammation.
Acute inflammatory responses can result from a wide range of diseases and
naturally occurring events such as stroke and myocardial infarction. Common
medical
procedures can also lead to localized and systemic inflammation. Left
untreated
inflammation can result in Significant tissue loss and may ultimately lead to
multi-system
failure and death. Interfering with the inflammatory response after injury may
be one
method to reduce tissue loss.
Inflammatory diseases and acute inflammatory responses resulting from tissue
injury, however, cannot be explained by cellular events alone. Accumulating
evidence
.supports a major role for the serum innate response or complement system in
inflammation.
Studies to date have looked at tissue injury resulting from ischemia and
reperfusion as one
type of inflammatory disorder that is complement dependent. For example, in
the rat
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myocardial model of reperfusion injury, pretreatment of the rats with the
soluble form of the
complement type 1 receptor dramatically reduced injury. Understanding how
complement
activation contributes to an inflammatory response is an area of active
investigation.
Inflammatory diseases or disorders are potentially life-threatening, costly,
and
affect a large number of people every year. Thus, effective treatments of
inflammatory diseases
or disorders are needed.
4. Summary of the Invention
In one aspect, the invention features isolated natural immunoglobulins (IgMs).
In
one embodiment, the antibody is produced by ATCC Accession Number PTA-3507. In
another
embodiment, the antibody has a light chain variable region comprising the
amino acid sequence
shown as SEQ ID NO: 8. In yet another embodiment, the antibody has a heavy
chain variable
region comprising the amino acid sequence shown as SEQ ID NO: 2.
In another aspect, the invention features IgM inhibitors and pharmaceutical
preparations thereof. In one embodiment, the IgM inhibitor is a peptide that
specifically binds to a
natural IgM and thereby blocks binding to the antigen and/or complement
activation. In one
embodiment, the peptide includes the following consensus sequence:
xNNNxNNxNNNN (SEQ
ID NO: 14). Certain inhibitory peptides are provided as SEQ ID NOs: 16, 18,
20, 22, 24, 26, 28,
30, 32, 34, 36, and 38. Inhibitory peptides may be modified, for example to
increase in vivo half-
life or bioavailability. Inhibitory peptides may also be labeled to facilitate
detection.
In another aspect, the invention features nucleic acids encoding peptides that
specifically bind to natural IgM antibodies, as well as vectors and host cells
for expressing the
peptides. Certain nucleic acids are provided as SEQ NOs: 13, 15, 17, 19, 21,
23, 25, 27, 29, 31,
33, 35 and 37.
In a further aspect, the invention features methods of treating an
inflammatory
disease in a subject by administering to the subject a pharmaceutical
composition comprising an
IgM inhibitor as disclosed herein.
In yet other aspects, the invention features method of detecting, diagnosing
or
monitoring inflammatory diseases in a subject using labeled inhibitory
antibodies.
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The invention as claimed relates to:
- an inhibitor of natural IgM antibodies, wherein the inhibitor is an isolated
antibody or antigen-binding fragment thereof that binds to a self-antigen,
inhibits binding of a
natural IgM antibody to the self-antigen, and blocks complement activation,
wherein the
amino acid sequence of the self-antigen is SEQ ID NO: 38;
- the use of an inhibitor as described herein for the preparation of a
medicament
for treating a complement-dependent inflammatory condition triggered by
binding of natural
IgM antibodies; and
- a pharmaceutical composition comprising the inhibitor as described herein,
and a pharmaceutically acceptable excipient.
Other features and advantages of the invention will be apparent based on the
following Detailed Description and Claims.
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5. Brief Description of the Drawings
Figure 1 shows an IgM heavy chain sequence of I3-1 hybridoma 22A5. (A) shows
= the IgMcm-22 (or 22A5 1gM) heavy chain nucleic acid sequence (SEQ ID NO:
1) and (B).
shows the amino acid sequence corresponding to the heavy chain sequence of SEQ
ID NO: 1
(SEQ JD NO: 2). Framework regions (FVWR) and complementarity-determining
regions
(CDR) are indicated above the nucleotides.
Figure 2 shows an IgM light chain sequence of B-1 hybridoma 22A5. (A) shows
the IgMcm'22 (or 22A5 IgM) light chain nucleic acid sequence (SEQ ID NO: 7)
and (B)
shows the amino acid sequence corresponding to the light chain sequence of SEQ
ID NO: 7
(SEQ ID NO: 8), Framework- regions (FVWR) and complementarity-determining
regions
(CDR) are indicated above the nucleotides.
Figure 3 is a bar graph depicting changes in intestinal permeability of inbred
mice
after intestinal ischemia and reperfusion or no injury (sham). WT represents
parent strain
for Cr2-/- mice. Cr24- was reconstituted with pooled IgG or IgM or saline
control. Pooled
IgM or IgG (0.5 mg) was administered intravenously approximately 1 hour before
treatment. Values are means + standard error, n equals the number of mice in
experimental
groups.
Figure 4 demonstrates reconstitution of I/R. injury in antibody deficient mice
(RAG- .
1) by pooled IgM from a single B-1 cell hybridoma clone. IgM or saline was
injected
= intravenously 30 minutes before initial laparotomy. At the end of
reperfusion, blood is
obtained and permeability index is calculated as the ratio of 1251 counts of
dried intestine
versus that of blood. Values represent means* standard error, n equals the
numbers of mice
used in experimental groups. 1 = WT plus normal saline; 2 .= RAG plus normal
saline; 3 =
RAG plus IgM hybridoma CM-22; 4 = WT sham control.
Figure 'S is a schematic diagram of the proposed role for complement and =
complement receptors in positive selection of peritoneal B-1 lymphocytes.
Figure 6A is a graph showing the ELISA screening of M-13 phage-display library
for IgmCM-22-specific peptides. Symbols: a-P1 clone; X-P2 clone, o-P7 clone; 0-
P8 clone.
=
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The plate was coated with a solution of IgMcm-22 before addition of varying
concentrations of phage-
clones. The results are representative of at least three independent
experiments.
Figure 6B is a bar graph showing that the synthetic peptide P8 inhibits IgmCM-
22
binding of phage clone P8. ELISA was performed with varying concentrations of
the synthetic peptide
P8 added to the Iguic¨ m-22-coated plate prior to the addition of 5x1On PFU
phage. The results are
representative of at least three independent experiments.
-22
Figure 6C is a bar graph showing specific binding of the P8 peptide to IgmCM.
The
ELISA plates were coated with 50 ug/m1 solution of P8 peptide, followed by
addition of Ig
mCM-22 or
IgMcm-75 at 1 or 10 ug/ml. IgM binding was detected with a biotinylated rat
anti-mouse IgM followed
by streptavidin-phosphatase and color reaction. The results are representative
of at least three
independent experiments.
Figure 7A is a series of photomicrographs showing that the P8 peptide blocked
IgMcm-22 mediated injury in vivo. Two upper panels (i and ii) are
representative sections (stained with
Haematoxylin and Eosin) prepared following RI treatment in RAG-14- mice with
IgMcm-22 alone or
mixed with P8 peptide, respectively. Two lower panels (iii and iv) are
representative sections prepared
from wild type mice treated for intestinal reperfusion injury, which received
either saline or peptide P8
5 minutes prior to reperfiision. Arrows indicate pathologic features of
injury. Magnification 200x.
Figure 7B is a scatter plot indicating the mean pathology score of each group
of treated
animals. Each symbol represents the score from one animal. Control group is WT
mice pretreated with
a control peptide (ACGMPYVRIPTA; SEQ ID NO: 61) at a similar dose as the
peptide P8. * indicates
statistical significance determined by Student t test of the P8-treated versus
untreated groups (p<0.05).
Figure 7C is a series of photomicrographs showing the absence of IgM and
complement C3 or C4 within the microvilli of P8-treated animals.
Representative cryosections of
intestinal tissues were harvested following intestinal RI. Panels i-viii are
IgMcm-22 reconstituted RAG-14"
mice without pretreatment with P8 (panels i-iv) or with P8 (panels v-viii).
Panels represent cryosections
from the intestines of WT without P8 (panels ix-xii) or pretreated with P8
(panels xiii-xvi). Sections (i,
v, vii, ix, xiii, xv) were stained with anti-IgM-biotin followed by
Streptavidin-Alexa-568 (red) and
counterstained with DAPI (violet). Panels (ii, x, xiv) were stained with anti-
C4-Alexa 488/FITC (green)
and panels (iv, viii, xii, xvi) were stained with anti-C3-FITC (green).
Magnification 400x.
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Figure 8A is an immunoblot showing the immune precipitation of reperfusion
injury (RI) specific antigens. Detection of a unique band (arrow) at
approximately 250 kDa
on a SDS-PAGE (10%). Size markers are indicated on the left. Intestinal
lysates were
prepared from RAG-14" mice reconstituted with IgMcm-22 and either sham control
(no
ischernia) or subjected to ischemia followed by reperfusion for 0 or 15 min.
Figure 8B is a series of graphs showing results of in vitro binding assays of
IgMcm-
22 to the isoforms of non-muscle myosin heavy chain-H (NMHC-H). ELISA plates
were
coated with monoclonal antibodies for 3 different isoforms of NMHC-II (upper
left:
isoform A, upper right: isoforrn B, lower left: isoforrn C and lower right:
anti-pan myosin
antibody). Bound myosin heavy chain from intestinal lysates was detected by Ig
or
mCM-22
IgMcm-31. The results represent mean standard error of OD 405 nm units and
are
representative of triplicate samples.
Figure 8C is a photomicrograph and a scatter plot showing the restoration of
RI
injury by anti-pan:myosin antibody in RAG-14" mice. RAG-14- mice were
reconstituted
with affinity purified anti-pan myosin followed by RI surgery. The left panels
represents
morphologies of RAG-14- animals with saline control and with anti-pan myosin
treatment.
The right panel is the pathology scores of intestinal injury. The scatter plot
(right panel)
represents the pathology scores where each symbol represents a single animal.
Figure 9A is a graph showing the surface plasmon resonance for the self-
peptide
N2. Binding isotherms for samples of the self-peptide N2 with concentration
from 10.5 M
to 120 M injected over the tmcm-22-coupled surface.
Figure 9B is a graph showing the surface plasmon resonance for a control
peptide.
Binding isotherm for a same-length, random-sequence control peptide
(AGCMPYVRIPTA;
SEQ ID NO: 62), injected at a concentration of 117 p.I\.4 over the IglAcm-22-
coupled surface.
Figure 9C is a graph showing the nonlinear curve fitting with a 1:1 Langmuir
binding isotherm to the steady-state response levels for the injection showed
in Figure 9A
(x2=10).
Figure 9D is a graph showing the binding isotherm for the injection of the
self-
peptide N2 at 120 M over a surface coupled with the control IgMcm-31.
Figure 10A is a series of photomicrographs showing that the N2 self-peptide
blocking RI in RAG-14" mice. Two upper panels show representative sections
prepared
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following RI treatment in RAG-14" mice with IgMcm-22 alone or mixed with N2
self-
peptide. Two lower panels are representative sections prepared from WT mice
treated for
intestinal RI, which received either saline or N2 peptide 5 minutes prior to
reperfusion.
Figure 10B is a scatter plot indicating the mean pathology score of each group
of
5 treated animals. Each symbol represents a single mouse. * indicates a
statistical
significance bases on a Student t test.
Figure 10C is a series of photomicrographs showing the prevention of the
activation of classical pathway of complement in intestinal RI by the self-
peptide N2.
Representative cryosections of intestinal tissues were harvested following
intestinal RI and
10 treated with an antibody specific for the mouse IgM, C4 or C3 (400x
magnification).
IgMcm-22-reconstituted RAG-14- mice without pretreatment with the self-peptide
N2 are in
panels i-iv or with the self-peptide N2 are in panels v-viii. Wild type mice
without
pretreatment with the self-peptide N2 are in panels ix-xii or with
pretreatment with the self-
peptide N2 are in panels xiii-xvi. The tissue in panels i, iii, v, vii, ix,
xiii, xi, xv were
15 stained with anti-IgM-biotin followed by Streptavidin-Alexa-568 (red)
and counterstained
with DAPI (violet). Panels ii, vi, x, and xiv were stained with anti-C4-FITC
(green).
Panels iv, viii, xii, xvi were stained with anti-C3-FITC (green).
Figure 11 shows the (A) nucleic acid sequence (SEQ ID NO: 47; Genbank
Accession no. NM 022410) and (B) amino acid sequence (SEQ ID NO: 48; Genbank
20 Accession no. NP_071855) of mouse non-muscle myosin heavy chain II-A
(mNMTIC-IIA).
Figure 12 shows the (A) nucleic acid sequence (SEQ ID NO: 49; Genbank
Accession no. NM 002473) and (B) amino acid sequence (SEQ ID NO: 50; Genbank
Accession no. NP 002464) of human non-muscle myosin heavy chain II-A (11NMHC-
TIA).
Figure 13 shows the (A) nucleic acid sequence (SEQ ID NO: 51; Genbank
25 Accession no. NM_175260) and (B) amino acid sequence (SEQ ID NO: 52;
Genbank
Accession no. NP 780469) of mouse non-muscle myosin heavy chain
(inNMHC-JIB).
Figure 14 shows the (A) nucleic acid sequence (SEQ ID NO: 53; Genbank
= Accession no. NM 005964) and (B) amino acid sequence (SEQ ID NO: 54;
Genbank
Accession no. NP 005955) of human non-muscle myosin heavy chain II-B (hNMHC-
ITB).
30 Figure 15 shows the (A) nucleic acid sequence (SEQ ID NO: 55; Genbank
Accession no. AY363100) and (B) amino acid sequence (SEQ ID NO: 56; Genbank
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Accession no. AAQ24173) of mouse non-muscle myosin heavy chain II-C (mNMHC-
IIC).
(C) shows the nucleic acid sequence (SEQ ID No: 57; Genbank Accession no.
NM_028021) and (D) shows the amino acid sequence (SEQ ID NO: 58; Genbank
Accesion
no. NP 079005) of another non-muscle myosin heavy chain II-C.
Figure 16 shows the (A) nucleic acid sequence (SEQ ID NO: 59; Genbank
Accession no. NM 024729) and (B) amino acid sequence (SEQ ID NO: 60; Genbank
Accession no. NP_079005) of human non-muscle myosin heavy chain IT-C (liNMHC-
11C).
6. Detailed Description
6.1. Definitions:
For convenience, certain terms employed in the specification, examples, and
appended claims are provided. Unless defined otherwise, all technical and
scientific terms
used herein have the same meaning as commonly understood by one of ordinary
skill in the
art to which this invention belongs.
"A" and "an" are used herein to refer to one or to more than one (i.e., to at
least one)
of the grammatical object of the article. By way of example, "an element"
means one
element or more than one element.
"Amino acid" is used herein to refer to either natural or synthetic amino
acids,
including glycine and D or L optical isomers, and amino acid analogs and
peptidomimetics.
"Antibody" is used herein to refer to binding molecules including
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen-binding site. Immunoglobulin molecules
useful in the
invention can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass.
Native
antibodies and immunoglobulins are usually heterotetrameric glycoproteins of
about
150,000 daltons, composed of two identical light chains and two identical
heavy chains.
Each heavy chain has at one end a variable domain followed by a number of
constant
domains. Each light chain has a variable domain at one end and a constant
domain at its
other end. Antibodies include, but are not limited to, polyclonal, monoclonal,
bispecific,
chimeric, partially or fully humanized antibodies, fully human antibodies
(i.e., generated in
a transgenic mouse expressing human immunoglobulin genes), camel antibodies,
and anti-
idiotypic antibodies. An antibody, or generally any molecule, "binds
specifically" to an
antigen (or other molecule) if the antibody binds preferentially to the
antigen, and, e.g., has
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less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with another
molecule.
The terms "antibody" and "immunoglobulin" are used interchangeably.
"Antibody fragment" or "antibody portion" are used herein to refer to any
derivative
of an antibody which is less than full-length. In exemplary embodiments, the
antibody
fragment retains at least a significant portion of the full-length antibody's
specific binding
ability. Examples of antibody fragments include, but are not limited to, Fab,
Fab', F(abI)2,
scFv, Fv, dsFAr diabody, minibody, Fd fragments, and single chain antibodies.
The
antibody fragment may be produced by any means. For instance, the antibody
fragment
may be enzymatically or chemically produced by fragmentation of an intact
antibody, it
may be recombinantly produced from a gene encoding the partial antibody
sequence, or it
may be wholly or partially synthetically produced. The antibody fragment may
optionally
be a single chain antibody fragment. Alternatively, the fragment may comprise
multiple
chains which are linked together, for instance, by disulfide linkages. The
fragment may
also optionally be a multimolecular complex. A functional antibody fragment
will typically
comprise at least about 50 amino acids and more typically will comprise at
least about 200
amino acids.
"Antigen-binding site" is used herein to refer to the variable domain of a
heavy
chain associated with the variable domain of a light chain.
"Bind" or "binding" are used herein to refer to detectable relationships or
associations (e.g. biochemical interactions) between molecules.
"Cells," "host cells" or "recombinant host cells" are terms used
interchangeably
herein. It is understood that such terms refer not only to the particular
subject cell but to the
progeny or potential progeny of such a cell. Because certain modifications may
occur in
succeeding generations due to either mutation or environmental influences,
such progeny
may not, in fact, be identical to the parent cell, but are still included
within the scope of the
term as used herein.
"Comprise" and "comprising" are used in the inclusive, open sense, meaning
that
additional elements may be included.
"Consensus sequence" is used herein to refer to the sequence formed from the
most
frequently occurring amino acids (or nucleotides) in a family of related
sequences (See.
e.g., Winnaker, From Genes to Clones, 1987). In a family of proteins, each
position in the
consensus sequence is occupied by the amino acid occurring most frequently at
that
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position in the family. If two amino acids occur equally frequently, either
can be included
in the consensus sequence. A "consensus framework" refers to the framework
region in the
consensus immunoglobulin sequence.
A "conservative amino acid substitution" is one in which the amino acid
residue is
replaced with an amino acid residue having a similar side chain. Families of
amino acid
residues having similar side chains have been defined in the art. These
families include
amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic
side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-
branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino
acid residue in
a natural immunoglobulin can be preferably replaced with another amino acid
residue from
the same side chain family. Alternatively, in another embodiment, mutations
can be
introduced randomly along all or part of a natural immunoglobulin coding
sequence, such
as by saturation mutageriesis, and the resultant mutants can be screened for
biological
activity.
"Detectable label" is used herein to refer to a molecule capable of detection,
including, but not limited to, radioactive isotopes, fluorophores,
chemiluminescent
moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,
dyes, metal
ions, ligands (e.g., biotin or haptens) and the like. "Fluorophore" refers to
a substance or a
portion thereof which is capable of exhibiting fluorescence in the detectable
range.
Particular examples of labels which may be used under the invention include
fluorescein,
rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, beta-
galactosidase, and
horseradish peroxidase.
"Inhibitor" or "IgM inhibitor" or "antagonist" as used herein refers to an
agent that
reduces or blocks (completely or partially) an interaction between a natural
antibody and
another molecule involved in an inflammatory cascade. An inhibitor may
antagonize one
or more of the following activities of a natural IgM: (i) inhibit or reduce an
interaction (e.g.,
binding) between the IgM and an ischemia-specific antigen; (ii) inhibit or
reduce an
interaction (e.g., binding) between the natural IgM and a component of the
complement
pathway, e.g., Clq; (iii) neutralize the natural IgM by, e.g., sequestering
the
immunoglobulin and/or targeting its degradation; or (iv) inhibit or reduce
production of the
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natural IgM e.g., blocks synthesis, assembly, and/or posttranslational
modifications of the
IgM. The inhibitor can bt. orotein or a peptide, an antibody or fragment
thereof (e.g., an
anti-idiotypic antibody), a modified antibody, a carbohydrate, a glycoprotein,
or a small
organic molecule.
"Interaction" refers to a physical association between two or more molecules,
e.g.,
binding. The interaction may be direct or indirect.
"Inflammatory disease" is used herein to refer to a disease or disorder that
is caused
or contributed to by a complicated set of functional and cellular adjustments
involving
acute or chronic changes in microcirculation, movement of fluids, and influx
and activation
of inflammatory cells (e.g., leukocytes) and complement, and included
autoimmune
diseases. Examples of such diseases and conditions include, but are not
limited to:
reperfusion injury, ischemia injury, stroke, autoimmune hemolytic anemia,
idiopathic
thrombocytopenic ptupura, rheumatoid arthritis, celiac disease, hyper-IgM
immunodeficiency, arteriosclerosis, coronary artery disease, sepsis,
myocarditis,
encephalitis, transplant rejection, hepatitis, thyroiditis (e.g. Hashimoto's
thyroiditis, Graves
disease), osteoporosis, polymyositis, dermatomyositis, Type I diabetes, gout,
dermatitis,
alopecia areata, systemic lupus erythematosus, lichen sclerosis, ulcerative
colitis, diabetic
retinopathy, pelvic inflammatory disease, periodontal disease, arthritis,
juvenile chronic
arthritis (e.g. chronic iridocyclitis), psoriasis, osteoporosis, nephropathy
in diabetes
mellitus, asthma, pelvic inflammatory disease, chronic inflammatory liver
disease, chronic
inflammatory lung disease, lung fibrosis, liver fibrosis, rheumatoid
arthritis, chronic
inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis,
liver fibrosis,
Crohn's disease, ulcerative colitis, burns, and other acute and chronic
inflammatory
diseases of the Central Nervous System (CNS; e.g. multiple sclerosis),
gastrointestinal
system, the skin and associated structures, the immune system, the hepato-
biliary system, or
any site in the body where pathology can occur with an inflammatory component.
An "isolated" molecule, e.g., an isolated IgM, refers to a condition of being
separate
or purified from other molecules present in the natural environment.
"Natural IgM" is used herein to refer to an IgM antibody that is naturally
produced
in a mammal (e.g., a human). They have a pentameric ring structure wherein the
individual
monomers resemble IgGs thereby having two light (K or k) chains and two heavy
(p)
chains. Further, the heavy chains contain an additional CH4 domain. The
monomers form a
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pentamer by disulfide bonds between adjacent heavy chains. The pentameric ring
is closed
by the disulfide bonding between a J chain and two heavy chains. Because of
its high
number of antigen binding sites, a natural IgM antibody is an effective
agglutinator of
antigen. Production of natural IgM antibodies in a subject are important in
the initial
activation of B-cells, macrophages, and the complement system. IgM is the
first
immunoglobulin synthesized in an antibody response.
"Nucleic acid" is used herein to refer to polynucleotides such as
deoxyribonucleic
acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should
also be
understood to include, as equivalents, analogs of either RNA or DNA made from
nucleotide
analogs, and, as applicable to the embodiment being described, single (sense
or antisense)
and double-stranded polynucleotides.
"Operatively linked" is used herein to refer to a juxtaposition wherein the
components so described are in a relationship permitting them to function in
their intended
manner. For example, a coding sequence is "operably linked" to another coding
sequence
when RNA polymerase will transcribe the two coding sequences into a single
mRNA,
which is then translated into a single polypeptide having amino acids derived
from both
coding sequences. The coding sequences need not be contiguous to one another
so long as
the expressed sequences ultimately process to produce the desired protein. An
expression
control sequence operatively linked to a coding sequence is ligated such that
expression of
the coding sequence is achieved under conditions compatible with the
expression control
sequences. As used herein, the term "expression control sequences" refers to
nucleic acid
sequences that regulate the expression of a nucleic acid sequence to which it
is operatively
linked. Expression control sequences are operatively linked to a nucleic acid
sequence
when the expression control sequences control and regulate the transcription
and, as
appropriate, translation of the nucleic acid sequence. Thus, expression
control sequences
can include appropriate promoters, enhancers, transcription terminators, a
start codon (i.e.,
ATG) in front of a protein-encoding gene, splicing signals for introns,
maintenance of the
correct reading frame of that gene to permit proper translation of the mRNA,
and stop
codons. The term "control sequences" is intended to include, at a minimum,
components
whose presence can influence expression, and can also include additional
components
whose presence is advantageous, for example, leader sequences and fusion
partner
sequences. Expression control sequences can include a promoter.
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"Patient", "subject" or "host" are used herein to refer to either a human or a
non-
human mammal.
"Peptide" is used herein to refer to a polymer of amino acids of relatively
short
length (e.g. less than 50 amino acids). The polymer may be linear or branched,
it may
comprise modified amino acids, and it may be interrupted by non-amino acids.
The term
also encompasses an amino acid polymer that has been modified; for example,
disulfide
bond formation, glycosylation, lipidation, acetylation, phosphorylation, or
any other
manipulation, such as conjugation with a labeling component.
"Promoter" is used herein to refer to a minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter elements
which are
sufficient to render promoter-dependent gene expression controllable for cell-
type specific,
tissue-specific, or inducible by external signals or agents; such elements may
be located in
the 5' or 3' regions of the of a polynucleotide sequence. Both constitutive
and inducible
promoters, are included in the invention (see e.g., Bitter et al., Methods in
Enzymology
153:516-544, 1987). For example, when cloning in bacterial systems, inducible
promoters
such as pL of bacteriophage, plac, ptrp, ptac (ptrp-lac hybrid promoter) and
the like may be
used. When cloning in mammalian cell systems, promoters derived from the
genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
reh-ovirus long terminal repeat; the adenovirus late promoter; the vaccinia
virus 7.5K
promoter) may be used. Promoters produced by recombinant DNA or synthetic
techniques
may also be used to provide for transcription of the nucleic acid sequences of
the invention.
Tissue-specific regulatory elements may be used. Including, for example,
regulatory
elements from genes or viruses that are differentially expressed in different
tissues.
"Specifically binds" is used herein to refer to the interaction between two
molecules
to form a complex that is relatively stable under physiologic conditions. The
term is used
herein in reference to various molecules, including, for example, the
interaction of an
antibody and an antigen (e.g. a peptide). Specific binding can be
characterized by a
dissociation constant of at least about 1x10-6 M, generally at least about
1x104 M, usually
at least about 1x10-8 M, and particularly at least about 1x10-9 M or lx 1 0-1
M or greater.
Methods for determining whether two molecules specifically bind are well known
and
include, for example, equilibrium dialysis, surface plasmon resonance, and the
like.
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"Stringency hybridization" or "hybridizes under low stringency, medium
stringency,
high stringency, or very high stringency conditions" is used herein to
describes conditions
for hybridization and washing. Guidance for performing hybridization reactions
can be
found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-
6:3.6. Aqueous and non-aqueous methods are
described in that reference and either can be used. Specific hybridization
conditions
referred to herein are as follows: 1) low stringency hybridization conditions
in 6X sodium
chloride/sodium citrate (SSC) at about 45 C, followed by two washes in 0.2X
SSC, 0.1%
SDS at least at 50 C (the temperature of the washes can 'be increased to 55 C
for low
stringency conditions); 2) medium stringency hybridization conditions in 6X
SSC at about
45 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 C; 3) high
stringency
hybridization conditions in 6X SSC at about 45 C, followed by one or more
washes in 0.2X
= SSC, 0.1% SDS at 65 C; and preferably 4) very high stringency
hybridization conditions
are 0.5M sodium phosphate, 7% SDS at 65 C, followed by one or more washes at
0.2X
SSC, 1% SDS at 65 C. Very high stringency conditions (4) are the preferred
conditions
and the ones that should be used unless otherwise specified. Calculations of
homology or
sequence identity between sequences (the terms are used interchangeably
herein) are
performed as follows.
To determine the percent identity of two amino acid sequences, or of two
nucleic
acid sequences, the sequences are aligned for optimal comparison purposes
(e.g., gaps can
be introduced in one or both of a first and a second amino acid or nucleic
acid sequence
for optimal alignment and non-homologous sequences can be disregarded for
comparison
purposes). In a preferred embodiment, the length of a reference sequence
aligned for
comparison purposes is at least 30%, preferably at least 40%, more preferably
at least
50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length
of the
reference sequence. The amino acid residues or nucleotides at corresponding
amino acid
positions or nucleotide positions are then compared. When a position in the
first sequence
is occupied by the same amino acid residue or nucleotide as the corresponding
position in
the second sequence, then the molecules are identical at that position.
The percent identity between the two sequences is a function of the number of
identical positions shared by the sequences and the percent homology between
two
sequences is a function of the number of conserved positions shared by the
sequences,
taking into account the number of gaps, and the length of each gap, which need
to be
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introduced for optimal alignment of the two sequences. The comparison of
sequences and
determination of percent identity and/or homology between two sequences can be
accomplished using a mathematical algorithm. In a preferred embodiment, the
percent
identity between two amino acid sequences is determined using the Needleman
and
Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been
incorporated into
the GAP program in the GCG software package (available on the world wide web
with
the extension gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet another
preferred embodiment, the percent identity between two nucleotide sequences is
determined using the GAP program in the GCG software package (available on the
world
wide web with the extension gcg.com), using a NWSgapdna CMP matrix and a gap
weight of 40, 50, 60, 70; or 80 and a length weight of 1, 2, 3,4, 5, or 6. A
particularly
preferred set of parameters (and the one that should be used unless otherwise
specified) are
a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of
4, and a
frame shift gap penalty of 5.
The percent identity and/or homology between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W. Miller
((1989)
CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version
2.0),
using a PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4.
"Treating" is used herein to refer to any treatment of, or prevention of, or
inhibition
of a disorder or disease in a subject and includes by way of example: (a)
preventing the
disease or disorder from occurring in a subject that may be predisposed to the
disease or
disorder, but has not yet been diagnosed as having it; (b) inhibiting the
disease or disorder,
i.e., arresting its progression; or (c) relieving or ameliorating the disease
or disorder, i.e.,
causing regression. Thus, treating as used herein includes, for example,
repair and
regeneration of damaged or injured tissue or cells at the site of injury or
prophylactic
treatments to prevent damage, e.g., before surgery.
"Vector" as used herein refers to a nucleic acid molecule, which is capable of
transporting another nucleic acid to which it has been operatively linked and
can include a
plasmid, cosmid, or viral vector. One type of preferred vector is an episome,
i.e., a nucleic
acid capable of extra-chromosomal replication. Preferred vectors are those
capable of
autonomous replication and/or expression of nucleic acids to which they are
linked.
Vectors may be capable of directing the expression of genes to which they are
operatively
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linked. A vector may also be capable of integrating into the host DNA.. In the
present
specification, "plasmid" and "vector" are used interchangeably as a plasmid (a
circular
arrangement of double stranded DNA) is the most commonly used form of a
vector.
However, the invention is intended to include such other forms of vectors
which serve
equivalent functions and which become known in the art subsequently hereto.
Viral
vectors include, e.g., replication defective retroviruses, adenoviru.ses and
adeno-
associated viruses.
6.2 Natural Ig111 antibodies
The present invention is based, at least in part, on the identification of
natural
immuno globulins (Ig), in particular natural IgMs. Certain IgMs may be
obtained from the
hybridoma that has been deposited with the American Type Culture Collection
and
provided Accession Number PTA-3507.
The nucleotide sequence of the heavy chain variable region of the IgM produced
from hybridoma PTA-3507, IgMcm-22 (also referred to as 22A5 IgM) is shown in
Figure
1A (SEQ ID NO: 1), and the amino acid sequence is shown in Figure 1B (SEQ ID
NO: 2).
The CDR1 domain of the heavy chain variable region corresponds to amino acids
31 to
35 of SEQ ID NO: 2 (SEQ ID NO: 4), which is encoded by nucleotides 91-105 of
SEQ ID
NO: 1 (SEQ ID NO: 3), and the CDR2 domain of the heavy chain variable region
corresponds to amino acids 50 to 66 of SEQ ID NO: 2 (SEQ ID NO: 6), which is
encoded
by nucleotides 148-198 of SEQ ID NO: 1 (SEQ ID NO: 5).
The nucleotide sequence of the light chain variable region of IgMcm-22 is
shown in
Figure 2A (SEQ ID NO: 7), and the amino acid sequence is shown in Figure 2B
(SEQ ID
NO: 8). The CDR1 domain of the light chain variable region corresponds to
amino acids
23 to 37 of SEQ ID NO: 8 (SEQ ID NO: 10), which is encoded by nucleotides 67-
111 of
SEQ ID NO: 7 (SEQ ID NO: 9), and the CDR2 domain of the light chain variable
region
corresponds to amino acids 53 to 59 of SEQ ID NO: 8 (SEQ ID NO: 12), which is
encoded by nucleotides 157 to 177 of SEQ ID NO: 7 (SEQ ID NO: 11). Due to the
degeneracy of the genetic code, other nucleotide sequences can encode the
amino acid
sequences listed herein.
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=
The nucleic acid compositions of the present invention, while often in a
native
sequence (except for modified restriction sites and the like), from either
cDNA, genomic
or mixtures may be mutated, in accordance with standard techniques. For coding
sequences, these mutations, may affect the amino acid sequence as desired. In
particular,
nucleotide sequences substantially identical to or derived from native V, D,
J, constant,
switches and other such sequences described herein are contemplated.
For example, an isolated nucleic acid can comprise an IgMCM-22 (or 22A5 IgM)
heavy chain variable region nucleotide sequence having a nucleotide sequence
as shown
in Figure lA (SEQ ID NO: 1), or a sequence, which is at least 80%, 90%, 95%,
96%,
97%, 98%, or 99% identical to SEQ ID NO: I. A nucleic acid molecule may
comprise the
heavy chain CDR1 nucleotide sequence of SEQ ID NO: 3, or a portion thereof.
Further, the
nucleic acid molecule may comprise the heavy chain CDR2 nucleotide sequence of
SEQ ID
NO: 5, or a portion thereof. In an exemplary embodiment, the nucleic acid
molecule
comprises a heavy chain CDR1 nucleotide sequence of SEQ ID NO: 3, or portion
thereof,
and a heavy chain CDR2 nucleotide sequence of SEQ ID NO: 5, or portion
thereof. The
nucleic acid molecules of the present invention may comprise heavy chain
sequences, e.g.
SEQ ID NO: I, SEQ ID NO: 3, SEQ ID NO: 5, or combinations thereof, or
encompass
nucleotides having at least 80%, 90%, 95%, 96%, 97 %, 98%, and 99% sequence
identity to
SEQ ID NOs: 1, 3 or 5. Further, the nucleic acid molecules of the present
invention may
comprise heavy chain sequences, which hybridize under stringent conditions,
e.g. low,
medium, high or very high stringency conditions, to SEQ ID NOs: 1, 3 or 5.
In another embodiment, the invention features nucleic acid molecules having at
least
80%, 90%, 95%, 96%, 97 %, 98%, and 99% sequence identity with a nucleic acid
molecule
encoding a heavy chain polypeptide, e.g., a heavy chain polypeptide of SEQ ID
NOs: 2, 4
or 6. The invention also features nucleic acid molecules which hybridize to
nucleic acid
sequences encoding a heavy chain variable region of a natural antibody or
portion thereof,
e.g., a heavy chain variable region of SEQ ID NO: 2,4 or 6.
In another embodiment, the isolated nucleic acid encodes a IgMcm -22 (22A5
IgM)
light chain variable region nucleotide sequence having a nucleotide sequence
as shown in
Figure 2A (SEQ ED NO: 7), or a sequence at least 80%, 90%, 95%, 96%, 97%, 98%,
99%
identical to SEQ ID NO: 7. The nucleic acid molecule may comprise the light
chain CDR1
nucleotide sequence of SEQ ID NO: 9, or a portion thereof. In another
preferred
embodiment, the nucleic acid molecule may comprise the light chain CDR2
nucleotide
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WO 2005/085288 PCT/US2005/006276
sequence of SEQ ID NO: 11, or a portion thereof. In an exemplary embodiment,
the
nucleic acid molecule comprises a light chain CDR1 nucleotide sequence of SEQ
ID NO: 9,
or portion thereof, and a light chain CDR2 nucleotide sequence of SEQ ID NO:
11, or
portion thereof The nucleic acid molecules of the present invention may
comprise light
chain sequences, e.g. SEQ ID NOs: 7, 9 or 11, or combinations thereof, or
encompass
nucleotides having at least 80%, 90%, 95%, 96%, 97 %, 98%, and 99% sequence
identity to
SEQ ID NOs: 7, 9 or 11. Further nucleic acid molecules may comprise light
chain
sequences, which hybridize under stringent conditions, e.g. low, medium, high
or very high
stringency conditions, to SEQ ID NOs: 7, 9 or 11.
Nucleic acid molecules can have at least 80%, 90%, 95%, 96%, 97 %, 98% or 99%
sequence identity with a nucleic acid molecule encoding a light chain
polypeptide, e.g., a
light chain polypeptide of SEQ ID NOs: 8, 10, or 12. The invention also
features nucleic
acid molecules which hybridize to a nucleic acid sequence encoding a light
chain variable
region of a natural antibody or portion thereof, e.g., a light chain variable
region of SEQ ID
NOs: 8, 10 or 12.
In another embodiment, the invention provides an isolated nucleic acid
encoding a
heavy chain cDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, or
a
fragment or modified form thereof. This nucleic acid can encode only the CDR1
region
or can encode an entire antibody heavy chain variable region or a fragment
thereof. For
example, the nucleic acid can encode a heavy chain variable region having a
CDR2
domain comprising the amino acid sequence of SEQ ID NO: 6. In yet another
embodiment, the invention provides an isolated nucleic acid encoding a heavy
chain
CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, or a fragment
or
modified form thereof. This nucleic acid can encode only the CDR2 region or
can encode
an entire antibody heavy chain variable region or a fragment thereof. For
example, the
nucleic acid can encode a light chain variable region having a CDR1 domain
comprising
the amino acid sequence of SEQ ID NO: 4.
In still another embodiment, the invention provides an isolated nucleic acid
encoding a light chain CDR1 domain comprising the amino acid sequence of SEQ
ID
NO: 10, or a fragment or modified form thereof. This nucleic acid can encode
only the
CDR1 region or can encode an entire antibody light chain variable region. For
example,
the nucleic acid can encode a light chain variable region having a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 12. The isolated nucleic acid
can
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also encode a light chain CDR2 domain comprising the amino acid sequence of
SEQ ID
NO: 12, or a fragment or modified foul), thereof This nucleic acid can encode
only the
CDR2 region or can encode an entire antibody light chain variable region. For
example,
the nucleic acid can encode a light chain variable region having a CDRI domain
comprising the amino acid sequence of SEQ ID NO: 10.
The nucleic acid encoding the heavy or light chain variable region can be of
murine or human origin, or can comprise a combination of murine and human
amino acid
sequences. For example, the nucleic acid can encode a heavy chain variable
region
comprising the CDR1 of SEQ ID NO: 2 (SEQ ID NO: 4) and/or the CDR2 of SEQ ID
NO: 2 (SEQ ID NO: 6), and a human framework sequence. In addition, the nucleic
acid
can encode a light chain variable region comprising the CDR1 of SEQ ID NO: 8
(SEQ ID
NO: 10) and/or the CDR2 of SEQ ID NO: 8 (SEQ ID NO: 12), and a human framework
sequence. The invention further encompasses vectors containing the above-
described
nucleic acids and host cells containing the expression vectors.
The invention also features polypeptides and fragments of the Ig
mCM-22 heavy
chain variable regions and/or light chain variable regions. In exemplary
embodiments, the
isolated polypeptides comprise, for example, the amino acid sequences of SEQ
ID NOs: 8,
10, or 12, or fragments or combinations thereof; or SEQ ID NO: 2, 4, or 6, or
fragments or
combinations thereof. The polypeptides of the present invention include
polypeptides
having at least, but not more than 20, 10, 5, 4, 3, 2, or 1 amino acid that
differs from SEQ
ID NOs: 8, 10, 12, 2, 4 or 6. Exemplary polypeptides are polypeptides that
retain biological
activity, e.g., the ability to bind an ischemia-specific antigen, and/or the
ability to bind
complement. In another embodiment, the polypeptides comprise polypeptides
having at
least 80%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with a light
chain
variable region, or portion thereof, e.g. a light chain variable region
polypeptide of SEQ
ID NOs: 8, 10, or 12. In another embodiment, the polypeptides comprise
polypeptides
having at least 80%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with a
heavy
chain variable region, or portion thereof, e.g. a heavy chain variable region
polypeptide of
SEQ ID NOs: 2, 4, or 6. In another embodiment, the invention features a
polypeptide
comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 2, further
comprising an IRES sequence.
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6.3 Inhibitors of Natural Igilf Antibodies
6.3.1 Peptide Inhibitors of Natural IgM Antibodies
The invention further features IgM inhibitors. In one embodiment, the IgM
inhibitor is a peptide that specifically binds to a natural IgM and thereby
blocks binding to
the antigen. Such peptides can include, but are not limited to, the asparagine-
rich peptides
described in Table 1 below.
Table 1: Amino acid sequences of natural IgM antibody-binding peptides
SEQ ID SEQUENCE Name
NO:
14 xNNNxNNxNNNN Asparagine-rich
Consensus
16 NrNNNNGNYTYRN P1
18 ANTRNGATNNNM P2
20 CDSSCDSVGNCN P3
22 WNNNGRNACNAN P4
94 HNSTSNGCNDNV P5
26 NSNSRYNSNSNN P6
98 = = SN P7
30 NGNNVNGNRNNN P8
32 NVANHNNSNHGN P9
34 SYNNNNHVSNRN P10
The peptides can also include certain "self-peptides" as described in Table 2
below.
Table 2: Amino acid sequences of self-peptides
SEQ ID SEQUENCE Name
NO:
36 LMKNMDPLNDNI Self-1
38 LMICNMDPLNDNV Self-2 ("N-2")
As described in more detail in the Exemplification, self peptides bind to the
natural
IgM antibody igmcm-22.
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In addition to the peptides described above, the present invention encompasses
modified peptides whose activity may be identified and/or assayed using a
variety of
methods well known to the skilled artisan. For example, binding of the peptide
to the IgM
may be detected using biological assays, Western blotting,
immunoprecipitation, or
immonocytochernical techniques, such as those described below. In particular,
the
'
biological activity (e.g., the ability to a bind natural IgM antibody) of a
modified peptide
can be characterized relative to that of P8 (SEQ ID NO: 30) or N2 (SEQ ID NO:
38).
Such modified peptides, when designed to retain at least one activity of the
naturally-occurring form of the protein, are considered "functional
equivalents" of the
peptides described in more detail herein. Such modified peptides may be
produced, for
instance, by amino acid substitution, deletion, or addition, which
substitutions may consist
in whole or part by conservative amino acid substitutions.
For instance, it is reasonable to expect that an isolated conservative amino
acid
substitution, such as replacement of a leucine with an isoleucine or valine,
an aspartate with
a glutamate, or a threonine with a serine, will not have a major effect on the
biological
activity of the resulting molecule. Whether a change in the amino acid
sequence of a
peptide results in a functional homolog may be readily determined by assessing
the ability
of the variant peptide to produce a response similar to that of the wild-type
peptide (e.g.
ability to bind natural IgM antibodies). Peptides in which more than one
replacement has
taken place may readily be tested in the same manner.
Mutagenesis of the peptide may give rise to homologs, which have improved in
vivo
half-lives relative to the corresponding wild-type peptide. For example, the
altered peptide
may be rendered more stable to proteolytic degradation or other cellular
processes which
result in destruction or inactivation of the protein.
The amino acid sequences for a population of peptide homologs can be aligned,
preferably to promote the highest homology possible. Such a population of
variants may
include, for example, homologs from one or more species, or homologs from the
same
species but which differ due to mutation. Amino acids which appear at each
position of the
aligned sequences are selected to create a degenerate set of combinatorial
sequences. In
certain embodiments, the combinatorial library is produced by way of a
degenerate library
of genes encoding a library of polypeptides which each include at least a
portion of =
potential peptide sequences. For instance, a mixture of synthetic
oligonucleotides may be
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enzymatically ligated into gene sequences such that the degenerate set of
potential
nucleotide sequences are expressible as individual polypeptides, or
alternatively, as a set of
larger fusion proteins (e.g., for phage display).
There are many ways by which the library of potential homologs may be
generated
from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate
gene
sequence may be carried out in an automatic DNA synthesizer, and the synthetic
genes may
then be ligated into an appropriate vector for expression. One purpose of a
degenerate set
of genes is to provide, in one mixture, all of the sequences encoding the
desired set of
potential peptide sequences. The synthesis of degenerate oligonucleotides is
well known in
the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.,
(1981)
Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,
Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem.
53:323;
Itakura etal., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.
11:477). Such
techniques have been employed in the directed evolution of other proteins
(see, for
example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS
USA
89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,
(1990) PNAS USA
87: 6378-6382; as well as U.S. Patent Nos: 5,223,409, 5,198,346, and
5,096,815).
Alternatively, other forms of mutagenesis may be utilized to generate a
combinatorial library. For example, peptide homologs may be generated and
isolated from
a library by screening using, for example, alanine scanning mutagenesis and
the like (Ruf et
al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.
269:3095-3099;
Balint etal., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J.
Biochem. 218:597-
601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.,
(1991)
Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-
1085), by
linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown
et al.,
(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316);
by
saturation mutagenesis (Meyers etal., (1986) Science 232:613); by PCR
mutagenesis
(Leung et al., (1989) Method Cell Mol. Biol. 1:11-19); or by random
mutagenesis (Miller et
al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring
Harbor, NY;
and Greener et al., (1994) Strategies in Mol. Biol. 7:32-34).
A wide range of techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations and truncations, and for
screening cDNA
libraries for gene products having a certain property (e.g., the ability to
bind a natural IgM
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. WO 2005/085288 PCT/US2005/006276
antibody). Such techniques will be generally adaptable for rapid screening of
the gene
libraries generated by the combinatorial mutagenesis of peptide homologs. The
most
widely used techniques for screening large gene libraries typically comprises
cloning the
=
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates relatively easy isolation of
the vector
encoding the gene whose product was detected. Each of the illustrative assays
described
below are amenable to high through-put analysis as necessary to screen large
numbers of
degenerate sequences created by combinatorial mutagenesis techniques.
In an illustrative embodiment of a screening assay, candidate combinatorial
gene
products are passed over a column containing beads having attached to it the
binding
protein, such as an IgM or portion thereof. Those candidate combinatorial gene
products
that are retained on the column may be further characterized for binding to
IgMs in a
manner that could be useful in blocking natural IgM antibody binding and
treating
inflammatory diseases.
In another example, the gene library may be expressed as a fusion protein on
the
surface of a viral particle. For instance, in the filamentous phage system,
foreign peptide
sequences may be expressed on the surface of infectious phage, thereby
conferring two
benefits. First, because these phage may be applied to affinity matrices at
very high
concentrations, a large number of phage may be screened at one time. Second,
because
each infectious phage displays the combinatorial gene product on its surface,
if a particular
phage is recovered from an affinity matrix in low yield, the phage may be
amplified by
another round of infection. The group of almost identical E. coli filamentous
phages M13,
fd, and fl are most often used in phage display libraries, as either of the
phage gIII or gVIII
coat proteins may be used to generate fusion proteins without disrupting the
ultimate
packaging of the viral particle (Ladner et al., PCT publication WO 90/02909;
Garrard et al.,
PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chem. 267:16007-
16010;
Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al., (1991) Nature
352:624-628;
and Barbas et al., (1992) PNAS USA 89:4457-4461). Other phage coat proteins
may be
used as appropriate.
The invention also provides for mimetics (e.g., non-peptide agents) which are
able
to mimic binding of the authentic peptide to a natural IgM antibody. For
example, the
critical residues of a peptide which are involved in molecular recognition of
a natural IgM
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antibody may be determined and used to generate peptidomimetics that bind to a
natural IgM antibody.
The peptidomimetic may then be used as an inhibitor of the wild-type protein
by binding to the natural
IgM antibodies and covering up the critical residues needed for interaction
with the wild-type protein,
thereby preventing interaction of the protein and the natural IgM antibody.
Peptidomimetic compounds
may be generated which mimic those residues in binding to the natural IgM
antibody. For instance, non-
hydrolyzable peptide analogs of such residues may be generated using
benzodiazepine (e.g., see
Freidinger et al., in Peptides: Chemistry and Biology, G.R. Marshall ed.,
ESCOM Publisher: Leiden,
Netherlands, 1988), azepine (e.g., see Huffman et al., in Peptides: Chemistry
and Biology, G.R. Marshall
ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam
rings (Garvey et al., in
Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988),
keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem. 29:295;
and Ewenson et al., in
Peptides: Structure and Function (Proceedings of the 9th American Peptide
Symposium) Pierce
Chemical Co. Rockland, IL, 1985), 13-turn dipeptide cores (Nagai et al.,
(1985) Tetrahedron Lett 26:647;
and Sato et al., (1986) J Chem Soc Perkin Trans 1:1231), and P-aminoalcohols
(Gordon et al., (1985)
Biochem Biophys Res Comtnun 126:419; and Dann et al., (1986) Biochem Biophys
Res Commun
13471).
6.3.2 Nucleic acids Encoding Peptide Inhibitors
The invention also features nucleic acids, which encode the peptides discussed
above.
Exemplary nucleic acids are provided in Table 3.
Table 3: Nucleic acids encoding natural IgM antibody-binding peptides
SEQ SEQUENCE Name
ID
NO:
13 NNN AAY AAY AAY NNN AAY AAY NNN AAY AAY AAY Aparagine-
AAY rich
Consensus
15 TAY AAY AAY AAY AAY GGN AA.Y TAY ACN TAY MGN P1
AAY
17 GCN AAY ACN MGN AAY GGN GCN ACN AAY AAY AAY P2
ATG
19 TGY GAY WSN WSN TGY GAY WSN GTN GUN AAY TGY P3
AAY
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SEQ SEQUENCE Name
ID
NO:
21 TOG AAY AA'S_T AAY GGN MGN AAY GCN TGY AAY GCN P4
AAY
23 CAY AAY WSN ACN WSN AAY GGN TGY AAY GAY AAY P5
GIN
25 AAY WSN AAY WSN MGN TAN AAN WSN AAY WSN AAY P6
AAY
27 AAR MGN AAY AAY CAY AAY AAY CAY AAY MGN WSN P7
AAY
29 AAY GGN AAY AAY GTN AAY GGN AAY MGN AAY AAY P8
AAY
31 AAY GTN GCN AAY CAY AAY AAY WSN AAY CAY GGN P9
AAY
33 WSN TAY AAY AAY AAY AAY CAY GTN WSN AAY MGN P10
AAY
35 YTN ATG AAR AAY ATG GAY CCN YTN AAY GAY AAY Self-1
ATH
37 YTN ATG AAR AAY ATG GAY CCN YTN AAY GAY AAY Self-2
GTN
The isolated nucleic acids in Table 3 reflect degeneracy in the genetic code.
In
particular, an "R" corresponds to a base that may be a A or G; a "S"
corresponds to a base
that may be a G or C; a "V" corresponds to a base that may be an A, C or G; a
corresponds to a base that may be a C or T; a "W" corresponds to a base that
may be an A
or T; a "D" corresponds to a base that may be an A, G or T; a "M" corresponds
to a base
that may be an A or C; a "H" corresponds to a base that may be an A, C or T; a
"N"
corresponds to a base that may be an A, C, G or T; a "K" corresponds to a base
that may be
a G or T and a "B" corresponds to a base that may be a C, G or T.
It is expected that DNA sequence polymorphisms that lead to changes in the
amino
acid sequences of the subject proteins will exist among mammalian cells. One
skilled in the
art will appreciate that these variations in one or more nucleotides (from
less than 1% up to
about 3 or 5% or possibly more of the nucleotides) of the nucleic acids
encoding a
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particular peptide of the invention may exist among individuals of a given
species due to
natural allelic variation. Any and all such nucleotide variations and
resulting amino acid
polymoiphisms are within the scope of this invention. Preferred nucleic acids
encode a
peptide, which is at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%
homologous
or more with an amino acid sequence of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26,
28, 30, 32,
34, 36, 38 or another peptide of the invention. Nucleic acids which encode
peptides having
an activity of a peptide of the invention and having at least about 60%, 70%,
80%, 85%,
90%, 95%, 98%, 99% homology or more with SEQ ID NO: 14, 16, 18, 20, 22, 24,
26, 28,
30, 32, 34, 36, 38, or another peptide of the invention are also within the
scope of the
invention.
Bias in codon choice within genes in a single species appears related to the
level of
expression of the protein encoded by that gene. Accordingly, the invention
encompasses
nucleic acid sequences which have been optimized for improved expression in a
host cell
by altering the frequency of codon usage in the nucleic acid sequence to
approach the
frequency of preferred codon usage of the host cell. Due to codon degeneracy,
it is possible
to optimize the nucleotide sequence without effecting the amino acid sequence
of an
encoded polypeptide. Accordingly, the instant invention relates to any
nucleotide sequence
that encodes the peptides set forth in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34,
36,38 or other peptides of the invention.
Nucleic acids within the scope of the invention may also contain linker
sequences,
modified restriction endonuclease sites and other sequences useful for
molecular cloning,
expression or purification of such recombinant polypeptides.
A nucleic acid encoding a peptide of the invention may be obtained from mRNA
or
genomic DNA from any organism in accordance with protocols described herein,
as well as
those generally known to those skilled in the art. A cDNA encoding a peptide
of the
invention, for example, may be obtained by isolating total mRNA from an
organism, e.g. a
bacteria, virus, mammal, etc. Double stranded cDNAs may then be prepared from
the total
mRNA, and subsequently inserted into a suitable plasmid or bacteriophage
vector using any
one of a number of known techniques. A gene encoding a peptide of the
invention may
also be cloned using established polymerase chain reaction techniques in
accordance with
the nucleotide sequence information provided by the invention.
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In another aspect of the invention, the subject nucleic acid is provided in an
expression vector comprising a nucleotide sequence encoding a peptide of the
invention and
operably linked to at least one regulatory sequence. It should be understood
that the design
of the expression vector may depend on such factors as the choice of the host
cell to be
transformed and/or the type of protein desired to be expressed. Moreover, the
vector's copy
number, the ability to control that copy number and the expression of any
other protein
encoded by the vector, such as antibiotic markers, should also be considered.
As will be apparent, the subject gene constructs may be used to cause
expression of
a peptide of the invention in cells propagated in culture, e.g., to produce
proteins or
polypeptides, including fusion proteins or polypeptides, for purification.
This invention also pertains to a host cell transfected with a recombinant
gene in
order to express a peptide of the invention. The host cell may be any
prokaryotic or
eukaryotic cell. For example, a polypeptide of the present invention may be
expressed in
bacterial cells, such as E. coli, insect cells (baculovirus), yeast, or
mammalian cells. Other
suitable host cells are known to those skilled in the art. Additionally, the
host cell may be
supplemented with tRNA molecules not typically found in the host so as to
optimize
expression of the peptide. Other methods suitable for maximizing expression of
the peptide
will be known to those in the art.
6.3.3 Methods of Producing Peptide Inhibitors
Peptide inhibitors may be synthesized, for example, chemically, ribosomally in
a
cell free system, or ribosomally within a cell. Chemical synthesis of peptides
of the
invention may be carried out using a variety of art recognized methods,
including stepwise
solid phase synthesis, semi-synthesis through the confomiationally-assisted re-
ligation of
peptide fragments, enzymatic ligation of cloned or synthetic peptide segments,
and
chemical ligation. Merrifield et al. in J. Am. Chem. Soc., Volume 85, page
2149 (1964), by
Houghten et al. in Proc. Natl. Acad. Sci. USA, Volume 82, page 5132 (1985),
and by
Stewart and Young in Solid Phase Peptide Synthesis, Pierce Chem. Co, Rockford,
Ill.
(1984). Native chemical ligation employs a chemoselective reaction of two
unprotected
peptide segments to produce a transient thioester-linked intennediate. The
transient
thioester-linked intermediate then spontaneously undergoes a rearrangement to
provide the
full length ligation product having a native peptide bond at the ligation
site. Full length
ligation products are chemically identical to proteins produced by cell free
synthesis. Full
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length ligation products may be refolded and/or oxidized, as allowed, to form
native
disulfide-containing protein molecules. (see e.g., U.S. Patent Nos. 6,184,344
and 6,174,530;
and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller,
et al., Science
(1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p
616; L. H.
Huang, etal., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int.
J. Pept. Prot.
Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol.
264, p 90; R.
E. Offord, "Chemical Approaches to Protein Engineering", in Protein Design and
the
Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds.,
(Plenum Press,
New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992):
vol. 267, p
3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang,
et al., Proc.
Natl. Acad. Sci. USA (1994) 91:12544-12548; M. Schnlzer, etal., Science
(1992): vol.,
3256, p 221; and K. Akaji, etal., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).
In another variation, peptide production may be achieved using in vitro
translation
systems. An in vitro translation systems is, generally, a translation system
which is a cell-
free extract containing at least the minimum elements necessary for
translation of an RNA
molecule into a protein. An in vitro translation system typically comprises at
least
ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved
in
translation, e.g., elF2, eIF3, the cap-binding (CB) complex, comprising the
cap-binding
protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in
vitro translation
systems are well known in the art and include commercially available kits.
Examples of in
vitro translation systems include eukaryotic lysates, such as rabbit
reticulocyte lysates,
rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ
extracts.
Lysates are commercially available from manufacturers such as Promega Corp.,
Madison,
Wis.; Stratagene, La Jolla, Calif.; Amerslaarn, Arlington Heights, Ill.; and
G113CO/BRL,
Grand Island, N.Y. In vitro translation systems typically comprise
macromolecules, such as
enzymes, translation, initiation and elongation factors, chemical reagents,
and ribosomes.
In addition, an in vitro transcription system may be used. Such systems
typically comprise
at least an RNA polymerase holoenzyme, ribonucleotides and any necessary
transcription
initiation, elongation and termination factors. In vitro transcription and
translation may be
carried out within in the same reaction to produce peptides from one or more
isolated
DNAs.
Nucleic acids encoding peptide inhibitors may be expressed in vitro by DNA
transfer into a suitable host cell. Expression of peptides may be facilitated
by inserting the
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nucleic acids encoding the peptides into a vector, such as a plasmid, virus or
other vehicle known
in the art that has been manipulated by insertion or incorporation of the
natural antibody-binding
peptide genetic sequences. Such vectors contain a promoter sequence which
facilitates the
efficient transcription of the inserted genetic sequence of the host. The
vector typically contains
an origin of replication, a promoter, as well as specific genes which allow
phenotypic selection of
the transformed cells. Vectors suitable for use in the present invention
include, but are not limited
to the T7-based expression vector for expression in bacteria (Rosenberg, et
al., Gene, 56:125,
1987), the pMSXND expression vector for expression in mammalian cells (Lee and
Nathans,
J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression
in insect cells.
The DNA segment can be present in the vector operably linked to regulatory
elements, for
example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
Nucleic acids encoding peptide inhibitors may be expressed in either
prokaryotes
or eukaryotes. Hosts can include microbial, yeast, insect, and mammalian
organisms. Methods of
expressing DNA sequences having eukaryotic or viral sequences in prokaryotes
are well known in
the art. Biologically functional viral and plasmid DNA vectors capable of
expression and
replication in a host are known in the art. Such vectors can incorporate DNA
sequences of the
invention. Methods which are well known to those skilled in the art can be
used to construct
vectors containing the natural antibody-binding peptide coding sequence and
appropriate
transcriptional/translational control signals. These methods include in vitro
recombinant DNA
techniques, synthetic techniques, and in vivo recombination/genetic
techniques. (See, for
example, the techniques described in Maniatis et al., 1989 Molecular Cloning A
Laboratory
Manual, Cold Spring Harbor Laboratory, N.Y.)
A variety of host-expression vector systems may be utilized. These include but
are not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage
DNA, plasmid DNA, or cosmid DNA expression vectors; yeast transformed with
recombinant
yeast expression vectors; plant cell systems infected with recombinant virus
expression vectors
(e.g, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems
infected with
recombinant virus expression vectors (e.g., baculovirus); or animal cell
systems infected with
recombinant virus expression vectors (e.g., retroviruses,
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adenovirus, vaccinia virus), or transformed animal cell systems engineered for
stable
expression.
Depending on the host/vector system utilized, any of a number of suitable
transcription and translation elements, including constitutive and inducible
promoters,
transcription enhancer elements, transcription terminators, etc. may be used
in the
expression vector (see e.g., Bitter et al., 1987, Methods in Enzymology
153:516-544). For
example, when cloning in bacterial systems, inducible promoters such as pL of
bacteriophage 7, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may
be used. When
cloning in mammalian cell systems, promoters derived from the genome of
mammalian
cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the
retrovirus long
terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K
promoter) may be
used. Promoters produced by recombinant DNA or synthetic techniques may also
be used.
In yeast, a number of vectors containing constitutive or inducible promoters
may be
used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988,
Ed. Ausubel
et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al.,
1987, Expression
and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman,
319 87,
Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II,
IRL Press,
Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast,
Methods in
Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684;
and The
Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et al., Cold
Spring
Harbor Press, Vols. I and H. A constitutive yeast promoter such as ADH or LEU2
or an
inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R.
Rothstein In:
DNA Cloning Vol, 11, A Practical Approach, Ed. D M Glover, 1986, IRL Press,
Wash.,
D.C.). Alternatively, vectors may be used which promote integration of foreign
DNA
sequences into the yeast chromosome.
Eukaryotic systems, and preferably mammalian expression systems, allow for
proper post-translational modifications of expressed mammalian proteins to
occur.
Eukaryotic cells which possess the cellular machinery for proper processing of
the primary
transcript, glycosylation, phosphorylation, and advantageously, plasma
membrane insertion
of the gene product may be used as host cells.
Mammalian cell systems which utilize recombinant viruses or viral elements to
direct expression may be engineered. For example, when using adenovirus
expression
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vectors, a natural antibody-binding peptide coding sequence may be ligated to
an
adenovirus transcription/-translation control complex, e.g., the late promoter
and tripartite
leader sequence. Alternatively, the vaccinia virus 7.5K promoter may be used.
(e.g., see,
Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et
al., 1984, J.
\Tirol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79:
4927-4931). Of
particular interest are vectors based on bovine papilloma virus which have the
ability to
replicate as extrachromosomal elements (Sarver, et al., 1981, Mol. Cell. Biol.
1: 486).
Shortly after entry of this DNA into mouse cells, the plasmid replicates to
about 100 to 200
copies per cell. Transcription of the inserted cDNA does not require
integration of the
plasmid into the host's chromosome, thereby yielding a high level of
expression. These
vectors can be used for stable expression by including a selectable marker in
the plasmid,
such as, for example, the neo gene. Alternatively, the retroviral genome can
be modified
for use as a vector capable of introducing and directing the expression of a
natural
antibody-binding peptide gene in host cells (Cone & Mulligan, 1984, Proc.
Natl. Acad. Sci.
USA 81:6349-6353). High level expression may also be achieved using inducible
promoters, including, but not limited to, the metallothionine HA promoter and
heat shock
promoters.
For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. Rather than using expression vectors which contain viral origins of
replication,
host cells can be transformed with a cDNA controlled by appropriate expression
control
elements (e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation
sites, etc.) and a selectable marker. The selectable marker in the recombinant
plasmid
confers resistance to the selection and allows cells to stably integrate the
plasmid into their
chromosomes and grow to form foci which in turn can be cloned and expanded
into cell
lines. For example, following the introduction of foreign DNA, engineered
cells may be
allowed to grow for 1-2 days in an enriched media, and then are switched to a
selective
media. A number of selection systems may be used, including but not limited to
the herpes
simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11: 223),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci.
USA 48:
2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:
817) genes can
be employed in tk-, hgprt- or aprf cells respectively. Also, antimetabolite
resistance can be
used as the basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et
al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl.
Acad. Sci. USA
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78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan &
Berg, 1981,
Proc. Natl. Acad Sci. USA 78: 2072; neo, which confers resistance to the
aminoglycoside
G-418 (Colben-e-Garapin, et al., 1981, J. Mol. Biol. 150: 1); and hygro, which
confers
resistance to hygrornycin (Santerre, et al., 1984, Gene 30: 147) genes.
Additional selectable
genes include trpB, which allows cells to utilize indole in place of
tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hattinan & Mulligan,
1988, Proc. Natl.
Acad. Sci. USA 85: 8047); and ODC (omithine decarboxylase) which confers
resistance to
the omithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO
(McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold
Spring
Harbor Laboratory ed.).
For stable recombinant cell lines, suitable cell types include but are not
limited to
cells of the following types: NIH 3T3 (Murine), C2C12, L6, and P19. C2C12 and
L6
myoblasts will differentiate spontaneously in culture and form myotubes
depending on the
particular growth conditions (Yaffe and Saxel, 1977; Yaffe, 1968) P19 is an
embryonic
carcinoma cell line. Such cells are described, for example, in the Cell Line
Catalog of the
American Type Culture Collection (ATCC). These cells can be stably transformed
by a
method known to the skilled artisan. See, for example, Ausubel et al.,
Introduction of DNA
Into Mammalian Cells, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
sections 9.5.1-9.5.6 (John Wiley & Sons, Inc. 1995). "Stable" transformation
in the context
of the invention means that the cells are immortal to the extent of having
gone through at
least 50 divisions.
When the host is a eukaryote, such methods of transfection of DNA as calcium
phosphate co-precipitates, conventional mechanical procedures such as
microinjection,
elech-oporation, insertion of a plasmid encased in liposomes, or virus vectors
may be used.
Eukaryotic cells can also be cotransformed with DNA sequences encoding natural
antibody-binding peptides, and a second foreign DNA molecule encoding a
selectable
phenotype, such as the herpes simplex thymidine kinase gene. Another method is
to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma
virus, to
transiently infect or transform eukaryotic cells and express the protein. (see
for example,
Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
To interact with natural antibodies or for isolation and purification, natural
antibody-binding proteins may need to be secreted from the host cell.
Accordingly a signal
sequence may be used to direct the peptide out of the host cell where it is
synthesized.
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Typically, the signal sequence is positioned in the coding region of nucleic
acid sequence,
or directly at the 5 end of the coding region. Many signal sequences have been
identified,
and any that are functional in the selected host cell may be used.
Accordingly, the signal
sequence may be homologous or heterologous to the polypeptide. Additionally,
the signal
sequence may be chemically synthesized using recombinant DNA techniques well
known
in the art.
The amount of peptide produced in the host cell can be evaluated using
standard
methods known in the art. Such methods include, without limitation, Western
blot analysis,
SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis,
HPLC
separation, immunoprecipitation, and/or activity assays such as DNA binding
gel shift
assays.
When natural antibody-binding peptides are secreted from the host cells, the
majority of the peptide will likely be found in the cell culture medium. If,
however, the
peptide is not secreted, it will be present in the cytoplasm (for eukaryotic,
Gram-positive
bacteria, and insect host cells) or in the periplasm (for Gram-negative
bacteria host cells).
If the natural antibody-binding peptide remains in the intracellular space,
the host
cells are typically first disrupted mechanically or osmotically to release the
cytoplasmic
contents into a buffered solution. The peptide is then isolated from this
solution.
Purification of the peptide from solution can thereafter be accomplished using
a variety of
techniques. If the peptide has been synthesized such that it contains a tag
such as
hexahistidine or other small peptides at either its carboxyl or amino
terminus, it may be
purified in a one-step process by passing the solution through an affinity
column where the
column matrix has a high affinity for the tag or for the peptide directly
(i.e., a monoclonal
antibody). For example, polyhistidine binds with great affinity and
specificity to nickel,
thus an affinity column of nickel (such as the Qiagen nickel columns) can be
used for
purification. (See, for example, Ausubel et al., eds., Current Protocols in
Molecular
Biology, John Wiley & Sons, New York, 1994).
Where, on the other hand, the peptide has no tag and it is not practical to
use an
antibody to purify the peptide, other well known procedures for purification
can be used.
Such procedures include, without limitation, ion exchange chromatography,
molecular
sieve chromatography, HPLC, native gel electrophoresis in combination with gel
elution,
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and preparative isoelectric focusing ("Isoprime" machine/technique, Hoefer
Scientific). In some cases,
two or more of these techniques may be combined to achieve increased purity.
If it is anticipated that the peptide will be found primarily in the
periplasmic space of the
bacteria or the cytoplasm of eukaryotic cells, the contents of the periplasm
or cytoplasm, including
inclusion bodies (e.g., Gram-negative bacteria) if the processed peptide has
formed such complexes, can
be extracted from the host cell using any standard technique known to the
skilled artisan. For example,
the host cells can be lysed to release the contents of the periplasm by the
use of a French press,
homogenization, and/or sonication. The homogenate can then be centrifuged.
6.3.4 Antibody Inhibitors of Natural IgM Antibodies
IgM inhibitors may also be antibodies that compete with natural IgMs in
binding to
antigen. Methods of producing antibodies are well known in the art. For
example, a monoclonal
antibody against a target (e.g., a pathogenic immunoglobulin or an ischemia
specific antigen on a cell)
can be produced by a variety of techniques, including conventional monoclonal
antibody methodology
e.g., the standard somatic cell hybridization technique of Kohler and
Milstein, Nature 256:495 (1975).
Although somatic cell hybridization procedures are preferred, in principle,
other techniques for
producing monoclonal antibody can be employed e.g., viral or oncogenic
transformation of B
lymphocytes. The preferred animal system for preparing hybridomas is the
murine system. Hybridoma
production in the mouse is a very well-established procedure. Immunization
protocols and techniques
for isolation of immunized splenocytes for fusion are known in the art. Fusion
partners (e.g., murine
myeloma cells) and fusion procedures are also known.
Human monoclonal antibodies can be generated using transgenic mice carrying
the
human immunoglobulin genes rather than mouse immunoglobulin genes. Splenocytes
from these
transgenic mice immunized with the antigen of interest are used to produce
hybridomas that secrete human
mAbs with specific affinities for epitopes from a human protein (see, e.g.,
Wood et al. International
Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741;
Lonberg et al. International
Application WO 92/03918; Kay et al. International Application 92/03917;
Lonberg, N. et al. 1994 Nature 3
68:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et
al. 1994 Proc. Natl. Acad.
Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immuno. 17:33-40; Tuaillon
etal. 1993 PNAS
90:3720-3724; Bruggeman et al. 1991 Eur. J. Immunol 21:1323-1326). In one
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embodiment, hybridomas can be generated from human CD5+, B-1 cells.
Alternatively,
"humanized" murine hybridomas can be used that recognize cross-reactive
"ischernic
antigen".
Monoclonal antibodies can also be generated by other methods known to those
skilled in the art of recombinant DNA technology. An alternative method,
referred to as the
"combinatorial antibody display" method, has been developed to identify and
isolate
antibody fragments having a particular antigen specificity, and can be
utilized to produce
monoclonal antibodies (for descriptions of combinatorial antibody display see
e.g., Sastry et
al. 1989 PNAS 86:5,728; Huse et al. 1989 Science 246:1275; and Orlandi et al.
1989 PNAS
86:3833). After immunizing an animal with an immunogen as described above, the
antibody repertoire of the resulting B-cell pool is cloned. Methods are
generally known for
obtaining the DNA sequence of the variable regions of a diverse population of
immuno globulin molecules by using a mixture of oligomer primers and PCR. For
instance,
mixed oligonucleotide primers corresponding to the 5' leader (signal peptide)
sequences
and/or framework 1 (FR1) sequences, as well as primer to a conserved 3'
constant region
primer can be used for PCR amplification of the heavy and light chain variable
regions
from a number of murine antibodies (Larrick et al., 1991, Biotechniques 11:152-
156). A
similar strategy can also been used to amplify human heavy and light chain
variable regions
from human antibodies (Larrick et al., 1991, Methods: Conzpanion to Methods in
Enzymology 2:106-110).
In an illustrative embodiment, RNA is isolated from B lymphocytes, for
example,
peripheral blood cells, bone marrow, or spleen preparations, using standard
protocols (e.g.,
U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et
al., PNAS
(1989) 86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-
strand cDNA is
synthesized using primers specific for the constant region of the heavy
chain(s) and each of
the K. and 2. light chains, as well as primers for the signal sequence. Using
variable region
PCR primers, the variable regions of both heavy and light chains are
amplified, each alone
or in combination, and ligated into appropriate vectors for further
manipulation in
generating the display packages. Oligonucleotide primers useful in
amplification protocols
may be unique or degenerate or incorporate inosine at degenerate positions.
Restriction
endonuclease recognition sequences may also be incorporated into the primers
to allow for
the cloning of the amplified fragment into a vector in a predetermined reading
frame for
expression.
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The V-gene library cloned from the immunization-derived antibody repertoire
can
be expressed by a population of display packages, preferably derived from
filamentous
phage; to folio an antibody display library. Ideally, the display package
comprises a system
that allows the sampling of very large variegated antibody display libraries,
rapid sorting
after each affinity separation round, and easy isolation of the antibody gene
from purified
display packages. In addition to commercially available kits for generating
phage display
libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01;
and the Stratagene SurfZAPTM phage display kit, catalog no. 240612), examples
of
methods and reagents particularly amenable for use in generating a variegated
antibody
display library can be found in, for example, Ladner et al. U.S. Patent No.
5,223,409; Kang
et al. International Publication No. WO 92/18619; Dower et al. International
Publication
No. WO 91/17271; Winter et al. International Publication WO 92/20791; Marldand
et al.
International Publication No. WO 92/15679; Breitling et al. International
Publication WO
93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard
et al.
International Publication No. WO 92/09690; Ladner et al. International
Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992)
HUMa71
Antibody Hybridornas 3:81-85; Huse etal. (1989) Science 246:1275-1281;
Griffths et al.
(1993) Ell4B0 J12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;
Clackson et
al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad
etal.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nue. Acid Res.
19:4133-
4137; and Barbas et all. (1991) PNAS 88:7978-7982.
In certain embodiments, the V region domains of heavy and light chains can be
expressed on the same polypeptide, joined by a flexible linker to form a
single-chain Fv
fragment, and the scFV gene subsequently cloned into the desired expression
vector or
phage genome. As generally described in McCafferty et all., Nature (1990)
348:552-554,
complete VH and VL domains of an antibody, joined by a flexible (G1y4-Ser)3
linker can be
used to produce a single chain antibody which can render the display package
separable
based on antigen affinity. Isolated scFV antibodies immunoreactive with the
antigen can
subsequently be formulated into a pharmaceutical preparation for use in the
subject method.
Once displayed on the surface of a display package (e.g., filamentous phage),
the
antibody library is screened with the target antigen, or peptide fragment
thereof, to identify
and isolate packages that express an antibody having specificity for the
target antigen.
Nucleic acid encoding the selected antibody can be recovered from the display
package
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(e.g., from the phage genome) and subcloned into other expression vectors by
standard
recombinant DNA techniques.
Specific antibody molecules with high affinities for a surface protein can be
made
according to methods known to those in the art, e.g., methods involving
screening of
libraries (Ladner, R.C., et al., US. Patent 5,233,409; Ladner, R.C., et al.,
US. Patent
5,403,484). Further, these libraries can be used in screens to obtain binding
determinants
that are mimeties of the structural determinants of antibodies.
In particular, the Fv binding surface of a particular antibody molecule
interacts with
its target ligand according to principles of protein-protein interactions,
hence sequence data
for VII and VL (the latter of which may be of the lc or X chain type) can be
used in protein
engineering techniques known to those with skill in the art. Details of the
protein surface
that comprises the binding determinants can be obtained from antibody sequence
information, by a modeling procedure using previously determined three-
dimensional
structures from other antibodies obtained from NMR studies or crystallographic
data. See
for example Bajorath, J. and S. Sheriff, 1996, Proteins: Struct., Funct., and
Genet. 24 (2),
152-157; Webster, D.M. and A. R. Rees, 1995, "Molecular modeling of antibody-
combining sites," in S. Paul, Ed., Methods in Molecular Biol. 51, Antibody
Engineering
Protocols, Humana Press, Totowa, NJ, pp 17-49; and Johnson, G., Wu, T.T. and
E.A.
Kabat, 1995, "Seqhunt: A program to screen aligned nucleotide and amino acid
sequences,"
in Methods in Molecular Biol. 51, op. cit., pp 1-15.
In one embodiment, a variegated peptide library is expressed by a population
of
display packages to form a peptide display library. Ideally, the display
package comprises a
system that allows the sampling of very large variegated peptide display
libraries, rapid
sorting after each affinity separation round, and easy isolation of the
peptide-encoding gene
from purified display packages. Peptide display libraries can be in, e.g.,
prokaryotic
organisms and viruses, which can be amplified quickly, are relatively easy to
manipulate,
and which allow the creation of large number of clones. Preferred display
packages
include, for example, vegetative bacterial cells, bacterial spores, and most
preferably,
bacterial viruses (especially DNA viruses). However, the present invention
also
contemplates the use of eukaryotic cells, including yeast and their spores, as
potential
display packages. Phage display libraries are described above.
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Other techniques include affinity chromatography with an appropriate
'receptor,
e.g., a target antigen, followed by identification of the isolated binding
agents or ligands by
conventional techniques (e.g., mass spectrometry and NMR). Preferably, the
soluble
receptor is conjugated to a label (e.g., fluorophores, colorirnetric enzymes,
radioisotopes, or
luminescent compounds) that can be detected to indicate ligand binding.
Alternatively,
immobilized compounds can be selectively released and allowed to diffuse
through a
membrane to interact with a receptor.
Combinatorial libraries of compounds can also be synthesized with "tags" to
encode
the identity of each member of the library (see, e.g., W.C. Still et al.,
International
Application WO 94/08051). In general, this method features the use of inert
but readily
detectable tags that are attached to the solid support or to the compounds.
When an active
compound is detected, the identity of the compound is determined by
identification of the
unique accompanying tag. This tagging method permits the synthesis of large
libraries of
compounds which can be identified at very low levels among the total set of
all compounds
in the library.
An antibody of the present invention can be one in which the variable region,
or a
portion thereof, e.g., the complementarity determining regions (CDR or CDRs),
are
generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-
grafted, and
humanized antibodies are within the invention. Antibodies generated in a non-
human
organism, e.g., a rat or mouse, and then modified, e.g., in the variable
framework or
constant region, to decrease antigenicity in a human are within the invention.
Any
modification is within the scope of the invention so long as the antibody has
at least one
antigen binding portion.
Chimeric antibodies (e.g. mouse-human monoclonal antibodies) can be produced
by
recombinant DNA techniques known in the art. For example, a gene encoding the
Fc
constant region of a murine (or other species) monoclonal antibody molecule is
digested
with restriction enzymes to remove the region encoding the murine Fc, and the
equivalent
portion of a gene encoding a human Fc constant region is substituted. (see
Robinson et al.,
International Patent Publication PCT/US86/02269; Akira, et al., European
Patent
Application 184,187; Taniguchi, M., European Patent Application 171,496;
Morrison et al.,
European Patent Application 173,494; Neuberger et al., International
Application WO
86/01533; Cabilly etal. U.S. Patent No. 4,816,567; Cabilly et al., European
Patent
Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al.
(1987) PNAS
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84:3439-3443; Liu at al., 1987, J. Inununol. 139:3521-3526; Sun et al. (1987)
PNAS
84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985)
Nature
314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559).
A chimeric antibody can be further humanized by replacing sequences of the Fv
variable region which are not directly involved in antigen binding with
equivalent
sequences from human Fv variable regions. General methods for generating
humanized
antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 by Oi
et al., 1986,
BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US
5,693,762,
the contents of all of which are hereby incorporated by reference. Those
methods include
isolating, manipulating, and expressing the nucleic acid sequences that encode
all or part of
immunoglobulin Fv variable regions from at least one of a'heavy or light
chain: Sources of
such nucleic acid are well known to those skilled in the art and, for example,
may be
obtained from 7E3, An anti-GPIVLI, antibody producing hybridoma. The
recombinant
DNA encoding the chimeric antibody, or fragment thereof, can then be cloned
into an
appropriate expression vector. Suitable humanized antibodies can alternatively
be produced
by CDR substitution. U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-
525;
Verhoeyan at al. 1988 Science 239:1534; and Beidler at al. 1988 J Immunol.
141:4053-
4060.
Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR
substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be
replaced.
See e.g., U.S. Patent 5,225,539; Jones etal. 1986 Nature 321:552-525;
Verhoeyan et al.
1988 Science 239:1534; Beidler etal. 1988 J. Immunol. 141:4053-4060; Winter US
5,225,539.
Winter describes a CDR-grafting method which may be used to prepare the
humanized
antibodies of the present invention (UK Patent Application GB 2188638A, filed
on
March 26, 1987; Winter US 5,225,539).
A humanized or CDR-grafted antibody will have at least one or two but
generally
all recipient CDRs (Of heavy and/or light immunoglobulin chains) replaced with
a donor
CDR. Preferably, the donor will be a rodent antibody, e.g., A rat or mouse
antibody, and the
recipient will be a human framework or a human consensus framework. Typically,
the
. immunoglobulin providing the CDRs is called the "donor" and the
immunoglobulin
providing the framework is called the "acceptor." In one embodiment, the donor
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immunoglobulin is a non-human (e.g., rodent). The acceptor framework can be a
naturally-
occurring (e.g., a human) framework or a consensus framework, or a sequence
about 85%
or higher, preferably 90%, 95%, 99% or higher identical thereto.
All of the CDRs of a particular antibody may be replaced with at least a
portion of a
non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It
is
only necessary to replace the number of CDRs required for binding of the
humanized
antibody to the Fe receptor.
Also within the scope of the invention are chimeric and humanized antibodies
in
which specific amino acids have been substituted, deleted or added. In
particular, preferred
humanized antibodies have amino acid substitutions in the framework region,
such as to
improve binding to the antigen. For example, a humanized antibody will have
framework
residues identical to the donor framework residue or to another amino acid
other than the
recipient framework residue. As another example, in a humanized antibody
having mouse
CDRs, amino acids located in the human framework region can be replaced with
the amino
acids located at the corresponding positions in the mouse antibody. Such
substitutions are
known to improve binding of humanized antibodies to the antigen in some
instances.
Antibody fragments of the invention are obtained using conventional procedures
known to those with skill in the art. For example, digestion of an antibody
with pepsin
yields F(ab1)2 fragments and multiple small fragments. Mercaptoethanol
reduction of an
antibody yields individual heavy and light chains. Digestion of an antibody
with papain
yields individual Fab fragments and the Fe fragment.
In another aspect, the invention also features a modified natural
immunoglobulin,
e.g., which functions as an agonist (mimetic) or as an antagonist. Preferably
the modified
natural immunoglobulin, e.g., modified pathogenic immunoglobulin, functions as
an
antagonist of complement activation. Variants of the pathogenic immunoglobulin
can be
generated by mutagenesis, e.g., discrete point mutation, the insertion or
deletion of
sequences or the truncation of a pathogenic immunoglobulin. An agonist of the
natural
immunoglobulin can retain substantially the same, or a subset, of the
biological activities of
the naturally occurring form of the protein. An antagonist of a natural
immunoglobulin can
inhibit one or more of the activities of the naturally occurring form of the
pathogenic
immunoglobulin by, for example, being capable of binding to an ischemic
specific antigen,
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but incapable of activating a complement pathway. Thus, specific biological
effects can be
elicited by treatment with a variant of limited function.
In one embodiment, the site within the natural immunoglobulin (e.g., a
pathogenic
IgM) that binds Clq can be mutated such that it is no longer capable of
binding Clq. For
example, the CH2 domain of an IgG and the CH4 domain of an IgM, which are
known to
contain binding sites for Clq, can be mutated (see WO 94/29351). For example,
the
carboxyl terminal half of the CH2 domain of an IgG (residues 231 to 239,
preferably.
within 234 to 239), which appear to mediate Clq binding and subsequent
complement
activation, can be mutated. As another example, Wright et al. have
demonstrated that a
single nucleotide change in the IgM constant region domain renders the
antibody defective
in initiating complement-dependent cytolysis. The single nucleotide change
results in the
encoding of a serine residue, rather than the normal proline residue, at amino
acid position
436 in the third constant domain (Wright etal. 1988, J. BioL Chem. 263:
11221). The
amino acid substitutions that can be made to antibodies in order to alter
complement .
binding or activity are well known in the art (see for example, Wright et al.
1988, J. Bid.
Chetn. 263: 11221; Shulman et al. (1986), Proc. Natl. Acad. Sci. USA 83: 7678-
7682; Arya
et al., (1994) J. ImmunoL 253: 1206-1212; Poon et al., (1995) J. Biol. Chem.
270: 8571-
8577. Accordingly, in one embodiment, the antibodies of the present invention
have a mutation
that alters complement binding or activity. Antibodies in which amino acids
have been added,
deleted, or substituted are referred to herein as modified antibodies or
altered antibodies.
As will be appreciated by the skilled artisan, the methods used for causing
such changes in
nucleotide or amino acid sequence will vary depending upon the desired
results.
Variants of a natural immunoglobulin can be identified by screening
combinatorial
libraries of mutants, e.g., truncation mutants, of a natural immunoglobulin
for agonist or
antagonist activity.
Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of
a
natural immunoglobulin coding sequence can be used to generate a variegated
population of
fragments for screening and subsequent selection of variants of this protein.
Variants in
which a cysteine residue is added or deleted or in which a residue that is
glycosylated is
added or deleted are particularly preferred.
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Methods for screening gene products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for gene products
having a
selected property. Recursive ensemble mutagenesis (REM), a technique which
enhances the
frequency of functional mutants in the libraries, can be used in combination
with the
screening assays to identify variants (Arkin and Yourvan (1992) Proc. Natl.
Acad. Sci. USA
89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
Cell based assays can be exploited to analyze a variegated library. For
example, a
library of expression vectors can be transfected into a cell line, e.g., a
cell line, which
ordinarily responds to the protein in a substrate-dependent manner. Plasmid
DNA can then
be recovered from the cells which score for inhibition, or alternatively,
potentiation of
signaling by the pathogenic immunoglobulin-substrate, and the individual
clones further
characterized.
The invention also features a method of making a natural immunoglobulin, e.g.,
a
pathogenic immunoglobulin having a non-wild type activity, e.g., an
antagonist, agonist, or
super agonist of a naturally occurring pathogenic immunoglobulin. The method
includes:
altering the sequence of a natural immunoglobulin, e.g., by substitution or
deletion of one
or more residues of a non-conserved region, a domain or residue disclosed
herein, and
testing the altered polypeptide for the desired activity.
Further, the invention features a method of making a fragment or analog of a
natural
immunoglobulin, e.g., a pathogenic immunoglobulin having an altered biological
activity of
a naturally occurring pathogenic immunoglobulin. The method includes: altering
the
sequence, e.g., by substitution or deletion of one or more residues, of a
pathogenic
immunoglobulin, e.g., altering the sequence of a non-conserved region, or a
domain or
residue described herein, and testing the altered pol3peptide for the desired
activity. In an
exemplary embodiment, the modified natural immunoglobulin may have a reduced
ability
to activate complement. For example, one or more of the amino acid residues
involved in
complement binding and/or activation are mutated.
In certain embodiment, the modified natural antibody may comprise at least the
CDR1 region of SEQ ID NO: 8 (SEQ ID NO: 10), or antigen binding portions
thereof,
and/or at least the CDR2 region of SEQ ID NO: 8 (SEQ ID NO: 12), or antigen
binding
portions thereof. In another embodiment, the modified antibody may comprise at
least the
CDR1 region of SEQ ID NO: 2 (SEQ ID NO: 4), or antigen binding portions
thereof,
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and/or at least the CDR2 region of SEQ ID NO: 2 (SEQ ID NO: 6), or antigen
binding
portions thereof In an exemplary embodiment, the modified antibody comprises
the CDR1
region of SEQ ID NO: 8 (SEQ ID NO: 10) and the CDR2 region of SEQ ID NO: 8
(SEQ
ID NO: 12) or antigen binding portions thereof. In another exemplary
embodiment, the
modified antibody comprises the CDR1 region of SEQ ID NO: 2 (SEQ ID NO: 4) and
the
CDR2 region of SEQ ID NO: 2 (SEQ ID NO: 6) or antigen binding portions
thereof. The
modified antibody may also comprise the CDR1 region of SEQ ID NO: 8 (SEQ ID
NO: 10)
and the CDR2 region of SEQ ID NO: 8 (SEQ ID NO: 12) and the modified antibody
comprises the CDR1 region of SEQ ID NO: 2 (SEQ ID NO: 4) and the CDR2 region
of
SEQ ID NO: 2 (SEQ ID NO: 6) or antigen binding portions thereof
The modified natural antibody can be a human antibody having a binding
affinity to
the ischemic-specific antigen, similar, e.g., greater than, less than, or
equal to, the binding
affinity of the antibody produced by the hybridoma deposited with the ATCC,
having the
accession number PTA-3507. In another embodiment, the natural antibody can be
a non-
human antibody, e.g., a cow, goat, mouse, rat, sheep, pig, or rabbit. In an
exemplary
embodiment, the non-human antibody is a murine antibody. The natural antibody
may also
be a recombinant antibody. In an exemplary embodiment, the natural antibody is
a
humanized antibody. The modified natural antibody may be an IgG or IgM
antibody. In
another embodiment, the isolated natural immunoglobulin possess the same
antigenic
specificity as the immunoglobulin produced by the hybridoma deposited with the
ATCC,
having accession number PTA-3507.
6.4 Screening Assay to Identib, Additional Inhibitors
Other inhibitors of an interaction between a natural IgM antibody and an
antigen or
a component of the complement pathway may beidentified from one or more (e.g.,
a
plurality of) test compounds, comprising (i) providing a reaction mixture
which includes
the natural IgIVI antibody and the antigen or the component of the complement
pathway
under conditions that allow binding of the natural IgM antibody and the
antigen or the
component of the complement pathway to occur; (ii) contacting the natural IgM
antibody
and the antigen or the component of the complement pathway with one or more
test
compounds (e.g., members of a combinatorial library); and (iii) detecting any
changes in
binding of the natural IgM antibody and the antigen or the component of the
complement in
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the presence of a given test compound relative to that detected in the absence
of the test
compound. A change (e.g., decrease) in the level of binding between the
natural IgM
antibody and the antigen or the component of the complement pathway in the
presence of
the test compound relative to that detected in the absence of the test
compound indicates
that the test compound is an inhibitor of the interaction between the natural
IgM antibody
and the antigen or the component of the complement pathway.
The method can further include pre-treating the natural IgM antibodies with
one or
more test compounds. The pre-treated natural IgM antibodies can then be
injected into mice
deficient in natural immunoglobulins.
In certain embodiments, the methods is performed in vitro. In an exemplary
embodiment, the contacting step is effected in vivo. hi an exemplary
embodiment, the
antigen is myosin. In other embodiments, the antigen is an endothelial tissue
or lysate
obtained from a subject e.g., a human patient with reperfitsion or ischemic
injury. In
another exemplary embodiment, the component of the complement pathway is a
component
of the classical pathway of complement. In a further exemplary embodiment, the
component of the complement pathway is a Cl molecule or a subunit thereof
(e.g., Clq).
In exemplary embodiments, either the natural IgM antibody or the antigen (or
both)
is labeled with a detectable signal, e.g., fluorophores, colorimetric enzymes,
radioisotopes,
luminescent compounds, and the like. The method can further include repeating
at least
one step, e.g., the contacting step with a second or subsequent member or
members of the
library.
In an exemplary embodiment, a plurality of test compounds, e.g., library
members,
is tested. The plurality of test compounds, e.g., library members, can include
at least 10,
102, 103, 104, 105, 106, 107, or 108 compounds. In a preferred embodiment, the
plurality of
test compounds, e.g., library members, share a structural or functional
characteristic. The
test compound can be a peptide or a small organic molecule.
In one embodiment, the inhibitor is a small organic molecule that may be
identified
in a combinatorial library. In one embodiment, the invention provides
libraries of
inhibitors. The synthesis of combinatorial libraries is well known in the art
and has been
reviewed (see, e.g., E.M. Gordon et al., J. Med. Chem. (1994) 37:1385-1401 ;
DeWitt, S.
H.; Czarnik, A. W. Ace. Chem. Res. (1996) 29:114; Armstrong, R. W.; Combs, A.
P.;
Tempest, P. A.; Brown, S. D.; Keating, T. A. Acc. Chem. Res. (1996) 29:123;
Ellman, J. A.
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Ace. Chem. Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V. Ace.
Chem.
Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelle et al.
Trends Anal.
Chem. (1995) 14:83; Chen et al. J.. Am. Chem. Soc. (1994) 116:2661; U.S.
Patents
5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. W092/10092,
W093/09668,
W091/07087, W093/20242, W094/08051).
Libraries of compounds of the invention can be prepared according to a variety
of
methods, some of which are known in the art. For example, a "split-pool"
strategy can be
implemented in the following way: beads of a functionalized polymeric support
are
placed in a plurality of reaction vessels; a variety of polymeric supports
suitable for solid-
phase peptide synthesis are known, and some are commercially available (for
examples,
see, e.g., M. Bodansky "Principles of Peptide Synthesis", 2nd edition,
Springer-Verlag,
Berlin (1993)). To each aliquot of beads is added a solution of a different
activated amino
acid, and the reactions are allowed to proceed to yield a plurality of
immobilized amino
acids, one in each reaction vessel. The aliquots of derivatized beads are then
washed,
"pooled" (i.e., recombined), and the pool of beads is again divided, with each
aliquot
being placed in a separate reaction vessel. Another activated amino acid is
then added to
each aliquot of beads. The cycle of synthesis is repeated until a desired
peptide length is
obtained. The amino acid residues added at each synthesis cycle can be
randomly
selected; alternatively, amino acids can be selected to provide a "biased"
library, e.g., a
library in which certain portions of the inhibitor are selected non-randomly,
e.g., to
provide an inhibitor having known structural similarity or homology to a known
peptide
capable of interacting with an antibody, e.g., the an anti-idiotypic antibody
antigen
binding site. It will be appreciated that a wide variety of peptidic,
peptidomimetic, or non-
peptidic compounds can be readily generated in this way.
The "split-pool" strategy results in a library of peptides, e.g., inhibitors,
which can
be used to prepare a library of test compounds of the invention. In another
illustrative
synthesis, a "diversomer library" is created by the method of Hobbs DeWitt et
al. (Proc.
Natl. Acad. Sci. USA 90:6909 (1993)). Other synthesis methods, including the
"tea-bag"
technique of Houghten (see, e.g., Houghten et al., Nature 354:84-86 (1991))
can also be
used to synthesize libraries of compounds according to the subject invention.
Libraries of
compounds can be screened to determine whether any members of the library have
a
desired activity, and, if so, to identify the active species. Methods of
screening
combinatorial libraries have been described (see, e.g., Gordon et al., J Med.
Chem.,
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supra). Soluble compound libraries can be screened by affinity chromatography
with an
appropriate receptor to isolate ligands for the receptor, followed by
identification of the
isolated ligands by conventional techniques (e.g., mass spectrometry, NMR, and
the.
like). Immobilized compounds can be screened by contacting the compounds with
a
soluble receptor; preferably, the soluble receptor is conjugated to a label
(e.g.,
fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds, and
the like)
that can be detected to indicate ligand binding. Alternatively, immobilized
compounds
can be selectively released and allowed to diffuse through a membrane to
interact with a
receptor. Exemplary assays useful for screening the libraries of the invention
are
described below.
In one embodiment, compounds of the invention can be screened for the ability
to
interact with a natural immunoglobulin by assaying the activity of each
compound to bind
directly to the immunoglobulin or to inhibit an interaction between the
immunoglobulin and
an ischemic antigen, e.g., by incubating the test compound with an
immunoglobulin and a
lysate, e.g., an endothelial cell lysate, e.g., in one well of a multiwell
plate, such as a
standard 96-well microtiter plate. In this embodiment, the activity of each
individual
compound can be determined. A well or wells having no test compound can be
used as a
control. After incubation, the activity of each test compound can be
determined by
assaying each well. Thus, the activities of a plurality of test compounds can
be determined
in parallel.
6.5 Modified Inhibitors and Pharmaceutical and Diagnostic
Preparations
IgM inhibitors may be modified, for example to increase solubility and/or
facilitate
purification, identification, detection, and/or structural characterization.
Exemplary
modifications, include, for example, addition of: glutathione S-transferase
(GST), protein
A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding
protein, HA, myc,
poly-arginine, poly-His, poly-His-Asp or FLAG fusion proteins and tags. In
various
embodiments, an IgM inhibitors may comprise one or more heterologous fusions.
For
example, peptides may contain multiple copies of the same fusion domain or may
contain
fusions to two or more different domains. The fusions may occur at the N-
terminus of the
peptide, at the C-terminus of the peptide, or at both the N- and C-terminus of
the peptide. It
is also within the scope of the invention to include linker sequences between
a peptide of
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the invention and the fusion domain in order to facilitate construction of the
fusion protein
or to optimize protein expression or structural constraints of the fusion
protein. In another
embodiment, the peptide may be constructed so as to contain protease cleavage
sites
between the fusion peptide and peptide of the invention in order to remove the
tag after
protein expression or thereafter. Examples of suitable endoproteases, include,
for example,
Factor Xa and TEV proteases.
Techniques for making fusion genes are well known. Essentially, the joining of
various DNA fragments coding for different polypeptide sequences is performed
in
accordance with conventional techniques, employing blunt-ended or stagger-
ended termini
for ligation, restriction enzyme digestion to provide for appropriate termini,
filling-in of
cohesive ends as appropriate, alkaline phosphatase treatment to avoid
undesirable joining,
and enzymatic ligation. In another embodiment, the fusion gene may be
synthesized by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
amplification of gene fragments may be carried out using anchor primers which
give rise to
complementary overhangs between two consecutive gene fragments, which may
subsequently be annealed to generate a chimeric gene sequence (see, for
example, Current
Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
IgM inhibitors may be chemically modified based on linkage to a polymer. The
polymer is typically water soluble so that the inhibitor to which it is
attached does not
precipitate in an aqueous environment, such as a physiological environment.
The polymer
may have a single reactive group, such as an active ester for acylation or an
aldehyde for
alk-ylation, so that the degree of polymerization may be controlled. A
preferred reactive
aldehyde is polyethylene glycol propionaldehyde, which is water stable, or
mono Cl -C10
alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714). The
polymer may be
branched or unbranched. Preferably, for therapeutic use of the end-product
preparation, the
polymer will be pharmaceutically acceptable. The water soluble polymer, or
mixture
thereof if desired, may be selected from the group consisting of, for example,
polyethylene
glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other
carbohydrate
based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene
glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol.
IgM inhibitors may be labeled, for example with an isotopic label to
facilitate its
detection using nuclear magnetic resonance or another applicable technique.
Exemplary
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isotopic labels include radioisotopic labels such as, for example, potassium-
40 (40K),
carbon-14 (14C), tritium (3H), sulphur-35 (35S), phosphorus-32 (32P),
technetium-99m
(99mTc), thallium-201 (201T1), gallium-67 (67Ga), indium-111 (1111n), iodine-
123 (1231),
iodine-131 (131I), yttrium-90 (90Y), samarium-153 (153Sm), rhenium-186 ('86R
e), rhenium-
188 (188Re), dysprosium-165 (165Dy) and holmium-166 (166H0) .
The isotopic label may
also be an atom with non zero nuclear spin, including, for example, hydrogen-1
(1H),
hydrogen-2 (2H), hydrogen-3 (3H), phosphorous-31 (31P), sodium-23 (23Na),
nitrogen-14
(14N), nitrogen-15 (15N), carbon-13 (13C) and fluorine-19 (19F). In certain
embodiments, the
inhibitor is uniformly labeled with an isotopic label, for example, wherein at
least 50%,
70%, 80%, 90%, 95%, or 98% of the inhibitor is labeled. In other embodiments,
the
isotopic label is located in one or more specific locations within the
inhibitor, for example,
the label may be specifically incorporated into one or more of the leucine
residues of a
peptide. A single inhibitor may comprise two or more different isotopic
labels, for
example, a peptide may comprise both 15N and 13C labeling.
Inhibitors may be labeled with a fluorescent label. In an exemplary
embodiment, an
inhibitor is fused to a heterologous polypeptide sequence which produces a
detectable
fluorescent signal, including, for example, green fluorescent protein (GFP),
enhanced green
fluorescent protein (EGFP), Renilla renifornzis green fluorescent protein,
GFPmut2,
GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent
protein
(ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent
protein from
discosoma (dsRED).
Toxicity and therapeutic efficacy of natural antibody inhibitors including
natural
IgM antibody-binding peptides or modified natural IgM antibodies can be
determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio LD50/ED50.
Natural antibody inhibitors which exhibit large therapeutic effects are
preferred. While
natural antibody inhibitors or natural antibody-binding peptides that exhibit
toxic side
effects may be used, care should be taken to design a delivery system that
targets such
peptides or modified antibodies to the site of affected tissue in order to
minimize potential
damage to uninfected cells and, thereby, reduce side effects.
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The data obtained from the cell culture assays and animal studies can be used
in
foimulating a range of dosage for use in humans. The dosage of a natural
antibody
inhibitor or a natural antibody-binding peptides lies preferably within a
range of circulating
concentrations that include the ED50 with little or no toxicity. The dosage
may vary within
this range depending upon the dosage form employed and the route of
administration
=
utilized. For any inhibitor or peptide used in the method of the invention,
the
therapeutically effective dose can be estimated initially from cell culture
assays. A dose
may be formulated in animal models to achieve a circulating plasma
concentration range
that includes the IC50 (i.e., the concentration of the test compound which
achieves a half-
maximal inhibition of symptoms) as determined in cell culture. Such
information can be
used to more accurately determine useful doses in humans. Levels in plasma may
be
measured, for example, by high performance liquid chromatography.
In another embodiment, a single bolus of a natural antibody inhibitor
including a
natural IgM antibody-binding peptide and modified natural IgM antibodies is
administered
prior to, contemporaneously with, or subsequent to a tissue injury. Typically
a single dose
injection will be a few hours, a few days or a few weeks after tissue injury.
The present
invention is based in part upon the discovery that a natural IgM antibody
inhibitor
preventsreperfusion injury. A single unit dosage delivery can be immediately
adjacent to
the site of injury or can be, for example, to a vessel that drains or flows to
the site of injury.
90 A natural IgM antibody inhibitor such as natural IgM antibody-binding
peptide Or
modified natural IgM antibody is administered initially at a point in time
prior to the time of
damage of the target organ or tissue. This may be a useful approach in
subjects who are
determined to be at risk for reperfusion injury, such as those with a history
of reperfusion
injury or those about to undergo surgery.
In yet another embodiment, a single bolus of a natural IgM antibody inhibitor
can be
followed by subsequence administrations of a natural IgM antibody inhibitor as
continuous
infusions or additional single bolus deliveries. The inhibitor may be
administer in
sequential exposures over a period of hours, days, weeks, months or years. In
addition, it is
contemplated that additional therapeutic agents can be combined with,
administered prior to
or subsequent to administration of a natural antibody-binding peptide or
another natural
antibody inhibitor. Other therapeutic agents that may be administered with an
natural IgM
antibody inhibitor include, but are not limited to, anti-coagulation agents
and complement
inhibitors.
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=
60412-4434D1
The subject inhibitors may be provided in pharmaceutically acceptable carriers
or
formulated for a variety of modes of administration, including systemic and
topical or
localized administration. Techniques and formulations generally may be found
in
Rernmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. In
certain
embodiments, the inhibitor is provided for transmucosal or transdermal
delivery. For such
administration, penetrants appropriate to the bather to be permeated are used
in the
formulation with the polypeptide. Such penetrants are generally known in the
art, and
include, for example, for transmucosal administration bile salts and fusidic
acid derivatives.
In addition, detergents may be used to facilitate permeation. Transmucosal
administration
may be through nasal sprays or using suppositories. For topical
administration, the
inhibitors of the invention are formulated into ointments, salves, gels, or
creams as
generally known in the art.
The pharmaceutical compositions according to the invention are prepared by
bringing a natural IgM antibody inhibitors into a form suitable for
administration to a
subject using carriers, excipients and additives or auxiliaries. Frequently
used carriers or
auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol
and other
sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its
derivatives, animal and
vegetable oils, polyethylene glycols and solvents, such as sterile water,
alcohols, glycerol
and polyhydric alcohols. Intravenous vehicles include fluid and nutrient
replenishers.
Preservatives include antimicrobial, anti-oxidants, chelating agents and inert
gases. Other
pharmaceutically acceptablecarriers include aqueous solutions, non-toxic
excipients,
including salts, preservatives, buffers and the like, as described, for
instance, in
Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.,
1405-1412,
1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington:
American
Pharmaceutical Association (1975). The pH and exact concentration of the
various =
components of the pharmaceutical composition are adjusted according to routine
skills
in the art. See Goodman and Gilman's The Pharmacological Basis for
Therapeutics (7th ed.).
The pharmaceutical compositions are preferably prepared and administered in
dose
units. Solid dose units are tablets, capsules and suppositories and including,
for example,
alginate based pH dependent release gel caps. For treatment of a subject,
depending on
activity of the compound, manner of administration, nature and severity of the
disorder, age
and body weight of the subject, different daily doses are necessary. Under
certain
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=
circumstances, however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form
of an individual dose unit or by several smaller dose units and also by
multiple
administration of subdivided doses at specific intervals.
The pharmaceutical compositions according to the invention may be administered
locally or systemically in a therapeutically effective dose. Amounts effective
for this use
will, of course, depend on the severity of the disease and the weight and
general state of the
subject. As discussed above, dosages used in vitro may provide useful guidance
in the
amounts useful for in situ administration of the pharmaceutical composition,
and animal
models may be used to determine effective dosages for treatment of particular
disorders.
Various considerations are described, e.g., in Langer, Science, 249: 1527,
(1990); Gilman et
al. (eds.) (1990) .
In one embodiment, the invention provides a pharmaceutical composition useful
for
administering a natural antibody-binding peptide to a subject in need of such
treatment.
"Administering" the pharmaceutical composition of the invention may be
accomplished by
any means known to the skilled artisan. Preferably a "subject" refers to a
Mammal, most
preferably a human.
The natural IgM antibody inhibitor can be administered parenterally,
enterically, by
injection, rapid infusion, nasopharyngeal absorption, dermal absorption,
rectally and orally.
Pharmaceutically acceptable carrier preparations for parenteral administration
include
sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-
aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil,
and injectable organic esters such as ethyl oleate. Carriers for occlusive
dressings can be
used to increase skin permeability and enhance antigen absorption. Liquid
dosage forms
for oral administration may generally comprise a liposome solution containing
the liquid
dosage form. Suitable solid or liquid pharmaceutical preparation forms are,
for example,
granules, powders, tablets, coated tablets, (micro)capsules, suppositories,
syrups,
emulsions, suspensions, creams, aerosols, drops or injectable solution in
ampule form and
also preparations with protracted release of active compounds, in whose
preparation
= 30 excipients and additives and/or auxiliaries such as disintegrants,
binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners and elixirs containing
inert diluents
commonly used in the art, such as purified water. Where the disease or
disorder is a
= gastrointestinal disorder oral formulations or suppository formulations
are preferred.
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Sterile injectable solutions can be prepared by incorporating a natural
antibody-
binding peptide in the required amount (e.g., about 10 Lig to about 10 mg/kg)
in an
appropriate solvent and then sterilizing, such as by sterile filtration.
Further, powders can be
prepared by standard techniques such as freeze drying or vacuum drying.
In another embodiment, a natural IgM antibody inhibitor is prepared with a ,
biodegradable carrier for sustained release characteristics for either
sustained release in the
GI tract or for target organ implantation with long term active agent release
characteristics
to the intended site of activity. Biodegradable polymers include, for example,
ethylene
vinyl acetate, polyanhydrides, polyglycolic acids, polylactic acids, collagen,
polyorthoesters, and poly acetic acid. Liposomal formulation can also be used.
Another means of delivering natural IgM antibody inhibitor (e.g., a natural
IgM
antibody-binding peptide) is by delivering host cells that express natural
antibody-binding
peptides to a site or tissue in need of repair. Alternatively, the cells may
be delivered in
conjunction with various delivery vehicles, including biocompatible
biodegradable or non-
biodegradable sponges (e.g., collagen, or other extracellular matrix
materials), cotton,
polyglycolic acid, cat gut sutures, cellulose, gelatin, dextran, polyamide, a
polyester, a
polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a
polytetrafluorethylene, or a nitrocellulose compound formed into a three-
dimensional
structure (see, for example, 'U.S. Pat. No. 5,858,721 to Naughton et al.
Any route of administration compatible with the active principle can be used.
The
preferred is parenteral administration, such as subcutaneous, intramuscular or
intravenous
injection. The dose of the active ingredient to be administered depends on the
basis of the
medical prescriptions according to age, weight and the individual response of
the patient.
The daily non-weighted dosage for the patient can be between about 2.5-5.0
mg/Kg,
e.g., about 2.5-3.0 mg/Kg, about 3.0-3.5 mg/Kg, about 3.5-4.0 mg/Kg, about 4.0-
4.5
mg/Kg, and about 4.5-5.0 mg/Kg. =
The pharmaceutical composition for parenteral administration can be prepared
in an
injectable form comprising the active principle and a suitable vehicle.
Vehicles for the
parenteral administration are well known in the art and comprise, for example,
water, saline
solution, Ringer solution and/or dextrose.
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The vehicle can contain small amounts of excipients in order to maintain the
stability and isotonicity of the pharmaceutical preparation.
The preparation of the cited solutions can be carried out according to the
ordinary
modalities.
The present invention has been described with reference to the specific
embodiments, but the content of the description comprises all modifications
and
substitutions which can be brought by a person skilled in the art without
extending beyond
the meaning and purpose of the claims. The compositions may, if desired, be
presented in a
pack or dispenser device which may contain one or more unit dosage forms
containing the
active ingredient. The pack may for example comprise metal or plastic foil,
such as a
blister pack. The pack or dispenser device may be accompanied by instructions
for
administration.
6.6 Diseases and conditions that can be treated with natural
Igllf antibody inhibitors
IgM inhibitors, such as natural IgM antibody-binding peptides or modified
natural
IgM antibodies, may be used for treating a number of inflammatory diseases and
conditions
that are triggered by binding of natural IgM antibodies. For instance, the
inhibitors may be
used to treat inflammatory diseases or cOnditions such as reperfusion injury,
ischemia
injury, stroke, autoimmune hemolytic anemia, idiopathic thrombocytopenic
purpura,
rheumatoid arthritis, celiac disease, hyper-IgM immunodeficiency,
arteriosclerosis,
coronary artery disease, sepsis, myocarditis, encephalitis, transplant
rejection, hepatitis,
thyroiditis (e.g., Hashimoto's thyroiditis, Graves disease), osteoporosis,
polymyositis,
dermatomyositis, Type I diabetes, gout, dermatitis, alopecia areata, systemic
lupus
erythematosus, lichen sclerosis, ulcerative colitis, diabetic retinopathy,
pelvic inflammatory
disease, periodontal disease, arthritis, juvenile chronic arthritis (e.g.,
chronic iridocyclitis),
psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic
inflammatory
disease, chronic inflammatory liver disease, chronic inflammatory lung
disease, lung
fibrosis, liver fibrosis, rheumatoid arthritis, chronic inflammatory liver
disease, chronic
inflammatory lung disease, lung fibrosis, liver fibrosis, Crohn's disease,
ulcerative colitis,
bum injury (or thermal injury), and other acute and chronic inflammatory
diseases of the
Central Nervous System (CNS; e.g., multiple sclerosis), gastrointestinal
system, the skin
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and associated structures, the immune system, the hepato-biliary system, or
any site in the
body where pathology can occur with an inflammatory component.
An inflammatory condition such as reperfusion or ischemic injury may result
following a naturally occurring episode, e.g., as a stroke or myocardial
infarction.
Reperfusion or ischemic injury may also occur during and/or following a
surgical
procedure. Exemplary surgical procedures that cause can cause injury include a
vessel-
corrective technique selected from the group consisting of angioplasty,
stenting procedure,
atherectomy, and bypass surgery. In an exemplary embodiment, reperfusion or
ischemic
injury occurs in a cardiovascular tissue, such as the heart.
In addition, diseases or conditions that are triggered by binding of natural
IgM
antibodies may be treated or prevented in a subject by removing from the
subject or
inactivating a natural or pathogenic IgM and/or B cells producing the
pathogenic
immunoglobulin (e.g., B-I cells as described herein), thereby reducing the
amount of the
pathogenic immunoglobulin and/or B cells present in the subject.
The methods described herein may comprise removing from the subject or
inactivating a pathogenic immunoglobulin, e.g., a pathogenic IgM as described
herein,
and/or B-cells producing the pathogenic IgM (e.g., B-1 cells as described
herein), thereby
reducing the amount of the pathogenic immunoglobulin and/or B cells present in
the
subject.
In one embodiment, the removing or inactivating step is performed ex vivo. The
pathogenic immunoglobulins or B cells can be removed by hemoperfusion.
Alternatively,
the B cells can be removed using a B cell-specific antibody (e.g., an anti-B-1
antibody or an
anti-CD5 antibody or anti-CD 11 G/CD 18). The pathogenic immunoglobulin, e.g.,
an
IgM, can be removed by contacting blood from a subject with an immobilized
antigen (e.g.,
an ischemia-specific antigen) or an immobilized anti-idiotypic antibody. The
removing or
inactivating step of the pathogenic immunoglobulin may be performed by
administering an
anti-idiotypic antibody to the subject. In another embodiment, the removing or
inactivating
step of the B cell is performed by administering to the subject a B cell
targeting moiety
(e.g., an antibody or an antigen binding fragment thereof, or an antigen)
coupled to a toxin,
e.g., ricin or diphteria toxin. The subject is a mammal, e.g., a rodent (e.g.,
a mouse) or a
primate (e.g., a human). In a exemplary embodiment, the subject has sustained
a
reperfusion or ischemic injury following a naturally occurring episode, e.g.,
as a stroke, and
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the removing step is carried out within minutes, one to five hours, five to
ten hours, ten to
twenty hours, one to five days, following the naturally occurring episode. In
another
exemplary embodiment, the reperfusion or ischemie injury occurs in a
cardiovascular
tissue, e.g., the heart, and the reperfusion or ischemic injury is prevented
and/or decreased
by, removing from the subject, the pathogenic immunoglobulin, and/or the B
cells, prior to,
during, and/or following the surgical procedure. For example, the removing
step can be
carried out at least one to five hours, five to ten hours, ten to twenty
hours, or one, two or
three days prior to the surgical procedure. The removing step can also be
continued for
appropriate time intervals during and after the surgical procedure.
6.7 Diagnostic Assays
The invention further provides a method for detecting the presence of a
natural IgM
antibody in a biological sample. Detection of a natural IgM antibody in a
subject,
particularly a mammal, and especially a human, will provide a diagnostic
method for
diagnosis of an inflammatory disease or condition in the subject. In general,
the method
involves contacting the biological sample with a compound or an agent capable
of detecting
natural IgM antibody of the invention or a nucleic acid of the invention in
the sample. The
term "biological sample" when used in reference to a diagnostic assay is
intended to include
tissues, cells and biological fluids isolated from a subject, as well as
tissues, cells and fluids
present within a subject.
The detection method of the invention may be used to detect the presence of a
natural IgIVI antibody or a nucleic acid of the invention in a biological
sample in vitro as
well as in vivo. For example, in vitro techniques for detection of a nucleic
acid of the
invention include Northern hybridizations and in situ hybridizations. In vitro
techniques for
detection of polypeptides of the invention include enzyme linked immunosorbent
assays
(ELISAs), Western blots, immunoprecipitations, inununofluoreseenee,
radioimmunoassays
and competitive binding assays.
Nucleic acids for diagnosis may be obtained from an infected individual's
cells and
tissues, such as bone, blood, muscle, cartilage, and skin. Nucleic acids,
e.g., DNA and
RNA, may be used directly for detection or may be amplified, e.g.,
enzymatically by using
PCR or other amplification technique, prior to analysis. Using amplification,
characterization of the species and strain of prokaryote present in an
individual, may be
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made by an analysis of the genotype of the prokaryote gene. Deletions and
insertions can
be detected by a change in size of the amplified product in comparison to the
genotype of a
reference sequence. Point mutations can be identified by hybridizing a nucleic
acid, e.g.,
amplified DNA, to a nucleic acid of the invention, which nucleic acid may be
labeled.
Perfectly matched sequences can be distinguished from mismatched duplexes by
RNase
digestion or by differences in melting temperatures. DNA sequence differences
may also
be detected by alterations in the electrophoretic mobility of the DNA
fragments in gels,
with or without denaturing agents, or by direct DNA sequencing. See, e.g.
Myers et al.,
Science, 230: 1242 (1985). Sequence changes at specific locations also may be
revealed by
nuclease protection assays, such as RNase and S1 protection or a chemical
cleavage
method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401
(1985).
Agents for detecting a nucleic acid of the invention, e.g., comprising the
sequence
set forth in a subject nucleic acid sequence, include labeled nucleic acid
probes capable of
hybridizing to a nucleic acid of the invention. The nucleic acid probe can
comprise, for
example, the full length sequence of a nucleic acid of the invention, or an
equivalent
thereof, or a portion thereof, such as an oligonucleotide of at least 15, 30,
50, 100, 250 or
500 nucleotides in length and sufficient to specifically hybridize under
stringent conditions
to a subject nucleic acid sequence, or the complement thereof Agents for
detecting a
polypeptide of the invention, e.g., comprising an amino acid sequence of a
subject amino
acid sequence, include labeled anti-antibodies capable of binding to a natural
IgM antibody
of the invention. Anti-idiotypic antibodies may be polyclonal, or
alternatively, monoclonal.
An intact anti-idiotypic antibody, or a fragment thereof can be used. Labeling
the probe or
antibody also encompasses direct labeling of the probe or antibody by coupling
(e.g.,
physically linking) a detectable substance to the probe or antibody, as well
as indirect
labeling of the probe or antibody by reactivity with another reagent that is
directly labeled.
Examples of indirect labeling include detection of a primary antibody using a
fluorescently
labeled secondary antibody and end-labeling of a DNA probe with biotin such
that it can be
detected with fluorescently labeled streptavidin.
In certain embodiments, detection of a nucleic acid of the invention in a
biological
sample involves the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g.
U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al.
(1988) Science
241:1077-1080; and Nakazawa et al. (1994) PNAS 91:360-364), the latter of
which can be
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particularly useful for distinguishing between orthologs of polynucleotides of
the invention
(see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can
include the
steps of collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic,
mRNA or both) from the cells of the sample, contacting the nucleic acid sample
with one or
more primers which specifically hybridize to a nucleic acid of the invention
under
conditions such that hybridization and amplification of the polynucleotide (if
present)
occurs, and detecting the presence or absence of an amplification product, or
detecting the
size of the amplification product and comparing the length to a control
sample.
In one aspect, the present invention contemplates a method for detecting the
presence of a natural IgM antibody in a sample, the method comprising: (a)
providing a
sample to be tested for the presence of a natural IgM antibody; (b) contacting
the sample
with an anti-idiotypic antibody reactive against about eight consecutive amino
acid residues
of a subject amino acid sequence from such species under conditions which
permit
association between the anti-idiotypic antibody and its ligand; and (c)
detecting interaction
of the anti-idiotypic antibody with its ligand, thereby detecting the presence
of a natural
IgM antibody in the sample.
In another aspect, the present invention contemplates a method for detecting
the
presence of a natural IgM antibody in a sample, the method comprising: (a)
providing a
sample to be tested for the presence of a natural IgM antibody; (b) contacting
the sample
with an anti-idiotypic antibody that binds specifically to a polypeptide of
the invention from
such species under conditions which permit association between the anti-
idiotypic antibody
and its ligand; and (c) detecting interaction of the anti-idiotypic antibody
with its ligand,
thereby detecting the presence of such species in the sample.
In yet another example, the present invention contemplates a method for
diagnosing
a patient suffering from an inflammatory disease or condition related to the
presence of a
natural Ig1\4 antibody, comprising: (a) obtaining a biological sample from a
patient; (b)
detecting the presence or absence of a polypeptide of the invention, e.g., a
natural Ig114
antibody, or a nucleic acid encoding a polypeptide of the invention, in the
sample; and (c)
diagnosing a patient suffering from such an inflammatory disease or condition
based on the
presence of a polypeptide of the invention, or a nucleic acid encoding a
polypeptide of the
invention, in the patient sample.
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The diagnostic assays of the invention may also be used to monitor the
effectiveness
of a treatment in an individual suffering from an inflammatory disease or
condition related
to a natural IgM antibody. For example, the presence and/or amount of a
nucleic acid of
the invention or a polypeptide of the invention can be detected in an
individual suffering
from an inflammatory disease or condition related to a natural IgM antibody
before and
after treatment with a natural IgM antibody therapeutic agent. Any change in
the level of a
polynucleotide or polypeptide of the invention after treatment of the
individual with the
therapeutic agent can provide information about the effectiveness of the
treatment course.
In particular, no change, or a decrease, in the level of a polynucleotide or
polypeptide of the
invention present in the biological sample will indicate that the therapeutic
is successfully
combating such disease or disorder.
Alternatively, polypeptides of the invention, e.g., natural IgM antibodies,
can be
detected in vivo in a subject by introducing into the subject a labeled
antibody specific for a
polypeptide of the invention, e.g., an anti-idiotypic antibody to detect
natural IgM
antibodies. For example, the anti-idiotypic antibody can be labeled with a
radionuclide
marker whose presence and location in a subject can be detected by standard
imaging
techniques.
A "radionuclide" refers to molecule that is capable of generating a detectable
image
that can be detected either by the naked eye or using an appropriate
instrument, e.g.
positron emission tomography (PET), and single photon emission tomography
(SPECT).
Radionuclides useful within the present disclosure include penetrating photon
emitters
including gamma emitters and X-ray emitters. These rays accompany nuclear
transfoiniation such as electron capture, beta emission and isomeric
transition.
Radionuclides useful include those with photons between 80 and 400 keV and
positron
producers, 511 keV annihilation photons and acceptable radiation doses due to
absorbed
photons, particles and half life. Radionuclides include radioactive isotopes
of an element.
-,
Examples of radionuclides include 123I 125
, 1 99mTc, isF, 68Ga, 62cu, Hi/11,131i, 186-
e,
'"Re,
90y, 212Bi, 211At, 89sr, 166H0, 153sm, 67cu, "Cu, 100F,d, 212pb, 109pd, 67Ga,
94Tc, 105- -1,
95RU,
177Lu, 170L - u
"C, and 76Br.
In one embodiment, an anti-idiotypic antibody that recognizes a natural IgM
antibody of the present invention may be labeled with 99mTc. 99'Tc, a commonly
used
radionuclide in Nuclear Medicine, combines desirable physical properties with
a 6 hr half-
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life and a 140-KeV gamma energy (85% as gamma photons) and widespread
availability,
since it can readily be eluted from molybdenum generators.
The imaging agents of the disclosure may be used in the following manner. An
effective amount of the imaging agent (from 1 to 50 mCi) may be combined with
a
pharmaceutically acceptable carrier for use in imaging studies. In accordance
with the
disclosure, "an effective amount" of the imaging agent of the disclosure is
defined as an
amount sufficient to yield an acceptable image using equipment which is
available for
clinical use. An effective amount of the imaging agent of the disclosure may
be
administered in more than one injection. Effective amounts of the imaging
agent of the
disclosure will vary according to factors such as the degree of susceptibility
of the
individual, the age, sex, and weight of the individual, idiosyncratic
responses of the
individual and dosimetry. Effective amounts of the imaging agent of the
disclosure will
also vary according to instrument and film-related factors. Optimization of
such factors is
well within the level of skill of a person skilled in the art.
The amount of imaging agent used for diagnostic purposes and the duration of
the
imaging study will depend upon the nature and severity of the condition being
treated, on
the nature of therapeutic treatments which the patient has undergone, and on
the
idiosyncratic responses of the patient. Ultimately, the attending physician
will decide the
amount of imaging agent to administer to each individual patient and the
duration of the
imaging study.
The pharmaceutically acceptable cattier for an imaging agent of the disclosure
may
include any and all solvents, dispersion media, coatings, antibacterial and
antifimgal agents,
isotonic agents, absorption delaying agents, and the like. The use of such
media and agents
for pharmaceutically active substances is well known in the art. The imaging
agent of the
disclosure may further be administered to an individual in an appropriate
diluent or
adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier
such as
human serum albumin or liposomes. Supplementary active compounds can also be
incorporated into the imaging agent of the disclosure. Pharmaceutically
acceptable
diluents; include saline and aqueous buffer solutions. Adjuvants contemplated
herein
include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether
and
nhexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin
inhibitor,
diethylpyrocarbonate, and trasylol. Liposomes include water-in-oil-in-water
CGF
emulsions as well as conventional liposomes (Strejan et al. (1984)J.
Neuroimmunol. 7, 27).
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In one embodiment, the imaging agent of the disclosure is administered
parenterally
as injections (intravenous, intramuscular or subcutaneous). The imaging agent
may be
formulated as a sterile, pyrogen-free, parenterally acceptable aqueous
solution. The
preparation of such parenterally acceptable solutions, having due regard to p1-
I, isotonicity,
stability, and the like, is within the skill in the art. Certain
pharmaceutical compositions of
this disclosure suitable for parenteral administration comprise one or more
imaging agents
in combination with one or more pharmaceutically acceptable sterile powders
which may
be reconstituted into sterile injectable solutions or dispersions just prior
to use, which may
contain antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic
with the blood of the intended recipient or suspending or thickening agents. A
formulation
for injection should contain, in addition to the cardiovascular imaging agent,
an isotonic
vehicle such as sodium chloride solution, Ringer's solution, dextrose
solution, dextrose and
sodium chloride solution, lactated Ringer's solution, dextran solution,
sorbitol solution, a
solution containing polyvinyl alcohol, or an osmotically balanced solution
comprising a
surfactant and a viscosity-enhancing agent, or other vehicle as known in the
art. The
formulation used in the present disclosure may also contain stabilizers,
preservatives,
buffers, antioxidants, or other additives known to those of skill in the art.
The invention also encompasses kits for detecting the presence of a natural
IgM
antibody in a biological sample. For example, the kit can comprise a labeled
compound or
agent capable of detecting a polynucleotide or polyp eptide of the invention
in a biological
sample; means for determining the amount of a natural IgM antibody in the
sample; and
means for comparing the amount of a natural IgM antibody in the sample with a
standard.
An unlabeled compound may also be provided with instructions for labeling the
compound.
The compound or agent can be packaged in a suitable container_ The kit can
further
comprise instructions for using the kit to detect a pc:lb/nucleotide or
polypeptide of the
invention.
Exemplification
The invention, having been generally described, may be more readily understood
by
reference to the following examples, which are included merely for purposes of
illustration
of certain aspects and embodiments of the present invention, and are not
intended to limit
the invention in any way.
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Example 1: Mechanism of Ischemia-Reperfusion Injury
This Example shows that mice deficient in the complement system were resistant
to
ischemia-reperfusion injury.
To examine the mechanism of ischemia-reperfusion injury, mice deficient in
complement C3 were treated in the hindlimb model. The C3-/- mice were
partially
=
protected from injury based on an approximate 50% reduction in permeability
index (see
Weiser et al. (1996) J. Exp. Med. 1857-1864). Thus, complement C3 is essential
for
induction of full injury in this murine model.
The experiments in Weiser et al. did not identify how complement was
activated.
The serum complement system can be activated by at least three distinct
pathways,
classical, lectin or alternative. Knowing which pathway is involved, is
important as it
suggests a mechanism for injury. For example, the classical pathways is
activated very
efficiently by IgM and IgG isotypes of immunoglobulin or by the serum
recognition protein
C-reactive protein. Whereas, the lectin pathway is activated following
recognition of
specific carbohydrates such as mannan by mannan binding lectin (MBL) (Epstein
et al.,
(1996) Inununo18, 29-35). In both pathways, complement C4 is required in
forming an
enzyme complex with C2 that catalyzes cleavage of the central component C3. By
contrast,
the alternative pathway activates spontaneously leading to conversion of C3 to
its active
form (C3b) and attachment to foreign-or self-tissues. The pathway is tightly
regulated
as all host cells express inhibitors of amplification of the complement
pathway by
inactivating, or displacing the C3 convertase (Muller-Eberhard, H.J., (1988)
Ann. Rev.
Bioehein. 57, 321-347). One approach for determining the pathway involved is
use of mice
deficient in C4, i.e., cannot form C3 convertase via classical or lectin
pathways. Comparison
of mice deficient in either C3 or C4 with wild type (WT) controls in the
hindlimb model,
revealed that C4 was also required for induction of full injury (Weiser et al.
supra). This
finding was important as it suggested that antibody or MBL might be involved.
Example 2: Natural IgM Mediates Ischemia Reperfusion (I/R) Injury
This Example shows that mice deficient in immunoglobulin were resistant to
ischemia-reperfusion injury.
To determine if antibody was involved in mediating I/R injury, mice totally
deficient in immunoglobulin, RAG2-/- (recombinase activating gene-2 deficient)
were
characterized along with the complement deficient animals in the intestinal
model.
Significantly, the RAG-2-/- mice were protected to a similar level as observed
in the
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=
complement deficient animals (Weiser et al. supra). Since the RAG2-/- animals
are also
missing mature lymphocytes, it was important to determine that the pathogenic
effect
was antibody dependent (Shinkai et al. (1992) Cell 68, 855-867). To confirm
that
injury was mediated by serum antibody, the deficient animals were
reconstituted
with either normal mouse sera (Weiser et al. supra) or purified IgM (William_s
et al.
(1999) J. Appl. Physiol 86; 938-42). In both cases, the reconstituted RAG-2-1-
mice were no longer protected and injury was restored. In the latter
experiments, a
model of intestinal injury was used as in this model, injury is thought to be
mediated primarily by complement.
The interpretation of these results is that during the period of ischemia,
neoantigens are either expressed or exposed on the endothelial cell surface.
Circulating
IgMs appear to recognize the new determinant, bind and activate classical
pathway of
complement. While the nature of the antigen is not known, IgM rather than IgG
seems to
be primarily responsible for activation of complement as reconstitution of
deficient mice
with pooled IgG did not significantly restore injury in the mice. An
alternative
hypothesis is that there is another initial event such as the MBL pathway that
recognizes
the altered endothelial surface, induces low level complement activation which
in turn
exposes new antigenic sites and the pathway is amplified by binding of IgM.
Example 3: Pathogenic IgM is a Product of B-1 cells
Since a major fraction of circulating IgM is thought to represent natural
antibody,
i.e. product of rearranged germline genes, it is possible that mice bearing
deficiencies in
the B-1 fraction of lymphocytes might also be protected. B-1 cells have a
distinct
phenotype from more conventional B-2 cells in that they express low levels of
Ig19 and
CD23 and a major fraction express the cell surface protein CD5 (Hardy et al.,
(19 94)
Immunol. Rev.: 137, 91; Kantor etal. (1993) Annu. Rev. Inununol. 11, 501-538,
1993. B-1
cells are also distinguished by reduced circulation in mice, limited frequency
in the
peripheral lymph nodes and spleen and are primarily localized within the
peritoneal cavity.
To examine a role for B-1 cells as a source of pathogenic IgM, antibody-
deficient mice
(RAG-2-/-) were reconstituted with 5 X 105 peritoneal B-1 cells and rested
approximately
30 days before treatment. Circulating IgM levels reach a near normal range
within a month
following adoptive transfer. Characterization of the B-1 cell reconstituted
mice in the
intestinal ischemia model confirmed that B-1 cells were a major source of
pathogenic IgM
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(see Williams et al. (1999) supra). This was an important observation because
the
repertoire of B-1 cell natural antibody is considerably more limited than
would be expected
for conventional B-2 cells. Therefore, it is possible that the pathogenic
antibody represents a
product of the germline.
Example 4: Cr2-/- Mice are protected from Ischemia Reperfusion Injury
The initial characterization of Cr2-/- knockout mice revealed an approximate
50%
reduction in the frequency of B-la or CD5 + B-1 cells (Abeam et al. (1996)
Immunity 4:
251-262). Although characterization of another strain of Cr2-deficient mice
did not identify
a similar reduction (Molina et al. (1996)Proc. Natl. Acad. Sci. USA 93, 3357-
3361).
Whether the difference in frequency of CD5 + cells was due to variation in
strain
background or environmental differences is not known. Despite the reduced
frequency of B-
1 a cells in the Cr2-/- mice, circulating levels of IgM were within the normal
range. These
findings suggested that the repertoire of IgM might be different in the Cr2-
deficient
animals. To test this hypothesis, mice in the intestinal PR model were
characterized.
Surprisingly, the Cr2-/- mice were equally protected as the complete-antibody
deficient
mice (Figure 3). Comparison of survival over a five-day period following
treatment in the
intestinal model demonstrated a significant increase in mortality of the WT
compared to
Cr2-deficient animals. Consistent with an increased mortality, a dramatic
reduction in injury
was observed in tissue sections harvested from treated WT or Cr2-/- deficient
mice.
Extensive injury to the mucosal layer of the intestine was observed in WT mice
or
Cr2-/- mice reconstituted with pooled IgM or B-1 cells. By contrast, tissue
sections
isolated from treated Cr2-/- mice were similar to that of sham controls. Thus,
despite
normal circulating levels of IgM, the Cr2-deficient mice were protected from
injury. These
results not only confirm the importance of 13-1 cells as a source of
pathogenic antibody but
suggest that the complement system is somehow involved in formation or
maintenance of the
repertoire of natural antibody. For example, complement may be involved in
positive
selection of B-1 cells.
Example 5: Identification of Pathogenic IgMs
This Example describes the generation of a specific hybridoma clone from
normal B-1
cells and the identification of one clone that produces a pathogenic IgM. The
pathogenic
IgM was shown to restore injury in vivo to antibody deficient mice.
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Studies in mice bearing a deficiency in complement receptors CD21/CD35,
revealed
that the mice were missing the pathogenic antibody. This finding was
unexpected because
they have a normal level of IgM in their blood. These findings led to the
hypothesis that a
special population of B cells termed B-1 cells are responsible for secreting
the pathogenic
IgM. For example, engraftment of the receptor deficient mice (Cr2-/-) with B-1
cells from
normal mice restored injury, confirming the importance of B-I cells. To
identify the specific
antibody or antibodies responsible for injury, a panel of hybridoma clones
were constructed
from an enriched pool of peritoneal B-1 cells harvested from normal mice. The
general
approach for preparing hybridomas from enriched fraction of peritoneal cells
includes
harvesting peritoneal cells from mice treated 7 days earlier with IL-10 and
subsequently
enriched for CD23 negative B cells by negative selection with magnetic beads.
Enriched B
cells are analyzed by FACS following staining with IgM, Mac-1 and CD23
specific Mab.
The enriched population is further activated by culturing with LPS for 24
hours. Activated
cells are hybridized with fusion partner myeloma cells in the presence of PEG
and grown in
HAT-selective medium. Hybridomas are screened for IgM secreting clones by
ELISA , and
positive wells are expanded for purification of IgM.
Twenty-two IgM-secreting hybridoma clones were analyzed by pooling an equal
amount of IgM product from each of the clones. Treatment of antibody-deficient
mice with
the pooled IgM restored injury similar to that seen with pooled IgM from
serum. This
finding confirmed that the pathogenic IgM was among the twenty-two hybridomas
produced. By dividing the pools into two fractions, i.e., 1-11 and 12-22, and
treatment
mice with the two fractions, the pathogenic antibody was found to fractionate
with the pool
that included clone # 22. Finally, mice were reconstituted with either clone
17 or 22.
Clone 22 restored injury whereas the other clones did not (see Figure 4).
Example 6: Complement involvement in B-1 cell selection
Two different models have been proposed to explain the development of B-1
cells.
The lineage hypothesis proposes that B-1 cells develop in early fetal life as
a distinct
population (Kantor et al. (1993) supra). Alternatively, B-1 cells develop from
the same
progenitors as conventional B cells but depending on their environment, i.e.,
encounter with
antigen, they develop into B-1 or retain the B-2 cell phenotype (Wortis, H.H.
(1992) Int.
Rev. Immunol. 8, 235; Clarke, J. (1998) Exp. Med. 187, 1325-1334).
Irrespective of their
origin, it is known that B-1 cells are not replenished from adult bone marrow
at the same
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frequency as B-2 cells and that their phenotype is more similar to that of
early fetal liver B cells or neonatal
bone marrow (BM) cells. Consistent with an early origin, their repertoire
tends to be biased towards
expression of more proximal VH genes and N-nucleotide addition is limited (Gu
et al. (1990) EMBO J 9,
2133; Feeney, J. (1990) Exp. Med 172, 1377). It seems reasonable that given
the reduced replenishment
by adult BM stem cells, B-1 cells are self-renewed and that antigen
stimulation might be important in their
renewal, expansion or even initial selection (Hayakawa et al., (1986)Eur. J
Immunol. 16, 1313). Indeed
inherent to the conventional model, B-1 cells must be antigen selected.
Evidence in support of a B-cell receptor (BCR) signaling requirement for
positive
selection of B-1 cells comes from mice bearing mutations that alter BCR
signaling. For example,
impairment of BCR signaling through CD 19, vav, or Btk dramatically affects
development of B-1 cells.
By contrast, loss of negative selection such as in CD22- or SHIP-1 deficient
mice can lead to an increase in
B-1 cell frequency (O'Keefe etal. (1996) Science 274, 798-801; Shultz et al.
(1993) Cell 73, 1445). Recent,
elegant studies with mice bearing two distinct Ig transgenes, VIII 2 (B-1 cell
phenotype) or VHB1-8 (B-2
cell phenotype) support the view that B-1 cells are positively selected by
self-antigens. For example,
B cells expressing VH12 either alone or together with B1-8 developed a B-1
cell phenotype. Whereas, few
if any B cells were identified that expressed the B1-8 transgene only. Thus,
these results suggested that
encounter of transgenic B cells with self-PtC resulted in expansion of those
expressing VH12. Selection of
B-1 cells was recently reported by Hardy et al. (1994) Immunol. Rev. 137, 91).
In their model, B cells
expressing an immunoglobulin transgene specific for Thy 1.1 were selected and
expanded in mice
expressing the cognate antigen. By contrast, transgene +B-1 cells were not
found in mice that expressed
the alternative allotype Thy 1.2.
Where does complement fit into B-1 cell development? The overall reduction in
B-1 a cell
frequency and the more specific loss of B-1 cells expressing IgM involved in
I/R injury suggests a role for
CD21/CD35 in either positive selection or maintenance of B-1 a cells. One
possible role for complement is
that it enhances BCR signaling on encounter with cognate antigen. Biochemical
studies and analysis of
CD21/CD35 deficient mice demonstrate the importance of co-receptor signaling
in activation and survival
of conventional B cells (Carroll, M.C., (1998)Ann. Rev. Immunol. 16, 545-568;
Fearon et al. (1995)Annu.
Rev. Immunol. 13, 127-149). It is very likely that B-1 cells likewise utilize
co-receptor signaling to enhance
the BCR signal. For example, bacteria express typical B-1
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cell antigens such as phosphoryl choline and it is not unreasonable that
coating of bacteria
with complement ligand C3d would enhance crosslinking of the co-receptor with
the BCR
and enhance overall signaling. Thus, antigens expressed at lower
concentrations might
require complement enhancement in order for the cognate B-cell to recognize it
and expand
or be positively selected. Another role for complement receptors is in
localizing antigen on
follicular dendritic cells (FDC) within the lymphoid compartment. However,
since the
major population of B-1 cells occupy the peritoneal tissues it is not clear if
they would
encounter FDC within lymphoid structures. The actual site or sites in which B-
I cells
undergo positive selection are not known. It is possible that they must
encounter cognate
antigen in early fetal development or in neonatal BM. If this is the case, it
might be expected
that complement receptors on stromal cells within these compartments bind
antigen for
presentation to B cells. It is possible that complement receptors could
participate in both
stages of development. First, they might enhance antigens signaling in initial
positive
selection. Secondly, as selected B-1 cells are replenished at peripheral
sites, complement
receptors might again be involved in enhancement of BCR signaling.
Figure 5 is a schematic diagram of the proposed role for complement and
complement receptors in positive selection of peritoneal B-1 lymphocytes. The
interaction of complement-ligand coated antigens (self- and non-self) results
in co-ligation
of the CD21/CD19 co-receptor and BCR on the cell surface leading to enhanced
signaling
and positive selection.
Example 7: Materials and Methods for Examples 8-11
Phage display peptide library and peptide synthesis
A 12-mer M-13 phage display library (New England Biolab, MA) was screened by
4 rounds with MBL-beads coated with Igmcm-22 and 2 rounds with IgMcm-75
according to
the manufacturer's recommendation. Phage clones were selected from the
enriched pool
and the nucleotide sequence of the relevant phage gene determined for at least
ten clones.
Selected peptides were synthesized with purity>95% in Harvard Proteomic Core
or New
England Peptide, Inc. (Gardner, MA).
Binding assays
ELISA was performed as described earlier (Zhang et al. (2004) PNAS USA
101:3886-91). Briefly, IgM binding to phage or phage-specific peptides was
determined by
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coating a 96-well plate with saturating amounts of antigen. Subsequent to
blocking, IgM was added (1
or 10 ig/ml) for 2 hr at 37 C. Plates were washed and then developed with
alkaline phosphatase-labeled
goat anti-mouse IgM (Sigma, MO). Binding of IgM to NMFIC-II was determined by
culturing 96-well
plates previously coated with specific rabbit antibody (NMI-IC-HA & B; Covance
Research Products;
NMHC-II C a gift from Dr. Adelstein, NHLBI, NTH, Bethesda, MD) or pan-myosin
Hc (Sigma, MO) with
intestinal lysates prepared from IgMcm-22 reconstituted RAG-14- mice either
sham treated or treated for
ischemia as described (Zhang et al. (2004) PNAS USA 101:3886-91). Lysates were
prepared as described
for immune precipitation (see below). Alkaline-phosphatase labeled goat anti-
mouse IgM (Sigma, MO)
was then used to detect bound IgM.
Intestinal RI model
Surgical protocol for RI was performed as previously described (Zhang et at.
(2004)
PNAS USA 101:3886-91). Briefly, a laparotomy is performed, and a microclip
(125g pressure, Roboz,
MD) was applied to the superior mesenteric artery and bilateral circulation
limited with silk sutures
flanking a 20 cm segment of the jejunum. After 40 minutes of ischemia, the
microclip was removed, and
reperfusion of the mesenteric vasculature was confirmed by the return of
pulsation to the vascular arcade
and a change to pink color. The incision was closed, and all animals kept warm
for 3 hours. Reconstituted
RAG-14' animals received either IgM mixed with peptide or saline in 0.2 ml
volume intravenously 30 min
before the initial laparotomy. WT animals were treated with saline or peptide
i.v. 5 minutes prior to
reperfusion. At the end of reperfusion, the ischemic segment of the jejunum
was harvested and the
central 4 cm was cut for pathological analysis.
Histopathology and immuno-histochemistry analysis
Cryostat sections of intestinal tissues were stained by hematoxylin and eosin
(H&E) and
examined by light microscopy for mucosa! damage. Pathology score was assessed
based on procedure by
Chiu (Chiu et al, Arch Surg 101:484-488, 1970; Chiu, et at, Arch Surg 101:478-
483, 1970) that included
direct inspection of all microvilli over a 4 cm stretch ofjejuneum as
described. Zhang etal. (2004) PNAS
USA 101:3886-91. For immuno-fluorescence, cryosections fixed with 4% (w/v)
paraformaldehyde were
incubated for varying periods with either biotin-labeled anti-mouse IgM
(Becton Dickinson, CA) followed
by 1 hour with streptavidin-Alexa-568 (1: 500 dilution, Molecular Probes, OR).
C4 deposition was
detected by staining with FITC-labeled rabbit anti-huC4c (DAKO, CO),
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followed by anti-rabbit-Alexa 488 (Molecular Probes, OR). The specificity of
anti-C4c
staining was confirmed by staining serial sections with biotin-labeled anti-
mouse C4 for 1
hour followed by streptavidin-FITC (Becton Dickinson, CA). C3 deposition was
detected =
by treating with FITC-labeled anti-C3 (DAKO, CO). Sections were mounted in
Anti-fade
Mounting Medium with DAPI (Molecular Probes, OR).
SPR analysis of peptide binding to antibody
An IgM (IgMCM-22 or IgMcm-31'
) antibody was immobilized by amine coupling in a
BiaCore SPR CM5Tm chip flowcell at a density of 33,400 response units (RU) ¨33
ng/mm2
as described. Vorup-Jensen et al, PNAS USA 100: 1873-1878, 2003. Briefly, a
reference
flow cell was prepared by coupling of ethanolamine-HC1. Peptides, diluted in
PBS running
buffer, were flowed separately over the IgM-coupled surface and the reference
at a rate of
10 I/min at 25 C and with the data collection rate at 10 Hz. The injection
phase had a
duration of 240 s (end of injection phases are marked by arrow heads in
Figures 9A, B and
D). Binding isotherms were derived by subtracting the response in the
reference cell from
the response of the IgM-coupled surface. Following each run, the surface was
regenerated
by injecting 40 gl 0.05% (v/v) polyoxyethylenesorbitan monolaureate/PBS.
Immune precipitation
Frozen tissues were homogenized in a lysis buffer containing detergent and a
cocktail of enzyme inhibitors. A sample of lysate is analyzed for total
protein content (Bio-
Rad kit) to insure similar levels of protein for analysis. Lysates are mixed
with sepharose
beads coated with rat anti-mouse IgM for 1 hr at 4 C. Subsequently, beads were
pelleted
gently, washed in lysis buffer and then boiled in SDS-sample buffer under
reducing
conditions to elute bound complexes. Samples were fractionated on 6% (w/v)
polyacrylamide SDS gels and subsequently fixed and then stained with either
coomassie
blue or silver stain to identify protein bands.
Protein identification by tandem mass spectrometry
Individual Coomassie Blue-stained bands were excised from SDS-gels, destained,
and subjected to enzyme digestion as described previously. Borodovsky et al,
Chem Biol 9:
1149-1159, 2002. The peptides were separated using a nanoflow liquid coupled
chromatography system (Waters Cap LC) and amino acid sequences determined by
tandem
mass spectrometer (Q-TOF micro, Waters, MA). MS/MS data were processed and
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subjected to database searches using Mascot (Matrixscience) against Swissprot,
TREMBL/New or the NCBY non-redundant database.
Example 8: Identification of asparagine-rich peptides that bind natural IgM
antibody
5M-2
We previously identified a hybridoma clone of a natural IgM antibody (IgMc 2)
that binds ischemic tissue in the intestinal RI model, which support our
hypothesis that
ischemic tissue was altered relative to normal tissue and that neo-epitopes
expressed during
ischemia were targets for an innate response to self. To characterize the
ligand bound by
pathogenic IgMC4-22, a M-I 3 phage-display library of random 12-mer amino acid
sequences was screened using beads coated with the specific IgM.
After four rounds of specific screening and two rounds with a control IgM
(clone
IgMcm:15), ten phage clones were isolated and the nucleotide sequence of the
relevant M-13
gene sequenced. Notably, all ten clones contain sequences rich in asparagine.
Five of the
clones were selected for a relative binding assay with IgM2m-22 and one of
these clones, P8,
which bound with the highest efficiency was selected for further study (Table
4 and Figure
6A).
Table 4: Phage displayed peptides bind to IgmCM-22
Phage Clone Sequence SEQ ID NO:
P1 YNNNNGNYTYRN 16
P2 ANTRNGATNNNM 18
P3 CDSSCDSVGNCN 20
P4 WNNNGRNACNAN 22
P5 HNSTSNGCNDNV 24
P6 NSNSRYNSNSNN 26
P7 KRNNI-INNHNRSN 28
P8 NGNNVNGNRNNN 30
P9 NVANHNNSNHGN 32
P10 SYNNNNHVSNRN 34
Asparagine-rich Consensus xNNNxNNxNNNN 14
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A 12- amino acid peptide (P8) was synthesized based on the phage sequence and
assayed for inhibition of phage P8 binding to IgMcm"22 (Figure 6B). Titration
of increasing
amounts of P8 peptide yielded 50% inhibition at an estimated concentration of
10 umol.
This assay indicates a reasonable overall avidity of binding based on multiple
binding sites
expressed on the phage surface. This result suggested that IgmCM-22
binding to phage PS
was specific for the peptide region and that the synthetic peptide could be
used as a
mimotope for the actual antigen. To further characterize binding of P8 peptide
to IgMcNA-22
ELISA plates were coated with the peptide and tested with IgMcm"22 or control
IgMcm"75 for
binding (Figure 6C). At the lower concentration of 1 jig/ml, neither IgM bound
above
background. However, at 10 jig/ml, significantly more IgMcm-22 bound than
IgMcm:75.
Together, the three results suggest that peptide P8 binds specifically to
IgMavl"22 and can be
used for identification of the actual antigen.
Example 9: Asparagine-rich peptide P8 blocks intestinal RI
Previous studies had demonstrated that intestinal RI in RAG-14" mice was IgM-
dependent and that IgMcm"22 alone was sufficient to restore injury. As
expected,
reconstitution of RAG-14- mice with IgMem-22 but not saline prior to
reperfusion resulted in
RI (Figure 7A(i) and Figure 7B). By contrast, mixing of igmcm-22 with P8 prior
to injec-tion
in ischemic mice significantly blocked apparent injury (mean pathology score 6
3 versa s
31 13; p<0.001) (Figure 7Aii and Figure 7B). Previous titration of peptide
with Ig4CM-22
suggested an optimal concentration of 10 IIM of P8 was sufficient to block 50-
100 lag of
IgMem"22 (0.1-0.2 jtM).
Immunohistological analyses of serial sections of reperfiised intestinal
tissue
(jejuneum) following RI identified co-localization of IgM and complement C4
and C3
within the microvilli in RAG-14" mice reconstituted with Igmcm-22 (Figure 7Ci-
iv). By
contrast, sections prepared from mice receiving P8 showed no evidence of IgM
or
complement binding (Figure 7Cv-viii). No binding of IgM or complement was
observed in
Ig4cm-22 reconstituted sham controls, nor RAG-I1 mice reconstituted with
control IgMc.m-
31 or RAG- 14" mice reconstituted with saline only (Zhang et al. (2004) PNAS
USA
101:3886-91). Thus, P8 blocks the binding of IgMcm"22 and the induction of
injury in vi-vo.
The identification of a single natural IgM antibody that could initiate RI in
RAG-14-
mice led to the general question of the number of possible neo-epitopes
expressed on
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ischemic tissues and the corresponding number of pathogenic clones of IgM in
the
repertoire of wild type (WT) mice. It might be predicted that the number of
antibodies is
limited based on the current understanding that the repertoire of natural IgMs
is relatively
small. Herzenberg et al, Inzmwzol Today 14: 79-83, discussion 88-90, 1993;
Arnold et al, J
Exp Med 179: 1585-1595, 1994.. Moreover, ligands of natural IgM antibodies are
considered highly conserved structures and also are probably limited in
number. To test if
P8 represented a mimotope for a major self-antigen, WT mice were pretreated
with P8
(approximately 10 piM) five minutes prior to reperfusion in the intestinal
model. Analysis
of jejuneum tissues of mice treated with saline or a control peptide prior to
reperfusion
identified significant injury to the microvilli as expected (Figure 7Aiii). By
contrast,
pretreatment of WT mice with P8 five minutes prior to reperfusion blocked
apparent injury
(mean pathology score 5+3 versus 24+16 and 23+19; p<0.005 and 0.027,
respectively)
(Figure 7A(iv) and Figure 7B). As expected, IgM, C4 and C3 co-localized within
microvilli of RI treated WT mice (Figure 7Cix-xii). By contrast, no apparent
deposits of
IgM or complement were observed in reperfused tissues of mice administered P8
(Figure
7Cxiii-xvi). These results suggest that the number of key epitopes required to
initiate RI is
limited as a single peptide blocks injury and deposition of IgM and
complement.
Example 10: Immunoprecipitation of self-peptides with IgMcm-22
Using the amino acid sequence of PS, a homology search of the genomic database
revealed no exact matches. Therefore, an immune-precipitation approach was
used to
identify the ischemia antigen/antigens in RAG-1I- mice reconstituted with
IgMcm-22.
RAG-11- mice were reconstituted with an optimal amount of IgMCM-22, treated
for
intestinal ischemia and reperfused for varying lengths of time, i.e., 0
minutes or 15 minutes
before harvesting of tissues. Immune complexes of IgM-antigen were isolated
from lysates
of jejuneum at the varying time points and fractionated by SDS-PAGE under
reducing
conditions. Analysis of the stained gels indicated common bands at lower
molecular weight
for all time points (Figure SA). However, at 15 minutes, a band at high
molecular weight
(>200 kD) was identified (Figure 8A).
Protein bands were excised from stained gels, enzymatically digested and
peptides =
analyzed by Tandem Mass Spec as described. Kocks et al, Mol Cell Proteonzics
2: 1188-
1197, 2003. Analysis of eluted peptides indicated that the common bands at
approximately
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25, 50 and 75 kDa represented immunoglohnlin light chain (Lc), and IgG heavy
chain (Hc)
and IgM Re, respectively. Analysis of the high molecular weight band yielded
peptide
sequences homologous to non-muscle myosin heavy chain (NMHC) type II isoforms
A and
C (Table 5).
10
Table 5: Mass Spectrometry Results
Matched proteins Mass
Spectroscopy sequenced peptides
Mouse non muscle myosin heavy chain LI-A VVFQEFR (MS-1; SEQ ID NO: 39)
(gi/20137006; GenBankTM Accession NO: CNGVLEGIR (MS-2; SEQ ID NO: 40)
NP 071855) KFDQLLAEEK (MS-3; SEQ ID NO: 41)
total score=130; peptides matched=6 KFDQLLAEEK
EQADFAIEALAK (MS-4; SEQ ID NO: 42)
QLLQANPILEAFGNAK (MS-5; SEQ ID
NO: 43)
Mouse non muscle myosin heavy chain CNGVLEGLR.
TI-C (g1,733638127; GenBankTM Accession VKPLLQVTR (MS-6; SEQ ID NO: 44)
NO: AAQ24173) KFDQLLAEEK
total score 133; peptides matched=7 KFDQLLAEEK
EQADFALEALAK
LAQAEEQLEQESR (MS-7; SEQ ID NO:
45)
QLLQANPILEAFGNAK (MS-8; SEQ ID
NO: 46)
*Score is -10XLog (P), where P is the probability that the observed match is
a random event. Individual ion scores >53 indicate identity or extensive
homology (p<0.05).
In similar experiments using lysates prepared fiom WT mice treated for 3 hours
in
intestinal RI, a similar size band at 200 kD was also observed and sequence
analysis
identified NMHC-A and C peptides.
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Three forms of type II NMHC have been identified (A, B and C) in the mouse and
human genome. Golomb et al, J Biol Chem 279: 2800-2808, 2004; Kelley et al, J
Cell Biol
134: 675-687, 1996. All eukaryotic cells express type II NMHC but the
distribution of the
three isoforms varies. NMHC-II A and B are approximately 85% homologous;
wherea.s
NMHC-II C is approximately 65% similar. Golomb et al, J Biol Chem 279: 2800-
280S,
2004. The three isotypes are highly conserved among mice and humans.
To confirm the binding of IgmCM-22
to type II NMHC, an ELISA approach was
used. Plates were coated with antibody specific for each of the three forms of
NMHC cif
with a pan-myosin antibody to capture the relevant antigen from lysates
prepared from
jejuneum of RAG-14" mice. Subsequently, I
gMcm-22 or IgMcm-31 were added and then
developed with a labeled anti-mouse IgM antibody. Above background binding of
IgIVICM-
22 but not IgMcm-3I to all three of the isoforms of NMHC-II was observed
(Figure 8B). The
combined sequence analysis and ELISA results show that IgMcm-22 recognizes a
conserved
region of the type II NMHC.
To determine whether myosin is exposed to circulating antibody following
ischemia, RAG-14- mice were reconstitute with a purified IgG fraction of
rabbit anti-pan
myosin heavy chain. Analysis of tissues of sham treated RAG-14- following
reconstitution
with the rabbit IgG mice showed no evidence of injury or deposition of IgG. By
contrast,
ischemic RAG-14" mice reconstituted with the pan-myosin IgG prior to
reperfiision
developed significant RI compared to saline controls (33 11 versus 11 8,
p<0.028) (Figure
8C). Accordingly, myosin is exposed to antibody in circulation following
ischemia.
Comparison of the sequences of the three NMHC-II isoforms with the P8 peptide
sequence identified one region of apparent homology (Table 6). All three
isoforms include
a motif of NxxxxNxNx that has similarity with the P8 sequence. A 12- amino
acid ser-
peptide (N2) sequence (NMHC-II C isoform) was prepared for further study.
Table 6: Conserved homologous sequence in NMHC-II A-C
Phage Clone Sequence
P8 NGNNVNGNRNNN (SEQ ID NO:30)
Consensus xNNNx(N/D)NxN(N/D)N(N/V) (SEQ ID NO:14)
NMHC-II Sequence
Mouse-11A (542-556) LMKNMDPLNDI (SEQ ID NO:36)
Human-IA (585-596) LMKNMDPLNDI
Mouse-JIB (592-603) LMKNMDPLNDNV (N2; SEQ ID NO:38)
Human-BB (592-603) LMKNMDPLNDNV
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Mouse-IIC (607-619) LMKNMDPLNDNV (N2; SEQ ID NO:38)
Human-TIC (611-622) LMKNMDPLNDNV
To test that this region bound IgMcm-22 surface, plasmon resonance analysis
was
used (Figure 9). N2 peptide was injected over a surface coupled with IgmCM-22
(Figure 9A)
and generated a robust response, which corresponded to a KID of 123+61 1.LM
(mean+SD,
n=2) as calculated from the steady-state response levels (Figure 9C). In
contrast, no
binding was observed when a control peptide was injected over the specific IgM-
coupled
surface (Figure 9B) or when the N2 peptide was injected over a surface coupled
with the
IgMcm-3I control (Figure 9D).
Example 11: Self-peptide N2 blocks intestinal RI
To test the functional binding of N2 with pathogenic IgM, approximately 100
nmoles of the peptide (or saline control) was mixed with IgmCM-22
prior to reconstitution of
RAG-14- mice and treatment in the RI model. Analysis of histology of tissue
sections
prepared from the reperfused jejuneum of IgMCM-22_ and saline-treated mice
identified
injury and deposition of IgM and complement as expected (Figure 5Ai and 5B).
By
contrast, mixing the N2 peptide with IgMCM-22 prior to reperfusion was
protective from
injury (mean pathology score 13+8 versus 31+10; p<0.049) (Figure 10Aii and
10B). In
addition, no deposition of IgM and complement was observed in reperfused
jejuneum when
IgMCM-22 was mixed with the N2 peptide prior to injection in RAG-14_ mice
(Figure 10Ci-
viii). Thus, as observed with the synthetic peptide P8, the self-peptide N2
blocked
functional binding of IgMcm-22 in vivo.
To test if self-peptide N2 represents the major self-epitope in intestinal RI,
WT mice
were treated with approximately 40 p,M of the synthetic peptide P8 prior to
reperfusion in
the intestinal model. Histological analysis of tissue sections of saline
treated WT mice
identified injury and deposition of IgM and complement as expected (Figure
10Aiii and
10Cix-xii). By contrast, treatment of WT mice with self-peptide N2 blocked
both injury
(mean pathology score 8+5 versus 22+17) and deposition of IgM and complement
(Figure
10Aiv; Figure 10B; Figure 10Cxiii-xvi). These results suggest that a conserved
region
within type II NMEIC proteins represents the major epitope for binding of
natural IgM
following ischemia in the intestinal model.
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60412-4434D1
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, many equivalents to the specific embodiments of
the invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
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SEQUENCE LISTING
<110> Immune Disease Institute, Inc, President and Fellows of Harvard
College, The Brigham and Women's Hospital, Inc. .
<120> NATURAL IGM ANTIBODIES AND INHIBITORS THEREOF
<130> 60412-4434D1
<140> CA 2,560,066
<141> 2005-03-01
<150> 60/588,648
<151> 2004-07-16
<150> 60/549,123
<151> 2004-03-01
<160> 65
<170> PatentIn Ver. 3.3
<210> 1
<211> 402
<212> DNA
.<213> Mus musculus
<400> 1
caggttcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatt 60
tcctgcaaag cttctggcta cgcattcagt agctactgga tgaactgggt gaagcagagg 120
cctggaaagg gtcttgagtg gattggacag atttatcctg gagatggtga tactaactac 180
aacggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagagaagat 300
tactacggta gtgactggta cttcgatgtc tggggcacag ggaccacggt caccgtctcc 360
tcaggtaagc tggctttttt ctttctgcac attccattct ga 402
<210> 2
<211> 133
<212> PRT
<213> Mus musculus
<400> 2
Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15'
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Gin Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
CA 02812132 2013-04-04
Ala Arg Glu Asp Tyr Tyr Gly Ser Asp Trp Tyr Phe Asp Val Trp Gly
100 105 110
Thr Gly Thr Thr Val Thr Val Ser Ser Gly Lys Leu Ala Phe Phe Phe
115 120 125
Leu His Ile Pro Phe
130
<210> 3
<211> 15
<212> DNA
<213> Mus musculus
<400> 3
agctactgga tgaac 15
<210> 4
<211> 5
<212> PRT
<213> Mus musculus
<400> 4
Ser Tyr Trp Met Asn
1 5
<210> 5
<211> 57
<212> DNA
<213> Mus musculus
,<400> 5
cagatttatc ctggagatgg tgatactaac tacaacggaa agttcaaggg caaggcc 57
<210> 6
<211> 17
<212> PRT
<213> Mus musculus
<400> 6
Gin Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 7
<211> 324
<212> DNA
<213> Mus musculus
<400> 7
attgtgatga cccagtctgc tgcttcctta gctgtatctc tggggcagag ggccaccatc 60
tcatacaggg ccagcaaaag tgtcagtaca tctggctata gttatatgca ctggaaccaa 120
cagaaaccag gacagccacc cagactcctc atctatcttg tatccaacct agaatctggg 180
gtccctgcca ggttcagtgg cagtgggtct gggacagact tcaccctcaa catccatcct 240
76
CA 02812132 2013-04-04
gtggaggagg aggatgctgc aacctattac tgtcagcaca ttagggagct tacacgttcg 300
gaggggggac caagctggaa ataa 324
<210> 8
<211> 107
<212> PRT
<213> Mus musculus
<400> 8
Ile Val Met Thr Gin Ser Ala Ala Ser Leu Ala Val Ser Leu Gly Gin
1 5 10 15
Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly
20 25 30
Tyr Ser Tyr Met His Trp Asn Gin Gin Lys Pro Gly Gin Pro Pro Arg
35 40 45
Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro
65 70= 75 80
Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gin His Ile Arg Glu
85 90 95
Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys
100 105
<210>9
<211> 45
<212> DNA
<213> Mus musculus
<400> 9
agggccagca aaagtgtcag tacatctggc tatagttata tgcac 45
<210> 10
<211> 15
<212> PRT
<213> Mus musculus
<400> 10
Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His
1 5 10 15
<210> 11
<211> 21
<212> DNA
<213> Mus musculus
<400> 11
cttgtatcca acctagaatc t 21
<210> 12
<211> 7
77
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<213> Mus musculus
<400> 12
Leu Val Ser Asn Leu Glu Ser
1 5
<210> 13
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified base
<222> (1)..(3)
<223> a, c, g or t
<220>
<221> modified base
<222> (13)..(15)
<223> a, c, g or t
<220>
<221> modified base
<222> (22)..(24)
<223> a, c, g or t
<400> 13
nnnaayaaya aynnnaayaa ynnnaayaay aayaay 36
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<221> MOD RES
<222> (1)
<223> Variable amino acid
<220>
<221> MOD RES
<222> (5)¨
<223> Variable amino acid
78
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<220>
<221> MOD RES
<222> (8)¨
<223> Variable amino acid
<400> 14
Xaa Asn Asn Asn Xaa Asn Asn Xaa Asn Asn Asn Asn
1 5 10
<210> 15
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (18)
<223>a, c, g or t
<220>
<221> modified_base
<222> (27)
<223> a, c, g or t
<220>
<221> modified_base
<222> (33)
<223> a, c, g or t
<400> 15
tayaayaaya ayaayggnaa ytayacntay mgnaay 36
<210> 16
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 16
Tyr Asn Asn Asn Asn Gly Asn Tyr Thr Tyr Arg Asn
1 5 10
<210> 17
<211> 36
<212> DNA
<213> Artificial Sequence
79
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<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified base
<222> (3)
<223> a, c, g or t
<220>
<221> modified_base
<222> (9)
<223> a, c, g or t
<220>
<221> modified base
<222> (12)
<223> a, c, g or t
<220>
<221> modified base
<222> (18)
<223> a, c, g or t
<220>
<221> modified_base
<222> (21)
<223> a, c, g or t
<220>
<221> modified_base
<222> (24)
<223> a, c, g or t
<400> 17
gcnaayacnm gnaayggngc nacnaayaay aayatg 36
<210> 18
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 18
Ala Asn Thr Arg Asn Gly Ala Thr Asn Asn Asn Net
1 5 10
<210> 19
<211> 36
<212> DNA
<213> Artificial Sequence
CA 02812132 2013-04-04
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (9)
<223> a, c, g or t
<220>
<221> modified_base
<222> (12)
<223> a, c, g or t
<220>
<221> modified_base
<222> (21)
<223> a, c, g or t
<220>
<221> modified_base
<222> (24)
<223> a, c, g or t
<220>
<221> modified_base
<222> (27)
<223> a, c, g or t
<400> 19
tgygaywsnw sntgygayws ngtnggnaay tgyaay 36
<210> 20
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 20
Cys Asp Ser Ser Cys Asp Ser Val Gly Asn Cys Asn
1 5 10
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
81
CA 02812132 2013-04-04
<220>
<221> modified_base
<222> (15)
<223> a, c, g or t
<220>
<221> modified_base
<222> (18)
<223> a, c, g or t
<220>
<221> modified_base
<222> (24)
<223> a, c, g or t
<220>
<221> modified_base
<222> (33)
<223> a, c, g or t
<400> 21
tggaayaaya ayggnmgnaa ygcntgyaay gcnaay 36
<210> 22
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 22
Trp Asn Asn Asn Gly Arg Asn Ala Cys Asn Ala Asn
1 5 10
<210> 23
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (9)
<223> a, c, g or t
<220>
<221> modified_base
<222> (12)
<223> a, c, g or t
82
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<220>
<221> modified base
<222> (15)
<223> a, c, g or t
<220>
<221> modified base
<222> (21)
<223> a, c, g or t
<220>
<221> modified base
<222> (36)
<223> a, c, g or t
<400> 23
cayaaywsna cnwsnaaygg ntgyaaygay aaygtn 36
<210> 24
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 24
His Asn Ser Thr Ser Asn Gly Cys Asn Asp Asn Val
1 5 10
<210> 25
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified base
<222> (6)
<223> a, c, g or t
<220>
<221> modified base
<222> (12)
<223> a, c, g or t
<220>
<221> modified base
<222> (15)
<223> a, c, g or t
83
CA 02812132 2013-04-04
<220>
<221> modified base
<222> (18)
<223> a, c, g or t
<220>
<221> modified base
<222> (21)
<223> a, c, g or t
<220>
<221> modified base
<222> (24)
<223> a; c, g or t
<220>
<221> modified _base
<222> (30)
<223> a, c, g or t
<400> 25
aaywsnaayw snmgntanaa nwsnaaywsn aayaay 36
<210> 26
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 26
Asn Ser Asn Ser Arg Tyr Asn Ser Asn Ser Asn Asn
1 5 10
<210> 27
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified base
<222> (6)
<223> a, c, g or t
<220>
<221> modified_base
<222> (30)
<223> a, c, g or t
84
CA 02812132 2013-04-04
<220>
<221> modified_base
<222> (33)
<223> a, c, g or t
<400> 27
aarmgnaaya aycayaayaa ycayaaymgn wsnaay 36
<210> 28
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 28
Lys Arg Asn Asn His Asn Asn His Asn Arg Ser Asn
1 5 10
<210> 29
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (6)
<223> a, c, g or t
<220>
<221> modified_base
<222> (15)
<223> a, c, g or t
<220>
<221> modified_base
<222> (21)
<223> a, c, g or t
<220>
<221> modified_base
<222> (27)
<223> a, c, g or t
<400> 29
aayggnaaya aygtnaaygg naaymgnaay aayaay 36
CA 02812132 2013-04-04
<210> 30
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 30
Asn Gly Asn Asn Val Asn Gly Asn Arg Asn Asn Asn
1 5 10
<210> 31
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (6)
<223> a, c, g or t
<220>
<221> modified_base
<222> (9)
<223> a, c, g or t
<220>
<221> modified_base
<222> (24)
<223> a, c, g or t
<220>
<221> modified_base
<222> (33)
<223> a, c, g or t
<400> 31
aaygtngcna aycayaayaa ywsnaaycay ggnaay 36
<210> 32
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
86
CA 02812132 2013-04-04
<400> 32
Asn Val Ala Asn His Asn Asn Ser Asn His Gly Asn
1 5 10
<210> 33
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified base
<222> (3)
<223> a, c, g or t
<220>
<221> modified base
<222> (24)
<223> a, c, g or t
<220>
<221> modified base
<222> (27)
<223> a, c, g or t
<220>
<221> modified base
<222> (33)
<223> a, c, g or t
<400> 33
wsntayaaya ayaayaayca ygtnwsnaay mgnaay 36
<210> 34
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 34
Ser Tyr Asn Asn Asn Asn His Val Ser Asn Arg Asn
1 5 10
<210> 35
<211> 36
<212> DNA
<213> Artificial Sequence
87
CA 02812132 2013-04-04
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (3)
<223> a, c, g or t
<220>
<221> modified_base
<222> (21)
<223> a, c, q or t
<220>
<221> modified_base
<222> (24)
<223>a, c, g or t
<400> 35
ytnatgaara ayatggaycc nytnaaygay aayath 36
<210> 36
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
, <223> Description of Artificial Sequence: Synthetic
peptide
<400> 36
Leu Met Lys Asn Met Asp Pro Leu Asn Asp Asn Ile
1 5 10
<210> 37
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
sequence
<220>
<221> modified_base
<222> (3)
<223> a, c, q or t
<220>
<221> modified_base
<222> (21)
<223> a, c, g or t
88
CA 02812132 2013-04-04
op
<220>
<221> modified base
<222> (24)
<223> a, c, g or t
<220>
<221> modified base
<222> (36)
<223> a, c, g or t
<400> 37
ytnatgaara ayatggaycc nytnaaygay aaygtn 36
=
<210> 38
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 38
Leu Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val
1 5 10
<210> 39
<211> 7
<212> PRT
<213> Mus musculus
<400> 39
Val Val Phe Gin Glu Phe Arg
1 5
<210> 40
<211> 9
<212> PRT
<213> Mus musculus
<400> 40
Cys Asn Gly Val Leu Glu Gly Ile Arg
1 5
<210> 41
<211> 10
<212> PRT
<213> Mus musculus
<400> 41
Lys Phe Asp Gin Leu Leu Ala Glu Glu Lys
1 5 10
89
CA 02812132 2013-04-04
0
<210> 42
<211> 12
<212> PRT
<213> Mus musculus
<400> 42
Glu Gin Ala Asp Phe Ala Ile Glu Ala Leu Ala Lys
1 5 10
<210> 43
<211> 16
<212> PRT
<213> Mus musculus
<400> 43
Gin Leu Leu Gin Ala Asn Pro Ile Leu Glu Ala Phe Gly Asn Ala Lys
1 5 10 15
<210> 44
<211> 9
<212> PRT
<213> Mus musculus
<400> 44
Val Lys Pro Leu Leu Gin Val Thr Arg
1 5
<210> 45
<211> 13
<212> PRT
<213> Mus musculus
<400> 45
Leu Ala Gin Ala Glu Glu Gin Leu Glu Gin Glu Ser Arg
1 5 10
<210> 46
<211> 16
<212> PRT
<213> Mus musculus
<400> 46
Gin Leu Leu Gin Ala Asn Pro Ile Leu Glu Ala Phe Gly Asn Ala Lys
1 5 10 15
<210> 47
<211> 7355
<212> DNA
<213> Mus musculus
<400> 47
tgggcagggc acggaaggct caagaacctg acctgctgca gcttccagtc tcgcgttcgc 60
16
o8f7E oqpbuebppb qopobbqp42 ebpubpoqob 4o5eebppbe ebbqbebpoo b5qqopb4o5
ozD,E bpDbqq6p6b e5bebbee6P eoo6bqobec 5Tebepo4o5 e6po6e4pbe obo6bpoo4D
09Ec be5qp644eb pooPbqbeo4 oppbe3P334 ou6P6bbeb6 qobpe36oqb opopbeubpb
00EE b44bebbpob bebeDbeebe 5bebbbe4bo D4006pbebe ebbqoaeboo ep4eb4euo5
CiZE bpbopobppo epbepogobP Poo6b400bP beeooqbPeb ebb-ebb-26pp bb4eo400pe
ogTE ooP4opoq.qp pb4obeqbeb eoPbePbb4o bqoe2Pbppb ebbpeoobbq oPpeobqopp
ozTE 6poppb6p6b qeo4p34pbp ope6bebbe6 b4Dpepbpp6 qobeepabbe bpoppopb4.6
090E bppopb4436 eob4obeebe obboopfio6e be6bebbebb e6b44beobe bbe6443ee6
.
000E beopgeoe2b eob2ob4ebe Pb2PbuPbeb oobbpobqoo pq.bpoobqob obpbbpbbeb
0176z bebbeb54bb bpoobbe654 oopb4poob4 oqebp5b2bb qobebbeobe ebeebobpop
oggz bqogb000bb booqobebbe bl.obbeboob qb4b4obebp ppbp66obbe 36qDbpobeb
038 bp31436pob 4pEreebpbeo ar4P3436e3 q3qbeob4e0 opbpbb4ebp bepe5-4366e.
09L
Pbebeobq obb434eppe ebebEboqqb bpepopb4o2 ebbobbpbbe poo5P44b4o
ooLz beb4pb5ebq poebeegepo qoePbqqbqo 000bppoqbb pPoopo44o4 obbeb64b.54
of79z bpobbqopeo 8ob4ob5eoq ooeqbobqob qbqopebbeb porqooqbbe eblepobooe
oggz b4obeopeob pobbebepoo b4440065pe bbeoobbi.00 24obbbbpob 4obqo3bbeo
OZSZ 344pbb44P0 4poq.b4pboo poqpbepb4o oebbbobebe ebbebb400e opobpi.obqb
0917z ebbeo.5.650o qq.o.44b4bee pDbebeopbb o4po5oopq5 qopepobeop bqqobebblq
o0D,z oobepeo4pb quo4obgbqb 4bo5epobee obbqebbqeo ggobbbeebo oporpooqose
of7Ez op000po400 4p6ebqeq5b ebuobboo4q 0P66epoqqo .4601.660p3p p33344-4o6b
08ZZ beooboob40 4ebboo4eob bbeb4googb obb4epob4o bob4o6eooe bb4ob4bb44
ozzz opob000ebb 4oeppobboo bbeebeetPb Tpoopeoopq qpoi.Pobqob o54boq.qo2p
ogTz DOD3PP3DU4 pPb6a6446D Poo5b4eoqo bpeoobbqob epbe5bevop q5qobeoebb
OOTZ 04640e4b0D 4464p3bb5y p66o3oP6ep oq40354664 poeqpeobeo e6pbo3464P
OVOZ ebb43bb4be eo3ebb443b 6q4uo4ebb3 4ebbqb4ebb ebbqb4obe b4o4346444
0861 bpeopbeoqo o4be33Poq4 0.6-436oupob.oqpopeoebo eebqqoopop 6.64.2o2t6pe
0z6T b4pbgobbqb eboebqobap eq.e4oebbgb beeobboobq eqopooTeqq. pabqp444p5
0981 1.366Peop5t. eeb4ouP36p Poop62ebuo 344beepopo poo3eo6.6.6e o5pbbeD6qb
008T b4.66pp6ebb 46344obe5e eppb4pe3ob bue4004446 64ob4befibe b4a6p43b4o
ObLT pobb4o042o bbeoopoogb bbobboobpe bebg4pogoo Ebb o5
3bpob400eb
oggT b400bbo44o eboquoqi.op eb5qbPbo4e obbbebebob pooporbpbbe b5eobebb4o
0z91 oq.poqqbqeo OPDPOOPPOq 4b4obeobP3 bqobppbpbo PEDOPOP4OP poTeob464o
09ST beDbeboqqo p4aee6404p 6q4434Pbe6 444 66436o qe3e6b4304 ebbbo4e444
00ST eo4Dobobbb eobbpbe2oD ebeeD26.643 4obuePpeeo quobo44obq 6.64005;36D
oppi og4b4e6bo6 ebTeqope4o 5bpPoob64q op5bebT4po p644.4opb4o 66eobe5eep
08E1 40u6o5o55 ee6pD64bge qopbebeobb bq6bpuoqeo boeopooeoq ooqeobbebe
ozET ope34qopb3 oeb464pPoq P6Mb4opqo 3pp33qb466 epppoqobqo bppeoePoeb
09Z1 bo3bqe334b obbeopebqo epeebbobeb beebeeog43 3b44e3eep6 .644obeo44o
0OZT oqbbbbqoqo 4po4bbbo64 ob4qp6b6qe buobeb4266 ebeoppguq6 b54p44epbP
of/TT b4po3bbebb gpPop5p55p oog4b4pop6 bePoPbbeob pobbb4opoq. p3o2o45oPo
0801 6bbopepoqb 4Do4-40.63oP 4uevoeeoPq epobPb6446 looqoqpbqo pfieptqopeo
OZOT epbebbo3b6 6.6q3464364 33 3E3; 34p3poo443 opb6obebbp beeepobeep
096 Dboo424obq boqoqeeube bb.404404e4 43ebeb4gpo pepa64.6044 bqqpop4obb
006 qepoq.bgpbq 4qoPeoqp4b 044eDqq-ePP 455044'2504 ogooqoppoe b4ep6ep5qb
OD'8 bopbeepobo peebbqqqoo bbebeq.00qe g000ppoobb eopqobqobe o5b3bebb4g
08L 0ebb666voo pbbpp6pp3b pbepoPopoq 33433bb4b4 ppeobbqope 4bp334p346
on eeebeeoDeo eebebeoe62 Pbbbeobebb 4o4bebbbb6 oeDbqbqopq epo4b2ogeb
099 epbbbooebb Pobqeb4p3b Pb6o3P433b qoP4ebe3po gpooboeqoq. PDPODOOPOD
oog b4p5pb3e3b bPbPpbepob 5bPeoe4b4p bp.b.b4.504eb ebbebpoqoe qoqpo33b4o
urG 3pe6eeqe44 3o3ee34P3q. bb4bqbqoqq. bqoobbeoq4 e43oP3pl3q P4.40655234
= 08f7 3p43pleb35 pbbPeoqoop P3e3b4ob4b bo44obeebo Pe343pb4b3 po4obebeob
ozt7 bqeoebbebb 4bbeepoq.oq qbP2303P33 32Pbqebee6 pool.eopboe 6bepopebqb
ogE beebqbbeeb epbbbqeebp beqbbgobeb elbqq.poobb ebeebebbbq bbebbpbbee
ooc oqoobeq.obe ooPebqqqab bqeebPeobe ooTloob4bb 54eq.bb4qbp ebepo3b4ob
ot,z bb4opb4obp p333bb4ob3 33pe4Pe342 3443pepeeq. ebb4b4eq34 opegbppopb
081 eob43bbe3b eo43554Poo pogbeeoboa bb4004bbe3 4q515po3oe5 eobbbo3oo4
ozT 4bq33b3beo opebeebboq opooqbeePb etoqobobeb opoboo5obo ob0000p000
VO-VO-ETOZ ZETZT8Z0 VD
Z6
0069 e5bee3eopq qopfyi.oq5P.E, 34qabeTeqo qoepoopoo6 4.6qobv5D45 Bqbb6eeDo6
0D,e9 55b4De5bpq poqoqoo646 bqoqobabqb qpPopofiebb 4abbbeeop4 EleutPoobp
09/.9 obbqoobbbe Dbqqa2b6be Pqq5b4Ppq4 4P4P4PePOO pabbbpqpqa p3bpo3p434
0n9 qobb4E633.4 qopbeopupo b6eppb-eqp4 peo3e6beD4 pobbbeopbp opeppobbal
0999 o3e3bb43pq booPbeopbb ep3ofq4333 433304333p bleupbqbET. 343463oeee
0099 4e0q0P63343 0436qoq3pb 4E643235e 6bb;044poo 43e33o3334 eebbb4o4oe
Of7S9 3bbeib4p3ee be3b466643 46404oDbet, beebeo34bb q424o335b6 403344330D ,
08179 obb4e4o3ob qopei.eqop eeqbboepeo epqepobbqq 5q36gooppo ogbooppoqb
0z179 3p334bga5b oqopeboopo 4pooeqqb45 24be4oqop5 buoqabe4e6 4444ebupeq
09E9 utpoqq04b4 444P434ePb 4e4pq4qqbe b2popo5b24 bebb4qppob bobbbeebep
00E9 365645Elq5.6 boeobpppbq pobqqopqop q_bg4oq55bp oepoo2b4e5 boobbuoqob
09 bqobbepo45 ebeeepoe4P 5404pebpbq qqopouqpoo qop45uopq4 oDobeob4o6
0819 q043DT4o55 qqeobbbubo 3pq4pobbeu oDo4o34poo poqbgpoob 43.435qopece
019 Eqqop6qopo 3POP001030 PP00000430 0'400640435 53PPPOPM4 pabobbpoop
0909 Beo5qp040.4 qobebbegee 5.436poMpe opEgeboobb ElbqebpobPP Pqbboeboqb
0009 bpbbebDebe oqabqopbo6 b4Dpo5beee bboqqbqqep booboqDpb4 bpqbqqqeop
0P68 b4oppElbaq bobbebloee popefieeb4o 334obpo4b6 eb3b33ep64 p4ob4pb4ob
0888 Daefiebppeo 084pbpu664 obeb4bobeo Bqobeebboo boe3-400bqu poobBbobeo
U8S 3obbebbeb2 eb43bbebbe be4obeabbo eee44obe3b eeb4035300 e3o4bpbbee
09L8 oeb4o5beo3 ebbeeo4qbe oeebboboee bbubbobubo ebbebbqbbe obqDbqobgb
ping oebb2e6405 ee5e2bebbo ebb3DB3b4b be3b2eop4o obpo5b2o3b obpbbueope
0f79s bsbo22Debb 43bPoeebbe aq.obPoPob 44eee23obb ebbqqoobbo boTepoqopb
088 beeou4epeo 34b2up46qo Eqbpbebb4p p-e5thob4ob ppoo5bpeog obpbbeeoPu
0zgs beoobouebb 4obea5eaeb obo542e5e5 gesbubeop ofopoofmob oppb54o3Pp
09fi8 bqopeboopo -2.2-egeeeooe boTebeobqo opPoobbueb ?e6qab5oqu 5opeoqe54o
00; babbopoupo bbbpopubbP 56E16438Pb 5p6be55435 goopbqqeob DooE162654o
0pEg p6o5bobppb e5bebeqqe3 5eqopobb6b speobbqbpo bpoeeooboq efipbTetqob
08Z8 b4obpboebb boeebbeoPe opobbeoqbp bpsqobqbob Etqabqobeo bbqopPbbpb
OZZg beo54obepq Tebqubpboo E6P6E.Te3bp bepbqobpeb ee5e6qeebe EbeppobbPo
0918 bobbqop4pb ebbebbb000 goobabobae pubopbbwb PbbbobTeo6 qopMePbge
00Tg beopobbepq qobppbbob4 obeopeeoqe pobbpbepE6 booppEtueqP PoobpoPoeb
()PDS 44-eopoupa, Pbbgoopbbe eb4a4e5bqe be.664pee2b eeoboeoboo bbgeoobbqe
08617 ooqobobeob eebbebefqe bbebb4obeb bobbebb4eb ebbbobqbEle oebeD45bqo
0Z617 beDbeebeeb eebebbebob ebepeebgeb bboobbbeob goqebbbobe b4qqbeopob
098P beeb4v035b vobqopeebq 6bp6bwobo o43.6Poob4 eBbubbopDp Bbeo54obpb
00817 4eb5ebbqo6 0bbe5b43be opoebpp54? b5 b56 Pobuobebb4 qpobboobpe
017Lp opT5pbe5b qobbopoo4 5.45pbeeobb 5-4bopb4pbb Peooqobpbq eoqpoebbub
089D, bqebebbopo booqqbpobe PoPuo4obbo bee&go6pbe 0b6ue6ceo6P bbgepabbET)
09t7 5Pbqqobobb bpopb5.4peo qp4o6p6beP poe6Pbbep5 Pb4boopb5e Eq.o6626qa6
098f, ebou5q6o.6 ebbe6?obq2 462Poo55pq oqepoPE)put, ebbuboobeq op4o6pooP5
oogD, 3446eubpub eobee8epue bbqooPepoq ombobebeob boo P bbqopebqql,
0D,7 bqobqoopbo e6bqa5e5bP obpobqoffo eppbsepoef, eubpbbqobe eqeboewob
08E17 qobbqbbepb ebbet4q3b6 obppobebqo obbPpbbqqo ebbppbpoqq obbebbobeP
up7 bobbebbebe 3bqoubet64 pob4bbbel5 4bEopb5ef6 Tebpebepbe pb4popboop
09Z17 b4b6e330bq popqoboeDo 5o4pbPo5ep 5p6b44opuo bobppoobbe bfipbbpbbeb
00n, bebbqobea6 efibbeaqq.Do 4peepPebeb 4ebbab6qeb pobeeogo6P eopeolyebqo
0PT17 o5eb4obppb eobbooeebe bbebElepoqo 6446ebbeop peoe6beoq4 obeapogbeb
080f7 b405381.040 443ebbeebo e44Dbeepbe opqbeeobeo eb4o4bepob e4qopqp4bb
uo ope.64546po e5b4Dee544 15b2o5435ee opeogbbpeo eboobb4De2 bopepbobqb
096E obp52beb55 eb3beo4gbe Poqbbeob4o eube2ob4ob Po6obbpbb4 bbppbePpbo
006E beuEobpbe 34Dpbobbbe Ppbb2e3b43 bqopobelePb qUeb4eeop bb4obbpbb
osc bbobe642e5 ebbqDoppbP obeebbbe 5,254qoppeg obPeeeqbbb obeeboebeD
08LE bebb4qbeo4 ebeof&gobe 6bebb45q36 Ecepeoqpeob ePbeobbebq PEIPHeooge
uti be3o35bebq =oebpeoo 5.bbov.b5e6 5qoTo2Eyee5 eebqoo4uob ebqb5ebb2o
099E 625qbobepb o4b5-86.4obe 662Dbuopo6 ;of:60'8034o u661.3.bouoe bbubbqobeb
009 ep2beeb4D6 3bbeb.64o6e bbebebbbqo qebbboepeb upbeebeboo beeeqeebbe
0178 304006e605 ebboqbebbq poebbebbeo oqobebqoqo qebpoqopee Ã643pebe5o
VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
tgcttttgaa agaaaaaaaa aggttttatt tttcccttct tgtagtaagt gctctagttc 6960
tgggtgtctt cactgccttg ccctggaact gtgtttagaa gagagtagct tgccctacaa 7020
tgtctacact ggtcgctgag ttccctgcgc actgcacctc actgtttgta aatgctgtga 7080
ttaggttccc ttatggcagg aaggcttttt ttttcttttt ttttttcttt tctttttttt 7140
ttttttaaag gaaaaccagt caaatcatga agccacatac gctagagaag ctgaatccag 7200
gtcccaaagg cgctgtcata aaggagcaag tgggacccgc accccttttt ttatataata 7260
caagtgcctt agcatgtgtc gcagctgtca ccactacagt aagctggttt acagatgttt 7320
ccactgagcg tcacaataaa gagtaccatg tccta 7355
<210> 48
<211> 1960 -
<212> PRT
<213> Mus musculus
<400> 48
Met Ala Gin Gin Ala Ala Asp Lys Tyr Leu Tyr Val Asp Lys Asn Phe
1 5 10 15
Ile Asn Asn Pro Leu Ala Gin Ala Asp Trp Ala Ala Lys Lys Leu Val
20 25 30
Trp Val Pro Ser Ser Lys Asn Gly Phe Glu Pro Ala Ser Leu Lys Glu
35 40 45
Glu Val Gly Glu Glu Ala Ile Val Glu Leu Val Glu Asn Gly Lys Lys
50 55 60
Val Lys Val Asn Lys Asp Asp Ile Gin Lys Met Asn Pro Pro Lys Phe
65 70 75 80
Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu Asn Glu Ala Ser
85 90 95
Val Leu His Asn Leu Lys Glu Arg Tyr Tyr Ser Gly Leu Ile Tyr Thr
100 105 110
Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr Lys Asn Leu Pro
115 120 125
Ile Tyr Ser Glu Glu Ile Val Glu Met Tyr Lys Gly Lys Lys Arg His
130 135 140
Glu Met Pro Pro His Ile Tyr Ala Ile Thr Asp Thr Ala Tyr Arg Ser
145 150 155 160
-Met Met Gin Asp Arg Glu Asp Gin Ser Ile Leu Cys Thr Gly Glu Ser
165 170 175
Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile Gin Tyr Leu Ala
180 185 190
His Val Ala Ser Ser His Lys Ser Lys Lys Asp Gin Gly Glu Leu Glu
195 200 205
Arg Gin Leu Leu Gin Ala Asn Pro Ile Leu Glu Ala Phe Gly Asn Ala
210 215 220
Lys Thr Val Lys Asn Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg
225 230 235 240
Ile Asn Phe Asp Val Asn Gly Tyr Ile Val Gly Ala Asn Ile Glu Thr
245 250 255
Tyr Leu Leu Glu Lys Her Arg Ala Ile Arg Gln Ala, Lys Glu Glu Arg
260 265 270
Thr Phe His Ile Phe Tyr Tyr Leu Leu Ser Gly Ala Gly Glu His Leu
275 280 285
Lys Thr Asp Leu Leu Leu Glu Pro Tyr Asn Lys Tyr Arg Phe Leu Ser
290 295 300
Asn Gly His Val Thr Ile Pro Gly Gin Gin Asp Lys Asp Met Phe Gin
305 310 315 320
93
CA 02812132 2013-04-04
Glu Thr Met Glu Ala Met Arg Ile Met Gly Ile Pro Glu Asp Glu Gin
325 330 335
Met Gly Leu Leu Arg Val Ile Ser Gly Val Leu Gin Leu Gly Asn Ile
340 345 350
Ala Phe Lys Lys Glu Arg Asn Thr Asp Gin Ala Ser Met Pro Asp Asn
355 360 365
Thr Ala Ala Gin Lys Val Ser His Leu Leu Gly Ile Asn Val Thr Asp
370 375 380
Phe Thr Arg Gly Ile Leu Thr Pro Arg Ile Lys Val Gly Arg Asp Tyr
385 390 395 400
Val Gin Lys Ala Gin Thr Lys Glu Gin Ala Asp Phe Ala Ile Glu Ala
405 410 415
Leu Ala Lys Ala Thr Tyr Glu Arg Met Phe Arg Trp Leu Val Leu Arg
420 425 430
Ile Asn Lys Ala Leu Asp Lys Thr Lys Arg Gin Gly Ala Ser Phe Ile
435 440 445
Gly Ile Leu Asp Ile Ala Gly Phe Glu Ile Phe Asp Leu Asn Ser Phe
450 455 460
Glu Gin Leu Cys Ile Asn Tyr Thr Asn Glu Lys Leu Gin Gin Leu Phe
465 470 475 480
Asn His Thr Met Phe Ile Leu Glu Gin Glu Glu Tyr Gin Arg Glu Gly
485 490 495
Ile Glu Trp Asn Phe Ile Asp Phe Gly Leu Asp Leu Gin Pro Cys Ile
500 505 510
Asp Leu Ile Glu Lys Pro Ala Gly Pro Pro Gly Ile Leu Ala Leu Leu
515 520 525
Asp Glu Glu Cys Trp Phe Pro Lys Ala Thr Asp Lys Ser Phe Val Glu
530 535 540
Lys Val Val Gin Glu Gin Gly Thr His Pro Lys Phe Gin Lys Pro Lys
545 550 555 560
Gin Leu Lys Asp Lys Ala Asp .Phe Cys Ile Ile His Tyr Ala Gly Lys
565 570 575
Val Asp Tyr Lys Ala Asp Glu Trp Leu Met Lys Asn Met Asp Pro Leu
580 585 590
Asn Asp Asn Ile Ala Thr Leu Leu His Gin Ser Ser Asp Lys Phe Val
595 600 605
Ser Glu Leu Trp Lys Asp Val Asp Arg Ile Ile Gly Leu Asp Gin Val
610 615 620
Ala Gly Met Ser Glu Thr Ala Leu Pro Gly Ala Phe Lys Thr Arg Lys
625 630 635 640
Gly Met She Arg Thr Val Gly Gin Leu Tyr Lys Glu Gin Leu Ala Lys
645 650 655
Leu Met Ala Thr Leu Arg Asn Thr Asn Pro Asn Phe Val Arg Cys Ile
660 665 670
Ile Pro Asn His Glu Lys Lys Ala Gly Lys Leu Asp Pro His Leu Val
675 680 685
Leu Asp Gin Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile Cys
690 695 700
Arg Gin Gly Phe Pro Asn Arg Val Val She Gin Glu Phe Arg Gin Arg
705 710 715 720
Tyr Glu Ile Leu Thr Pro Asn Ser Ile Pro Lys Gly She Met Asp Gly
725 730 735
Lys Gin Ala Cys Val Leu Met Ile Lys Ala Leu Glu Leu Asp Ser Asn
740 745 750
Leu Tyr Arg Ile Gly Gin Ser Lys Val She She Arg Ala Gly Val Leu
755 760 765
94
CA 02812132 2013-04-04
Ala His Leu Glu Glu Glu Arg Asp Leu Lys Ile Thr Asp Val Ile Ile
770 775 780
Gly Phe Gin Ala Cys Cys Arg Gly Tyr Leu Ala Arg Lys Ala Phe Ala
785 790 795 800
Lys Arg Gin Gin Gin Leu Thr Ala Met Lys Val Leu Gin Arg Asn Cys
805 810 815
Ala Ala Tyr Leu Arg Leu Arg Asn Trp Gin Trp Trp Arg Leu Phe Thr
820 825 830
Lys Val Lys Pro Leu Leu Asn Ser Ile Arg His Glu Asp Glu Leu Leu
835 840 845
Ala Lys Glu Ala Glu Leu Thr Lys Val Arg Glu Lys His Leu Ala Ala
850 855 860
Glu Asn Arg Leu Thr Glu Met Glu Thr Met Gin Ser Gin Leu Met Ala
865 870 875 880
Glu Lys Leu Gin Leu Gin Glu Gin Leu Gin Ala Glu Thr Glu Leu Cys
885 890 895
Ala Glu Ala Glu Glu Leu Arg Ala Arg Leu Thr Ala Lys Lys Gin Glu
900 905 910
Leu Glu Glu Ile Cys His Asp Leu Glu Ala Arg Val Glu Glu Glu Glu
915 920 925
Glu Arg Cys Gin Tyr Leu Gin Ala Glu Lys Lys Lys Met Gin Gin Asn
930 935 940
Ile Gin Glu Leu Glu Glu Gin Leu Glu Glu Glu Glu Ser Ala Arg Gin
945 950 955 960
Lys Leu Gin Leu Glu Lys Val Thr Thr Glu Ala Lys Leu Lys Lys Leu
965 970 975
Glu Glu Asp Gin Ile Ile Met Glu Asp Gin Asn Cys Lys Leu Ala Lys
980 985 990
Glu Lys Lys Leu Leu Glu Asp Arg Val Ala Glu Phe Thr Thr Asn Leu
995 1000 1005
Met Glu Glu Glu Glu Lys Ser Lys Ser Leu Ala Lys Leu Lys Asn Lys
1010 1015 1020
His Glu Ala Met Ile Thr Asp Leu Glu Glu Arg Leu Arg Arg Glu Glu
1025 1030 1035 1040
Lys Gin Arg Gin Glu Leu Glu Lys Thr Arg Arg Lys Leu Glu Gly Asp
1045 1050 1055
Ser Thr Asp Leu Ser Asp Gin Ile Ala Glu Leu Gin Ala Gin Ile Ala
1060 1065 1070
Glu Leu Lys Met Gin Leu Ala Lys Lys Glu Glu Glu Leu Gin Ala Ala
1075 1080 1085
Leu Ala Arg Val Glu Glu Glu Ala Ala Gin Lys Asn Met Ala Leu Lys
1090 1095 1100
Lys Ile Arg Glu Leu Glu Thr Gin Ile Ser Glu Leu Gin Glu Asp Leu
1105 1110 1115 1120
Glu Ser Glu Arg Ala Ser Arg Asn Lys Ala Glu Lys Gin Lys Arg Asp
1125 1130 1135
Leu Gly Glu Glu Leu Glu Ala Leu Lys Thr Glu Leu Glu Asp Thr Leu
1140 1145 1150
Asp Ser Thr Ala Ala Gin Gin Glu Leu Arg Ser Lys Arg Glu Gin Glu
1155 1160 1165
Val Ser Ile Leu Lys Lys Thr Leu Glu Asp Glu Ala Lys Thr His Glu
1170 1175 1180
Ala Gin Ile Gin Glu Met Arg Gin Lys His Ser Gin Ala Val Glu Glu
1185 1190 1195 1200
Leu Ala Asp Gin Leu Glu Gin Thr Lys Axg Val Lys Ala Thr Leu Glu
1205 , 1210 1215
CA 02812132 2013-04-04
Lys Ala Lys Gin Thr Leu Glu Asn Glu Arg Gly Glu Leu Ala Asn Glu
1220 1225 1230
Val Lys Ala Leu Leu Gin Gly Lys Gly Asp Ser Glu His Lys Arg Lys
1235 1240 1245
Lys Val Glu Ala Gin Leu Gin Glu Leu Gin Val Lys Phe Ser Glu Gly
1250 1255 1260
Glu Arg Val Arg Thr Glu Leu Ala Asp Lys Val Thr Lys Leu Gin Val
1265 1270 1275 1280
Glu Leu Asp Ser Val Thr Gly Leu Leu Ser Gin Ser Asp Ser Lys Ser
1285 1290 1295
Ser Lys Leu Thr Lys Asp Phe Ser Ala Leu Glu Ser Gin Leu Gin Asp
1300 1305 1310
Thr Gin Glu Leu Leu Gin Glu Glu Asn Arg Gin Lys Leu Ser Leu Ser
1315 1320 1325
Thr Lys Leu Lys Gin Met Glu Asp Glu Lys Asn Ser Phe Arg Glu Gin
1330 1335 1340
Leu Glu Glu Glu Glu Glu Ala Lys Arg Asn Leu Glu Lys Gin Ile Ala
1345 1350 1355 1360
Thr Leu His Ala Gin Val Thr Asp Met Lys Lys Lys Met Glu Asp Gly
1365 1370 1375
Val Gly Cys Leu Glu Thr Ala Glu Glu Ala Lys Arg Arg Leu Gin Lys
1380 1385 1390
Asp Leu Glu Gly Leu Ser Gin Arg Leu Glu Glu Lys Val Ala Ala Tyr
1395 1400 1405
Asp Lys Leu Glu Lys Thr Lys Thr Arg Leu Gin Gin Glu Leu Asp Asp
1410 1415 1420
Leu Leu Val Asp Leu Asp His Gin Arg Gin Ser Val Ser Asn Leu Glu
1425 1430 1435 1440
Lys Lys Gin Lys Lys Phe Asp Gin Leu Leu Ala Glu Glu Lys Thr Ile
1445 1450 1455
Ser Ala Lys Tyr Ala Glu Glu Arg Asp Arg Ala Glu Ala Glu Ala Arg
1460 1465 1470
Glu Lys Glu Thr Lys Ala Leu Ser Leu Ala Arg Ala Leu Glu Glu Ala
1475 1480 1485
Met Glu Gin Lys Ala Glu Leu Glu Arg Leu Asn Lys Gin Phe Arg Thr
1490 1495 1500
Glu Met Glu Asp Leu Met Ser Ser Lys Asp Asp Val Gly Lys Ser Val
1505 1510 1515 1520
His Glu Leu Glu Lys Ser Lys Arg Ala Leu Glu Gin Gin Val Glu Glu
1525 1530 1535
Met Lys Thr Gin Leu Glu Glu Leu Glu Asp Glu Leu Gin Ala Thr Glu
1540 1545 1550
Asp Ala Lys Leu Arg Leu Glu Val Asn Leu Gin Ala Met Lys Ala Gin
1555 1560 1565
Phe Glu Arg Asp Leu Gin Gly Arg Asp Glu Gin Ser Glu Glu Lys Lys
1570 1575 1580
Lys Gin Leu Val Arg Gin Val Arg Glu Met Glu Ala Glu Leu Glu Asp
1585 1590 1595 1600
Glu Arg Lys Gin Arg Ser Met Ala Met Ala Ala Arg Lys Lys Leu Glu
1605 1610 1615
Met Asp Leu Lys Asp Leu Glu Ala His Ile Asp Thr Ala Asn Lys Asn
1620 1625 1630
Arg Glu Glu Ala Ile Lys Gin Leu Arg Lys Leu Gin Ala Gin Met Lys
1635 1640 1645
Asp Cys Met Arg Glu Leu Asp Asp Thr Arg Ala Ser Arg Glu Glu Ile
1650 1655 1660
96
CA 02812132 2013-04-04
Leu Ala Gin Ala Lys Glu Asn Glu Lys Lys Leu Lys Ser Met Glu Ala
1665 1670 1675 1680
Glu Met Ile Gin Leu Gin Glu Glu Leu Ala Ala Ala Glu Arg Ala Lys
1685 1690 1695
Arg Gin Ala Gin Gin Glu Arg Asp Glu Leu Ala Asp Glu Ile Ala Asn
1700 1705 1710
Ser Ser Gly Lys Gly Ala Leu Ala Leu Glu Glu Lys Arg Arg Leu Glu
1715 1720 1725
Ala Arg Ile Ala Leu Leu Glu Glu Glu Leu Glu Glu Glu Gin Gly Asn
1730 1735 1740
Thr Glu Leu Ile Asn Asp Arg Leu Lys Lys Ala Asn Leu Gin Ile Asp
1745 1750 1755 1760
Gin Ile Asn Thr Asp Leu Asn Leu Glu Arg Ser His Ala Gin Lys Asn
1765 1770 1775
Glu Asn Ala Arg Gin Gin Leu Glu Arg Gin Asn Lys Glu Leu Lys Ala
1780 1785 1790
Lys Leu Gin Glu Met Glu Ser Ala Val Lys Ser Lys Tyr Lys Ala Ser
1795 1800 1805
Ile Ala Ala Leu Glu Ala Lys Ile Ala Gin Leu Glu Glu Gin Leu Asp
1810 1815 1820
Asn Glu Thr Lys Glu Arg Gin Ala Ala Ser Lys Gin Val Arg Arg Thr
1825 1830 1835 1840
Glu Lys Lys Leu Lys Asp Val Leu Leu Gin Val Glu Asp Glu Arg Arg
1845 1850 1855 =
Asn Ala Glu Gin Phe Lys Asp Gin Ala Asp Lys Ala Ser Thr Arg Leu
1860 1865 1870
Lys Gin Leu Lys Arg Gin Leu Glu Glu Ala Glu Glu Glu Ala Gin Arg
1875. =1880 1885
Ala Asn Ala Ser Arg Arg Lys Leu Gin Arg Glu Leu Glu Asp Ala Thr
1890 1895 1900
Glu Thr Ala Asp Ala Met Asn Arg Glu Val Ser Ser Leu Lys Asn Lys
1905 1910 1915 1920
Leu Arg Arg Gly Asp Leu Pro Phe Val Val Thr Arg Arg Ile Val Arg
1925 1930 1935
Lys Gly Thr Gly Asp Cys Ser Asp Glu Glu Val Asp Gly Lys Ala Asp
1940 1945 1950
Gly Ala Asp Ala Lys Ala Ala Glu
1955 1960
<210> 49
<211> 7474
<212> DNA
<213> Homo sapiens
<400> 49
atacgactca ctatagggcg atcaggtgct ggaaagaagg ctaagcaagg ctgacctgct 60
gcagctcccg cctcgtgcgc tcgccccacc cggccgccgc ccgagcgctc gagaaagtcc 120
tctcgggaga agcagcgcct gttcccgggg cagatccagg ttcaggtcct ggctataagt 180
caccatggca cagcaagctg ccgataagta tctotatgtg gataaaaact tcatcaacaa 240
tccgctggcc caggccgact gggctgccaa gaagctggta tgggtgcctt ccgacaagag 300
tggctttgag ccagccagcc tcaaggagga ggtgggcgaa gaggccatcg tggagctggt 360
ggagaatggg aagaaggtga aggtgaacaa ggatgacatc cagaagatga acccgcccaa 420
gttctccaag gtggaggaca tggcagagct cacgtgcctc aacgaagcct cggtgctgca 480
caacctcaag gagcgttact actcagggct catctacacc tattcaggcc tgttctgtgt 540
ggtcatcaat ccttacaaga acctgcccat ctactctgaa gagattgtgg aaatgtacaa 600
gggcaagaag aggcacgaga tgccccctca catctatgcc atcacagaca ccgcctacag.660
97
86
080P 4DbeeDbeoo 4beeobeoeb Da4beppbeD 404qobbbpo eb4boeepeb b43bebbqbb
ont7 pobqobeeop 23466ppop6 op66.4obP6v puo6364.5ob obpbE,6bbeb pueoqq6ueo
096E qbbpobqobp bbeobqpbeo bobbPbbqbe ePbePobobe Poeobebboq opbbb6epPo
006E bbbtob4ob4 obqbbeeb4b bebo22oobb 4oba6bb6bb DbPbopubeb bqD4oPbeDb
om3E pe2obbeebp bogopeepo6 PePb4665o6 epboubpo6e b6-436eo6p5 536.6436e66
08L p6b4boobbe peoqpeobpP bPobbPb4pb ebbpooqpbp poobbpEopo 3pe6pp33bb
onc ebbebbebb4 popubeebee b4op4eoPPb 4.6beE6Pobp bqbopeepoq bbpoqo6ebb
099 po5po3obqo 5eoepoq4p6 64o602p265 e66446p5po ePPe54o6o6 6ebpqobebe
009E ebb.66q4pop bbboeeebPo BuTbebqobv ePqePbbepo 44obqbo6eb qpqbebbqoo
ot,sE ebpebbeop4 ospb4oqoqe beoqpqppbb 4obubbbooq efreebppo4o po55qepee6
086E eebpoop6qo 6Pe5be6pe6 646p6poo66 4opo50066p pogo62b6P6 6e6eaebuPo
OE obbgobeobq ebupp436E6 bo6aTebeop obbepoqobp bpobogutcep opbobeoqop
09EE pbecuopqop bpbbbebbqo bepbbooboo opbspbpbbq DbebBeobo beobeubpb.6
00u e5,55poboo4 3obo6u,beub 64qoe5qopo qebqpeobbe bqpobpeope bueoqp6peo
ovz obpqaobpbe eqoqppe5e6 bebbebeebe oep4opeepo eoopoqqbeb qobe4pebea
08ft fyeebbgobq peeebeeeeb bepoobb4ob peob4oeubp oppb&ebbqo oqeogebeob
0TE p6bebbebbq obeppee6qo buTtobbEto opopubqbbv ebebbqpbpo bqobpe6u3.6
09H boopbDbebe bbubbebbeb bqabeobubb pb44obe5be oo;bz 54ebee6
ocw eebee62bbo bbeobqopeo 6eop.6qpb3b eb52bbebbe aye56q5b6e 3p5.6e6eq.3p
Of76Z Pbqppob1.34 p6e6ep6pqg Pebbeobps6 pepoboou64 pob000bb6o oqp6eb6e64
088Z obbpb3ob4b 4b4obaboae ePbuobbeop 43buobebbp D64peyeabg4 ee2.52623b5
OZ8Z gPogob?p4o q5eo.64o5ou .6256qpbe66 peo4D66epe e6P66ob4D5 54o5e3.6ePE,
09L fy4b6q3but
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00Lz 2Dbqob4D5o obe2o4b5ee ooep4gal.o.6 bob54b64bp obbq3ppb5D 64pEcepbqop
0v9z p4o36q.D6D5 qoe2Moficeo oqopq6beeb q.Poobopeqq. obea5Po5Po bbobeepo64
08SZ 44Pbbupp66 po0b6400e4 3bbbbp3b43 bq3pbbeop4 qbbbequaqp o4boubooPo
ozu qefyeebqoo? bpbobubbeb 6e56qopepo obbqobgb45 boobqboo44 oqqa4bpeep
09T7 bebeop66.44 eoboopqbqo guTo6Poe5o 4o6Pb643oo 6Peeegebqe oqobqbobqb
ouz obbsofyeebb boeb64eal4 466fieepooq 4p3o4peeu3 p43ub433qu beb42426eb
ovu eo55oq4g5p 5.5e30qq.34.6 b45bbPpeep poo4305.6bp opboob4o4e 4booqeobbb
08ZZ Pb o645 Eoepobgobo 5i.o5repoe66 gob4bogoqp oboopPb643 beeobboDE6
ozu pubeutPfioe poPeo.000qe oqeobqoboo 4bqq.i.oepoo 33ee53eDe2 b6e.b43b3eq.
091Z 066.4e5go62 poo564D6eo 62b5eepPqq. 4ofreob6E64 b4opp6poqq. 53eo.666epb
00-c Baboebepoq 4DoMbEo3o 543eo533P5 P.E6oqbqpob boob54b6p3 oubbqoDbbo
0170Z 420qeobooe .664.64eb5ee bb4b43bebb 3qp4bi.44be eoe5qoqoo4 bepoeop4ob
0861 40"epeop53g epeepe54up bqopoogebb Teop25ee5q e54355.45e5 pebqpbeeeo
pqgv.56455p eobboobquq. Depoqeqqeo Egoqqq.p.bqo bppeoebbep bqobeDbeep
0981 Dabeeoepoq. qbeeppopeo ppeobbbeob e5be3b4e54 bbeebubbgb p4gobebueo
oogi pbooeoobep poopoqgbbq. ofiqbebbebo pbbqobqopo bbqoqquobb boop000bbp
OfiLT obeopbpebe Egqeoqopeb ogPobqpoob eob433eb3g pobbqq4oPb oquoggpepb
0891 biLeboq.eob bbebobobea pe4b25bebb epbebbqooq. up44b4e33p p2o2po4go
0z91 40.6p35p364 obeebubque 0020Pq4PPO Teo54b4a5.2 a5.544-4534 pee6qoqubq
09gT qq34p5e634 qobboobqqe pebbqopqeb 6b3qe34433 qopbobbbpo bbpbueopeb
OOST e2opbbqp4o 5be2peepqe obob4ob4bh gob54o5opq qb4ebbo5ab 4e4poeEob5
ovvi puopbfq.qop b5E,60-4Poob 444pubi.obb Pobebeupgo .2.6.2o6066pe &Boo-450244
08ET P565oe555q. 6.6Peo4pobo boopoPoqop 4pubbpbpoo epq.44Pboop fy4b4peq.e4
OZET 555443qoge 333 5i5
eepD3.6q3.62 opoeeoeboo 354-233q535 be3pebqo23
09-E ee6bobp6be ebeeo.44045 3qeou23563 qoEreoqqoqq. 5555eoqp4e oi.55.6o543.5
00ZI 403E664ePP obpbbpbET.5 eopoqeobbb qPqqubbubq poo55e664u oDp6e66eop
oviT 44.54-eop55e .eoebbeobeo bbboopoqeo peo4bos,Dub frTeepoqbqo oqqabooeqp
0801 ueopeoe46D 35ebfq.q.54o p4oTe5oop6 ppbqopeobe bebbqabbbb qpq6qopqoq.
OZOT e44Pq0-440q. p3p334q33p bbope6pe66 epoobppoob pamob-46o 431.Pepbebb
096 41.44oqeqqo ebp.644E,pee Dobebbq4.64 4p3e4o5b4e 204b4pbq44 oe2o4eoboq
006 qpoqq.epeob 53qqabo3oq DoqoeD-26-1. pebeeb4boo e6peoD5oep 565pqqoD55
ovg eb54op4poo 30P2ODUPD b43b4o5pob 636e5b'4Dbe bobbbepoeb beebeepbeb
08L upopo5343o 4b3bbi.5pe3 bo5b4oquq6 eoaqeoqbbe 2622opeoue beb53ebe2o
5bqobe5bqo le7fq..5643-2 obqbqqoqs,D oqeeoqpbep beb3opbeep bqebqpqbeb
VO-VO-ETOZ ZETZT8Z0 VD
66
t7L171. eeee
eveeeeeeve 2822884054 54800e2525
0f91,L PPP4PPOPOq PObElq.OPOO 4444642620 244466405e 2462024320 3204640623
08EL 5445464e0-5 eq400b4b2P eP4PP4e4P4 44444D0000 :2400020004 DePP-b45Pb4
ozu 6266222020 4640644620 004E620442 2640406222 0240364242 0540622224
09ZL P425462254 ppqpae0404 02PPOPPe55 2224444T44 4444420652 2622065024
00ZL 4026264420 5436424246 4444b40Pe4 0044P4b440 P444Peb3ob 446540E0-eq.
0D,1L 0365402030 0460405236 2626226244 3644400640 2030344046 2600442222
080L 4P64222422 4644044400 0044444404 4446622422 2206222644 44424E620e
140L 0540020064 03222224;3 624246,2030 2644500205 4002444664 6652600066
0969 6402662434 3240464644 0426366203 3430066062 0066662624 4606262006
0069 2026303522 0004544026 6522446532 233423242u 2002E66644 340POPPODO
(:189 404406646e 0044006206 2005622266 4062204E56 5036406622 0620200.540
08L9 30064006E0 2562600244 4004000030 0000642206 3522040364 2222404425
OZL9 4004000404 6406004530 2006052066 2000464000 2000040033 2E64626526
0999 5E6664E666 4065225202 06E6806464 0400200400 5663000652 0626622620
0099 04650E6540 4056420004 0200040065 5440000640 4002020204 0026466460
0p,s9 2020422064 4464054440 4440040040 0402000005 0520020054 0460854000
08179 3006204504 6e05000005 e66045e455 44440056e5 4086204403 444484042e
0n.9 6424044446 252036E662 6666300306 6235662062 606E264464 262202062e
09E9 2220054403 0400062040 0306620520 026058625u 0552000665 0005662026
00E9 P.E.PPPOOP4P 6434226204 0540000400 0406200440 0004206406 3040044066
OT7Z9 4442065585 4454464E62 0040004436 0004P3303D 0460000500 3000640040
0819 3400642028 6464064026 6644044038 3333434336 0236206030 3852030443
019 0004005802 0020252026 2026542653 2586400580 6300403304 0062242260
0909 0640082800 5b2640ba6 42660E2220 6642624662 6E26026004 0664eb6bb0
0009 06366E2266 3006542260 3603006463 4.644460054 3025656060 5620405220
OP6S 8262284000 4062046286 0503225420 06426036bo 2.52.640203.6 0266266406
088s 260E068364 3282563060 0040050820 0E6E052300 6526626226 3355266266
0Z8S 405206506e 20405E062e 6400600024 3420662232 600E620025 6220245206
09Ls 8606632266 25E0626386 4266465235 4064064.642 .662254062e 6226260026
OOLS 6046064568 3822064006 2366233636 2562230262 5p-22026643 6236266266
OP9S 4052320E144 2622005626 0430360320 4200400662 2324682334 6220464020
08ss 6662664262 6620643622 3456224406 2562202262 006022E640 6205205603
oess 06422.62632 2628620006 0200620635 2664008254 0026002022 042E1200260
09; 4252064008 2006622622 6405600260 2203264058 6502022055 6206256266
0017S 2564368552 6626640620 3063420600 3652664046 0660622686 626244505b
OPES 4000626522 206506206e 0820050325 26026405.63 0626426650 525E805200
08ZS 366200506e 2005463626 6063062065 4382662652 0544523042 6426260366
ozs 2E54205262 86405225221260225252 2200652000 5640042626 5254604040
09Is 0606030202 6485643626 0635420640 2E6226426e 0006583640 6226605406
001s 202234233 6225025550 022E820220 0650302604 2080606625 5400255286
Of7OS 4008664262 6640522622 6600060065 4620664260 406062062e 6526260266
086 2554062520 5526532626 6605365802 6204554058 0528522522 6255260525
0Z617 2062608666 0065623640 026E606250 4452000E62 2542006620 6300820466
098P 256435605.4 0522006425 2260020066 2053062602 6526640626 2256306206
008' 0252263252 5526646620 6206252400 0655052800 4522626630 6250200464 ,
0f7L' 6268806663 6326426522 0040625384 1002668664 2626502050 0436206220
089, 22040E6052 5640586606 6226205265 3800622552 5640006650 0055305046
0Z9P 4040552200 2626622626 2600065265 06526405E6 0026050625 6252064236
09; 2200640403 2002522626 5266055300 4058008644 3582522520 5226226266
00SP 3008205460 6068683063 6200803266 4002664664 0543026025 6405266236
012,t,f7 2053066060 2682008522 5266405820 8502300500 6646528685 6260805606
0E3E17 2006264006 662E640026 6286200405 2256262264 6626525405 4028256400
0n, 5455664E35 2386586638 6226222885 3232630bb4 5683336123 0133323063
09ZP 4862052262 5640022020 6820355266 86.52.6.62662 6640520626 6600440044
00n, 8262262602 6585545583 6223435883 3835863306 2643628523 6630226256
0vu, 2662064064 0686580402 0266205406 8000452564 0506004044 0266220020
VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
<210> 50
<211> 1960
<212> PRT
<213> Homo sapiens
<400> 50
Met Ala Gin Gin Ala Ala Asp Lys Tyr Leu Tyr Val Asp Lys Asn Phe
1 5 10 15
Ile Asn Asn Pro Leu Ala Gin Ala Asp Trp Ala Ala Lys Lys Leu Val
20 25 30
Trp Val Pro Ser Asp Lys Ser Gly Phe Glu Pro Ala Ser Leu Lys Glu
35 40 45
Glu Val Gly Glu Glu Ala Ile Val Glu Leu Val Glu Asn Gly Lys Lys
50 55 60
Val Lys Val Asn Lys Asp Asp Ile Gin Lys Met Asn Pro Pro Lys Phe
65 70 75 80
Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu Asn Glu Ala Ser
85 90 95
Val Leu His Asn Leu Lys Glu Arg Tyr Tyr Ser Gly Leu Ile Tyr Thr
100 105 110
Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr Lys Asn Leu Pro
115 120 125
Ile Tyr Ser Glu Glu Ile Val Glu Met Tyr Lys Gly Lys Lys Arg His
130 135 140
Glu Met Pro Pro His Ile Tyr Ala Ile Thr Asp Thr Ala Tyr Arg Ser
145 150 155 160
Met Met Gin Asp Arg Glu Asp Gin Ser Ile Leu Cys Thr Gly Glu Ser
165 170 175
Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile Gin Tyr Leu Ala
180 185 190
Tyr Val Ala Ser Ser His Lys Ser Lys Lys Asp Gin Gly Glu Leu Glu
195 200 205
Arg Gin Leu Leu Gin Ala Asn Pro Ile Leu Glu Ala Phe Gly Asn Ala
210 215 220
Lys Thr Val Lys Asn Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg
225 230 235 240
Ile Asn Phe Asp Val Asn Gly Tyr Ile Val Gly Ala Asn Ile Glu Thr
245 250 255
Tyr Leu Leu Glu Lys Ser Arg Ala Ile Arg Gin Ala Lys Glu Glu Arg
260 265 270
Thr Phe His Ile Phe Tyr Tyr Leu Leu Ser Gly Ala Gly Glu His Leu
275 280 285
Lys Thr Asp Leu Leu Leu Glu Pro Tyr Asn Lys Tyr Arg Phe Leu Ser
290 295 300
Asn Gly His Val Thr Ile Pro Gly Gin Gin Asp Lys Asp Met Phe Gin
305 310 315 320
Glu Thr Met Glu Ala Met Arg Ile Met Gly Ile Pro Glu Glu Glu Gin
325 330 335
Met Gly Leu Leu Arg Val Ile Ser Gly Val Leu Gin Leu Gly Asn Ile
340 345 350
Val Phe Lys Lys Glu Arg Asn Thr Asp Gin Ala Ser Met Pro Asp Asn
355 360 365
Thr Ala Ala Gin Lys Val Ser His Leu Leu Gly Ile Asn Val Thr Asp
370 375 380
Phe Thr Arg Gly Ile Leu Thr Pro Arg Ile Lys Val Gly Arg Asp Tyr
385 390 395 400
100
CA 02812132 2013-04-04
Val Gin Lys Ala Gin Thr Lys Giu Gin Ala Asp Phe Ala Ile Giu Ala
405 410 415
Leu Ala Lys Ala Thr Tyr Glu Arg Met Phe Arg Trp Leu Val Leu Arg
420 425 430
Ile Asn Lys Ala Leu Asp Lys Thr Lys Arg Gin Gly Ala Ser Phe Ile
435 440 445
Gly Ile Leu Asp Ile Ala Gly Phe Glu Ile Phe Asp Leu Asn Ser Phe
450 455 460
Glu Gin Leu Cys Ile Asn Tyr Thr Asn Glu Lys Leu Gin Gin Leu Phe
465 470 475 480
Asn His Thr Met Phe Ile Leu Glu Gin Glu Glu Tyr Gin Arg Glu Gly
485 490 495
Ile Glu Trp Asn Phe Ile Asp Phe Gly Leu Asp Leu Gin Pro Cys Ile
500 505 510
Asp Leu Ile Glu Lys Pro Ala Gly Pro Pro Gly Ile Leu Ala Leu Leu
515 520 525
Asp Glu Glu Cys Trp Phe Pro Lys Ala Thr Asp Lys Ser Phe Val Glu
530 535 540
Lys Val Met Gin Glu Gin Gly Thr His Pro Lys Phe Gin Lys Pro Lys
545 550 555 560
Gin Leu Lys Asp Lys Ala Asp Phe Cys Ile Ile His Tyr Ala Gly Lys
565 570 575 .
Val Asp Tyr Lys Ala Asp Glu Trp Leu Met Lys Asn Met Asp Pro Leu
580 585 590
Asn Asp Asn Ile Ala Thr Leu Leu His Gin Ser Ser Asp Lys Phe Val
595 600 605
Ser Glu Leu Trp Lys Asp Val Asp Arg Ile Ile Gly Leu Asp Gin Val
610 615 620
Ala Gly Met Ser Glu Thr Ala Leu Pro Gly Ala Phe Lys Thr Arg Lys
625 630 635 640
Gly Met Phe Arg Thr Val Gly Gin Leu Tyr Lys Glu Gin Leu Ala Lys
645 650 655
Leu Met Ala Thr Leu Arg Asn Thr Asn Pro Asn Phe Val Arg Cys Ile
660 665 670
Ile Pro Asn His Glu Lys Lys Ala Gly Lys Leu Asp Pro His Leu Val
675 680 685
Leu Asp Gin Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile Cys
690 695 700
Arg Gin Gly Phe Pro Asn Arg Val Val Phe Gin Glu Phe Arg Gin Arg
705 710 715 720
Tyr Glu Ile Leu Thr Pro Asn Ser Ile Pro Lys Gly Phe Met Asp Gly
725 730 735
Lys Gin Ala Cys Val Leu Met Ile Lys Ala Leu Glu Leu Asp Ser Asn
740 745 750
Leu Tyr Arg Ile Gly Gin Ser Lys Val Phe Phe Arg Ala Gly Val Leu
755 760 765
Ala His Leu Glu Glu Glu Arg Asp Leu Lys Ile Thr Asp Val Ile Ile
770 775 780
Gly Phe Gin Ala Cys Cys Arg Gly Tyr Leu Ala Arg Lys Ala Phe Ala
785 790 795 800
Lys Arg Gin Gin Gin Leu Thr Ala Met Lys Val Leu Gln'Arg Asn Cys
805 810 815
Ala Ala Tyr Leu Lys Leu Arg Asn Trp Gin Trp Trp Arg Leu Phe Thr
820 825 830
Lys Val Lys Pro Leu Leu Gin Val Ser Arg Gin Glu Glu Glu Met Met
835 840 845
101
CA 02812132 2013-04-04
Ala Lys Glu Glu Glu Leu Val Lys Val Arg Glu Lys Gln Leu Ala Ala
850 855 860
Glu Asn Arg Leu Thr Glu Met Glu Thr Leu Gin Ser Gin Leu Met Ala
865 870 875 880
Glu Lys Leu Gin Leu Gin Glu Gin Leu Gin Ala Glu Thr Glu Leu Cys
885 890 895
Ala Glu Ala Glu Glu Leu Arg Ala Arg Leu Thr Ala Lys Lys Gin Glu
900 905 910
Leu Glu Glu Ile Cys His Asp Leu Glu Ala Arg Val Glu Glu Glu Glu
915 920 925
Glu Arg Cys Gin His Leu Gin Ala Glu Lys Lys Lys Met Gin Gin Asn
930 935 940
Ile Gin Glu Leu Glu Glu Gin Leu Glu Glu Glu Glu Ser Ala Arg Gin
945 950 955 . 960
Lys Leu Gin Leu Glu Lys Val Thr Thr Glu Ala Lys Leu Lys Lys Leu
965 970 975
Glu Glu Glu Gin Ile Ile Leu Glu Asp Gin Asn Cys Lys Leu Ala Lys
980 985 990
Glu Lys Lys Leu Leu Glu Asp Arg Ile Ala Glu Phe Thr Thr Asn Leu
995 1000 1005
Thr Glu Glu Glu Glu Lys Ser Lys Ser Leu Ala Lys Leu Lys Asn Lys
1010 1015 1020
His Glu Ala Met Ile Thr Asp Leu Glu Glu Arg Leu Arg Arg Glu Glu
1025 1030 1035 1040
Lys Gin Arg Gin Glu Leu Glu Lys Thr Arg Arg Lys Leu Glu Gly Asp
1045 1050 1055
Ser Thr Asp Leu Ser Asp Gin Ile Ala Glu Leu Gin Ala Gin Ile Ala
1060 1065 1070
Glu Leu Lys Met Gin Leu Ala Lys Lys Glu Glu Glu Leu Gin Ala Ala
1075 1080 1085
Leu Ala Arg Val Glu Glu Glu Ala Ala Gin Lys Asn Met Ala Leu Lys
1090 1095 1100
Lys Ile Arg Glu Leu Glu Ser Gin Ile Ser Glu Leu Gin Glu Asp Leu
1105 1110 1115 1120
Glu Ser Glu Arg Ala Ser Arg Asn Lys Ala Glu Lys Gin Lys Arg Asp
1125 1130 1135
Leu Gly Glu Glu Leu Glu Ala Leu Lys Thr Glu Leu Glu Asp Thr Leu
1140 1145 1150
Asp Ser Thr Ala Ala Gin Gin Glu Leu Arg Ser Lys Arg Glu Gin Glu
1155 1160 1165
Val Asn Ile Leu Lys Lys Thr Leu Glu Glu Glu Ala Lys Thr His Glu
1170 1175 1180
Ala Gin Ile Gin Glu Met Arg Gin Lys His Ser Gin Ala Val Glu Glu
1185 1190 1195 1200
Leu Ala Glu Gin Leu Glu Gin Thr Lys Arg Val Lys Ala Asn Leu Glu
1205 1210 1215
Lys Ala Lys Gin Thr Leu Glu Asn Glu Arg Gly Glu Leu Ala Asn Glu
1220 1225 1230
Val Lys Val Leu Leu Gin Gly Lys Gly Asp Ser Glu His Lys Arg Lys
1235 1240 1245 =
Lys Val Glu Ala Gin Leu Gin Glu Leu Gin Val Lys Phe Asn Glu Gly
1250 1255 1260
Glu Arg Val Arg Thr Glu Leu Ala Asp Lys Val Thr Lys Leu Gin Val
1265 1270 1275 1280
Glu Leu Asp Asn Val Thr Gly Leu Leu Ser Gin Ser Asp Ser Lys Ser
1285 1290 1295
102
CA 02812132 2013-04-04
Ser Lys Leu Thr Lys Asp Phe Ser Ala Leu Glu Ser Gin Leu Gin Asp
1300 1305 1310
Thr Gin Glu Leu Leu Gin Glu Glu Asn Arg Gin Lys Leu Ser Leu Ser
1315 1320 1325
Thr Lys Leu Lys Gin Val Glu Asp Glu Lys Asn Ser Phe Arg Glu Gin
1330 1335 1340
Leu Glu Glu Glu Glu Glu Ala Lys His Asn Leu Glu Lys Gin Ile Ala
1345 1350 1355 1360
Thr Leu His Ala Gin Val Ala Asp Met Lys Lys Lys Met Glu Asp Ser
1365 1370 1375
Val Gly Cys Leu Glu Thr Ala Glu Glu Val Lys Arg Lys Leu Gin Lys
1380 1385 1390
Asp Leu Glu Gly Leu Ser Gin Arg His Glu Glu Lys Val Ala Ala Tyr
1395 1400 1405
Asp Lys Leu Glu Lys Thr Lys Thr Arg Leu Gin Gin-Glu Leu Asp Asp
1410 1415 1420
Leu Leu Val Asp Leu Asp His Gin Arg Gin Ser Ala Cys Asn Leu Glu
1425 1430 1435 1440
Lys Lys Gin Lys Lys Phe Asp Gin Leu Leu Ala Glu Glu Lys Thr Ile
1445 1450 1455
Ser Ala Lys Tyr Ala Glu Glu Arg Asp Arg Ala Glu Ala Glu Ala Arg
1460 1465 1470
Glu Lys Glu Thr Lys Ala Leu Ser Leu Ala Arg Ala Leu Glu Glu Ala
1475 1480 1485
Met Glu Gin Lys Ala Glu Leu Glu Arg Leu Asn Lys Gin Phe Arg Thr
1490 1495 1500
Glu Met Glu Asp Leu Met Ser Ser Lys Asp Asp Val Gly Lys Ser Val
1505 1510 1515 1520
His Glu Leu Glu Lys Ser Lys Arg Ala Leu Glu Gin Gin Val Glu Glu
1525 1530 1535
Met Lys Thr Gin Leu Glu Glu Leu Glu Asp Glu Leu Gin Ala Thr Glu
1540 1545 1550
Asp Ala Lys Leu Arg Leu Glu Val Asn Leu Gin Ala Met Lys Ala Gin
1555 1560 1565
Phe Glu Arg Asp Leu Gin Gly Arg Asp Glu Gln Ser Glu Glu Lys Lys
1570 1575 1580
Lys Gin Leu Val Arg Gin Val Arg Glu Met Glu Ala Glu Leu Glu Asp
1585 1590 1595 1600
Glu Arg Lys Gin Arg Ser Met Ala Val Ala Ala Arg Lys Lys Leu Glu
1605 1610 1615
Met Asp Leu Lys Asp Leu Glu Ala His Ile Asp Ser Ala Asn Lys Asn
1620 1625 1630
Arg Asp Glu Ala Ile Lys Gin Leu Arg Lys Leu Gin Ala Gin Met Lys
1635 1640 1645
Asp Cys Met Arg Glu Leu Asp Asp Thr Arg Ala Ser Arg Glu Glu Ile
1650 1655 1660
Leu Ala Gin Ala Lys Glu Asn Glu Lys Lys Leu Lys Ser Met Glu Ala
1665 1670 1675 1680
Glu Met Ile Gin Leu Gin Glu Glu Leu Ala Ala Ala Glu Arg Ala Lys
1685 1690 1695 =
Arg Gin Ala Gin Gin Glu Arg Asp Glu Leu Ala Asp Glu Ile Ala Asn
1700 1705 1710
Ser Ser Gly Lys Gly Ala Leu Ala Leu Glu Glu Lys Arg Arg Leu Glu
1715 1720 1725
Ala Arg Ile Ala Gin Leu Glu Glu Glu Leu Glu Glu Glu Gin Gly Asn
1730 1735 1740
103
CA 02812132 2013-04-04
Thr Glu Leu Ile Asn Asp Arg Leu Lys Lys Ala Asn Leu Gin Ile Asp
1745 1750 1755 1760
Gin Ile Asn Thr Asp Leu Asn Leu Glu Arg Ser His Ala Gin Lys Asn
1765 1770 1775
Glu Asn Ala Arg Gin Gin Leu Glu Arg Gin Asn Lys Glu Leu Lys Val
1780 1785 1790
Lys Leu Gin Glu Met Glu Gly Thr Val Lys Ser Lys Tyr Lys Ala Ser
1795 1800 1805
Ile Thr Ala Leu Glu Ala Lys Ile Ala Gin Leu Glu Glu Gin Leu Asp
1810 1815 1820
Asn Glu Thr Lys Glu Arg Gin Ala Ala Cys Lys Gin Val Arg Arg Thr
1825 1830 1835 1840
Glu Lys Lys Leu Lys Asp Val Leu Leu Gin Val Asp Asp Glu Arg Atg
1845 1850 1855
Asn Ala Glu Gin Tyr Lys Asp Gin Ala Asp Lys Ala Ser Thr Arg Leu
1860 1865 1870
Lys Gin Leu Lys Arg Gin Leu Glu Glu Ala Glu Glu Glu Ala Gin Arg
1875 1880 1885
Ala Asn Ala Ser Arg Arg Lys Leu Gin Arg Glu Leu Glu Asp Ala Thr
1890 1895 1900
Glu Thr Ala Asp Ala Met Asn Arg Glu Val Ser Ser Leu Lys Asn Lys
1905 1910 1915 1920
Leu Arg Arg Gly Asp Leu Pro Phe Val Val Pro Arg Arg Met Ala Arg
1925 1930 1935 '
Lys Gly Ala Gly Asp Gly Ser Asp Glu Glu Val Asp Gly Lys Ala Asp
1940 1945 1950
Gly Ala Glu Ala Lys Pro Ala Glu
1955 1960
<210> 51
<211> 7666
<212> DNA
<213> Mus musculus
<400> 51
gtctttcctg ggagatgggc gcgcaaaccg accagtgggt ctgggggcgg cagtgatggg 60
cgtggagatg gcccaatgag ggtgggagtg ggtggggcag gcgcgagcag cagtgctaaa 120
ggagcccggc ggaggcagcg gtgggtttgg aattgagacg ctggatctgt ggtcgctgct 180
ggggacgtgt gccggcgcca ccatcttcgg ctgaagaggc aattactttt gggtccttct 240
gtttacaatg gcccagagaa ctggactgga ggatcccgag aggtatctct ttgtggacag 300
ggctgtcatc tacaaccctg ccactcaagc tgactggaca gctaaaaagc tggtgtggat 360
tccatcggaa cgccatggtt ttgaggcagc tagtattaaa gaagagcggg gcgatgaggt 420
tatggtggag ctggcagaga atgggaagaa agcaatggtc aacaaagatg acattcagaa 480
gatgaaccca ccaaagttct ccaaggtgga ggatatggca gagctgacat gcttgaacga 540
agcctctgtc ttacataatt tgaaggaccg ctactattca ggacttatct atacttactc 600
tggactcttc tgtgtggtga taaatcctta caagaacctt ccaatttact ctgagaatat 660
tattgaaatg tatagaggga agaaacgcca tgagatgcca ccacacatct acgccatatc 720
agagtctgct tacagatgca tgcttcaaga tcgtgaggac cagtcaattc tatgcacggg 780
tgaatcgggt gccgggaaga cagaaaatac caagaaagtc attcagtacc ttgcccacgt 840
tgcttcttct cacaaaggaa gaaaggacca taatattcct ggggaacttg aacggcagct 900
tttacaagca aatccaattc tggaatcctt tggaaatgcg aagactgtga aaaatgataa 960
ctcatctcgc tttggcaagt ttatccggat caactttgat gtaactggct atattgttgg 1020
ggccaacatt gaaacatacc ttctggaaaa gtctcgtgct gttcgtcaag ctaaagatga 1080
gcgtacattt catatctttt atcagttgct ctctggagca ggggaacacc tgaaatccga 1140
cttactcctg gaaggtttca acaactacag attcctctcc aatggctata ttcctattcc 1200
tggacagcaa gacaaggata acttccagga gaccatggaa gccatgcaca tcatgggctt 1260
104
gOT
oggp obbub4obee b4DbbboDub bbobebeebu 3b4p4o5D4o b43404e066 beeeebeebe
ozgp obb4qbqo6e opeboqqbee beebeoPeeb eububbqqoe epoqoqboqe beopbobeop
oggp eopebbqooe bbqbboeb4o oebqebbqoe ebeepeepbq obbopeebee opebeebebb
00c17 40beepubqe 4bo6bqopqb beebebbubb qobbobepob eb4obobbub bqb4ebbeep
oppp q3b4Deeebe epePoobeeb bebbmbeb ebaq.PPoebb 6bqopeb4eb oebbqbeeeb
08EP eebeeppeou bgobbgobeo opq.beobqoq. o554.4b4bbe obeebebbqo oeebeebbeo
ozEp obbebbebbe bbubbebbeo beobebbeoq. qopbepeebe ebebbebbeb bqobeobboo
09ZP 4ebboq.beob ebqopeubqo eeebeobboe oebebeebbe oqqopqabub beoppeoebb
00ZP upeqopeo4o qbeboqoqbb gobuobTebb uebobqqqbe eqqeqbbeee 5eebeb4obe
017TP ebeebb4ob4 poopeo4b4b qeeq.ebbqob ebquebeoug obeepeueob eeebeboobb
080t7 qobebpqbbb epqobbeoeb qbbbebeoqb bb cob poi.obebbeo
oqbbeopobi.
OZOD' ebb4obee6e ebbebeeoep bebeo4beb5 obbeebqbbe obeobqob4b beebqbbe64
096E b4bobbqobe bbeeoeepeb eoebebbqop bbbe3peepe ebeebebbqo peepobeeeo
066 4.465eeee6p beeDbebbqo be3bebooqq qDbpbbebbq Db3bPD2006 4eobbebe3e
opgE bebqeoebbe 33-4pbeoqob eeboob eeqoppebqe bbebqqp4ob bepbeeb4ob
08L 6p36bqbue
bbe3beb4bo eepeopoboD qoepbbeobe o4obeobeoe oop3ebeq3D
0LE opoebbpbbq Dbebeoebee bqoq.obeebb 4obebbebqb ebqqoebbbo pepeeoeeeb
oggE efq_obbee3e ebEyeeoqqob beepebqp-46 pfy4qqop6ee 66 c66
poboqeeepo
009E obbeD5gobe bbboeobg4b pee-440E064 eegeebeepe obqoeoebeb 4ebTebe6be
opgE beoobbqobc bbbbbeoggo bebbebbebb Pebeepoubq qbeopqbeue ogobubqebo
ogpE qbbe3e3bbe o6qobeb4ob oqebeooebb eobqoqeboo eepeeebbbb qe664o6eeb
opE bDeeepobbe eeebb4oueb beoeboqope eubebbebbe ebee644obo eebeebeqqo
ogEE e6bo4o4ebq ebqbeebeeo beeqeebbuo qeeee3obbq 4peeeepeob beeeebeebb
00EE ebeeb4obb4 obep4ogoog 4b4bebgobq quebopebee bbgeogoepe beeppbeeeo
opE ge4ggepepo 4geebeopeb pebob 4344bbebbe beebbgebee beeogeppeg
08TE obbebeobeo pbqbbeebeb b4obeob4ob Peepobboop bbbbbebbeb bebqebb4De
OZTE poeebeebeg opebbeogge peobobbeob qebeebeebe ebebgeebeo egoogebepo
ogoc eebbobebee bbebbebbeb bqbbbeopqb ebo4poubqe opqoqi.ebpb bebb4Deebb
000E pobeeepeoo b4ob43obbe pobebeb4e6 ebeebeobee bqoboqqoqo bebooebebo
Of76Z obeeobqoeu oePbeobbqo oqeqeebeeb ebeebbgobq obeobeopeo beebbobebb
088Z 4ebub6ebgq. obubbbbeeb bqbeeeepub uobeebebee ubqbbeebqo b4o6ebbebq
0Z8Z Ebeepeobbe 334peebeeb bubbepoboo oeb4beeoog poqoqoobee b456eebouo
09/2 qqoqbqbobb qbbqbeDbb.q. peoebobqob Peb400eq5p bbobqb4oPp bbDbe36qqo
OOLZ qbbeeeqqop bqbee4peep eepbeoeueb eeoobqqqop bbeeeb000b oqopeqobbe
opgz beob4e4b4o beeooqqqq.4.34eoquo4e4 eb4peoqeee eernebebe eebeebeebe
080Z 440e0505b4 44q.bebbi.ob p.5=444442 qebeepbebe oebbqqeebe qe4bqopeee
ozg poDebb4oee be44gobebo oqebqeebob eb4bqbo6be peeeobb4e5 5qeD44pbbe
ogp eeqoolqeqo bqeeDooqoe eqopqebebq eqebebeoeb eoqqeebbeo oqqqqbeqeb
oop booeuppopq qbbbbeoobo qbqoqebboo qebbbeebbq paq.bobboee 4b4oboqq.ob
opE epq.eboqobq boqopeoboo aebb4pueub bb4obbbobe ebeboeo4ue eopqq.eo4Po
083 64060o-450-4 4opeooppeu oppoeupboo qoqoeeobbq e64obeepoe oqoqoqbebb
ozz eeDe4p4obe o6b6o4600p eb3oqq.bqeo bbbeebeeoo eeeepegeob go4o6644gb
ogTz obDoebebqo ebTebbbwe oqbppoTebb qoqbbuqbqq Pebooebb4b oeb6eebb44
OOTZ 4Dbeb4obb4 b444ebeoub bp400gbeop pobgoog000 upobbgboue oebqeebqDb
Of7OZ 000ebbquDe e6ee64ebqo bbqbeb4e6e o66ee4e4oe bbqbbeebbb boboe4opoo
0861 4e0qeo6qpq qoebDobeue oebeee643P upoboboobp Ebeoqqqbee op4Deopoq4
OZ61 bbeeobubbe oqqbbqobee ePbqq.b4.44e opuee4ebeo eqobeeepoo oqqbbqobqe
0981 ebeebgebbq o3400066qo bqbobbqopo oo4eeDobq3 Debubebege bqopeboquo
0081 bqoopbeabi. 33e5.Eq.Dobb oqqopboqpi. qqopebbqbe bogeobbbeb ebobepougb
opLT ebbebbeabe bbqop4Poqq bT230E3PDD ueoqqbqobe DbeDbi.obee bebopeooeo
0891 eqopeoqeob 1.bgobuobeb 3q4poqope6 qobebqq444 eeebq.44-45b qab4bb
Toomeb5.54i, poqqooqqob ebbbeoo6oP eeopebbeqe bbqobobeee Teepqeoboo
ogsT ep4T6DqDbb goboqq4b44 abobefqeqo peqobeeeep bb44eobeeb bmbeobmq.
0001 ebgabbpobe beuppoubeo pobee2buop qbopq4ebb5 oDbbqqbbee o4eb6eDo3b
OPP' progoogeqo bbboqoeoql 525b4Pbqbq Pebqebbb34 Dbqaoe3ob4 34obeebPDP
08E1 05045P0202 2b26boo5qp poqopbepoo ebqDeoPeeb ebeffeeeee oqqqololeo
ozET epe.56-4qqbe o6lofq.62o; goTe4boqfre, epT4o5qpeo qoqooqebeb eeboeol.pqo
VO-VO-ETOZ ZETZT8Z0 VD
901
sninosnm snyq <EI>
Did <ZIZ>
9L6I <IIZ>
Zg <OTZ>
999L PUPPET'
PPEEPPPePP ePbPqreqqP0 qqeq4qeueg eepebeeqbq
0Z9L 44q4eb4eup Ebeqoqq4e4 uppoqquqbq 40444.4o4e4 eeeobqoubq OOPOPEIPOOP
09sL opubqqbqob bqouqbqque 54e4bqopoo qqqeb-4-44o4 eebbqq4bbb qeoupqbbqo
00gL eebeebbebq bqp4ppee44 4o6eeee44b E644q4DDeb boubeeeebb qob4bqebob
0f7f,/, b4booeb000 obeeoebqbe qqbqbeoqee ebbeoebqbe oqqeoeeebb obqebeqbee
08L b4beqoqoqo pe4b4eo4u4 ebqqabqbqo eb4oqqbeeq qeqb4uu4qo eupeequqpq
03EL obbqqqeqqe qeqeeeqqbq PPOP5PPq3P qbeoeqbeou beq4Dobqbe eebeoeqqeo
09z7_, ebqoeeebbe bqqbeqeb4q bbqqqq46qo lmeobe44ou beebqeeopq qbqbebebb4
00L 44opq4poo4 qqopoqq4qe 3q4obbe6q6 4bobbbobeo obqeoebqqb 4eub400b43
0pTL Tepobqebee beepqopbeq eqabbebTee bqbqq4beeD oqqbqbqbbe bbbqbqeqq4
080L 4beq4eqpeb bbopupeopo bpbqqqobqo pobbeoqqee pobT4o4pob beebqbobe
00L eb3434pobb eebeoboqob 4bbb544poo ubqbqoqopb eb4bebbeaq. 4eeb4boobq
0969 ee4eb44obb q3q,bqbebqo o4oqbbq6be poqqopeqbe peoqepobeo bbqbb5oboo
0069 bqbbeeqebe beebepobbq bebebbebeb ebeDbmbqpq obebeoqoqb eqeeb4bqee
0689 D4oqbooeoo poqbqbb4eo qqeeepqoop obgpopebqb bbbeepooqo leopobqopq
08L9 oqq666b4T4 eboqeobeDb eebbbbqqeb bqqqbbbqbb bbeqqobbob bq443bbbbe
0zL9 bebbbqopee obubqbeoqo qbqpeoqebq beqeebqbqb oqqqebqp4o 4b4bqq.beob
0999 400eP4OOPP bqbqqeoeeb eqqoqp4bbq poepe3DueD qqopT4Dope bqopobbqbq
0099 uo44bqbqp4 qbeqqopeqb beDeb4beqo ombqopobqu ob4b4obqob epqobqbooe
0D,g9 bbboopobqg bbbeeqoobb bqobbqbeop bqqbqqqopo bbeboqopeu peeubqqqq4
08D,9 opeubeuebe ebeeT4epeo ebqe444420 oebebeeoqu pqmeqbqeop 44gge4oTeo
0D,9 b4q.boqeoqo qbqqebepee eqqeqbobqe Debeeaeopq opepeqeqeq e4.4.444opo3
09E9 b4queoequb eqoebeoqqo bqopqeqeqo ebeobobqoo bbobqqqobb euqqoqbqbq
00E9 opeoqbeeeb beopq4opq4 ebepooqobe eobbquebbb pqabqpeoqb eobqopobeb
0179 44obqoqbeo peobeopp4e p4ouebepob beoebbebqo bqebqbbebe oqbbebbeoe
0819 obbeqeebeo Tepoopoepo beoupeoFbq euoqb4e6qb epoebee4be uebepeoebo
0z19 ebqebeoqbq obebeqpbo4 eobbbbbebq quouobqobe opb3bbDobb qoquboobee
0909 344044qqob eDqueopqbb obbbbobbeo qobbopeebe poqoqbeobe oqbbeboboo
0009 bebqoabbee bqueopbbub 4beboboe5o ebbqobubbb eeeopqopee 4bobboqoqe
0mq oboeeopbqb oupeopbbub eubbebqobb ebeebb4qbe oe6oeeeq4o beobeebqeb
088g be3obqeeqD bbeebebbqe beobubbeel eqbeoqebbo bquobbD46o beboebee64
0z8s q66eD54uoq qoTeeebeee bqoeeebeeb ebupeeboqb po4beqopee ouebobqobe
09Lg obubDbubbe epobeebbeo bebqqobeob ebbebbqobe obbbqqubee pobee6b4op
OOLS obeogogepo eqobbeepqq beeopqbeep qbeobbbbbe bbqobebbeo bqpbeepobb
0f79s eebqobubbe epeeeepebo bebbqobepb eobboobbqe eDebqbebee beboobqobb
oess bebbobebeo beobbqobeb uoupeebqoe oebebbqbbe obgoboe3De beeobooqq3
cqgg boDubqueoq obqobubbge pueobebeob ebbebbebbq obebbebeeb bqobeoeobo
09t7g 4e65obo6ee bb4pobo6bo beebeb4ebb qqbqobob4o qbeeubbqo4 pobobebeep
00f7s oboqebebqe b4obbqobeb oebebobebb ebbebeobDe DebDoboobb ebobebooqe
0IvEg oqopbbqobe bbubee3bqo beoqqoqqee ebebbeebe4 oqbebeebqo eeebeeeebq
08zg beeebeeeoo qeep4obqqq 44ebebqube be4o4eobeb o4obbebeeb eqpeebgbob
0zzs eoDe4qebbe ubgebupeob beoqqopepo bbq4obeobe eoqebqbeeb gebbboDobe
09-Eq evoeebob4o bbebo4ebeo qobbebbqoo ebbeebqope beqebebb4e beebeebeee
00's oqbDbbqb4b bbqouobbbo beoeeebbob ebqebbebbq obebeobbeb b43bebbb35
060S q56eobee-44 obqbbqobbo beebeeeebe ebqeebebbe b4ebubDope Eppb43Debb
08617 bpbutqqq6-2 opobbeeb4e oDbbebbqeo ee34beubb4 Do5oogo6e2 oobqebbebq
0z617 peopbbuDbq obeboebbeb bqDbeb52bb 436e3oopbb 35-42.6266e5 b4bbeobeo5
098f7 e6b4433bpb obeeboqeee bebq4o62bo epoqbouebe ebbbbqbgeb pebp2eq3qo
008p bpbqebqo32 beebb4eoeb EDE,Pboqqob eobueoepbu o66p5pEoqq 225526beeb
cmj, o55e564qop bbebbebqqo pobbbobobb qopoqoqobo bepeopeupb eep6e6p6po
VO-VO-ETOZ ZETZT8Z0 VO
CA 02812132 2013-04-04
<400> 52
Met Ala Gin Arg Thr Gly Leu Glu Asp Pro Glu Arg Tyr Leu Phe Val
1 5 10 15
Asp Arg Ala Val Ile Tyr Asn Pro Ala Thr Gin Ala Asp Trp Thr Ala
20 25 30
Lys Lys Leu Val Trp Ile Pro Ser Glu Arg His Gly Phe Glu Ala Ala
35 40 45
Ser Ile Lys Glu Glu Arg Gly Asp Glu Val Met Val Glu Leu Ala Glu
50 55 60
Asn Gly Lys Lys Ala Met Val Asn Lys Asp Asp Ile Gin Lys Met Asn
65 70 75 80
Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu
85 90 95
Asn Glu Ala Ser Val Leu His Asn Leu Lys Asp Arg Tyr Tyr Ser Gly
100 105 110
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr
115 120 125
Lys Asn Leu Pro Ile Tyr Ser Glu Asn Ile Ile Glu Met Tyr Arg Gly
130 135 140
Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ser Glu Ser
145 150 155 160
Ala Tyr Arg Cys Met Leu Gin Asp Arg Glu Asp Gin Ser Ile Leu Cys
165 170 175
Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile
180 185 190
Gin Tyr Leu Ala His Val Ala Ser Ser His Lys Gly Arg Lys Asp His
195 200 205
Asn Ile Pro Gly Glu Leu Glu Arg Gin Leu Leu Gin Ala Asn Pro Ile
210 215 220
Leu Glu Ser Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser
225 230 235 240
Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile
245 250 255
Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Val
260 265 270
Arg Gin Ala Lys Asp Glu Arg Thr Phe His Ile Phe Tyr Gin Leu Leu
275 280 285
Ser Gly Ala Gly Glu His Leu Lys Ser Asp Leu Leu Leu Glu Gly Phe
290 295 300
Asn Asn Tyr Arg Phe Leu Ser Asn Gly Tyr Ile Pro Ile Pro Gly Gin
305 310 315 320
Gin Asp Lys Asp Asn Phe Gin Glu Thr Met Glu Ala Met His Ile Met
325 330 335
Gly Phe Ser His Glu Glu Ile Leu Ser Met Leu Lys Val Val Ser Ser
340 345 350
Val Leu Gin Phe Gly Asn Ile Ser Phe Lys Lys Glu Arg Asn Thr Asp
355 360 365
Gin Ala Ser Met Pro Glu Asn Thr Val Ala Gin Lys Leu Cys His Leu
370 375 380
Leu Gly Met Asn Val Met Glu Phe Thr Arg Ala Ile Leu Thr Pro Arg
385 390 395 400
Ile Lys Val Gly Arg Asp Tyr Val Gin Lys Ala Gin Thr Lys Glu Gin
405 410 415
Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu
420 425 430
Phe Arg Trp Leu Val His Arg Ile Asn Lys Ala Leu Asp Arg Thr Lys
435 440 445
107
CA 02812132 2013-04-04
Arg Gin Gly Ala Ser Phe Ile Gly Ile Leu Asp Ile Ala Gly Phe Glu
450 455 460
Ile Phe Glu Leu Asn Ser Phe Glu Gin Leu Cys Ile Asn Tyr Thr Asn
465 470 475 480
Glu Lys Leu Gin Gin Leu Phe Asn His Thr Met Phe Ile Leu Glu Gin
485 490 495
Glu Glu Tyr Gin Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly
500 505 510
Leu Asp Leu Gin Pro Cys Ile Asp Leu Ile Glu Arg Pro Ala Asn Pro
515 520 525
Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala
530 535 540
Thr Asp Lys Thr Phe Val Glu Lys Leu Val Gin Glu Gin Gly Ser His
545 550 555 560
Ser Lys Phe Gin Lys Pro Arg Gin Leu Lys Asp Lys Ala Asp Phe Cys
565 570 575
Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Lys Ala Asp Glu Trp Leu
580 585 590
Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val Ala Thr Leu Leu His
595 600 605
Gin Ser Ser Asp Arg Phe Val Ala Glu Leu Trp Lys Asp Val Asp Arg
610 615 620
Ile Val Gly Leu Asp Gin Val Thr Gly Met Thr Glu Thr Ala Phe Gly
625 630 635 640
Ser Ala Tyr Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gin Leu
645 ' 650 655
Tyr Lys Glu Ser Leu Thr Lys Leu Met Ala Thr Leu Arg Asn Thr Asn
660 665 670
Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ala Gly
675 680 685
Lys Leu Asp Pro His Leu Val Leu Asp Gin Leu Arg Cys Asn Gly Val
690 695 700
Leu Glu Gly Ile Arg Ile Cys Arg Gin Gly Phe Pro Asn Arg Ile Val
705 710 715 720
Phe Gin Glu Phe Arg Gin Arg Tyr Glu Ile Leu Thr Pro Asn Ala Ile
725 730 735
Pro Lys Gly Phe Met Asp Gly Lys Gin Ala Cys Glu Arg Met Ile Arg
740 745 750
Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gin Ser Lys Ile
755 760 765
Phe Phe Arg Ala Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu
770 775 780
Lys Ile Thr Asp Ile Ile Ile Phe Phe Gin Ala Val Cys Arg Gly Tyr
785 790 795 800
Leu Ala Arg Lys Ala Phe Ala Lys Lys Gin Gin Gin Leu Ser Ala Leu
805 810 815
Lys Val Leu Gin Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg His Trp
820 825 830
Gin Trp Trp Arg Val Phe Thr Lys Val Lys Pro Leu Leu Gin Val Thr
835 840 845
Arg Gin Glu Glu Glu Leu Gin Ala Lys Asp Glu Glu Leu Leu Lys Val
850 855 860
Lys Glu Lys Gin Thr Lys Val Glu Gly Glu Leu Glu Glu Met Glu Arg
865 870 875 880
Lys His Gin Gin Leu Leu Glu Glu Lys Asn Ile Leu Ala Glu Gin Leu
885 890 895 =
108
CA 02812132 2013-04-04
Gin Ala Glu Thr Glu Leu Phe Ala Glu Ala Glu Glu Met Arg Ala Arg
900 905 910
Leu Ala Ala Lys Lys Gin Glu Leu Glu Glu Ile Leu His Asp Leu Glu
915 920 925
Ser Arg Val Glu Glu Glu Glu Glu Arg Asn Gin Ile Leu Gin Asn Glu
930 935 940
Lys Lys Lys Met Gin Ala His Ile Gin Asp Leu Glu Glu Gin Leu Asp
945 950 955 960
Glu Glu Glu Gly Ala Arg Gin Lys Leu Gin Leu Glu Lys Val Thr Ala
965 970 975
Glu Ala Lys Ile Lys Lys Met Glu Glu Glu Val Leu Leu Leu Glu Asp
980 985 990
Gin Asn Ser Lys Phe Ile Lys Glu Lys Lys Leu Met Glu Asp Arg Ile
995 1000 1005
Ala Glu Cys Ser Ser Gin Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn
1010 1015 1020
Leu Ala Lys Ile Arg Asn Lys Gin Glu Val Met Ile Ser Asp Leu Glu
1025 1030 1035 1040
Glu Arg Leu Lys Lys Glu Glu Lys Thr Arg Gin Glu Leu Glu Lys Ala
1045 1050 - 1055
Lys Arg Lys Leu Asp Gly Glu Thr Thr Asp Leu Gin Asp Gin lie Ala
1060 1065 1070
Glu Leu Gin Ala Gin Val Asp Glu Leu Lys Val Gin Leu Thr Lys Lys
1075 1080 1085 ,
Glu Glu Glu Leu Gln Gly Ala Leu Ala Arg Gly Asp Asp Glu Thr Leu
1090 1095 1100
His Lys Asn Asn Ala Leu Lys Val Ala Arg Glu Leu Gin Ala Gin Ile
1105 1110 1115 1120
Ala Glu Leu Gin Glu Asp Phe Glu Ser Glu Lys Ala Ser Arg Asn Lys
1125 1130 1135 .
Ala Glu Lys Gin Lys Arg Asp Leu Ser Glu Glu Leu Glu Ala Leu Lys
1140 1145 1150
Thr Glu Leu Glu Asp Thr Leu Asp Thr Thr Ala Ala Gin Gin Glu Leu
1155 1160 1165
Arg Thr Lys Arg Glu Gin Glu Val Ala Glu Leu Lys Lys Ala Leu Glu
1170 1175 1180
Asp Glu Thr Lys Asn His Glu Ala Gin lie Gin Asp Met Arg Gin Arg
1185 1190 1195 1200 '
His Ala Thr Ala Leu Glu Glu Leu Ser Glu Gin Leu Glu Gin Ala Lys
1205 1210 1215
Arg Phe Lys Ala Asn Leu Glu Lys Asn Lys Gin Gly Leu Glu Thr Asp
1220 1225 1230
Asn Lys Glu Leu Ala Cys Glu Val Lys Val Leu Gin Gin Val Lys Ala
1235 1240 1245
Glu Ser Glu His Lys Arg Lys Lys Leu Asp Ala Gin Val Gin Glu Leu
1250 1255 1260
His Ala Lys Val Ser Glu Gly Asp Arg Leu Arg Val Glu Leu Ala Glu
. 1265 1270 1275 1280
Lys Ala Asn Lys Leu Gin Asn Glu Leu Asp Asn Val Ser Thr Leu Leu
1285 1290 1295
Glu Glu Ala Glu Lys Lys Gly Ile Lys Phe Ala Lys Asp Ala Ala Gly
130Q 1305 1310
Leu Glu Ser Gin Leu Gin Asp Thr Gin Glu Leu Leu Gin Glu Glu Thr
1315 1320 1325
Arg Gin Lys Leu Asn Leu Ser Ser Arg Ile Arg Gin Leu Glu Glu Glu
1330 1335 1340
109
CA 02812132 2013-04-04
Lys Asn Ser Leu Gln Glu Gln Gln Glu Glu Glu Glu Glu Ala Arg Lys
1345 1350 1355 1360
Asn Leu Glu Lys Gln Val Leu Ala Leu Gin Ser Gln Leu Ala Asp Thr
1365 1370 1375
Lys Lys Lys Val Asp Asp Asp Leu Gly Thr Ile Glu Ser Leu Glu Glu
1380 1385 1390
Ala Lys Lys Lys Leu Leu Lys Asp Val Glu Ala Leu Ser Gln Arg Leu
1395 1400 1405
Glu Glu Lys Val Leu Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg
1410 1415 1420
Leu Gln Gln Glu Leu Asp Asp Leu Thr Val Asp Leu Asp His Gln Arg
1425 1430 1435 1440
Gln Ile Val Ser Asn Leu Glu Lys Lys Gln Lys Lys Phe Asp Gin Leu
1445 1450 1455
Leu Ala Glu Glu Lys Gly Ile Ser Ala Arg Tyr Ala Glu Glu Arg Asp
1460 1465 1470
Arg Ala Glu Ala Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu
1475 1480 1485
Ala Arg Ala Leu Glu Glu Ala Leu Glu Ala Lys Glu Glu Phe Glu Arg
1490 1495 1500
Gln Asn Lys Gln Leu Arg Ala Asp Met Glu Asp Leu Met Ser Ser Lys
1505 1510 1515 1520
Asp Asp Val Gly Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala
1525 1530 1535
Leu Glu Gln Gln Val Glu Glu Met Arg Thr Gln Leu Glu Glu Leu Glu
1540 1545 1550
Asp Glu Leu Gln Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn
1555 1560 1565
Met Gln Ala Met Lys Ala Gln Phe Glu Arg Asp Leu Gln Thr Arg Asp
1570 1575 1580
Glu Gln Asn Glu Glu Lys Lys Arg Leu Leu Leu Lys Gln Val Arg Glu
1585 1590 1595 1600
Leu Glu Ala Glu Leu Glu Asp Glu Arg Lys Gln Arg Ala Leu Ala Val
1605 1610 1615
Ala Ser Lys Lys Lys Met Glu Ile Asp Leu Lys Asp Leu Glu Ala Gln
1620 1625 1630
Ile Glu Ala Ala Asn Lys Ala Arg Asp Glu Val Ile Lys Gln Leu Arg
1635 1640 1645
Lys Leu Gln Ala Gln Met Lys Asp Tyr Gln Arg Glu Leu Glu Glu Ala
1650 1655 1660
Arg Ala Ser Arg Asp Glu Ile Phe Ala Gln Ser Lys Glu Ser Glu Lys
1665 1670 1675 1680
Lys Leu Lys Ser Leu Glu Ala Glu Ile Leu Gln Leu Gln Glu Glu Leu
1685 1690 1695
Ala Ser Ser Glu Arg Ala Arg Arg His Ala Glu Gln Glu Arg Asp Glu
1700 1705 1710
Leu Ala Asp Glu Ile Ala Asn Ser Ala Ser Gly Lys Ser Ala Leu Leu
1715 1720 1725
Asp Glu Lys Arg Arg Leu Glu Ala Arg Ile Ala Gln Leu Glu Glu Glu
1730 1735 1740
Leu Glu Glu Glu Gln Ser Asn Met Glu Leu Leu Asn Asp Arg Phe Arg
1745 1750 1755 1760
Lys Thr Thr Leu Gln Val Asp Thr Leu Asn Thr Glu Leu Ala Ala Glu
1765 1770 1775
Arg Ser Ala Ala Gln Lys Ser Asp Asn Ala Arg Gln Gln Leu Glu Arg
1780 1785 1790
110
CA 02812132 2013-04-04
=
Gin Asn Lys Glu Leu Lys Ala Lys Leu Gin Glu Leu Glu Gly Ala Val
1795 1800 1805
Lys Ser Lys Phe Lys Ala Thr Ile Ser Ala Leu Glu Ala Lys Ile Gly
1810 1815 1820
Gin Leu Glu Glu Gin Leu Glu Gin Glu Ala Lys Glu Arg Ala Ala Ala
1825 1830 1835 1840
Asn Lys Leu Val Arg Arg Thr Glu Lys Lys Leu Lys Glu Ile Phe Met
1845 1850 1855
Gin Val Glu Asp Glu Arg Arg His Ala Asp Gin Tyr Lys Glu Gin Met
1860 1865 1870
Glu Lys Ala Asn Ala Arg Met Lys Gin Leu Lys Arg Gin Leu Glu Glu
1875 1880 1885
Ala Glu Glu Glu Ala Thr Arg Ala Asn Ala Ser Arg Arg Lys Leu Gin
1890 1895 1900
Arg Glu Leu Asp Asp Ala Thr Glu Ala Asn Glu Gly Leu Ser Arg Glu
1905 1910 1915 1920
Val Ser Thr Leu Lys Asn Arg Leu Arg Arg Gly Gly Pro Ile Ser Phe
1925 1930 , 1935
Ser Ser Ser Arg Ser Gly Arg Arg Gin Leu His Ile Glu Gly Ala Ser
1940 1945 1950
Leu Glu Leu Ser Asp Asp Asp Thr Glu Ser Lys Thr Ser Asp Val Asn
1955 1960 1965
Asp Thr Gin Pro Pro Gin Ser Glu
1970 1975
<210> 53
<211> 7619
<212> DNA
<213> Homo sapiens
<400> 53
actgaggcgc tggatctgtg gtcgcggctg gggacgtgcg cccgcgccac catcttcggc 60
tgaagaggca attgcttttg gatcgttcca tttacaatgg cgcagagaac tggactcgag 120
gatccagaga ggtatctctt tgtggacagg gctgtcatct acaaccctgc cactcaagct 180
gattggacag ctaaaaagct agtgtggatt ccatcagaac gccatggttt tgaggcagct 240
agtatcaaag aagaacgggg agatgaagtt atggtggagt tggcagagaa tggaaagaaa 300
gcaatggtca acaaagatga tattcagaag atgaacccac ctaagttttc caaggtggag 360
gatatggcag aattgacatg cttgaatgaa gcttccgttt tacataatct gaaggatcgc 420
tactattcag gactaatcta tacttattct ggactcttct gtgtagttat aaacccttac 480
aagaatcttc caatttactc tgagaatatt attgaaatgt acagagggaa gaagcgtcat 540
gagatgcctc cacacatcta tgctatatct gaatctgctt acagatgcat gcttcaagat 600
cgtgaggacc agtcaattct ttgcacgggt gagtcaggtg ctgggaagac agaaaataca 660
aagaaagtta ttcagtacct tgcccatgtt gcttcttcac ataaaggaag aaaggaccat 720
aatattcctg gggaacttga acggcagctt ttgcaagcaa atccaattct ggaatcattt 780
ggaaatgcga agactgtgaa aaatgataac tcatctcgtt ttggcaaatt tattcggatc 840
aactttgatg taactggcta tatcgttggg gccaacattg aaacatacct tctggaaaag 900
tctcgtgctg ttcgtcaagc aaaagatgaa cgtacttttc atatctttta ccagttgtta 960
tctggagcag gagaacacct aaagtctgat ttgcttcttg aaggatttaa taactacagg 1020
tttctctcca atggctatat tcctattccg ggacagcaag acaaagataa tttccaggag 1080
accatggaag caatgcacat aatgggcttc tcccatgaag agattctgtc aatgcttaaa 1140
gtagtatctt cagtgctaca gtttggaaat atttctttca aaaaggagag aaatactgat 1200
caagcttcca tgccagaaaa tacagttgcg cagaagctct gccatcttct tgggatgaat 1260
gtgatggagt ttactcgggc catcctgact ccccggatca aggtcggccg agactatgtg 1320
caaaaagccc agaccaaaga acaggcagat tttgcagtag aagcattggc aaaagctacc 1380
tatgagcggc tctttcgctg gctcgttcat cgcatcaata aagctctgga taggaccaaa 1440
cgtcagggag catctttcat tggaatcctg gatattgctg gatttgaaat ttttgagctg 1500
1 1 1
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PS <0017>
suoTdes OWOH <ETZ>
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<OTZ>
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A
VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
Lys Lys Leu Val Trp Ile Pro Ser Glu Arg His Gly Phe Glu Ala Ala
35 40 45
Ser Ile Lys Glu Glu Arg Gly Asp Glu Val Met Val Glu Leu Ala Glu
50 55 60
Asn Gly Lys Lys Ala Met Val Asn Lys Asp Asp Ile Gin Lys Met Asn
65 70 75 80
Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu
85 90 95
Asn Glu Ala Ser Val Leu His Asn Leu Lys Asp Arg Tyr Tyr Ser Gly
100 105 110
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr
115 120 125
Lys Asn Leu Pro Ile Tyr Ser Glu Asn Ile Ile Glu Met Tyr Arg Gly
130 135 140
Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ser Glu Ser
145 150 155 160
Ala Tyr Arg Cys Met Leu Gin Asp Arg Glu Asp Gin Ser Ile Leu Cys
165 170 175
Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile
180 185 190
Gin Tyr Leu Ala His Val Ala Ser Ser His Lys Gly Arg Lys Asp His
195 200 205
Asn Ile Pro Gly Glu Leu Glu Arg Gin Leu Leu Gin Ala Asn Pro Ile
210 215 220
Leu Glu Ser Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser
225 230 235 . 240
Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile
245 250 255
Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Val
260 265 270
Arg Gin Ala Lys Asp Glu Arg Thr Phe His Ile Phe Tyr Gin Leu Leu
275 280 285
Ser Gly Ala Gly Glu His Leu Lys Ser Asp Leu Leu Leu Glu Gly Phe
290 295 300
Asn Asn Tyr Arg Phe Leu Ser Asn Gly Tyr Ile Pro Ile Pro Gly Gin
305 310 315 320
Gin Asp Lys Asp Asn Phe Gin Glu Thr Met Glu Ala Met His Ile Met
325 330 . 335
Gly Phe Ser His Glu Glu Ile Leu Ser Met Leu Lys Val Val Ser Ser
340 345 350
Val Leu Gln Phe Gly Asn Ile Ser Phe Lys Lys Glu Arg Asn Thr Asp
.355 360 365
Gin Ala Ser Met Pro Glu Asn Thr Val Ala Gin Lys Leu Cys His Leu
370 375 380
Leu Gly Met Asn Val Met Glu Phe Thr Arg Ala Ile Leu Thr Pro Arg
385 390 395 400
Ile Lys Val Gly Arg Asp Tyr Val Gin Lys Ala Gin Thr Lys Glu Gin
405 410 415 '
Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu
420 425 430
Phe Arg Trp Leu Val His Arg Ile Asn Lys Ala Leu Asp Arg Thr Lys
435 440 445
Arg Gin Gly Ala Ser Phe Ile Gly Ile Leu Asp Ile Ala Gly Phe Glu
450 455 460
Ile Phe Glu Leu Asn Ser Phe Glu Gin Leu Cys Ile Asn Tyr Thr Asn
465 470 475 480
114
CA 02812132 2013-04-04
. :
Glu Lys Leu Gin Gin Leu Phe Asn His Thr Met Phe Ile Leu Glu Gin
485 490 495
Glu Glu Tyr Gin Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly
500 505 510
Leu Asp Leu Gin Pro Cys Ile Asp Leu Ile Glu Arg Pro Ala Asn Pro
515 520 525
Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala
530 535 540
Thr Asp Lys Thr Phe Val Glu Lys Leu Val Gin Glu Gin Gly Ser His
545 550 555 560
Ser Lys Phe Gin Lys Pro Arg Gin Leu Lys Asp Lys Ala Asp Phe Cys
565 570 575
Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Lys Ala Asp Glu Trp Leu
580 585 590
Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val Ala Thr Leu Leu His
595 600 605
Gin Ser Ser Asp Arg Phe Val Ala Glu Leu Trp Lys Asp Val Asp Arg
610 615 620
Ile Val Gly Leu Asp Gin Val Thr Gly Met Thr Glu Thr Ala Phe Gly
625 630 635 640
Ser Ala Tyr Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gin Leu
645. 650 655
Tyr Lys Glu Ser Leu Thr Lys Leu Met Ala Thr Leu Arg Asn Thr Asn
660 665 670
Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ala Gly
675 680 685
Lys Leu Asp Pro His Leu Val Leu Asp Gin Leu Arg Cys Asn Gly Val
690 695 700
Leu Glu Gly Ile Arg Ile Cys Arg Gin Gly Phe Pro Asn Arg Ile Val
705 710 715 720
Phe Gin Glu Phe Arg Gin Arg Tyr Glu Ile Leu Thr Pro Asn Ala Ile
725 730 735
Pro Lys Gly Phe Met Asp Gly Lys Gin Ala Cys Glu Arg Met Ile Arg
740 745 750
Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gin Ser Lys Ile
755 760 765
Phe Phe Arg Ala Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu
770 775 780
Lys Ile Thr Asp Ile Ile Ile Phe Phe Gin Ala Val Cys Arg Gly Tyr
785 790 795 800
Leu Ala Arg Lys Ala Phe Ala Lys Lys Gin Gin Gin Leu Ser Ala Leu
805 810 815
Lys Val Leu Gin Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg His Trp
820 825 830
Gin Trp Trp Arg Val Phe Thr Lys Val Lys Pro Leu Leu Gin Val Thr
835 840 845
Arg Gin Glu Glu Glu Leu Gin Ala Lys Asp Glu Glu Leu Leu Lys Val
850 855 860
Lys Glu Lys Gin Thr Lys Val Glu Gly Glu Leu Glu Glu Met Glu Arg
865 870 875 880
Lys His Gin Gin Leu Leu Glu Glu Lys Asn Ile Leu Ala Glu Gin Leu
885 890 895
Gin Ala Glu Thr Glu Leu Phe Ala Glu Ala Glu Glu Met Arg Ala Arg
900 905 910
Leu Ala Ala Lys Lys Gin Glu Leu Glu Glu Ile Leu His Asp Leu Glu
915 920 925
115
CA 02812132 2013-04-04
Ser Arg Val Gila Glu Glu Glu Glu Arg Asn Gln Ile Leu Gln Asn Glu
930 935 940
Lys Lys Lys Met Gln Ala His Ile Gln Asp Leu Glu Glu Gln Leu Asp
945 950 955 960
Glu Glu Glu Gly Ala Arg Gln Lys Leu Gln Leu Glu Lys Val Thr Ala
965 970 975
Glu Ala Lys Ile Lys Lys Met Glu Glu Glu Ile Leu Leu Leu Glu Asp
980 985 990
Gln Asn Ser Lys She Ile Lys Glu Lys Lys Leu Met Glu Asp Arg Ile
995 1000 1005
Ala Glu Cys Ser Ser Gln Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn
1010 1015 1020
Leu Ala Lys Ile Arg Asn Lys Gln Glu Val Met Ile Ser Asp Leu Glu
1025 1030 1035 1040
Glu Arg Leu Lys Lys Glu Glu Lys Thr Arg Gln Glu Leu Glu Lys Ala
1045 1050 1055
Lys Arg Lys Leu Asp Gly Glu Thr Thr Asp Leu Gln Asp Gln Ile Ala
1060 1065 1070
Glu Leu Gln Ala Gln Ile Asp Glu Leu Lys Leu Gln Leu Ala Lys Lys
1075 1080 1085
Glu Glu Glu Leu Gln Gly Ala Leu Ala Arg Gly Asp Asp Glu Thr Leu
1090 1095 1100
His Lys Asn Asn Ala Leu Lys Val Val Arg Glu Leu Gln Ala Gln Ile
1105 1110 1115 1120
Ala Glu Leu Gln Glu Asp She Glu Ser Glu Lys Ala Ser Arg Asn Lys
1125 1130 1135
Ala Glu Lys Gln Lys Arg Asp Leu Ser Glu Glu Leu Glu Ala Leu Lys
1140 1145 1150
Thr Glu Leu Glu Asp Thr Leu Asp Thr Thr Ala Ala Gln Gln Glu Leu
1155 1160 1165
Arg Thr Lys Arg Glu Gln Glu Val Ala Glu Leu Lys Lys Ala Leu Glu
1170 1175 1180
Glu Glu Thr Lys Asn His Glu Ala Gln Ile Gln Asp Met Arg Gln Arg
1185 1190 1195 1200
His Ala Thr Ala Leu Glu Glu Leu Ser Glu Gln Leu Glu Gln Ala Lys
1205 1210 1215
Arg Phe Lys Ala Asn Leu Glu Lys Asn Lys Gln Gly Leu Glu Thr Asp
1220 1225 1230
Asn Lys Glu Leu Ala Cys Glu Val Lys Val Leu Gln Gln Val Lys Ala
1235 1240 1245
Glu Ser Glu His Lys Arg Lys Lys Leu Asp Ala Gln Val Gln Glu Leu
1250 1255 1260
His Ala Lys Val Ser Glu Gly Asp Arg Leu Arg Val Glu Leu Ala Glu
1265 1270 1275 1280
Lys Ala Ser Lys Leu Gln Asn Glu Leu Asp Asn Val Ser Thr Leu Leu
1285 1290 1295
Glu Glu Ala Glu Lys Lys Gly Ile Lys Phe Ala Lys Asp Ala Ala Ser
1300 1305 1310
Leu Glu Ser Gln Leu Gln Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr
1315 1320 1325
Arg Gln Lys Leu Asn Leu Ser Ser Arg Ile Arg Gln Leu Glu Glu Glu
1330 1335 1340
Lys Asn Ser Leu Gln Glu Gln Gln Glu Glu Glu Glu Glu Ala Arg Lys
1345 1350 1355 1360
Asn Leu Glu Lys Gln Val Leu Ala Leu Gln Ser Gln Leu Ala Asp Thr
1365 1370 1375
116
CA 02812132 2013-04-04
Lys Lys Lys Val Asp Asp Asp Leu Gly Thr Ile Glu Ser Leu Glu Glu
1380 1385 1390
Ala Lys Lys Lys Leu Leu Lys Asp Ala Glu Ala Leu Ser Gin Arg Leu
1395 1400 1405
Glu Glu Lys Ala Leu Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg
1410 1415 1420
Leu Gin Gin Glu Leu Asp Asp Leu Thr Val Asp Leu Asp His Gin Arg
1425 1430 1435 1440
Gin Val Ala Ser Asn Leu Glu Lys Lys Gin Lys Lys Phe Asp Gin Leu
1445 1450 1455
Leu Ala Glu Glu Lys Ser Ile Ser Ala Arg Tyr Ala Glu Glu Arg Asp
1460 1465 1470
Arg Ala Glu Ala Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu
1475 1480 1485
Ala Arg Ala Leu Glu Glu Ala Leu Glu Ala Lys Glu Glu Phe Glu Arg
1490 1495 1500
Gin Asn Lys Gin Leu Arg Ala Asp Met Glu Asp Leu Met Ser Ser Lys
1505 1510 1515 1520
Asp Asp Val Gly Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala
1525 1530 1535
Leu Glu Gin Gin Val Glu Glu Met Arg Thr Gin Leu Glu Glu Leu Glu
1540 1545 1550
Asp Glu Leu Gin Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn
1555 1560 1565
Met Gin Ala Met Lys Ala Gin Phe Glu Arg Asp Leu Gin Thr Arg Asp
1570 1575 1580
Glu Gin Asn Glu Glu Lys Lys Arg Leu Leu Ile Lys Gin Val Arg Glu
1585 1590 1595 1600
Leu Glu Ala Glu Leu Glu Asp Glu Arg Lys Gin Arg Ala Leu Ala Val
1605 1610 1615
Ala Ser Lys Lys Lys Met Glu Ile Asp Leu Lys Asp Leu Glu Ala Gin
1620 1625 1630
Ile Glu Ala Ala Asn Lys Ala Arg Asp Glu Val lie Lys Gin Leu Arg
1635 1640 1645
Lys Leu Gin Ala Gin Met Lys Asp Tyr Gin Arg Glu Leu Glu Glu Ala
1650 1655 1660
Arg Ala Ser Arg Asp Glu Ile Phe Ala Gin Ser Lys Glu Ser Glu Lys
1665 1670 1675 1680
Lys Leu Lys Ser Leu Glu Ala Glu Ile Leu Gin Leu Gin Glu Glu Leu
1685 1690 1695
Ala Ser Ser Glu Arg Ala Arg Arg His Ala Glu Gin Glu'Arg Asp Glu
1700 1705 1710
Leu Ala Asp Glu Ile Thr Asn Ser Ala Ser Gly Lys Ser Ala Leu Leu
1715 1720 1725
Asp Glu Lys Arg Arg Leu Glu Ala Arg Ile Ala Gin Leu Glu Glu Glu
1730 1735 1740
Leu Glu Glu Glu Gin Ser Asn Met Glu Leu Leu Asn Asp Arg Phe Arg
1745 1750 1755 1760
Lys Thr Thr Leu Gin Val Asp Thr Leu Asn Ala Glu Leu Ala Ala Glu
1765 1770 1775 .
Arg Ser Ala Ala Gin Lys Ser Asp Asn Ala Arg Gin Gin Leu Glu Arg
1780 1785 1790
Gin Asn Lys Glu Leu Lys Ala Lys Leu Gin Glu Leu Glu Gly Ala Val
1795 1800 1805
Lys Ser Lys Phe Lys Ala Thr Ile Ser Ala Leu Glu Ala Lys Ile Gly
1810 1815 1820
117
=
CA 02812132 2013-04-04
Gin Leu Glu Glu Gin Leu Glu Gin Glu Ala Lys Glu Arg Ala Ala Ala
1825 1830 1835 1840
Asn Lys Leu Val Arg Arg Thr Glu Lys Lys Leu Lys Glu Ile Phe Met
1845 1850 1855
Gin Val Glu Asp Glu Arg Arg His Ala Asp Gin Tyr Lys Glu Gin Met
1860 1865 1870
Glu Lys Ala Asn Ala Arg Met Lys Gin Leu Lys Arg Gin Leu Glu Glu
1875 1880 1885
Ala Glu Glu Glu Ala Thr Arg Ala Asn Ala Ser Arg Arg Lys Leu Gin
1890 1895 1900
Arg Glu Leu Asp Asp Ala Thr Glu Ala Asn Glu Gly Leu Ser Arg Glu
1905 1910 1915 1920
Val Ser Thr Leu Lys Asn Arg Leu Arg Arg Gly Gly Pro Ile Ser Phe
1925 1930 1935
Ser Ser Ser Arg Ser Gly Arg Arg Gin Leu His Leu Glu Gly Ala Ser
1940 1945 1950
Leu Glu Leu Ser Asp Asp Asp Thr Glu Ser Lys Thr Ser Asp Val Asn
1955 1960 1965
Glu Thr Gin Pro Pro Gin Ser Glu
1970 1975
<210> 55
<211> 6442
<212> DNA
<213> Mus musculus
<400> 55
ccttttctgt ccaggccgag gcctctggac cgccctgggc gccgaccatg gctgcagtga 60
ccatgtccgt gtctgggagg aaggtagcct ccaggCcagg cccggtgcct gaggcagccc 120
aatcgttcct ctacgcgccc cggacgccaa atgtaggtgg ccctggaggg ccacaggtgg 180
agtggacagc ccggcgcatg gtgtgggtgc cctcggaact gcatgggttc gaggcagcag 240
ccctgcggga tgaaggggag gaggaggcag aagtggagct ggcggagagt gggcgccgcc 300
tgcggctgcc cagggaccag atccagcgca tgaacccacc caagttcagc aaggcagaag 360
atatggctga gctcacctgc ctcaacgagg cctcggtcct gcacaacctg cgagaacgct 420
actactccgg gctcatttat acctactctg gcctcttctg tgtggtcatt aacccataca 480
agcagctgcc catctacacg gaggccattg ttgaaatgta ccggggcaag aagcgccatg 540
aggtgccacc tcacgtgtat gctgtgacgg agggcgcgta ccgcagcatg cttcaggatc 600
gtgaggatca atccattctc tgcacgggag agtctggcgc tgggaagacg gagaacacca 660
agaaggtcat ccagtacctg gcccatgtgg catcatctcc aaagggcagg aaggagcctg 720
gtgtccctgc ctccgtcagc accatgtctt atggggagct agagcgtcag cttcttcaag 780
ccaaccccat cctagaggcc tttggcaatg ccaagacagt gaagaacgac aactcttccc 840
gatttggcaa attcatccgc atcaactttg atattgctgg ctacatcgtg ggagcaaaca 900
tcgagaccta tctgttggag aagtcccggg ccatcagaca ggccaaggat gaatgcagct 960
tccatatctt ctaccagctg ctagggggcg ctggggagca gctaaaagct gacctccttc 1020
tggagccctg ttcccattat cgcttcctga ccaatgggcc ctcatcgtcc ccgggccagg 1080
agcgtgagtt attccaggag accctggagt ccctgcgtgt gctgggcctc ctcccagaag 1140
agatcactgc catgctgcgc actgtctctg ctgtcctcca gtttggcaac attgtcctga 1200
agaaagagcg caatacggac caagccacca tgcctgacaa cacagctgcc cagaagcttt 1260
gccgcctctt gggactcgga gtgaccgact tctccagagc ccttctcaca ccccgcatca 1320
aagtgggccg agattatgtt cagaaagcac aaaccaagga gcaggctgac tttgcgctgg 1380
aggctctggc caaagctacc tatgagcgcc tgttccgctg gctggttctg cggctcaacc 1440
gtgccctgga cagaagcccg cggcagggtg cctccttcct gggcatcctg gacatcgcgg 1500
gctttgagat cttccagctg aactccttcg agcagctgtg catcaactac accaacgaga 1560
agctacagca gctattcaac cacaccatgt tcgtgctgga gcaggaggag taccagcgag 1620
agggcatccc ctggaccttc ctagacttcg ggttggacct gcaaccttgc atcgacctca 1680
ttgagcgtcc ggccaaccct ccaggtctcc tggccctgct ggacgaggag tgctggttcc 1740
118
6-ET
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090E bqq.4eeebee b4ebeepobb ebeoPoo2bq Ebeebebbqo bepeqabepb eobb000bqb
opoz bbebbpb4o5 epbbqopeoo 526p.bPqabe bbeDa4pTeo bpobeobi.ob bebpp6pp6E,
OD'6Z b4butvo6qp bPobboobpo BqbebpPbee bbebo6fib4b pbapobbubb qobp5popbq
088Z 66456qobpb bgobebbeob eebb000bqo bfiqoebobob bbobDebebb eboobbebqo
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ogLz o236646pbo qUbsob4oe ubabbbqbee 51.5oi.ob3.5e beobeobeob qobpbbpoqq
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ugz oppobeeb4b bueo4poqq.b qobbPbbqbb gbeob6.43pe ayeo4o5e3 goop4opbqo
oesz 546qopue5e 5eo64p6.4b5 525'43;35-46 PEopD6pobpo 5pobbop6p5 -200;4'40E66
OZcZ e4b000bbqo peqobbbbop obbobeobbe 3344404bqb Pqvp-TepEto opoqbePpbq
09DrZ opebbbobeb b2.5bebbgob eaoabbi.Doq. bb6beabbbp oqi.04404eb PP0.5PPPDa6
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Of7Cz eobboebbqe 344Dbfibeep poggu4pEop pboopopo4D oqepebqeqo bobpDebooq.
08Z 82E623344
04004eo6Do epopoo44ob 5eeppbo454 042pboeq.eq bbbebDqoo4
ozzz bbbboeeqbq qbooqoepoo ebbqobqbbq poboboobEt bqobeeebbq obbbpbeebe
09-CZ bg234223o3 gq5oqe3.6go'booqb41q4b eqooDeeope oeeobeoob DeDDbb4po4
00-Tz poboobpeclo poqep6Peyo eloqabeobb bb4b4oebbo p44bgeebbq 6Dobopoopb
orioz opbbabbboo Eopobb4eb2 E0544pobob eeq.be2Db2b .5qobbabqbp qpobbbebbq
0861 5.4e65e-e5b4 oTetp64obe 3epi.34boqu 56peDbpb23 peaqqo5q4p oboobbqbqe
0F6T PoPbqeebqo popoe6b4v3 eebpebqe64 obbgbebgbu pabPepoe43 pEq.4bbpepb
0981 bpDb4e4p2o bqoD4bobeo 44oebpDbbe p4ebubDbqD 3226bepoop ba62334422
0081 POD0020052 obbb?Dbebb popoboqbbe pbebb45444 qoi.beepe.65 o23obbeepo
VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
ggctggaagc tgaggtgctg cgtctgcaag aggaacttgc tgcctcagac cgagcccgga 5220
ggcaggccca gcaagacaga gacgagatgg cagaggaggt ggccagtggc aatcttagca 5280
aggcagccac cctggaggaa aaacggcagc tggaggggcg actgagccag ttggaagagg 5340
agctggagga agaacagaac aactcggagc tgctcaagga ccattaccga aagctagtgc 5400
tacaggtcga gtccctcacc acagaactgt ctgccgaacg aagtttctca gccaaggccg 5460
agagtggacg gcagcagctg gagcggcaga tccaggaact gcgggcccgc ttgggtgaag 5520
aggatgctgg agcccgagcc aggcagaaaa tgctgatcgc tgctctggag tctaaactgg 5580
cccaggcaga ggagcagctg gagcaggaga gcagggagcg catcctctct ggcaagctgg 5640
tacgcagagc tgagaagcgg ctgaaggagg tagttcttca ggtggatgaa gagcgcaggg 5700
tggctgacca ggtccgggac cagctggaga aaagcaacct ccggctgaag cagctcaaga 5760
ggcagctgga ggaggcagag gaggaggcat ctcgggcaca ggctggtcgg aggcggctgc 5820
agcgggagct ggaggacgtc actgagtctg cagaatccat gaaccgggag gtgaccacgc 5880
tgaggaacag gctccggcgt ggcccactta cattcaccac acggactgtg cgccaggtgt 5940
tccggctgga agagggcgtg gcttctgacg aggaagaggc tgaaggagct gaacctggct 6000
ctgcaccagg ccaggagccg gaggctccgc cccctgccac accccaatga tccagtctgt 6060
cctagatgcc ccaaggacag agccctttcc agtgcccctc ctggtttgca ctttgaaatg 6120
gcactgtcct ctggcacttt ctggcattga tgaaccctcc tgggacccca ggacccctgc 6180
ccactggggg ccccaaacca aggagctggg tgggagggag gccatgatgg tctctcttgt 6240
tagagaaaca aaattgaacg tggatgtcaa gaatgtcctg tctgcaccta ttttcagcag 6300
gcctgtcccc tggagagggc aggcagggtg cttccatccc ctctcagtat cttgccctct 6360
tttttggggg gaagtggggt gtctgtgtgc tcatagggta atgctcatgg cccctcatgc 6420
tccagacact aaagaaataa aa 6442
<210> 56
<211> 2000
<212> PRT
<213> Mus musculus
<400> 56
Met Ala Ala Val Thr Met Ser Val Ser Gly Arg Lys Val Ala Ser Arg
1 5 10 15
Pro Gly Pro Val Pro, Glu Ala Ala Gln Ser Phe Leu Tyr Ala Pro Arg
20 25 30
Thr Pro Asn Val Gly Gly Pro Gly Gly Pro Gin Val Glu Trp Thr Ala
35 40 45
Arg Arg Met Val Trp Val Pro Ser Glu Leu His Gly Phe Glu Ala Ala
50 55 60
Ala Leu Arg Asp Glu Gly Glu Glu Glu Ala Glu Val Glu Leu Ala Glu
65 70 75 80
Ser Gly Arg Arg Leu Arg Leu Pro Arg Asp Gin Ile Gin Arg Met Asn
85 90 95
Pro Pro Lys Phe Ser Lys Ala Glu Asp Met Ala Glu Leu Thr Cys Leu
100 105 110
Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Tyr Ser Gly
115 120 125
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr
130 135 140
Lys Gin Leu Pro Ile Tyr Thr Glu Ala Ile Val Glu Met Tyr Arg Gly
145 150 155 160
Lys Lys Arg His Glu Val Pro Pro His Val Tyr Ala Val Thr Glu Gly
165 170 175
Ala Tyr Arg Ser Met Leu Gin Asp Arg Glu Asp Gin Ser Ile Leu Cys
180 185 190
Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile
195 200 205
120
CA 02812132 2013-04-04
Gin Tyr Leu Ala His Val Ala Ser Ser Pro Lys Gly Arg Lys Glu Pro
210 215 220
Gly Val Pro Ala Ser Val Ser Thr Met Ser Tyr Gly Glu Leu Glu Arg
225 230 235 240
Gin Leu Leu Gin Ala Asn Pro Ile Leu Glu Ala She Gly Asn Ala Lys
245 250 255
Thr Val Lys Asn Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile
260 265 270
Asn Phe Asp Ile Ala Gly Tyr Ile Val.Gly Ala Asn Ile Glu Thr Tyr
275 280 285
Leu Leu Glu Lys Ser Arg Ala Ile Arg Gin Ala Lys Asp Glu Cys Ser
290 295 300
Phe His Ile Phe Tyr Gin Leu Leu Gly Gly Ala Gly Glu Gin Leu Lys
305 310 315 320
Ala Asp Leu Leu Leu Glu Pro Cys Ser His Tyr Arg Phe Leu Thr Asn
325 330 335
Gly Pro Ser Ser Ser Pro Gly Gin Glu Arg Glu Leu Phe Gin Glu Thr
340 345 350
Leu Glu Ser Leu Arg Val Leu Gly Leu Leu Pro Glu Glu Ile Thr Ala
355 360 365
Met Leu Arg Thr Val Set Ala Val Leu Gin She Gly Asn Ile Val Leu
370 375 380
Lys Lys Glu Arg Asn Thr Asp Gin Ala Thr Met Pro Asp Asn Thr Ala
385 390 395 400
Ala Gin Lys Leu Cys Arg Leu Leu Gly Leu Gly Val Thr Asp She Ser
405 410 415
Arg Ala Leu Leu Thr Pro Arg Ile Lys Val Gly Arg Asp Tyr Val Gin
420 425 430
Lys Ala Gin Thr Lys Glu Gin Ala Asp Phe Ala Leu Glu Ala Leu Ala
435 440 - 445
Lys Ala Thr Tyr Glu Arg Leu Phe Arg Trp Leu Val Leu Arg Leu Asn
450 455 460
Arg Ala Leu Asp Arg Ser Pro Arg Gin Gly Ala Ser She Leu Gly Ile
465 470 475 480
Leu Asp Ile Ala Gly She Glu Ile Phe Gin Leu Asn Ser She Glu Gin
485 490 495
Leu Cys Ile Asn Tyr Thr Asn Glu Lys Leu Gin Gin Leu Phe Asn His
500 505 510
Thr Met Phe Val Leu Glu Gin Glu Glu Tyr Gin Arg Glu Gly Ile Pro
515 520 525
Trp Thr She Leu Asp She Gly Leu Asp Leu Gin Pro Cys Ile Asp Leu
530 535 540
Ile Glu Arg Pro Ala Asn Pro Pro Gly Leu Leu Ala Leu Lou Asp Glu
545 550 555 560
Glu Cys Trp She Pro Lys Ala Thr Asp Lys Ser Phe Val Glu Lys Val
565 570 575
Ala Gin Glu Gin Gly Ser His Pro Lys Phe Gin Arg Pro Arg Asn Leu
580 585 590
Arg Asp Gin Ala Asp She Ser Val Leu His Tyr Ala Gly Lys Val Asp
595 600 605
Tyr Lys Ala Ser Glu Trp Leu Met Lys Asn Met Asp Pro Leu Asn Asp
610 615 620
Asn Val Ala Ala Leu Leu His Gin Ser Thr Asp Arg Leu Thr Ala Glu
625 630 635 640
Ile Trp Lys Asp Val Glu Gly Ile Val Gly Lou Glu Gin Val Ser Ser
645 650 655
121
CA 02812132 2013-04-04
Leu Gly Asp Gly Pro Pro Gly Gly Arg Pro Arg Arg Gly Met Phe Arg
660 665 670
Thr Val Gly Gin Leu Tyr Lys Glu Ser Leu Ser Arg Leu Met Ala Thr
675 680 685
Leu Ser Asn Thr Asn Pro Ser Phe Val Arg Cys Ile Val Pro Asn His
690 695 700
Glu Lys Arg Ala Gly Lys Leu Glu Pro Arg Leu Val Leu Asp Gin Leu
705 710 715 720
Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile Cys Arg Gin Gly Phe
725 730 735
Pro Asn Arg Ile Leu Phe Gin Glu Phe Arg Gin Arg Tyr Glu Ile Leu
740 745 750
Thr Pro Asn Ala Ile Pro Lys Gly Phe Met Asp Gly Lys Gin Ala Cys
755 760 765
Glu Lys Met Ile Gin Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Val
770 775 780
Gly Gin Ser Lys Ile Phe Phe Arg Ala Gly Val Leu Ala Gin Leu Glu
785 790 795 800
Glu Glu Arg Asp Leu Lys Val Thr Asp Ile Ile Val Ser Phe Gin Ala
805 810 815
Ala Ala Arg Gly Tyr Leu Ala Arg Arg Ala Phe Gin Arg Arg Gin Gin
820 825 830
Gin Gin Ser Ala Leu Arg Val Met Gin Arg Asn Cys Ala Ala Tyr Leu
835 840 845
Lys Leu Arg Asn Trp Gin Trp Trp Arg Leu Phe Ile Lys Val Lys Pro
850 855 860
Leu Leu Gin Val Thr Arg Gin Asp Glu Val Leu Gin Ala Arg Ala Gin
865 870 875 880
Glu Leu Gin Lys Val Gin Glu Leu Gin Gin Gin Ser Ala Arg Glu Val
885 890 895
Gly Glu Leu Gin Gly Arg Val Ala Gin Leu Glu Glu Glu Arg Thr Arg
900 905 910
Leu Ala Glu Gin Leu Arg Ala Glu Ala Glu Leu Cys Ser Glu Ala Glu
915 920 925
Glu Thr Arg Ala Arg Leu Ala Ala Arg Lys Gin Glu Leu Glu Leu Val
930 935 940
Val Thr Glu Leu Glu Ala Arg Val Gly Glu Glu Glu Glu Cys Ser Arg
945 950 955 960
Gin Leu Gin Ser Glu Lys Lys Arg Leu Gin Gin His Ile Gin Glu Leu
965 970 975
Glu Ser His Leu Glu Ala Glu Glu Gly Ala Arg Gin Lys Leu Gin Leu
980 985 990
Glu Lys Val Thr Thr Glu Ala Lys Met Lys Lys Phe Glu Glu Asp Leu
995 1000 1005
Leu Leu Leu Glu Asp Gin Asn Ser Lys Leu Ser Lys Glu Arg Arg Leu
1010 1015 1020
Leu Glu Glu Arg Leu Ala Glu Phe Ser Ser Gin Ala Ala Glu Glu Glu
1025 1030 1035 1040 .
Glu Lys Val Lys Ser Leu. Asn Lys Leu Arg Leu Lys Tyr Glu Ala Thr
1045 1050 1055
Ile Ser Asp Met Glu Asp Arg Leu Lys Lys Glu Glu Lys Gly Arg Gin
1060 1065 1070
Glu Leu Glu Lys Leu Lys Arg Arg Leu Asp Gly Glu Ser Ser Glu Leu
1075 1080 1085
Gin Glu Gin Met Val Glu Gin Lys Gin Arg Ala Glu Glu Leu Leu Ala
1090 1095 1100
122
CA 02812132 2013-04-04
Gln Leu Gly Arg Lys Glu Asp Glu Leu Gln Ala Ala Leu Leu Arg Ala
1105 1110 1115 1120
Glu Glu Glu Gly Gly Ala Arg Ala Gln Leu Leu Lys Ser Leu Arg Glu
1125 1130 1135
Ala Gin Ala Gly Leu Ala Glu Ala Gln Glu Asp Leu Glu Ala Glu Arg
1140 1145 1150
Val Ala Arg Ala Lys Ala Glu Lys Gln Arg Arg Asp Leu Gly Glu Glu
1155 1160 1165
Leu Glu Ala Leu Arg Gly Glu Leu Glu Asp Thr Leu Asp Ser Thr Asn
1170 1175 1180
Ala Gin Gln Glu Leu Arg Ser Lys Arg Glu Gln Glu Val Thr Glu Leu
1185 1190 1195 1200
Lys Lys Ala Leu Glu Glu Glu Ser Arg Ala His Glu Val Ser Met Gln
1205 1210 1215
Glu Leu Arg Gln Arg His Ser Gln Ala Leu Val Glu Met Ala Glu Gln
1220 1225 1230
Leu Glu Gin Ala Arg Arg Gly Lys Gly Val Trp Glu Lys Thr Arg Leu
1235 1240 1245
Ser Leu Glu Ala Glu Val Ser Glu Leu Lys Ala Glu Leu Ser Ser Leu
1250 1255 1260
Gln Thr Ser Arg Gln Glu Gly Glu Gln Lys Arg Arg Arg Leu Glu Ser
1265 1270 1275 1280
Gln Leu Gln Glu Val Gln Gly Arg Ser Ser Asp Ser Glu Arg Ala Arg
1285 1290 1295
Ser Glu Ala Ala Glu Lys Leu Gln Arg Ala Gln Ala Glu Leu Glu Ser
1300 1305 1310
Val Ser Thr Ala Leu Ser Glu Ala Glu Ser Lys Ala Ile Arg Leu Gly
1315 1320 1325
Lys Glu Leu Ser Ser Ala Glu Ser Gln Leu His Asp Thr Gln Glu Leu
1330 1335 1340
Leu Gln Glu Glu Thr Arg Ala Lys Leu Ala Leu Gly Ser Arg Val Arg
1345 1350 1355 1360
Ala Leu Glu Ala Glu Ala Ala Gly Leu Arg Glu Gln Met Glu Glu Glu
1365 1370 1375
Val Val Ala Arg Glu Arg Ala Gly Arg Glu Leu Gln Ser Thr Gln Ala
1380 1385 1390
Gln Leu Ser Glu Trp Arg Arg Arg Gln Glu Glu Glu Ala Ala Val Leu
1395 1400 1405
Glu Ala Gly Glu Glu Ala Arg Arg Arg Ala Ala Arg Glu Ala Glu Thr
1410 1415 1420
Leu Thr Gln Arg Leu Ala Glu Lys Thr Glu Ala Val Glu Arg Leu Glu
1425 1430 1435 1440
Arg Ala Arg Arg Arg Leu Gln Gin Glu Leu Asp Asp Ala Thr Val Asp
1445 1450 1455
Leu Gly Gln Gln Lys Gln Leu Leu Ser Thr Leu Glu Lys Lys Gln Arg
1460 1465 1470
Lys Phe Asp Gln Lou Leu Ala Glu Glu Lys Ala Ala Val Leu Arg Ala
1475 1480 1485
Val Glu Asp Arg Glu Arg Ile Glu Ala Glu Gly Arg Glu Arg Glu Ala
1490 1495 = 1500
Arg Ala Leu Ser Leu Thr Arg Ala Leu Glu Glu Glu Gln Glu Ala Arg
1505 1510 1515 1520
Glu Glu Leu Glu Arg Gln Asn Arg Ala Leu Arg Ala Glu Leu Glu Ala
1525 1530 1535
Leu Leu Ser Ser Lys Asp Asp Val Gly Lys Asn Val His Glu Leu Glu
1540 1545 . 1550
123
CA 02812132 2013-04-04
Arg Ala Arg Lys Ala Ala Glu Gin Ala Ala Ser Asp Leu Arg Thr Gin
1555 1560 1565
Val Thr Glu Leu Glu Asp Glu Leu Thr Ala Ala Glu Asp Ala Lys Leu
1570 1515 1580
Arg Leu Glu Val Thr Val Gin Ala Leu Lys Ala Gin His Glu Arg Asp
1585 1590 1595 1600
Leu Gin Gly Arg Asp Asp Ala Gly Glu Glu Arg Arg Arg Gin Leu Ala
1605 1610 1615
Lys Gin Leu Arg Asp Ala Glu Val Glu Arg Asp Glu Glu Arg Lys Gin
1620 1625= 1630
Arg Ala Leu Ala Met Ala Ala Arg Lys Lys Leu Glu Leu Glu Leu Glu
1635 1640 1645
Glu Leu Lys Ala Gin Thr Ser Ala Ala Gly Gin Gly Lys Glu Glu Ala
1650 1655 1660
Val Lys Gin Leu Lys Lys Met Gin Val Gin Met Lys Glu Leu Trp Arg
1665 1670 1675 1680
Glu Val Glu Glu Thr Arg Ser Ser Arg Asp Glu Met Phe Thr Leu Ser
1685 1690 1695
Arg Glu Asn Glu Lys Lys Leu Lys Gly Leu Glu Ala Glu Val Leu Arg
1700 1705 1710
Leu Gin Glu Glu Leu Ala Ala Ser Asp Arg Ala Arg Arg Gin Ala Gin
1715 1720 1725
Gin Asp Arg Asp Glu Met Ala Glu Glu Val Ala Ser Gly Asn Leu Ser
1730 1735 1740
Lys Ala Ala Thr Leu Clu Glu Lys Arg Gin Leu Glu Gly Arg Leu Ser
1745 1750 1755 1760
Gin Leu Glu Glu Glu Leu Glu Glu Glu Gin Asn Asn Ser Glu Leu Leu
1765 1770 1775
Lys Asp His Tyr Arg Lys Leu Val Leu Gin Val Glu Ser Leu Thr Thr
1780 1785 1790
Glu Leu Ser Ala Glu Arg Ser Phe Ser Ala Lys Ala Glu Ser Gly Arg
1795 1800 1805
Gin Gin Leu Glu Arg Gin Ile Gin Glu Leu Arg Ala Arg Leu Gly Glu
1810 1815 1820
= Glu Asp Ala Gly Ala Arg Ala Arg Gin Lys Met Leu Ile Ala Ala Leu
1825 1830 1835 1840
Glu Ser Lys Leu Ala Gin Ala Glu Glu Gin Leu Glu Gin Glu Ser Arg
1845 1850 1855
Glu Arg Ile Leu Ser Gly Lys Leu Val Arg Arg Ala Glu Lys Arg Leu
1860 1865 1870
Lys Glu Val Val Leu Gin Val Asp Glu Glu Arg Arg Val Ala Asp Gin
1875 1880 1885
Val Arg Asp Gin Leu Glu Lys Ser Asn Leu Arg Lou Lys Gln Leu Lys
1890 1895 1900
Arg Gin Leu Glu Giu Ala Glu Glu Glu Ala Ser Arg Ala Gin Ala Gly
1905 1910 1915 1920
Arg Arg Arg Leu Gin Arg Glu Leu Glu Asp Val Thr Glu Ser Ala Glu
1925 1930 1935
Ser Met Asn Arg Glu Val Thr Thr Leu Arg Asn Arg Leu Arg Arg Gly
1940 1945 1950
Pro Leu Thr Phe Thr Thr Arg Thr Val Arg Gin Val Phe Arg Leu Glu
1955 1960 1965
Glu Gly Val Ala Ser Asp Glu Glu Glu Ala Glu Gly Ala Glu Pro Gly
1970 1975 1980
Ser Ala Pro Gly Gin Glu Pro Glu Ala Pro Pro Pro Ala Thr Pro Gin
1985 1990 1995 2000
124
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0819 bebegq.b1.43 4Dial.bbqeb 4epobbu665 ebbbqbbbqa bebbeeppee epopabbbbb
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0909 40Pabbqeee b4q4peob44 4bb1opqap3 ob4bp30444 poobeftoeb beep3ppb4e
0009 beqop4b4pg bpopqebgee ooppepeopb goo3poboo4 abbabboabe bbeopbbeop
0t76S eob-laqabbq ooeebqobeb beebqabbeb eebbeboebq al.qobb4b35 bbebeebb4D
088S bbo3q4b4bb ep3b3bqbqa ebboeoepoe 34423e4q.pe o3ob64bobb op4obbepee
08q bbebqoboeo oebqbbebbb opeebgpopq eubeobqoqb ebgpeo4boe bbe5b4obeb
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VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
<210> 58
<211> 1992
<212> PRT
<213> Mus musculus
<400> 58
Met Ala Ala Val Thr Met Ser Val Ser Gly Arg Lys Val Ala Ser Arg
1 5 10 15
Pro Gly Pro Val Pro Glu Ala Ala Gln Ser Phe Leu Tyr Ala Pro Arg
20 25 30
Thr Pro Asn Val Gly Gly Pro Gly Gly Pro Gln Val Glu Trp Thr Ala
35 40 45
Arg Arg Met Val Trp Val Pro Ser Glu Leu His Gly Phe Glu Ala Ala
50 55 60
Ala Leu Arg Asp Glu Gly Glu Glu Glu Ala Glu Val Glu Leu Ala Glu
65 70 75 80
Ser Gly Arg Arg Leu Arg Leu Pro Arg Asp Gln Ile Gln Arg Met Asn
85 90 95
Pro Pro Lys Phe Ser Lys Ala Glu Asp Met Ala Glu Leu Thr Cys Leu
100 105 110
Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Tyr Ser Gly
115 120 125
Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Ile Asn Pro Tyr
130 135 140
Lys Gln Leu Pro Ile Tyr Thr Glu Ala Ile Val Glu Met Tyr Arg Gly
145 150 155 160
Lys Lys Arg His Glu Val Pro Pro His Val Tyr Ala Val Thr Glu Gly
165 170 175 .
Ala Tyr Arg Ser Met Leu Gin Asp Arg Glu Asp Gln Ser Ile Leu Cys
180 185 190
Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile
195 200 205
Gln Tyr Leu Ala His Val Ala Ser Ser Pro Lys Gly Arg Lys Glu Pro
210 215 220
Gly Val Pro Gly Glu Leu Glu Arg Gln Leu Leu Gln Ala Asn Pro Ile
225 230 235 240
Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser
245 250 255
Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Ile Ala Gly Tyr Ile
260 265 270
Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Her Arg Ala Ile
275 280 285
Arg Gln Ala Lys Asp Glu Cys Ser Phe His Ile Phe Tyr Gln Leu Leu
290 295 300
Gly Gly Ala Gly Glu Gln Leu Lys Ala Asp Leu Leu Leu Glu Pro Cys
=
305 310 315 320
Ser His Tyr Arg Phe Leu Thr Asn Gly Pro Ser Ser Ser Pro Gly Gln
325 330 335
Glu Arg Glu Leu Phe Gln Glu Thr Leu Glu Ser Leu Arg Val Leu Gly
340 345 350
Leu Leu Pro Glu Glu Ile Thr Ala Met Leu Arg Thr Val Ser Ala Val
355 360 365
Leu Gln Phe Gly Asn Ile Val Leu Lys Lys Glu Arg Asn Thr Asp Gln
370 375 380
Ala Thr Met Pro Asp Asn Thr Ala Ala Gln Lys Leu Cys Arg Leu Leu
385 390 395 400
127
CA 02812132 2013-04-04
Gly Leu Gly Val Thr Asp Phe Ser Arg Ala Leu Leu Thr Pro Arg Ile
405 410 415
Lys Val Gly Arg Asp Tyr Val Gin Lys Ala Gin Thr Lys Glu Gin Ala
420 425 430
Asp Phe Ala Leu Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu Phe
435 440 445
Arg Trp Leu Val Leu Arg Leu Asn Arg Ala Leu Asp Arg Ser Pro Arg
450 455 460
Gin Giy Ala Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu Ile
465 470 475 480
Phe Gin Leu Asn Ser Phe Glu Gin Leu Cys Ile Asn Tyr Thr Asn Glu
485 490 495
Lys Leu Gin Gin Leu Phe Asn His Thr Met Phe Val Leu Glu Gin Glu
500 505 510
Glu Tyr Gin Arg Glu Gly Ile Pro Trp Thr Phe Leu Asp Phe Gly Leu
515 520 525
Asp Leu Gin Pro Cys Ile Asp Leu Ile Glu Arg Pro Ala Asn Pro Pro
530 535 540
Gly Leu Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala Thr
545 550 555 560
Asp Lys Ser Phe Val Glu Lys Val Ala Gin Glu Gin Gly Ser His Pro
565 570 575
Lys Phe Gin Arg Pro Arg Asn Leu Arg Asp Gin Ala Asp Phe Ser Val
580 585 590
Leu His Tyr Ala Gly Lys Val Asp Tyr Lys Ala Ser Glu Trp Leu Met
595 600 605
Lys Asn Met Asp Pro Leu Asn Asp Asn Val Ala Ala Leu Leu His Gin
610 615 620
Ser Thr Asp Arg Leu Thr Ala Glu Ile Trp Lys Asp Val Glu Gly Ile
625 630 635 640
Val Gly Leu Glu Gin Val Ser Ser Leu Gly Asp Gly Pro Pro Gly Gly
645 650 655
Arg Pro Arg Arg Gly Met Phe Arg Thr Val Gly Gin Leu Tyr Lys Glu
660 665 670
Ser Leu Ser Arg Leu Met Ala Thr Leu Ser Asn Thr Asn Pro Ser Phe
675 680 685
Val Arg Cys Ile Val Pro Asn His Glu Lys Arg Ala Gly Lys Leu Glu
690 695 700
Pro Arg Leu Val Leu Asp Gin Leu Arg Cys Asn Gly Val Leu Glu Gly
705 710 715 720
Ile Arg Ile Cys Arg Gin Gly Phe Pro Asn Arg Ile Leu Phe Gin Glu
725 730 735
Phe Arg Gin Arg Tyr Glu Ile Leu Thr Pro Asn Ala Ile Pro Lys Sly
740 745 750
Phe Met Asp Gly Lys Gin Ala Cys Glu Lys Met Ile Gin Ala Leu Glu
755 760 765
Leu Asp Pro Asn Leu Tyr Arg Val Gly Gin Ser Lys Ile Phe Phe Arg
770 775 780
Ala Gly Val Leu Ala Gin Leu Glu Glu Glu Arg Asp Leu Lys Val Thr
785 790 795 800
Asp Ile Ile Val Ser Phe Gin Ala Ala Ala Arg Gly Tyr Leu Ala Arg
805 810 815
Arg Ala Phe Gin Arg Arg Gin Gin Gin Gin Ser Ala Leu Arg Val Met
820 825 830
Gin Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg Asn Trp Gin Trp Trp
835 840 845
128
CA 02812132 2013-04-04
Arg Leu Phe Ile Lys Val Lys Pro Leu Leu Gin Val Thr Arg Gin Asp
850 855 860
Glu Val Leu Gin Ala Arg Ala Gin Glu Leu Gin Lys Val Gin Giu Leu
865 870 875 880
Gin Gin Gin Ser Ala Arg Glu Val Gly Glu Leu Gin Gly Arg Val Ala
885 890 895
Gin Leu Glu Glu Glu Arg Thr Arg Leu Ala Glu Gin Leu Arg Ala Glu
900 905 910
Ala Glu Leu Cys Ser Glu Ala Glu Giu Thr Arg Ala Arg Leu Ala Ala
915 920 925
Arg Lys Gin Glu Leu Glu Leu Val Val Thr Glu Leu Glu Ala Arg Val
930 935 940
Gly Glu Glu Glu Glu Cys Ser Arg Gin Leu Gin Ser Glu Lys Lys Arg
945 950 955 960
Leu Gin Gin His Ile Gin Glu Leu Glu Ser His Leu Glu Ala Glu Glu
965 970 975
Gly Ala Arg Gin Lys Leu Gin Leu Glu Lys Val Thr Thr Glu Ala Lys
980 985 990
Met Lys Lys Phe Glu Glu Asp Leu Leu Leu Leu Glu Asp Gin Asn Ser
995 1000 1005
Lys Leu Ser Lys Glu Arg Arg Leu Leu Glu Glu Arg Leu Ala Glu Phe
1010 1015 1020
Ser Ser Gin Ala Ala .Glu Glu Glu Glu Lys Val Lys Ser Leu Asn Lys
1025 1030 1035 1040
Leu Arg Leu Lys Tyr Glu Ala Thr Ile Ser Asp Met Glu Asp Arg Leu
1045 1050 1055
Lys Lys Glu Glu Lys Gly Arg Gin Glu Leu Glu Lys Leu-Lys Arg Arg
1060 1065 1070
Leu Asp Gly Glu Ser Ser Glu Leu Gin Glu Gin Met Val Glu Gin Lys
1075 1080 1085
Gin Arg Ala Glu Glu Leu Leu Ala Gin Leu Gly Arg Lys Glu Asp Glu
1090 1095 1100
Leu Gin Ala Ala Leu Leu Arg Ala Glu Glu Glu Gly Gly Ala Arg Ala
1105 1110 1115 1120
Gin Leu Leu Lys Ser Leu Arg Giu Ala Gin Ala Gly Leu Ala Giu Ala
1125 1130 1135
Gin Glu Asp Leu Glu Ala Glu Arg Val Ala Arg Ala Lys Ala Glu Lys
1140 1145 1150
Gin Arg Arg Asp Leu Gly Glu Glu Leu Glu Ala Leu Arg Gly Glu Leu
1155 1160 1165
Glu Asp Thr Leu Asp Ser Thr Asn Ala Gin Gin Giu Leu Arg Ser Lys
1170 1175 1180
Arg Giu Gin Glu Val Thr Glu Leu Lys Lys Ala Leu Glu Glu Glu Ser.
1185 1190 1195 1200
Arg Ala His Glu Val Ser Met Gin Glu Leu Arg Gin Arg His Ser Gin
1205 1210 1215
Ala Leu Val Glu Met Ala Glu Gin Leu Glu Gin Ala Arg Arg Gly Lys
1220 1225 1230
Gly Val Trp Glu Lys Thr Arg Leu Ser Leu Glu Ala Glu Val Ser Glu
1235 1240 1245
Leu Lys Ala Glu Leu Ser Ser Leu Gin Thr Ser Arg Gin Glu Gly Glu
1250 1255 1260
Gin Lys Arg Arg Arg Leu Glu Ser Gin Leu Gin Glu Val Gin Gly Arg
1265 1270 1275 1280
Ser Ser Asp Ser Glu Arg Ala Arg Ser Glu Ala Ala Glu Lys Leu Gin
1285 1290 1295
129
CA 02812132 2013-04-04
Arg Ala Gin Ala Glu Leu Glu Ser Val Ser Thr Ala Leu Ser Glu Ala
1300 1305 1310
Glu Ser Lys Ala Ile Arg Leu Gly Lys Glu Leu Ser Ser Ala Glu Ser
1315 1320 1325
Gln Leu His Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr Arg Ala Lys
1330 1335 1340
Leu Ala Leu Gly Ser Arg Val Arg Ala Leu Glu Ala Glu Ala Ala Gly
1345 1350 1355 1360
Leu Arg Glu Gln Met Glu Glu Glu Val Val Ala Arg Glu Arg Ala Gly
1365 1370 1375
Arg Glu Leu Gln Ser Thr Gin Ala Gln Leu Ser Glu Trp Arg Arg Arg
1380 1385 1390
Gln Glu Glu Glu Ala Ala Val Leu Glu Ala Gly Glu Glu Ala Arg Arg
1395 1400 1405
Arg Ala Ala Arg Glu Ala Glu Thr Leu Thr Gln Arg Leu Ala Glu Lys
1410 1415 1420
Thr Glu Ala Val Glu Arg Leu Glu Arg Ala Arg Arg Arg Leu Gln Gln
1425 1430 1.435 1440
Glu Leu Asp Asp Ala Thr Val Asp Leu Gly Gln Gln Lys Gln Leu Leu
1445 1450 1455
Ser Thr Leu Glu Lys Lys Gln Arg Lys Phe Asp Gln Leu Leu Ala Glu
1460 1465 1470
Glu Lys Ala Ala Val Leu Arg Ala Val Glu Asp Arg Glu Arg Ile Glu
1475 1480 1485
Ala Glu Gly Arg Glu Arg Glu Ala Arg Ala Leu Ser Leu Thr Arg Ala
1490 1495 1500
Leu Glu Glu Glu Gln Glu Ala Arg Glu Glu Leu Glu Arg Gln Asn Arg
1505 1510 1515 1520
Ala Leu Arg Ala Glu Leu Glu Ala Leu Leu Ser Ser Lys Asp Asp Val
1525 1530 1535
Gly Lys Asn Val His Glu Leu Glu Arg Ala Arg Lys Ala Ala Glu Gln
1540 1545 1550
Ala Ala Ser Asp Leu Arg Thr Gln Val Thr Glu Leu Glu Asp Glu Leu
1555 1560 1565
Thr Ala Ala Glu Asp Ala Lys Leu Arg Leu Glu Val Thr Val Gln Ala
1570 1575 1580
Leu Lys Ala Gln His Glu Arg Asp Leu Gln Gly Arg Asp Asp Ala Gly
1585 1590 1595 1600
Glu Glu Arg Arg Arg Gln Leu Ala Lys Gln Leu Arg Asp Ala Glu Val
1605 1610 1615 .
Glu Arg Asp Glu Glu Arg Lys Gln Arg Ala Leu Ala Met Ala Ala Arg
1620 1625 1630
Lys Lys Leu Glu Leu Glu Leu Glu Glu Leu Lys Ala Gln Thr Ser Ala
1635 1640 1645
Ala Gly Gln Gly Lys Glu Glu Ala Val Lys Gln Leu Lys Lys Met Gln
1650 1655 1660
Val Gln Met Lys Glu Leu Trp Arg Glu Val Glu Glu Thr Arg Ser Ser
1665 1670 1675 1680
Arg Asp Glu Met Phe Thr Leu Ser Arg Glu Asn Glu Lys Lys Leu Lys
1685 1690 1695
Gly Leu Glu Ala Glu Val Leu Arg Leu Gln Glu Glu Leu Ala Ala Ser
1700 1705 1710
Asp Arg Ala Arg Arg Gln Ala Gln Gln Asp Arg Asp Glu Met Ala Glu
1715 1720 1725
Glu Val Ala Ser Gly Asn Leu Ser Lys Ala Ala Thr Leu Glu Glu Lys
1730 1735 1740
130
CA 02812132 2013-04-04
Arg Gin Leu Glu Gly Arg Leu Ser Gin Leu Glu Glu Glu Leu Glu Glu
1745 1750 1755 1760
Glu Gin Asn Asn Ser Glu Leu Leu Lys Asp His Tyr Arg Lys Leu Val
1765 1770 1775
Leu Gin Val Glu Ser Leu Thr Thr Glu Leu Ser Ala Glu Arg Ser Phe
1780 1785 1790
Ser Ala Lys Ala Glu Ser Gly Arg Gin Gin Leu Glu Arg Gin Ile Gin
1795 1800 1805
Glu Leu Arg Ala Arg Leu Gly Glu Glu Asp Ala Gly Ala Arg Ala Arg
1810 1815 1820
Gin Lys Met Leu Ile Ala Ala Leu Glu'Ser Lys Leu Ala Gln Ala Glu
1825 1830 1835 1840
Glu Gin Leu Glu Gin Glu Ser Arg Glu Arg Ile Leu Ser Gly Lys Leu
1845 1850 1855
Val Arg Arg Ala Glu Lys Arg Leu Lys Glu Val Val Leu Gin Val Asp
1860 1865 1870
Glu Glu Arg Arg Val Ala Asp Gin Val Arg Asp Gin Leu Glu Lys Ser
1875 1880 1885
Asn Leu Arg Leu Lys Gin Leu Lys Arg Gin Leu Glu Glu Ala Glu Glu
1890 1895 1900
Glu Ala Ser Arg Ala Gin Ala Gly Arg Arg Arg Leu Gin Arg Glu Leu
1905 1910 1915 1920
Glu Asp Val Thr Glu Ser Ala Glu Ser Met Asn Arg Glu Val Thr Thr
1925 1930 1935
Leu Arg Asn Arg Leu Arg Arg Gly Pro Leu Thr Phe Thr Thr Arg Thr
1940 1945 1950
Val Arg Gin Val Phe Arg Leu Glu Glu Gly Val Ala Ser Asp Glu Glu
1955 1960 1965
Glu Ala Glu Gly Ala Glu Pro Gly Ser Ala Pro Gly Gin Glu Pro Glu
1970 1975 1980
Ala Pro Pro Pro Ala Thr Pro Gin
1985 1990
<210> 59
<211> 6786
<212> DNA
<213> Homo sapiens
<400> 59
ctctttctcc ccaggccgaa gcctcgggac ggccctggaa gccgaccatg gcagccgtga 60
ccatgtcggt gcccgggcgg aaggcgcccc ccaggccggg cccagtgccc gaggcggccc 120
agccgttcct gttcacgccc cgcgggccca gcgcgggtgg 'cgggcctggc tcgggcacct 180
ccccgcaggt ggagtggacg gcccggcgtc tcgtgtgggt gccttcggag cttcacgggt 240
tcgaggcggc ggcgctgcgg gacgaaggcg aggaggaggc ggaggtggag ctggcggaga 300
gcgggaggcg gctgcgactg ccgcgggacc agatccagcg catgaacccg cccaagttca 360
gcaaggccga ggacatggcc gagctgacct gcctcaacga ggcctcggtc ctgcacaacc 420
tccgggagcg gtactactcc ggcctcatct acacgtactc cggccttttc tgtgtggtca 480
tcaacccgta caagcagctt cccatctaca cagaagccat tgtggagatg taccggggca 540
agaagcgcca cgaggtgcca ccccacgtgt acgcagtgac cgagggggcc tatcggagca 600
tgctgcagga tcgtgaggac cagtccattc tctgcactgg agagtctgga gctgggaaga 660
cggaaaacac caagaaggtc atccagtacc tcgcccacgt ggcatcgtct ccaaagggca 720
ggaaggagcc gggtgtcccc ggtgagctgg agcggcagct gcttcaggcc aaccccatcc 780
tagaggcctt tggcaatgcc aagacagtga agaatgacaa ctcctcccga ttcggcaaat 840
tcatccgcat caactttgat gttgccgggt acatcgtggg cgccaacatt gagacctacc 900
tgctggagaa gtcgcgggcc atccgccagg ccaaggacga gtgcagcttc cacatcttct 960
accagctgct ggggggcgct ggagagcagc tcaaagccga cctcctcctc gagccctgct 1020
131
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08g po4opeqoab bobabqopPb bobeobqebq bb6pbqopob obebeobpob pobpoobo6u
OZGZ ebeop44pob bbeobo4obb 4ope4ebbbb opobqobeob 62poq4poqp 4bo4upg2oP
09T7Z boopo4bfyee bqoapbPbo6 EMPbeebbq obpopobb4o ogbbbbqobb booggoggo4
0oD. pbsp.ofyebpo ebbbqboboo eqpqoappoo Doebbqpepb Egobobbeop 4ebgebpe2e
OVEZ 5.464pobbeo bupbbbqebb qeo4qobbbp Poopo4epob qpeoppeoPb qopqebeboe
08ZZ Tebobeobbp oqqbebElpoo q4o4opqeob opeupopoq4 obbhupobo4 b4o4Pobooq.
. ozzz eobbbeb64o p46.6.6boepo 543634435e oD2b5qobqb 5405.6opoob ebb4o6pebb
09TZ boo5bbebee beboeopPeo poogfiggE,Db qbbpogbqqg gbepoopeep peopppbeog
OOTZ Deoepobb4e al_poboobeb 4opoi.bebbe epe4D4oneo ebatqbeoeb booqqbqe4b
opoz bbboq.boopo o5oobb4bbe ooepoobbo2 6065E14036u obpb45.Erepe ebbqDbbbbq
0861 5oqp05.66e6 6g5opb2Pub 54o4ebebpo 55pe64Db8o pe6Popo6p6 pDpeop4o5q.
OZ6T 400bpoboqb opeopEgeeb gogoopubb4 ppeeepp.64p bqobb4bebo pepobbaeou
0981 4Deboqb5ee obbbobouqo ppo434qb4b epg4oeboob beo4ebbbob 4popobbubo
008T obbobepogq. bPpooppeop BbobbbeobP 552poob2qb 5uebeElb4.64 Ti.boqbupou
OD'LT 5pop.po6bee boopq46.5qo bq5p6bebe 5b4DbqDoob .6q.Doqop65q. 00300OPPOO
0891 5boobbobeb oqpoqopebo quobqopobp ob4oppb343 obbqqqoebo gooqqoppbb
0Z91 4oppoq2abb beb4bob2po eq52bbebb2 obebb4a64.5 p4;642oop3 200ePo4404
09G1 o6eob2ob4o Bepbaboeep aeopqoP2pq 2obqo4Db2D beboqqDoqo peb435.2004
00gT qp4Pb2bqq.q obbbo5Dgeo ebbqopqeob bbqopqqopq DobobbeepD boDoppbeob
01717T poebbgi.pob bbopepal.po bobqoqqbbq obb4oboD44 oqopbobebo e4oppoobbe
08E1 2Dp5b4Doo6 .52.554obo63 qqoebqobbp peebb2243e beoDobe2eb 2o5454eqo2
ozET 6253p5bq4b eeeDgpobo4 poopeo4354 4po5e5oD34 0qqqe5bo25 4E6b6bq0eb
097T bbDqoDbo Dbqpqobepb Poepbqobeo popeoebqoD bqeopeoDbe poqpboDepe
00ZI ebbpeebebe beeb1.4o054 ge0e2obb4q 4beaDq344b epbeo434bb Tebbab4ob4
0171T eoo43.4e3qp EabbubDeo3 5eo44e5554 D34b55D5qo 63.4.5ebbqob Debebbeopq
0801 4D4D2Pbbbo 5,255200550 ooqDqpoqeD Ilbo30bboep D3ebgooqqb booeqoepoo
VO-VO-ETOZ ZETZT8Z0 VD
EET
09 SS OS
AID sTH naq n19 IeS old TEA daI TEA naq bay bay PTV 114.1, dcL TD
St7 017 SE
TPA UT D old l@S lIIL AID 19S AID cud AID AID AID PTV 39S cud AID
OE SZ OZ
bay 03d 3U aqd n9q cad uTD PTV PTV nTD old TEA old AT Old
ST OT
bay old Old ETV sic,' bay AID old TPA 1@S 4W -NI TPA PTV PTV 49W
09 <00f7>
suaTdes owoH <ETZ>
<ZTZ>
0661 <TTZ>
09 <OTZ>
98L9 oeBbqo
08L9 eb000epooq OPPPq.PUP.6P ob6q34obbe 004005P000 4000P44403 qbqbebbEEE
0ZL9 4Debb6q3q3 opagobebbo .1.4bbbeb-404 obqq.bblo44 333433 6;o 4obqe3p3bi..
0999 ob4obq3o1.o P34040303E bbbbeeoebb eabbbbeeeo b4400peeb4 4o3be3peo3
0099 qoobbeoppo buEbeoebob Eb43o4oeEb 4000pE3o4b ob40444E0o opoqqop4o4
op,09 oe3obe45oe 4o4oi.oepq3 4o6o4o4eeb be444qqoqe ofq.64o4o4o 4b65e6qoqo
ogp9 qoobb44o4-4 -404Eopeo4o 43o3eq3poq 4poqoq0004 opoqoqoqoq ogoqoqoqoq
0z179 oqoq.q.obgaq. poogqeopop Eobqbeopbe 04004'200P3 oqooqobbou befigbebbbe
09E9 EbbEqquaeb e4eq3q.E544 E4q534op34 qoqqqoaq.00 46qo3oqobe oTeooqqq5q.
009 b43bee3b54 qbeoeqbbbq oEbbbbbqqb -443fqqopeq obubbqoebb ueobbEbbbe
0f7z9 obbtobfibbo Beb5PbPOPO PPP40000P6 Tebbeepoqe opoqoppEob qopooqbbbq
ogT9 opeoopoopo qeqq4E3bbq oqoEobeob4 bbqeeebbqq. q000b4-43.44 oeb4o4000b
0Z19 opooeEbbuo ooqbqopoob bboe0000eb 643334334g op000bepoo bbbb4ebeoe
0909 eg3e6b4ebe opoo4b4000 E43p3eb4be op000epoob eopeopoo4b bbbeb4005e
0009 bboopoqeop bbbqoqb5b4 oobeoeobEE b5EbEobbEb beboeboo4E obbqbobbbE
03,6g bbebeqoebo oq.q.D4bbE33 b3b4bb3uo.5 003P3OPD44 30P0400000 5b353eb044
oggg 0bb33EE5bE 543p3P3oe5 4beEbqbo3e ubqeooqbeb 3p5boqbE5E opoqbqebEE
ozgg bb4obEbgb3 bE3b4o65e5 8 053065g bbeoqobbbo 00.4E365E65 EDBEEcobbE
09L0 bbebbqobeo 5Do6ee6q3.5 e3bEE04.5E5 oqqopEEEbb bEe5e5bqo6 eopEbbbooq.
on; obeopebqob 545bbE653b e5bE55E5bq b5Epoq3bq6 64bbebEEEq. lobbobeebE
(,9g bqobebeobo Erl.b.bqobeee 6b4oqpi_ooq eabobebEbe opEbEbEeob EbeqobEobe
08SS bbebqobbeo oobbqqbEE-4 oqbEbqqopo b4o6qqeoop b4Ebeeopoo b000bgb000
OZSS bbbbgobqeb bebbebqbbb gooboEbbbb oE43bebbeo ogEbeobboe ebbgobeobe
09170 obbobbbobe bebeobbeeo obe3434qq5 eobobebqob e34bq3bebe oeooebqoeo
0017; Mbebegbbeo bi.00qobqob eeobooegob 33E54e-234o 64obebeogo eeobEbEobE
op,gg bbebbebbqo bebEEbbebb q4bE355bbq opbobbbbEb bqobeo4bob EEbEbbebb4
ogzg oqqeoobeob bEeobeqqop epqbbTeEpo bEc4e,b2b4E5 eo65qube64 Ebbbooebbe
ozzg obeopobbeo bbobbo4obq booEbboqop boo5bq3pE6 bEbbeobqob bobqobqbbe
ogIg .64obbebbqo ob65uE343o bobEEEE54.5 eeEb65ogee bEopoqoqqo qpbebSebbb
0010 033.430E35o p3ebeb0e65 45bE6b6obb Teqo6Ebbee bqubeopobb eobqebeEpb
oog oqqobEobee bqbbobbebb Ebbeeobbbe pob5oobgo4 pobbqebEoq obbeEbqobE
086f7 5bebbqpbe5 ebbbebbqob Eebeeob000 bqobbqboob bqoqopobob eobEebbobe
0z6f7 bbebqebbbo bebbqbbebe ob4ebEbeb4 obEobEepob b4o5Eobbvb bobbebebEE
098P 6qbb4obbub qebgboobbb e36433eb4b obeb4eo6eo 4oEbee3l.o4 obbe3b4b43
008 Pb4bbEbb40 gbobi.obeeo obgebbeboo b535eoe543 bEbgebbebb goEebeoebq
bbeopobebo bq34ebgEe3 ob2ob52o2e be352q.bbb3 ob4o3beb3e 25b436a642
089 obqbabebep obbolbDeb ebbpPobeDb eb43b43236 bebbqobpbq abbbobqo3o
0z9D, bbboouebeo 6b3bebbqb ebbpbqboeo 6ftebbe3beb bubbebbq.De 35E63e3pb4
090f, 3u34643D3b 55343bbeb4 5D5.25.66oD5 650bea6be6 op655obebq 53eeb5e554
000 Eceobbboqqo e45qp6eo5b ee5256,2523 55qoqq36p3 opbqqqbpeo bobeobeebe
VO-VO-ETOZ ZETZT8Z0 VD
CA 02812132 2013-04-04
Phe Glu Ala Ala Ala Leu Arg Asp Glu Gly Glu Glu Glu Ala Glu Val
65 70 75 80
Glu Leu Ala Glu Ser Gly Arg Arg Leu Arg Leu Pro Arg Asp Gin Ile
85 90 95
Gin Arg Met Asn Pro Pro Lys Phe Ser Lys Ala Glu Asp Met Ala Glu
100 105 110
Leu Thr Cys Leu Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg
115 120 125
Tyr Tyr Ser Gly Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val
130 135 140
Ile Asn Pro Tyr Lys Gin Leu Pro Ile Tyr Thr Glu Ala Ile Val Glu
145 150 155 160
Met Tyr Arg Gly Lys Lys Arg His Glu Val Pro Pro His Val Tyr Ala
165 170 175
Val Thr Glu Gly Ala Tyr Arg Ser Met Leu Gin Asp Arg Glu Asp Gin
180 185 190
Ser Ile Leu Cys Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr
195 200 205
Lys Lys Val Ile Gin Tyr Leu Ala His Val Ala Ser Ser Pro Lys Gly
210 215 220
Arg Lys Glu Pro Gly Val Pro Gly Glu Leu Glu Arg Gin Leu Leu Gin
225 230 235 240
Ala Asn Pro Ile Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn
245 250 255
Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val
260 265 270
Ala Gly Tyr Ile Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys
275 280 285
Ser Arg Ala Ile Arg Gin Ala Lys Asp Glu Cys Ser Phe His Ile Phe
290 295 300
Tyr Gin Leu Leu Gly Gly Ala Gly Glu Gin Leu Lys Ala Asp Leu Leu
305 310 315 320
Leu Glu Pro Cys Ser His Tyr Arg Phe Leu Thr Asn Gly Pro Ser Ser
325 330 335
Ser Pro Gly Gin Glu Arg Glu Leu She Gin Glu Thr Leu Glu Ser Leu
340 345 350
Arg Val Leu Gly Phe Ser His Glu Glu Ile Ile Ser Met Leu Arg Met
355 360 365
Val Ser Ala Val Leu Gin Phe Gly Asn Ile Ala Leu Lys Arg Glu Arg
370 375 380
Asn Thr Asp Gin Ala Thr Met Pro Asp Asn Thr Ala Ala Gin Lys Leu
385 390 395 400
Cys Arg Leu Leu Gly Leu Gly Val Thr Asp She Ser Arg Ala Leu Leu
405 410 415
Thr Pro Arg Ile Lys Val Gly Arg Asp Tyr Val Gin Lys Ala Gin Thr
420 425 430
Lys Glu Gin Ala Asp Phe Ala Leu Glu Ala Leu Ala Lys Ala Thr Tyr
435 , 440 445
Glu Arg Leu Phe Arg Trp Leu Val Leu Arg Leu Asn Arg Ala Leu Asp
450 455 460
Arg Ser Pro Arg Gin Gly Ala Ser She Leu Gly Ile Leu Asp Ile Ala
465 470 475 . 480
Gly Phe Glu Ile Phe Gin Leu Asn Ser Phe Glu Gin Leu Cys Ile Asn
485 490 495
Tyr Thr Asn Glu Lys Leu Gin Gin Leu She Asn His Thr Met She Val
500 505 510
134
CA 02812132 2013-04-04
Leu Glu Gin Glu Glu Tyr Gin Arg Glu Gly Ile Pro Trp Thr Phe Leu
515 520 525
Asp Phe Gly Leu Asp Leu Gin Pro Cys Ile Asp Leu Ile Glu Arg Pro
530 535 540
Ala Asn Pro Pro Giy Leu Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe
545 550 555 560
Pro Lys Ala Thr Asp Lys Ser Phe Val Glu Lys Val Ala Gin Glu Gin
565 570 575
Gly Gly His Pro Lys Phe Gin Arg Pro Arg His Leu Arg Asp Gin Ala
580 585 590
Asp Phe Ser Val Leu His Tyr Ala Gly Lys Val Asp Tyr Lys Ala Asn
595 600 605
Glu Trp Leu Met Lys Asn Met Asp Pro Leu Asn Asp Asn Val Ala Ala
610 615 620
Leu Leu His Gin Ser Thr Asp Arg Leu Thr Ala Glu Ile Trp Lys Asp
625 630 635 640
Val Glu Gly Ile Val Gly Leu Glu Gin Val Ser Ser Leu Gly Asp Gly
645 650 655
Pro Pro Gly Gly Arg Pro Arg Arg Gly Met Phe Arg Thr Val Gly Gin .
660 665 670
Leu Tyr Lys Glu Ser Leu Ser Arg Leu Met Ala Thr Leu Ser Asn Thr
675 680 685
Asn Pro Ser Phe Val Arg Cys Ile Val Pro Asn His Glu Lys Arg Ala
690 695 700
Gly Lys Leu Glu Pro Arg Leu Val Leu Asp Gin Leu Arg Cys Asn Gly
705 710 715 720
Val Leu Glu Gly Ile Arg Ile Cys Arg Gin Gly Phe Pro Asn Arg Ile
725 730 735
Leu Phe Gin Glu Phe Arg Gin Arg Tyr Glu Ile Leu Thr Pro Asn Ala
740 745 750
Ile Pro Lys Gly Phe Met Asp Gly Lys Gin Ala Cys Glu Lys Met Ile
755 760 765
Gin Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Val Gly Gin Ser Lys
770 775 780
Ile Phe Phe Arg Ala Gly Val Leu Ala Gin Leu Glu Glu Glu Arg Asp
785 790 795 800
Leu Lys Val Thr Asp Ile Ile Val Ser Phe Gin Ala Ala Ala Arg Gly
805 810 815
Tyr Leu Ala Arg Arg Ala Phe Gin Lys Arg Gin Gin Gin Gin Ser Ala
820 825 830
Leu Arg Val Met Gin Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg His
835 840 845
Trp Gin Trp Trp Arg Leu Phe Thr Lys Val Lys Pro Leu Leu Gin Val
850 855 860
Thr Arg Gin Asp Glu Val Leu Gin Ala Arg Ala Gin Glu Leu Gin Lys
865 870 875 880
Val Gin Glu Leu Gin Gin Gin Ser Ala Arg Glu Val Gly Glu Leu Gin
885 890 895
Gly Arg Val Ala Gin Leu Glu Glu Glu Arg Ala Arg Leu Ala Glu Gin
900 905 910
Leu Arg Ala Glu Ala Glu Leu Cys Ala Glu Ala Glu Glu Thr Arg Gly
915 920 925
Arg Leu Ala Ala Arg Lys Gin Glu Leu Glu Leu Val Val Ser Glu Leu
930 935 940
Glu Ala Arg Val Gly Glu Glu Glu Glu Cys Ser Arg Gin Met Gin Thr
945 950 955 960
135
CA 02812132 2013-04-04
Glu Lys Lys Arg Leu Gin Gin His Ile Gin Glu Leu Glu Ala His Leu
965 970 975
Glu Ala Glu Glu Gly Ala Arg Gin Lys Leu Gin Leu Glu Lys Val Thr
980 985 990
Thr Glu Ala Lys Met Lys Lys Phe Glu Glu Asp Leu Leu Leu Leu Glu
995 1000 1005
Asp Gin Asn Ser Lys Leu Ser Lys Ser Gly Ser Cys Trp Lys Ile Val
1010 1015 1020
Trp Pro Ser Ser His Pro Arg Gin Leu Arg Arg Arg Arg Arg Ser Arg
1025 1030 1035 1040
Ala Ser Ile Ser Tyr Gly Ser Asn Met Arg Pro Gin Ser Gin Thr Trp
1045 1050 1055
Arg Asp Arg Leu Arg Lys Glu Glu Lys Gly Arg Gin Glu Leu Glu Lys
1060 1065, 1070
Leu Lys Arg Arg Leu Asp Gly Glu Ser Ser Glu Leu Gin Glu Gin Met
1075 1080 1085
Val Glu Gin Gin Gin Arg Ala Glu Glu Leu Arg Ala Gin Leu Gly Arg
1090 1095 1100
Lys Giu Glu Glu Leu Gin Ala Ala Leu Ala Arg Ala Glu Asp Glu Gly
1105 1110 1115 1120
Gly Ala Arg Ala Gin Leu Leu Lys Ser Leu Arg Glu Ala Gin Ala Ala
1125 1130 1135
Leu Ala Glu Ala Gin Glu Asp Leu Glu Ser Glu Arg Val Ala Arg Thr
1140 1145 1150
Lys Ala Glu Lys Gin Arg Arg Asp Leu Gly Glu Glu Leu Glu Ala Leu
1155 1160 1165 .
Arg GlY Glu Leu Giu Asp Thr Leu Asp Ser Thr Asn Ala Gin Gin Giu
1170 1175 1180
Leu Arg Ser Lys Arg Glu Gin Glu Val Thr Glu Leu Lys Lys Thr Leu
1185 1190 1195 1200
Glu Glu Glu Thr Arg Ile His Glu Ala Ala Val Gin Glu Leu Arg Gin
1205 1210 1215
Arg His Gly Gin Ala Leu Gly Glu Leu Ala Glu Gin Leu Glu Gin Ala
1220 1225 1230
Arg Arg Gly Lys Gly Ala Trp Glu Lys Thr Arg Leu Ala Leu Glu Ala
=
1235 1240 1245
Giu Val Ser Glu Leu Arg Ale Glu Leu Ser Ser Leu Gin Thr Ala Arg
1250 1255 1260
Gin Glu Gly Glu Gin Arg Arg Arg Arg Leu Glu Leu Gln Leu Gin Glu
1265 1270 1275 1280
Val Gin Gly Arg Ala Gly Asp Gly Glu Arg Ala Arg Ala Glu Ala Ala
1285 1290 1295
Glu Lys Leu Gin Arg Ala Gin Ala Giu Leu Glu Asn Val Ser Gly Ala
1300 1305 1310
Lou Asn Glu Ala Glu Ser Lys Thr Ile Arg Leu Ser Lys Glu Leu Ser
1315 1320 1325
Ser Thr Glu Ala Gin Leu His Asp Ala Gin Giu Leu Leu Gin Glu Glu
1330 1335 1340
Thr Arg Ala Lys Leu Ala Leu Gly Ser Arg Val Arg Ala Met Glu Ala
1345 1350 1355 1360
Glu Ala Ala Gly Leu Arg Glu Gin Leu Glu Glu Glu Ala Ala Ala Arg
1365 1370 1375
Glu Arg Ala Gly Arg Glu Leu Gin Thr Ala Gin Ala Gin Leu Ser Glu
1380 1385 1390
Trp Arg Arg Arg Gin Glu Glu Glu Ala Gly Ala Leu Glu Ala Gly Glu
1395 1400 1405
136
CA 02812132 2013-04-04
Glu Ala Arg Arg Arg Ala Ala Arg Glu Ala Glu Ala Leu Thr Gln Arg
1410 1415 1420
Leu Ala Glu Lys Thr Glu Thr Val Asp Arg Leu Glu Arg Gly Arg Arg
1425 1430 1435 1440
Arg Leu Gln Gln Glu Leu Asp Asp Ala Thr Met Asp Leu Glu Gln Gln
1445 1450 1455
Arg Gln Leu Val Ser Thr Leu Glu Lys Lys Gln Arg Lys Phe Asp Gln
1460 1465 1470
Leu Leu Ala Glu Glu Lys Ala Ala Val Leu Arg Ala Val Glu Glu Arg
1475 1480 1485
Glu Arg Ala Glu Ala Glu Gly Arg Glu Arg Glu Ala Arg Ala Leu Ser
1490 1495 1500
Leu Thr Arg Ala Leu Glu Glu Glu Gln Glu Ala Arg Glu Glu Leu Glu
1505 1510 1515 1520
Arg Gln Asn Arg Ala Leu Arg Ala Glu Leu Glu Ala Leu Leu Ser Ser
1525 1530 1535
Lys Asp Asp Val Gly Lys Ser Val His Glu Leu Glu Arg Ala Cys Arg
1540 1545 1550
Val Ala Glu Gln Ala Ala Asn Asp Leu Arg Ala Gln Val Thr Glu Leu
1555 1560 1565
Glu Asp Glu Leu Thr Ala Ala Glu Asp Ala Lys Leu Arg Leu Glu Val
1570 1575 1580
Thr Val Gln Ala Leu Lys Thr Gln His Glu Arg Asp Leu Gln Gly Arg
1585 1590 1595 1600
Asp Glu Ala Gly Glu Glu Arg Arg Arg Gln Leu Ala Lys Gln Leu Arg
1605 1610 1615
Asp Ala Glu Val Glu Arg Asp Glu Glu Arg Lys Gin Arg Thr Leu Ala
1620 1625 1630
Val Ala Ala Arg Lys Lys Leu Glu Gly Glu Leu Glu Glu Leu Lys Ala
1635 1640 1645
Gln Met Ala Ser Ala Gly Gln Gly Lys Glu Glu Ala Val Lys Gln Leu
1650 1655 1660
Arg Lys Met Gln Ala Gln Met Lys Glu Leu Trp Arg Glu Val Glu Glu
1665 1670 1675 1680
Thr Arg Thr Ser Arg Glu Glu Ile Phe Ser Gln Asn Arg Glu Ser Glu
1685 1690 1695
Lys Arg Leu Lys Gly Leu Glu Ala Glu Val Leu Arg Leu Gln Glu Glu
1700 1705 1710
Leu Ala Ala Ser Asp Arg Ala Arg Arg Gln Ala Gln Gln Asp Arg Asp
1715 1720 1725
Glu Met Ala Asp Glu Val Ala Asn Gly Asn Leu Ser Lys Ala Ala Ile
1730 1735 1740
Leu Glu Glu Lys Arg Gln Leu Glu Gly Arg Leu Gly Gln Leu Glu Glu
1745 1750, 1755 1760
Glu Leu Glu Glu Glu Gln Ser Asn Ser Glu Leu Leu Asn Asp Arg Tyr
1765 1770 1775
Arg Lys Leu Leu Leu Gln Val Glu Ser Leu Thr Thr Glu Leu Ser Ala
1780 1785 1790
Glu Arg Ser Phe Ser Ala Lys Ala Glu Ser Gly Arg Gln Gln Leu Glu
1795 1800 1805
Arg Gln Ile Gin Glu Leu Arg Gly Arg Leu Gly Glu Glu Asp Ala Gly
1810 1815 1820
Ala Arg Ala Arg His Lys Met Thr Ile Ala Ala Leu Glu Ser Lys Leu
1825 1830 1835 1840
Ala Gln Ala Glu Glu Gln Leu Glu Gln Glu Thr Arg Glu Arg Ile Leu
1845 1850 1855
137
CA 02812132 2013-04-04
Ser Gly Lys Leu Val Arg Arg Ala Glu Lys Arg Leu Lys Glu Val Val
1860 1865 1870
Leu Gin Val Glu Glu Glu Arg Arg Val Ala Asp Gin Leu Arg Asp Gin
1875 1880 1885
Leu Glu Lys Gly Asn Leu Arg Val Lys Gin Leu Lys Arg Gin Leu Glu
1890 1895 1900
Glu Ala Glu Glu Glu Ala Ser Arg Ala Gin Ala Gly Arg Arg Arg Leu
1905 1910 1915 1920
Gin Arg Glu Leu Glu Asp Val Thr Glu Ser Ala Glu Ser Met Asn Arg
1925 1930 1935
Glu Val Thr Thr Leu Arg Asn Arg Leu Arg Arg Gly Pro Leu Thr Phe
1940 1945 1950
Thr Thr Arg Thr Val Arg Gin Val Phe Arg Leu Glu Glu Gly Val Ala
1955 1960 1965
Ser Asp Glu Glu Ala Glu Glu Ala Gin Pro Gly Ser Gly Pro Ser Pro
1970 1975 1980 ,
Glu Pro Glu Gly Ser Pro Pro Ala His Pro Gin
1985 1990 1995
<210> 61
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 61
Ala Cys Gly Met Pro Tyr Val Arg Ile Pro Thr Ala
1 5 10
<210> 62
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 62
Ala Gly Cys Met Pro Tyr Val Arg Ile Pro Thr Ala
1 5 10
<210> 63
<211> 11
<212> PRT
<213> Homo sapiens
<400> 63
Leu Met Lys Asn Met Asp Pro Leu Asn Asp Ile
1 5 10
138
CA 02812132 2013-04-04
<210> 64
<211> 12
<212> PRT
<213> Homo sapiens
<400> 64
Leu Met Lys Asn Net Asp Pro Leu Asn Asp Asn Val
1 5 10
<210> 65
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
linker peptide
<400> 65
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
139
