U.S. patent application number 14/495020 was filed with the patent office on 2015-04-23 for methods and compositions for modulating tumor cell activity.
The applicant listed for this patent is National Research Council Of Canada. Invention is credited to Christiane Cantin, Anne E.G. Lenferink, Maureen O'Connor-McCourt.
Application Number | 20150111250 14/495020 |
Document ID | / |
Family ID | 37864593 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111250 |
Kind Code |
A1 |
O'Connor-McCourt; Maureen ;
et al. |
April 23, 2015 |
METHODS AND COMPOSITIONS FOR MODULATING TUMOR CELL ACTIVITY
Abstract
Antibodies which target clusterin, a protein involved in the
epithelial-to-mesenchymal transition of carcinoma cells, are
identified and characterized. The antibodies may be used to
modulate tumour cell activity through binding to clusterin.
Inventors: |
O'Connor-McCourt; Maureen;
(Beaconsfield, CA) ; Cantin; Christiane;
(Pierrefonds, CA) ; Lenferink; Anne E.G.;
(Lorraine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Research Council Of Canada |
Ottawa |
|
CA |
|
|
Family ID: |
37864593 |
Appl. No.: |
14/495020 |
Filed: |
September 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13268020 |
Oct 7, 2011 |
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14495020 |
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11991459 |
Mar 5, 2008 |
8044179 |
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PCT/CA2006/001505 |
Sep 13, 2006 |
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13268020 |
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60716086 |
Sep 13, 2005 |
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Current U.S.
Class: |
435/69.6 ;
435/320.1; 435/332; 536/23.53 |
Current CPC
Class: |
C07K 2317/34 20130101;
G01N 33/574 20130101; A61P 35/04 20180101; C07K 2317/56 20130101;
C07K 16/30 20130101; A61P 35/00 20180101; A61K 31/00 20130101; C07K
16/18 20130101; C07K 2317/76 20130101; C07K 2317/565 20130101 |
Class at
Publication: |
435/69.6 ;
536/23.53; 435/320.1; 435/332 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-35. (canceled)
36. A nucleic acid sequence that encodes a light chain variable
region and/or a heavy chain variable region of an antibody that
specifically binds clusterin, wherein the antibody is selected from
the group consisting of: a. an antibody comprising three
complementary determining regions of the light chain variable
region set forth in SEQ ID NO.:8 and three complementary
determining regions of the light chain variable region set forth in
SEQ ID NO.:20; b. an antibody comprising three complementary
determining regions of the light chain variable region set forth in
SEQ ID NO.:9 and three complementary determining regions of the
light chain variable region set forth in SEQ ID NO.:20; c. an
antibody comprising three complementary determining regions of the
light chain variable region set forth in SEQ ID NO.:10 and three
complementary determining regions of the light chain variable
region set forth in SEQ ID NO.:21; d. an antibody comprising three
complementary determining regions of the light chain variable
region set forth in SEQ ID NO.:11 and three complementary
determining regions of the light chain variable region set forth in
SEQ ID NO.:22, and; e. an antibody comprising three complementary
determining regions of the light chain variable region set forth in
SEQ ID NO.:12 and three complementary determining regions of the
light chain variable region set forth in SEQ ID NO.:23.
37. (canceled)
38. The nucleic acid of claim 36, wherein the antibody comprises:
a. a light chain variable region as set forth in SEQ ID NO.:8 and a
heavy chain variable region as set forth in SEQ ID NO.:20; b. a
light chain variable region as set forth in SEQ ID NO.:9 and a
heavy chain variable region as set forth in SEQ ID NO.:20; c. a
light chain variable region as set forth in SEQ ID NO.:10 and a
heavy chain variable region as set forth in SEQ ID NO.:21; d. a
light chain variable region as set forth in SEQ ID NO.:11 and a
heavy chain variable region as set forth in SEQ ID NO.:22 or; e. a
light chain variable region as set forth in SEQ ID NO.:12 and a
heavy chain variable region as set forth in SEQ ID NO.:23.
39. A vector or vectors comprising the nucleic acid of claim
36.
40. A cell comprising the nucleic acid of claim 36.
41. A method of making an antibody comprising culturing the cell of
claim 40 so that the antibody is produced.
42. A vector or vectors comprising the nucleic acid of claim
38.
43. A cell comprising the nucleic acid of claim 38.
44. A method of making an antibody comprising culturing the cell of
claim 43 so that the antibody is produced.
Description
PRIORITY CLAIM
[0001] This patent application is a continuation of U.S. Ser. No.
13/268,020 filed on Oct. 7, 2011 which is a divisional of U.S. Ser.
No. 11/991,459 filed on Mar. 5, 2008, now U.S. Pat. No. 8,044,179
issued on Oct. 25, 2011 which is a national stage filing under 35
U.S.C. .sctn.371 of international application No. PCT/CA2006/001505
filed on Sep. 13, 2006 which claimed priority to U.S. provisional
application No. 60/716,086 filed Sep. 13, 2005. The entire contents
of each of these priority applications are incorporated herein by
reference.
SEQUENCE LISTING
[0002] In accordance with 37 C.F.R. .sctn.1.52(e)(5), a Sequence
Listing in the form of a text file (entitled "Sequence Listing",
created on Sep. 24, 2014 of 80 kilobytes) is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to antibodies, peptides and small
molecules which bind clusterin, and their use in modulating tumor
cell activity.
BACKGROUND OF THE INVENTION
[0004] Carcinomas, the most common human malignancy, arise from
epithelial cells. Progression of epithelial cancers begins with the
disruption of cell-cell contacts as well as the acquisition of a
migratory (mesenchymal-like) phenotype. This phenomenon, which is
called an epithelial-to-mesenchymal transition (EMT), is considered
to be a crucial event in late stage tumor progression and
metastasis.
[0005] The secreted protein TGF-.beta. suppresses tumor growth
initially largely due to its growth inhibitory action on tumor
cells of epithelial origin, then at later stages promotes tumor
cell progression and metastasis. One mechanism by which TGF-.beta.
can promote tumor progression is through the induction of an
EMT.
[0006] Due to the dual role that TGF-.beta. plays in
carcinogenesis, direct inhibitors of TGF-.beta. may be risky since,
while they could benefit late stage tumors, they could also
accelerate preneoplastic lesions. A better therapeutic may be one
that inhibits the pro-oncogenic EMT-promoting action of TGF-.beta.,
while leaving the tumor suppressor growth-inhibitory action of
TGF-.beta. unaffected. To develop such an inhibitor it would be
necessary to identify the point at which there is a bifurcation of
the TGF-.beta. signaling pathway such that the mediators in one
branch of the pathway participate in the EMT response, but not the
growth inhibitory response to TGF-.beta.. Therapeutics that inhibit
mediators that lie exclusively in the EMT-promoting branch of the
TGF-.beta. signaling pathway will reduce metastasis while having
little or no effect on the acceleration of preneoplastic
lesions.
[0007] No TGF-.beta. signal pathway specific components have been
generally identified that promote or mediate the EMT-promoting
action of TGF-.beta., yet are not involved in the growth inhibitory
action of TGF-.beta..
[0008] In contrast, an endogenous protein (the YY1 nuclear factor)
has been identified that is able to interfere with (as opposed to
promote) the protumorigenic EMT action of TGF-.beta., while leaving
the tumor-suppressing action (growth inhibition) intact (Kurisaki
et al., 2004).
[0009] Inhibitors that target TGF-.beta. ligands, receptors and the
Smad signaling proteins are known. Specifically, soluble receptor
ectodomains, antibodies and other binding proteins are able to act
as antagonists by interacting with TGF-.beta. ligands and
sequestering them away from cell surface receptors. Small molecules
are available that inhibit the kinase activity of the Type I
TGF-.beta. receptor and endogenous inhibitors of the Smad signaling
proteins are also known. Since all of these signaling pathway
components are involved in both the pro- and anti-carcinogenic
actions of TGF-.beta., these inhibitors that target them may
benefit late stage tumors, however, they could also accelerate
preneoplastic lesions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A shows pictures of the morphology of JM01 cells and
expression of selected markers (E-cadherin (E-cad), .beta.-catenin
(.beta.-cat), Zona Occludens-1 (ZO-1) and F-actin) in the absence
(CTL panels) or presence of TGF-.beta. (TGF-.beta. panels).
[0011] FIG. 1B shows pictures of wounded JM01 cell layer in the
absence (CTL panels) or presence of TGF-.beta. (TGF-.beta. panels)
after 24 hrs of incubation.
[0012] FIG. 1C shows picture of wounded JM01 cell layer in the
absence (CTL panels) or presence of TGF-.beta. (TGF-.beta. panels)
in a black ink motility assay after 24 hrs of incubation.
[0013] FIG. 2A is a diagram representing changes in gene expression
induced by TGF-.beta. in JM01 cells at different time points (0.5,
1, 2, 4, 6, 12 and 24 hrs post-TGF-.beta. induction).
[0014] FIG. 2B is a diagram summarizing the number of genes in
which changes of expression is observed during the early stage
(0.5, 1 hr), middle stage (2, 4, 6 hr) and/or late stage (12, 24
hr) post-TGF-.beta. induction including genes that are common to
each of these stages.
[0015] FIG. 2C is a list of selected genes associated with a
mesenchymal tumor cell phenotype and with clinical tumor
progression.
[0016] FIG. 3A shows graphs and pictures of clusterin and
caveolin-1 gene expression upon induction of JM01 cells with
TGF-.beta.. The graph and picture on the left side illustrates
clusterin expression over time (0, 2, 4, 6, 12 and 24 hr) whereas
the graph and picture on the right side illustrates caveolin-1
expression over time (0, 2, 4, 6, 12 and 24 hr) as measured by
semi-quantitative PCR.
[0017] FIG. 3B is a picture of a Western blot performed on whole
cell lysates of JM01 cells treated with TGF-.beta. for 24 hrs and
illustrating clusterin and caveolin-1 protein levels upon induction
with TGF-.beta. (p-Clu: pre-clusterin; s-Clu: secreted mature
clusterin; Cav-1: caveolin-1).
[0018] FIG. 3C shows pictures of immunofluorescence microscopy data
illustrating the effect of TGF-.beta. induction on the expression
of clusterin and caveolin-1 in JM01 cells after 24 hrs of
treatment.
[0019] FIG. 4A shows pictures of immunofluorescence microscopy data
illustrating the localization of clusterin in JM01 cells upon
treatment with TGF-.beta. (TGF-.beta. panel) or in untreated cells
(CTL panel). The right side of the CTL and TGF-.beta. panels
represents Western blots performed on conditioned media harvested
from untreated cells or TGF-.beta.-treated JM01 cells using an
antibody raised against the C-terminus of the clusterin .beta.
chain.
[0020] FIG. 4B shows pictures of immunofluorescence microscopy data
of JM01 cells treated with TGF-.beta. for 24 hr (TGF-.beta.) or
with conditioned media obtained from JM01 untreated cells (CM CTL)
or from JM01 cells treated with TGF-.beta. (CM TGF-.beta.) in the
presence of an anti-TGF-.beta. antibody (Anti-TGF-.beta.), an
anti-clusterin antibody (Anti-clu) or without any antibody (left
panels). The right panel shows a picture of JM01 cells treated with
purified clusterin. The marker used for the immunofluorescence
microscopy assay is ZO-1.
[0021] FIG. 4C is an histogram representing the number of ZO-1
positive cells estimated from immunofluorescence microscopy
data.
[0022] FIG. 4D shows graphs of FACS analysis illustrating the level
of E-cadherin expression upon treatment of cells with clusterin
alone (clusterin), TGF-.beta. alone (TGF-.beta.), or with
TGF-.beta. and an anti-clusterin (TGF-.beta.1+anti-clu) in
comparison with control cells (CTL).
[0023] FIG. 5 shows pictures of 4T1 and DU145 wounded cells after
24 hrs of treatment with an anti-clusterin antibody (anti-clu) or
without treatment (CTL).
[0024] FIG. 6A shows pictures of JM01 cells treated with clusterin
alone (clusterin) or with TGF-.beta. in the absence or presence of
an anti-clusterin antibody (+anti-clu panel) or left untreated
(CTL) in a black ink motility assay.
[0025] FIG. 6B is an histogram showing the result of ink clearance
assays performed on cells treated with clusterin alone (clusterin)
or with TGF-.beta. in the absence or presence (+anti-clu) of an
anti-clusterin antibody. Results are expressed as ink
clearance/cell/24 hours relative to control cells to which a value
of 1 was attributed.
[0026] FIG. 6C is an histogram showing the results of cell growth
assay as measured by [.sup.3H]thymidine incorporation in cells
treated with clusterin alone (clusterin) or treated with TGF-.beta.
in the presence of an anti-clusterin antibody (+anti-clu panel), in
the presence of an anti-TGF-.beta. antibody (+anti-TGF-.beta.) or
in the absence of antibody as compared to untreated cells to which
a value of 100% was attributed.
[0027] FIG. 7 is a schematic illustrating the clusterin-dependent
and -independent TGF-.beta. pathway.
[0028] FIG. 8 shows pictures illustrating the effect of
anti-clusterin polyclonal antibodies on the motility of 4T1 cells
or JM01 cells in wound healing assays. 4T1 or JM01 cells were left
untreated (CTL) or treated with either TGF-.beta., an
anti-clusterin antibody (anti-clu), pre-immune sera of two rabbits
(Pre-Immune #9, Pre-Immune #10), sera of the same rabbits after
immunization with a clusterin peptide consisting of amino acids
421-437 (Immunized #9, Immunized #10). In addition to the tested
anti-clusterin sera, JM01 cells were also treated with
TGF-.beta..
[0029] FIG. 9A is a picture of Western blot experiments performed
after immunoprecipitation of recombinant human clusterin (500 ng)
with either 50 or 100 ng of selected anti-clusterin monoclonal
antibodies; i.e., the 8F6, 7B7, 16B5, 11E2, 7C12, 6E12, 20E11,
20G3, 18F4, 16C11, 21B12 and 7D6 monoclonal antibodies or the
commercial polyclonal C18 and monoclonal B5 antibodies and
SDS-PAGE. The first well was loaded with 500 ng of recombinant
human clusterin.
[0030] FIG. 9B is an histogram representing the data of ink
clearance assays performed on JM01 cells treated with TGF-.beta. in
the presence of the monoclonal anti-clusterin antibodies 8F6, 7B7,
16B5, 11E2, 7C12, 6E12, 20E11, 20G3, 18F4, 16C11, 21B12 and 7D6 or
the commercial polyclonal C18 and monoclonal B5 antibodies or left
untreated. Results are expressed as "Ink clearance/cell/24 hr"
relative to TGF-.beta.-treated JM01 cells without antibody (No mAb)
to which the value of 1 was attributed.
[0031] FIG. 10A is a schematic of an antibody competition assay
performed by SPR-biosensor, where a first antibody (mAb1) is
captured via a rabbit anti-mouse FC antibody (RAMFc) covalently
immobilized on the sensor chip. Recombinant human clusterin (Huclu)
is allowed to bind to the first antibody and a second antibody
(mAb2) is then flowed over the surface.
[0032] FIG. 10B is a schematic of another antibody competition
assay performed by SPR-biosensor, where a first antibody (mAb1) is
covalently immobilized on the sensor chip and where a solution of
recombinant human clusterin (Huclu) pre-incubated or not with a
second antibody (Ab2) is then flowed over the surface.
[0033] FIG. 11 is a table summarizing the results of the
competitions assays performed between selected antibodies.
[0034] FIG. 12 is a flow diagram illustrating the steps for the
isolation, sequencing and sequence analysis of the variable regions
of the monoclonal antibodies.
[0035] FIG. 13 shows the amino acid sequence of the variable
regions or the amino acid sequence of the frameworks and CDRs
sequence of monoclonal antibodies.
[0036] FIG. 14 shows the consensus CDR1 and CDR2 sequences obtained
from the alignment of the 16C11, 11 E2, 21B12 and 20E11 heavy chain
CDR1 or CDR2.
DETAILED DESCRIPTION OF THE INVENTION
[0037] FIG. 1: TGF-.beta. induces an epithelial to mesenchymal
transition (EMT) in JM01 cells.
[0038] (A) This transition is characterized by an elongated
morphology, the relocalization of the markers E-cadherin (E-cad),
.beta.-catenin (.beta.-Cat) and F-actin and the down-regulation of
the marker Zona Occludens-1 (ZO-1). (B) This morphology change is
accompanied by an increase in cell motility as shown in a wound
healing assay in which the cells' ability to migrate in to a
`scratch` area is monitored in the absence or presence of
TGF-.beta.. (C) A complementary black ink motility assay was also
used to visualize and quantify the motility of individual JM01
cells in the absence or presence of TGF-.beta.. The black ink which
is coated on the plastic sticks to the migrating cells, thereby
generating the white tracks. Both assays show that the presence of
TGF-.beta. increases the motility of the JM01 cells.
[0039] FIG. 2: Analysis of TGF-.beta.-induced gene expression
changes using microarray technology. (A) Extensive analysis of
microarray data obtained from 7 time-points (0.5, 1, 2, 4, 6, 12,
and 24 hrs) during the TGF-3 induction of the JM01 cell EMT allowed
for the identification of 328 genes that are modulated during the
early (0.5, 1 hr), middle (2, 4, 6 hr) or late (12, 24 hr) stages
of the transition. (B) Only 5 of these genes are affected over the
entire time-course. (C) By comparing our gene list with data on the
basal gene expression profiles of the NCI-60 cell line panel (some
of these cell lines exhibit a mesenchymal phenotype), and with
expression profiling data from clinical samples, we identified 15
genes from our list that are associated with a mesenchymal tumor
cell phenotype and with clinical tumor progression.
[0040] FIG. 3: Validation of the TGF-.beta. modulation of selected
gene expression and protein levels. (A) Semi-quantitative PCR
confirmed the TGF-.beta.-induced clusterin up-regulation and
caveolin-1 down-regulation thereby validating the microarray
analysis (microarray data shown below PCR results). (B) Western
blot analysis of whole cell lysates of JM01 cells treated for 24
hrs with TGF-.beta. demonstrated that these transcriptional changes
result in increased clusterin (p-clu=pre-clusterin; s-clu=secreted
mature clusterin) and decreased caveolin-1 (Cav-1) protein levels.
(C) Immunofluorescent microscopy of JM01 cells treated for 24 hrs
with TGF-3 further confirmed these changes in clusterin and
caveolin-1 protein levels through the visualization of these
proteins in the intact cell. Nuclei are stained blue, caveolin-1
and clusterin are stained green and the F-actin fibers are stained
red.
[0041] FIG. 4: Identification of secreted clusterin as a mediator
of the TGF-.beta. induced EMT. (A) Immunofluorescent microscopy
indicated that clusterin is localized to the secretory pathway in
JM01 cells and Western blot analysis of conditioned media (CM)
indicated that clusterin is secreted (s-clu). (B, C) JM01 cells
were treated for 24 hr with TGF-.beta., or CM taken from TGF-.beta.
treated JM01 cells, in the absence or presence of a antibody raised
against the C-terminus of the clusterin .beta. chain (anti-clu).
Using immunofluorescent microscopy of ZO-1 as a marker of the EMT
it was shown that the clusterin antibody blocks the induction of
the EMT by both TGF-.beta. and the CM indicating that secreted
clusterin is a necessary mediator in the TGF-.beta. EMT pathway.
Purified clusterin alone was also shown to promote the EMT
indicating that clusterin is not only necessary, but is sufficient
for EMT induction. (D) The induction of the EMT by clusterin alone
was further confirmed by using FACS analysis of the epithelial
marker E-cadherin to monitor the EMT.
[0042] FIG. 5: Clusterin acts as an EMT mediator in cell lines
other than the JM01 cells. 4T1 tumor cells (breast) and DU 145
tumor cells (prostate) were observed to secrete clusterin and
exhibit a motile phenotype in the absence of TGF-.beta.
stimulation. Using the wound healing assay to monitor the motility
of the 4T1 and DU145 cells, it was observed that a clusterin
antibody (anti-clu) inhibits the motility of these cells indicating
that clusterin is important for the maintenance of the TGF-.beta.
independent mesenchymal phenotype in these cells.
[0043] FIG. 6: Clusterin is a pivotal mediator in the pathway
leading to TGF-.beta. induction of EMT but not in the pathway
leading to TGF-.beta. growth inhibition. (A) Using the black ink
motility assay to monitor the EMT of the JM01 cells, it was
confirmed that a clusterin antibody blocks the TGF-.beta. induced
EMT and that clusterin alone promotes the EMT. (B) This result was
further confirmed by quantifying the motility change as area
cleared in the ink per cell. (C) In contrast, as monitored by the
incorporation of tritiated thymidine, it was shown that the
clusterin antibody does not block TGF-.beta. induced growth
inhibition and that clusterin alone does not promote growth
inhibition, indicating that clusterin is not a mediator in
TGF-.beta. growth inhibitory pathways.
[0044] FIG. 7: Clusterin is an essential mediator in a TGF-.beta.
tumor promoting pathway but not in its tumor suppressing pathway.
TGF-.beta. induces secretion of clusterin and antibodies raised
against the C-terminus of the clusterin .beta. chain block the
TGF-.beta.1 induced EMT, but not the growth inhibitory response of
the cells to TGF-.beta.. These results indicate that clusterin is a
necessary mediator in the TGF-.beta. EMT pathway but do not address
whether other TGF-.beta.-induced mediators act in concert with
clusterin to induce the EMT; that is, do not address the question
of whether clusterin alone mediates an EMT. The fact that purified
clusterin in the absence of TGF-.beta. also promotes an EMT
indicates that clusterin is sufficient to induce this
transition.
[0045] FIG. 8: Analysis of the neutralizing activity of
anti-clusterin polyclonal antibodies produced at BRI. Sera
collected from two rabbits (#9 and #10) immunized with a clusterin
peptide (a.a. 421-437) were confirmed to contain antibodies that
interact with the peptide using surface plasmon resonance (data not
shown), and were tested for their ability to inhibit cell motility
in a wound healing assay (1/25 dilution of rabbit serum). The mouse
mammary epithelial cell line, 4T1 (top), secretes clusterin and is
motile in the absence of TGF-.beta., whereas the JM01 cell line
(bottom) requires stimulation with TGF-.beta. to induce clusterin
production and cell motility. The sera of both rabbit #9 and #10
inhibit motility, with #10 serum being more potent. As expected,
the pre-immune sera of both rabbits does not affect motility. A
commercially available clusterin antibody is shown as a positive
control (anti-clu, Santa Cruz).
[0046] FIG. 9: Analysis of the activity of the anti-clusterin
monoclonal antibodies produced at BRI. (A) Immunoprecipitations of
recombinant human clusterin (500 ng) using either 50 or 100 ng of
each of 12 BRI-produced monoclonal antibodies (commercial
polyclonal (C18) and monoclonal (B5) antibodies were used as
positive controls). Samples were analyzed on a 12% reducing
SDS-PAGE. All antibodies were observed to interact with recombinant
clusterin by immunoprecipitation. (B) Assessment of the ability of
the 12 BRI-produced monoclonal antibodies to inhibit the TGF-b
induced motility of JM01 cells using the black ink motility assay
(commercial polyclonal (C18) and monoclonal (B5) antibodies were
used as positive controls). The bar graph shows the relative values
of the motility of the TGF-b treated BRI-JM01 cells in the presence
of the various antibodies. Five BRI-produced monoclonal antibodies
(21 B12, 20E11, 16C11, 16B5 and 11 E2) inhibit the TGF-b induced
motility of the BRI-JM01 cells. Values are expressed as the
clearance/cell/24 hr relative to that of the TGF-b treated
(control) cells. The * illustrates the cut-off value that was used
when assessing neutralizing ability. When using this cut-off value
in the black ink motility assay, there was a good agreement with
the evaluation of the neutralizing ability of the monoclonal
antibodies when using the wound healing motility assay (data not
shown).
[0047] FIG. 10: Two SPR-biosensor (Biacore) approaches to analysing
the relationship between the epitopes of antibodies. (A) In the
first approach, a rabbit anti-mouse Fc antibody (RAMFc) is
covalently immobilized on the sensor chip and one monoclonal
(termed Ab 1) is captured on the surface. After binding clusterin
to Ab1, the second monoclonal antibody (termed Ab 2) is flowed over
the surface. If the epitopes of the two antibodies are overlapping,
then Ab2 will not be able to bind to Ab1-bound clusterin. If the
two antibodies have unrelated epitopes, then Ab2 will be able to
bind to Ab1 -bound clusterin. (B) In the second approach, one
monoclonal (termed Ab 1) is covalently immobilized on the sensor
chip surface. Clusterin is then incubated with a second antibody
(monoclonal or polyclonal, termed Ab2) in solution and the complex
is then flowed over Ab1. If the epitopes of the two antibodies are
overlapping, then Ab2-bound clusterin will not be able to bind to
Ab1.
[0048] FIG. 11: Results of the analysis of the relationship of the
epitopes of the 5 EMT neutralizing BRI-produced anti-clusterin
monoclonals antibodies with each other, and with the peptide
epitopes of the C18, pAb#10 and B5 antibodies. This table
summarizes all the epitope mapping results obtained using the two
SPR-biosensor (Biacore) approaches. A blue + indicates that Ab1
competed with Ab2 for binding to clusterin in the first Biacore
approach (i.e. the ratio of RUs of Ab2 to RUs of bound clusterin
was 0.1 or less). A red + or +/- indicates that Ab2 competed with
Ab1 for binding to clusterin in the second Biacore approach (i.e.
the binding of clusterin to Ab1 was inhibited between 30-100% for
+, and between 10-30% for +/-, when preincubated with Ab2). It is
evident that all of the five neutralizing monolconal antibodies
(21B12, 20E11, 16C11, 16B5 and 11 E2) interact with the overlapping
peptide epitopes of pAb#10, pAbC18 and mAb B5 since they all
compete for each other, and for pAb#10, pAbC18 and mAb B5. *It
should be noted that all of the negative results from the first
approach (blue -) occurred when Ab 20E11 was used (either as Ab1 or
Ab2) indicating that this Ab did not behave well in that
experimental set up. Therefore, for Ab 20E11, conclusions are taken
primarily from the second experimental approach.
[0049] A first object of the invention is to identify a method for
inhibiting EMT in tumour cells without inhibiting the
tumour-suppressing activity of TGF-.beta..
[0050] A further object of the invention is to identify molecules
or compositions which may inhibit TGF-.beta.-induced EMT in tumour
cells without inhibiting the tumour-supressing activity of
TGF-.beta..
[0051] A first aspect of the invention provides for an agent having
a binding affinity for clusterin, wherein binding of the agent to
clusterin inhibits epithelial-to-mesenchymal transition in
carcinoma cells. In particular, the agent may bind to the
.beta.-subunit of clusterin, and more specifically, it may bind to
the C-terminal portion of the clusterin .beta.-subunit. The agent
may, for example, be an antibody, including a monoclonal or
polyclonal antibody.
[0052] A second aspect of the invention provides for a method for
modulating the activity of carcinoma cells, comprising the steps of
exposing the cells to an agent having a binding affinity for
clusterin.
[0053] A further aspect of the invention provides for the use of an
amino acid sequence in the generation of agents having a binding
affinity for clusterin, wherein the sequence comprises SEQ ID NO.:
4 or a portion thereof. In particular, the sequence may comprise
shorter portions of SEQ ID NO.: 4, including SEQ ID NO.: 1, SEQ ID
NO.: 2, SEQ ID NO.: 3, and SEQ ID NO.: 5.
[0054] A further aspect of the invention provides for a vaccine
comprising clusterin or a portion thereof which is involved in
epithelial-to-mesenchymal transition in carcinoma cells, and a
pharmaceutically suitable carrier. The portion of clusterin may
comprise SEQ ID NO.: 4 or a portion thereof.
[0055] A further aspect of the invention provides for the use of an
amino acid sequence in the preparation of a vaccine, wherein the
sequence comprises SEQ ID NO.: 4 or a portion thereof. In
particular, the sequence may comprise shorter portions of SEQ ID
NO.: 4, including SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, and
SEQ ID NO.: 5.
[0056] A further aspect of the invention provides for a nucleic
acid sequence that encodes at least one of SEQ ID NO.: 1 through
SEQ ID NO.: 30.
[0057] A further aspect of the invention provides for the use of an
agent with a binding affinity for clusterin as a diagnostic tool,
wherein binding of the agent to clusterin inhibits
epithelial-to-mesenchymal transition in carcinoma cells.
[0058] It is disclosed herein that clusterin is a therapeutic
target whose inhibition blocks EMT without preventing TGF-.beta.'s
anti-proliferative tumor suppressor action.
[0059] Clusterin was first identified as a protein possibly
involved in EMT using transcriptome analysis, then was analyzed to
identify potential binding sites within clusterin. Synthetic
peptides were created accordingly, and antibody preparations
directed against these peptides were produced or purchased.
Additionally, twelve monoclonal antibodies were isolated using
full-length recombinant clusterin as the antigen. Both the
anti-peptide antibody preparations and the twelve monoclonal
antibodies were confirmed to bind to recombinant clusterin. The
anti-peptide polyclonal antibody preparations and five of the
twelve monoclonal antibodies were shown to inhibit EMT. These five
neutralizing monoclonal antibodies were shown to interact with the
same peptide epitope as the anti-peptide antibodies.
[0060] Using semi-quantitative RT-PCR, Western blot and
immunofluorescent microscopy analysis, it was confirmed that
several of the EMT-associated transcriptional changes that were
detected by microarray analysis were reflected in changes in
message and protein abundance (clusterin and caveolin are shown in
FIG. 3). Anti-peptide antibodies were used to demonstrate that
clusterin is an essential EMT mediator that is not involved in
TGF-.beta.'s growth inhibitory pathways (FIGS. 4-6). These results
indicate that clusterin is an accessible therapeutic target whose
inhibition blocks EMT without preventing TGF-.beta.'s
anti-proliferative tumor suppressor action.
[0061] The epitope within clusterin that is important for the
generation of EMT-inhibiting agents was elucidated using
anti-peptide antibody preparations in neutralization assays. Two
different commercial polyclonal antibody preparations raised
against synthetic peptides corresponding to sections of the
C-terminus of the clusterin .beta. sub-unit were used. The first
antibody (from RDI Research Diagnostics Inc.) was raised against
the synthetic peptide corresponding to amino acids 421-437 of
clusterin (VEVSRKNPKFMETVAEK, SEQ ID NO 1) (termed RDI) and the
second antibody (from Santa Cruz Biotechnology Inc.) was raised
against the synthetic peptide corresponding to amino acids 432-443
of clusterin (ETVAEKALQ EYR, SEQ ID NO 2) (termed C-18). An
anti-peptide monoclonal antibody against the same peptide (SEQ ID
NO 2) was also purchased (termed B5). The overlap between these two
epitopes is shown below. The ability of these antibody preparations
to block EMT indicates the significance of the C-terminal portion
of the clusterin .beta. subunit in inducing EMT (FIG. 4-6, C-18
results shown; similar results obtained with RDI).
##STR00001##
[0062] Prediction of Putative Functional Subdomains in Clusterin
Based on Structural Bioinformatics
[0063] Generally, clusterin is thought to be a protein that is only
partially structured, containing molten globule fragments.
Additionally, it has been classified as an intrinsically disordered
protein. Clusterin is postulated to contain several independent
classes of binding sites capable of interacting with numerous other
binding partners.
[0064] The clusterin sequence was examined using bioinformatics
programs, namely: [0065] PredictProtein (Rost, 1996). [0066]
GenTHREADER (Jones, 1999). [0067] COILS (Lupas, 1996). [0068] PONDR
(Li et al., 1999)
[0069] The C-terminal fragment of the .beta.-subunit was identified
as a putative binding region. The fragment (a.a. 375-449, SEQ ID
NO.: 4), which starts after the second coiled-coil region, is
likely unfolded but has some propensity for .beta.-sheet
formation.
[0070] A synthetic peptide was produced corresponding to a.a.
421-437 of clusterin in order to generate polyclonal antibody
preparations at BRI that are similar to the commercial antibody 1
preparation (RDI) (these new polyclonal preparations are termed
pAb#9 and #10). Additionally, full-length human clusterin was
expressed in 293 cells and purified in order to use as antigen to
generate monoclonal antibodies against full-length human clusterin.
Twelve monoclonal antibodies were raised against full-length
clusterin and were demonstrated to interact with clusterin by
ELISA. These twelve antibodies are named 6E12, 7B7, 21B12, 20G3,
20E11, 18F4, 16C11, 16B5, 11E2, 8F6, 7D6, 7C12.
[0071] The polyclonal antibody preparations raised against the a.a.
421-437 epitope (pAb#9 and #10) were confirmed to inhibit the EMT
(FIG. 8).
[0072] All twelve monoclonal antibody preparations raised against
full-length human clusterin were confirmed to interact with
recombinant human clusterin as evidenced by their ability to
immunoprecipitate clusterin (FIG. 9A). Five of the twelve
monoclonals were shown to be able to neutralize the EMT promoting
action of clusterin in the black ink cell motility assay (FIG. 9B)
and the wound healing cell motility assay (not shown). The five
monoclonal antibodies that neutralize are 11E2, 21B12, 20E11,
16C11, 16B5.
[0073] Two Surface Plasmon Resonance (SPR)-based biosensor epitope
mapping assays (FIG. 10) were used to determine whether the five
neutralizing monoclonal antibodies generated using full-length
clusterin were interacting with the same clusterin peptide epitope
as the anti-peptide antibody preparations.
[0074] The two approaches that were used are described below:
[0075] 1) The monoclonal antibodes were individually captured on a
CM5 sensor chip surface on which a Rabbit-anti-Mouse Fc antibody
was covalently immobilized (when captured, the mAb is termed mAb1
in this experimental approach). Clusterin was then allowed to bind
to mAb1. Then all five monoclonal antibodies were sequentially
injected over mAb1 -bound clusterin (the injected mAb is termed
mAb2 in this experimental approach) in order to determine if both
mAb1 and mAb2 are able to interact with clusterin simultaneously
(FIG. 11). It was found that all of the five neutralizing mAbs
(except 20E11 in some cases) competed with each other for binding
to clusterin (when used both as mAb1 or as mAb2). Additionally,
they were found to compete with the C18, pAb#10 and B5 anti-peptide
antibodies, suggesting that the five neutralizing mAbs interact
with the overlapping peptide epitopes of pAb#10, pAbC18 and mAb B5.
It should be noted that, although Ab 20E11 appeared to have a
distinct epitope in some cases (when used either as mAb1 or mAb2),
this conclusion was not supported by the results of the second
experimental approach.
[0076] 2) The monoclonal antibodies were individually covalently
immobilized on a CM5 sensor chip surface using amine coupling (when
immobilized, the mAb is termed mAb1 in this experimental approach).
To demonstrate competition for binding to clusterin, an Ab (termed
Ab2 in this approach) was then incubated with clusterin prior to
injection of the complex over the mAb1 surface (FIG. 11).
[0077] It was confirmed that all of the five neutralizing mAbs
competed with each other for binding to clusterin, and with the
C18, pAb#10 and B5 anti-peptide antibodies. This confirms that the
five neutralizing mAbs interact with the overlapping peptide
epitopes of pAb#10, pAbC18 and mAb B5.
[0078] The hypervariable complementary determining regions (CDRs)
of all twelve monoclonal Abs were sequenced. Mammalian light- and
heavy-chain Igs contain conserved regions adjacent to the CDRs and
the use of appropriately designed oligonucleotide primer sets
enabled the CDRs to be specifically amplified using PCR (FIG. 12).
These products were then sequenced directly (SEQ ID NO 8-30; see
FIG. 13).
[0079] By aligning the CDR sequences of four out of the five
neutralizing monoclonal antibodies (11E2, 21B12, 20E11, 16C11), we
were able to determine a consensus sequence for VH CDR1 and CDR2 of
these anti-clusterin antibodies (see FIG. 14). The following
consensus sequences were determined: CDR-1: G-Y-S/T-F-T-X-Y-X (SEQ
ID NO.: 6) and CDR-2: I-N/D-P/T-Y/E-X-G-X-P/T (SEQ ID NO.: 7).
[0080] The antibodies or peptides that interact with the epitope of
clusterin defined here may be applied as therapeutics, i.e. they
may act as a therapeutic in their own right due to their intrinsic
ability to neutralize the EMT promoting activity of clusterin.
Additionally, these antibodies and peptides may be used as a
therapeutic due to their ability to target toxins, suicide genes or
other agents with anti-tumor activity to the vicinity of tumor
cells through their interaction with secreted clusterin.
[0081] Small molecules that interact with the epitope of clusterin
defined here may also act as therapeutics by blocking the EMT
promoting activity of clusterin. These antibodies, peptides and
small molecules that exert their therapeutic activity by
interacting with this clusterin epitope may exhibit less toxicity
or side-effects as compared to other agents that remove all
activities of clusterin, i.e. antisense or RNAi agents, since,
while the EMT activity of clusterin is neutralized when this
epitope is blocked, the other activities of clusterin may remain
intact.
[0082] Other applications of the antibodies and peptides that
interact with the epitope of clusterin defined here may be as 1)
non-imaging diagnostics, i,e, they may detect clusterin as a
biomarker in accessible body fluids or in tissue/tumor samples for
diagnostic and prognostic applications in cancer, and 2) imaging
diagnostics, i.e. they may be used to target contrast agents to
tumors for imaging in vivo due to their interaction with secreted
clusterin.
[0083] Antibodies comprising the heavy and light sequences
identified herein, antibodies comprising the CDRs (complementarity
determining regions) identified herein (FIG. 13), and antibodies
comprising the consensus sequences (FIG. 14) are expected to be
useful for the above-mentioned purposes.
[0084] Clusterin itself, or the portions thereof which contain the
epitope recognized by the antibodies and peptides discussed above,
may be used as a vaccine. Preferably, the clusterin should be
combined with a pharmaceutically suitable carrier. Clusterin or
epitope-containing portions of clusterin may also be used in the
generation of vaccines. Similarly, amino acid sequences having at
least 90% identity with SEQ ID NO. 4 or the clusterin epitope
identified herein will also be useful, since they are likely to
have similar functionality to the specific sequences identified
herein.
[0085] Cell Culture, Antibodies and Reagents
[0086] BRI-JM01 cells were isolated and characterized as described
(Lenferink et al., Breast Cancer Res., 6, R514-30 (2004)). Cells
were maintained at 37.degree. C. in a humidified, 5% CO.sub.2
atmosphere and cultured in DF/5% FBS (1:1 mixture of Ham's F12 and
Dulbecco's modified Eagles Medium (DMEM) with 5% Fetal Bovine Serum
(FBS) and antibiotics/antimicotics (both Wisent Inc.)).
[0087] Human recombinant TGF-.beta.1 and pan-TGF-.beta.
neutralizing antibody 1D11 were reconstituted according to the
manufacturer's instructions (R&D Systems). Purified human serum
clusterin was kindly provided by Dr M R Wilson (Wilson and
Easterbrook-Smith, 1992). Purified human recombinant clusterin was
produced in HEK-293 cells (general expression system described in
Durocher et al, 2002). Antibodies against the following proteins
were purchased and used in the indicated v/v dilutions: E-cadherin
(E-cad, anti-uvomorulin clone Decma-1; Sigma), Zona Occludens-1
(ZO-1; Chemicon), polyclonal antibodies raised against the
C-terminus of the human clusterin .beta. chain (clu.beta.; RDI and
Santa Cruz), and Caveolin-1 (cav-1; Santa Cruz). Horseradish
peroxidase (HRP) conjugated antibodies were obtained from Jackson
ImmunoResearch Laboratories Inc and Alexa-488 labeled antibodies
and Texas-red labeled phalloidin were purchased from Molecular
Probes. All experiments were carried out with 75-80% confluent
monolayers of BRI-JM01 cells in DF/5%. Where indicated, cells were
treated for 24 hr or 48 hr with TGF-.beta.1 or purified clusterin
at a final concentration of 100 .mu.M or 200 nM, respectively.
[0088] RNA Isolation and Labeling
[0089] Monolayers of BRI-JM01 cells were grown in the absence or
presence of TGF-.beta.1 for 30 min, 1, 2, 4, 6, 12 or 24 hr. PolyA+
mRNA was extracted (4.times.150 mm dishes per time point) using the
FastTrack.TM. 2.0 kit (Invitrogen) according to the manufacturer's
instructions. RNA was isolated and labeled according to Schade et
al., 2004.
[0090] Hybridization and Data Analysis
[0091] cDNA microarrays (15,264 sequence verified mouse ESTs) were
obtained from the University Health Network Microarray Center in
Toronto. Slides were hybridized with Cy3 or Cy5 labeled cDNA as
described (Enjalbert et al., 2003), scanned using a ScanArray 5000
(Perkin Elmer v2.11) at a 10-micron resolution and 16-bit TIFF
files were quantified using QuantArray software (Perkin Elmer,
v3.0). Microarray data normalization and analysis was performed as
described (Enjalbert et al., 2003).
[0092] Northern Blot and Semi-Quantitative RT-PCR (SQ-RT-PCR)
Analysis
[0093] For SQ-RT-PCR, 3-5 .mu.g of total RNA was amplified in a 20
.mu.l first-strand RT-PCR reaction using 50 U Superscript II
(Invitrogen) according to the manufacturer's guidelines with
modifications. Samples were preincubated (2 min, 42.degree. C.)
before adding Superscript II and the RNaseOUT treatment was
omitted. Samples were incubated (90 min, 42.degree. C.) and then
cooled on ice. Two .mu.l of first-strand reaction was added to the
PCR mix (2.5 U Taq polymerase (New England Biolabs), 10 .mu.M
forward/reversed primers) in a final volume of 50 .mu.l, which was
heated (2 min, 94.degree. C.) prior to PCR amplification. Primers
for the generation of the probes used for northern blot and
SQ-RT-PCR are listed in Table 1.
[0094] Western Blot Analysis
[0095] BRI-JM01 cells grown in 35 mm dishes were treated with
TGF-.beta.1 (24 hr). Cells were lysed in hot 2% SDS. Fifty .mu.g of
total protein or 30 .mu.l of conditioned medium was resolved by
SDS-PAGE (10%) under reducing conditions. Proteins were transferred
to nitrocellulose and membranes incubated with primary antibodies
(clu.beta., cav-1; 1/500) in TBS-T (20 mM Tris-HCl (pH 7.6), 137 mM
NaCl, 0.1% Tween 20 (v/v)) containing 5% non-fat milk (overnight,
4.degree. C.). Membranes were washed with TBS-T, incubated with
secondary HRP-conjugated antibody (1/20,000) in TBS-T+5% milk (1
hr), and washed with TBS-T. Immunoreactive bands were visualized
using Enhanced Chemiluminescence (ECL; Perkin Elmer).
[0096] Immunofluorescence Microscopy
[0097] BRI-JM01 cells were seeded in glass chamber slides (Lab-Tek)
and treated with purified clusterin or TGF-.beta.1 preincubated (30
min) with or without clu.beta. antibody (8 .mu.g/ml) or 1D11 (100
nM). Conditioned medium, obtained from non-treated and TGF-.beta.1
-treated BRI-JM01 cells (24 hr), was preincubated (30 min) with
these antibodies prior to incubation with non-treated BRI-JM01
cells. After 24 hr of exposure, cells were fixed with 4%
para-formaldehyde (10 min), rinsed twice (PBS), permeabilized (2
min, 0.2% Triton X-100 in PBS), rinsed again, and non-specific
sites were blocked with 10% FBS in PBS (40 min). Para-formaldehyde
fixed cells were then incubated (1 hr) with primary antibody
(E-cad, 1/200; ZO-1, 1/100; clu.beta., cav-1; 1/50) in PBS/10% FBS,
were rinsed (4.times. in PBS) and finally were incubated with
fluorescently conjugated secondary antibodies (Molecular Probes).
Simultaneously, F-actin filaments were labeled with Texas-red
labeled phalloidin (1/100) and nuclei were counterstained with 0.4
.mu.g/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma). Slides were
rinsed (PBS) and mounted using Prolong anti-fade (Molecular
Probes). Fluorescent images were captured using a Princeton
Instrument Coolsnap CCD digital camera mounted on Leitz Aristoplan
microscope and analyzed using Eclipse (Empix Imaging Inc.) and
Photoshop (Adobe) software.
[0098] Cell Proliferation Assays
[0099] BRI-JM01 cells (2.5.times.10.sup.4 cells/well) were seeded
in 24-well plates. The next day the medium was replenished and
purified clusterin, TGF-.beta.1, or TGF-.beta.1 pre-incubated for
30 min with 1D11 antibody (100 nM) or clu.beta. antibody (8
.mu.g/ml), was added to the cells. After 24 hr, cells were
pulse-labeled with 0.5 .mu.Ci/ml [.sup.3H]thymidine (Amersham),
rinsed (PBS, 4.degree. C.), trypsinized and [.sup.3H]thymidine
incorporation was evaluated by liquid scintillation counting.
[0100] Cell Motility Assays
[0101] Cells (2.times.10.sup.4 cells/well) were seeded in
ink-coated 12-well plates according to Al-Moustafa et al. (1999) in
the absence or presence of TGF-.beta.1, TGF-.beta.1+clu.beta.
antibody, or purified clusterin. Images were captured after 24 hr
using a Nikon Coolpix 995 digital camera mounted on Leitz
Aristoplan microscope and particle-free tracks were quantified
using ImageJ freeware.
[0102] Black Ink Motility Assay
[0103] Cells (2.times.10.sup.4 cells/well) were seeded in
ink-coated 12-well plates according to Al-Moustafa et al. (1999) in
the absence or presence of TGF-.beta.1, TGF-.beta.1+clu.beta.
antibody, or purified clusterin. Images were captured after 24 hr
using a Nikon Coolpix 995 digital camera mounted on Leitz
Aristoplan microscope and particle-free tracks were quantified
using ImageJ freeware.
[0104] Wound Healing Motility Assay
[0105] Confluent cell monolayers (12-well plates) were "wounded'
using a 2 .mu.L pipet tip. The medium was then replenished, to
remove cell debris, and the anti-clusterin mAbs were added (final
concentration of 4 .mu.g/mL) in the absence or presence of 100 pM
TGF-.beta.. Images of the wound were captured prior to and after 24
hr of incubation using a Nikon Coolpix 995 digital camera mounted
on Leitz Aristoplan microscope.
[0106] Polyclonal Antibody Production
[0107] The peptide (a.a. 421-437 of the clusterin protein) was
produced and purified at the University of Calgary. An extra
cysteine was added to the C-terminus of the peptide to facilitate
oriented coupling on the surface of the CM-5 sensor chips that were
used for screening of the rabbit antisera by surface plasmon
resonance (SPR, Biacore.TM. 2000). The peptide was coupled to
Keyhole Lympet Hemocyanin (KLH, Imject Mariculture KLH; Pierce)
using either glutaraldehyde (Sigma) or
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCL (Pierce) and
dialyzed against PBS (overnight at 4.degree. C.). The peptide
preparations that were conjugated by the two methods were mixed
(1:1). Pre-immune serum was drawn from two female New Zealand white
rabbits (10 ml), which were then injected with the KLH-coupled
peptide preparation (1.25 ug peptide per leg/0.5 ml Freund's
Incomplete Adjuvant or PBS). Animals were boosted (1.25 ug peptide
per leg/0.5 ml Freund's Incomplete Adjuvant or PBS) every third
week and serum was drawn (6 ml/kg) every 10 days after each boost
until the antibody titer did not increase, at which point the
animals were euthanized and exsanguinated.
[0108] Sera were tested for antibody activity using SPR. For this,
the peptide was coupled to a CM-5 sensor chip (Biacore Inc.) using
the Thiol coupling method (as described by the manufacturer) and
dilutions (1/50) of the pre-immune sera, the antibody-containing
sera and the commercially available anti-clusterin antibody (Santa
Cruz) were run over the peptide surface.
[0109] Monoclonal Antibody Production
[0110] Four BALB/c mice were injected subcutaneously (s.c.) and
intra-peritoneally (i.p.) with 35 .mu.g of purified human clusterin
emulsified in TiterMax adjuvant (Pierce). Animals were re-injected
i.p. three weeks later and the serum titer was assessed 10 days
later. Ten weeks later, responsive mice was boosted by i.p.
injections (50 .mu.g purified clusterin) and sacrificed three days
later. Spleen cells harvested, fused with NSO myeloma cells and
immediately plated (5.times.10.sup.4 cells/well in 96-well
microplates; Costar) in Iscove's medium supplemented with 20% FBS,
100 .mu.M hypoxanthine, 0.4 .mu.M aminopterin and 16 .mu.M
thymidine (HAT medium), murine IL-6 (1 ng/ml), penicillin (50 U/ml)
and streptomycin(50 .mu.g/ml). Supematants (10-20 days post-fusion)
were tested for anti-clusterin activity on immobilized purified
clusterin by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody
producing cells were cloned and retested twice for anti-clusterin
activity. Thirteen anti-clusterin antibody producing clones were
generated of which frozen stocks were prepared and a large-scale
antibody production was initiated.
[0111] SPR-Based Biosensor (Biacore) Epitope Mapping
[0112] Approach 1: [0113] Running buffer: [0114] HBS (20 mM Hepes
(pH7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) [0115] All
experiments were run at 5 .mu.L/min [0116] Standard amine coupling
of the anti mouse Fc immunoglobulin: [0117] Inject 35 .mu.L of a
mixture of 0.05M NHS and 0.2M EDC [0118] Inject antibodies diluted
in 10 mM NaAc pH5.0 at concentration of 30 .mu.g/mL until an
appropriate amount in captured [0119] Inject 35 .mu.L 1 M
ethanolamine-HCL pH8.5 [0120] Epitope mapping: [0121] Inject 25
.mu.L of mAb1 at a concentration of 25 or 50 .mu.g/mL. [0122]
Inject 25 .mu.L of a mixture of IgGI, IgG2a, IgG2b and IgG3 each
one at a concentration of 25 .mu.g/mL. [0123] Inject 25 .mu.L of
human recombinant clusterin at a concentration of 30 .mu.g/mL.
[0124] Inject 25 .mu.L of mAb2 at a concentration of 25 or 50
.mu.g/mL. [0125] Control: [0126] For each pair of antibodies, the
non-specific binding of mab2 was determined by repeating all
injections described in the epitope mapping section but injecting
running buffer instead of clusterin. [0127] The response (RU)
obtained 20 sec after the end of the mab2 injection in the control
was subtracted from the response obtained in the presence of
clusterin. [0128] Regeneration of the surface: [0129] At the end of
each cycle, inject 10 .mu.L of 20 mM glycine pH1.7 followed with 10
.mu.L of 100 mM HCl.
[0130] Approach 2: [0131] Running buffer: [0132] HBS (20 mM Hepes
(pH7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20) [0133] Standard
amine coupling of the antibodies: [0134] Inject 35 .mu.L of a
mixture of 0.05M NHS and 0.2M EDC [0135] Inject antibodies diluted
in 10 mM NaAc (pH4.5 or 5.0) at concentration raging from 20 to 80
.mu.g/mL until a appropriate amount in captured [0136] Inject 35
.mu.L 1M ethanolamine-HCl pH8.5 [0137] Preparation of control
surface [0138] Inject 35 .mu.L of a mixture of 0.05M NHS and 0.2M
EDC [0139] Inject 35 .mu.L 1M ethanolamine-HCl pH8.5 [0140]
Competition [0141] Mix human recombinant clusterin at 50 nM with
250 nM or 500 nM antibodies in PBS (without Mg++ and Ca++) [0142]
Prepare a tube with antibody alone [0143] Inject at a flow of 5
.mu.L/min, 25 .mu.L of clusterin alone, antibody alone or clusterin
preincubated with antibodies over the antibody and the control
surfaces. [0144] Subtract the response obtained for the antibody
alone solution from the response obtained for clusterin
preincubated with the same antibody. [0145] Calculate the % binding
inhibition by dividing the response obtained for the clusterin
preincubated with antibody by the response obtained for clusterin
alone. [0146] Regeneration solution [0147] At the end of each
cycle, inject 10 .mu.L of 10 mM HCl at a flow rate of 20
.mu.L/min
[0148] Immunoprecipitation
[0149] 50 or 100 ng of the various monoclonal antibodies or the
polyclonal antibody preparation (C18) was incubated with 20 .mu.L
of protein G slush (1:1 in PBS) overnight at 4.degree. C. Then 500
ng of human recombinant clusterin was added and the mixture was
incubated for another 2 hr at 4.degree. C. Immunocomplexes were
washed 3 times with 1 mL of buffer (150 nM NaCl, 50 mM Tris pH 8.0,
0.55% NP-40, 50 mM Na fluoride) and 20 .mu.L of reducing sample
buffer was added. Samples were boiled for 5 min prior to loading on
a 12% SDS-PAGE. Separated proteins were then transferred to
nitrocellulose and membranes were probed with anti-clusterin
antibodies as described.
[0150] Sequencing of the Monoclonal Antibody Variable Region
[0151] Total RNA was isolated from the 12 hybridomas and first
strand cDNA was prepared with reverse transcriptase and the Ig-3
constant region primer followed by amplification with the
appropriate Ig-5' primer. These primer sets used in conjunction
with KOD Hot Start DNA Polymerase specifically amplify the variable
regions of light- and heavy-chain cDNAs. PCR products can be
directly cloned with Novagen's pSTBlue-1 Perfectly Blunt.TM.
Cloning Kit or treated with the Single dA.TM. Tailing Kit and
cloned into the pSTBlue-1 AccepTor.TM. Vector. For details see FIG.
13.
TABLE-US-00001 TABLE 1 Primer sets used for the validation of some
of the 328 TGF-.beta. modulated genes in the BRI-JM01 cells. Gene
GeneBank# Reverse Forward size (bp) Eef1a1 AW556381
CTGGCTTCACTGCTCAGGT TGGCCAATTGAGACAAACAG 457 Clusterin AU041878
TGGTGAAAGCTGTTTGACTCTG AAGGCGGCTTTTATTGGATT 355 Integrin .alpha.6
AW556992 ATGTGCCATTGTTGCTTTGA CAAGCGATGAGCACTTTTGT 517 Caveolin-1
AU016590 GTGCAGGAAGGAGAGAATGG GCACACCAAGGAGATTGACC 247 Ptpn13
AW548343 CCTGCAATGGTTCTTGGTTT GGGAAAATCGATGTTGGAGA 300
14-3-3.sigma. AA410123 GGGCTGTTGGCTATCTCGTA AGAGACCGAGCTCAGAGGTG
297
[0152] Inclusion of a reference is neither an admission nor a
suggestion that it is relevant to the patentability of anything
disclosed herein
[0153] Bailey et al., Biochemistry. 2001; 40:11828-40
[0154] Dunker et al., J Mol Graph Model. 2001;19 (1):26-59
[0155] Li et al., Genome Inform. Ser. Workshop Genome Inform. 1999;
10: 30-
[0156] Jones, J. Mol. Biol. 1999; 287: 797-815
[0157] Lupas, Meth. in Enzym. 1996; 266: 513-525
[0158] Rost, Meth. in Enzym. 1996; 266: 525-539
[0159] Singh et al., Curr Opin Drug Discov Devel. 2004: 437-445
[0160] Al-Moustafa et al., Biotechniques. 1999: 60-62
[0161] Durocher et al Nucleic Acids Res 2002: E9
[0162] Enjalbert et al., Mol Biol Cell. 2003: 1460-1467
[0163] Schade et al., Mol Biol Cell 2004: 5492-5502
[0164] Wilson and Easterbrook-Smith, Biochim Biophys Acta 1992:
319-326
Sequence CWU 1
1
203117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
Thr Val Ala Glu 1 5 10 15 Lys 212PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 2Glu Thr Val Ala Glu Lys
Ala Leu Gln Glu Tyr Arg 1 5 10 36PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 3Glu Thr Val Ala Glu Lys 1
5 475PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val
Thr Thr Val Ala 1 5 10 15 Ser His Thr Ser Asp Ser Asp Val Pro Ser
Gly Val Thr Glu Val Val 20 25 30 Val Lys Leu Phe Asp Ser Asp Pro
Ile Thr Val Thr Val Pro Val Glu 35 40 45 Val Ser Arg Lys Asn Pro
Lys Phe Met Glu Thr Val Ala Glu Lys Ala 50 55 60 Leu Gln Glu Tyr
Arg Lys Lys His Arg Glu Glu 65 70 75 523PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Val
Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu 1 5 10
15 Lys Ala Leu Gln Glu Tyr Arg 20 68PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Gly
Tyr Xaa Phe Thr Xaa Tyr Xaa 1 5 78PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 7Ile Xaa Xaa Xaa Xaa Gly
Xaa Xaa 1 5 8106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic protein construct 8Glu Asn Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro
Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
9107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 9Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys
Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln
His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr
Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70
75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu
Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
10111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 10Asp Ile Val Leu Thr Leu Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys
Arg Ala Ser Gln Ser Val Asn Ser Ser 20 25 30 Asn Tyr Ser Tyr Met
His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu
Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Asn Ile His 65 70
75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser
Trp 85 90 95 Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110 11113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic protein construct 11Asp Ile Val Met
Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 35
40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val
Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ile Tyr Pro Arg Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys 12112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 12Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser
Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr
Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys 100 105 110
13112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 13Asp Val Leu Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Pro Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln
Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 14112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic protein construct 14Asp Val Val Leu
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95Thr His Ile Pro Arg Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 110 15113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 15Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser
Val Gly 1 5 10 15 Gln Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asn Ser 20 25 30 Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val
Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr
Asn Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110
Lys 16112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 16Asp Val Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asp Thr Tyr
Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln
Ser 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 17112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic protein construct 17Asp Val Leu Leu
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Asn Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110 18107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 18Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr
Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser
Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu
Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser
Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp
Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly
Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 19108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 19Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Thr Ala Ser
Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser
Ile Ser Ser Asn 20 25 30 Phe Leu His Trp Tyr Gln Gln Lys Pro Gly
Phe Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Thr Ser Asn Leu Pro
Ser Gly Val Pro Pro Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val
Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Leu Pro 85 90 95 Arg Thr
Phe Gly Ala Gly Thr Lys Leu Ala Leu Lys 100 105 20120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 20Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Asn Asn Gly
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly
Thr Pro Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Leu
Asn Ser Leu Leu Arg Leu Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Ser Val Thr Val Ser Ser 115 120 21118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 21Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro
Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly
Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly
Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe
Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn
Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg
Thr Gly Ser Ser Gly Tyr Phe Asp Cys Trp Gly Gln Gly Thr 100 105 110
Thr Leu Thr Val Ser Ser 115 22118PRTArtificial SequenceDescription
of Artificial Sequence Synthetic protein construct 22Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30 Gly Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp
Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp Gly Phe Leu Tyr Phe
Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser
115 23117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 23Glu Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Thr
Thr Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val
Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile
Asp Pro Ala Tyr Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln
Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70
75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Arg Tyr Asp Thr Ala Met Asp Tyr Trp Gly Gln
Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 24118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 24Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Gly Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Arg
Lys Ser Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln
Lys Ser 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp
Ser Gly Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Gly Ser Ser
Tyr Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Ala Val Ser Ala
115 25122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 25Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile
Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Thr Arg Ile Tyr Tyr Asp Tyr Gly Ser Trp Asp Gly Phe
Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115
120 26120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 26Gln Val Gln Leu Gln Gln Ser Gly Pro
Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser Asp Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile
Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ser Arg Asp Gly Asn Tyr Arg Tyr Tyr Thr Leu Asp Phe
Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
27119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 27Thr Cys Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile
Ser Thr Ile Gly Ser Tyr Thr Asp Tyr Pro Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Cys
Cys 85 90 95 Thr Arg Glu Asp Tyr Arg Tyr Ala Trp Phe Ala Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115
28120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 28Gln Val Gln Leu Gln Gln Ser Gly Pro
Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser Asp Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile
Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ser Arg Asp Gly Asn Tyr Arg Ser Tyr Thr Met Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
29115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein construct 29Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Phe Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ile Asn Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Ile 35 40 45 Ala Glu Ile
Ser Ser Gly Gly Ser Asp Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Leu 65 70
75 80 Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Ala 85 90 95 Arg Asp Gly Asn Trp Asp Gly Gly Ser Leu Thr Thr Gly
Ala Lys Ala 100 105 110 Pro Leu Ser 115 30118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
construct 30Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro
Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Leu Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly
Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly
Glu Pro Thr Tyr Val Asp Asp Phe 50 55 60 Lys Arg Arg Phe Ala Phe
Ser Leu Glu Thr Ser Ala Ser Ala Ala Tyr 65 70 75 80 Leu Gln Ile Asn
Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg
Asp Gly Ser Ser Thr Trp Phe Ser Tyr Trp Gly Gln Gly Thr 100 105 110
Leu Val Thr Val Ser Ala 115 3119DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 31ctggcttcac tgctcaggt
193220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32tggccaattg agacaaacag 203322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33tggtgaaagc tgtttgactc tg 223420DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 34aaggcggctt ttattggatt
203520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35atgtgccatt gttgctttga 203620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36caagcgatga gcacttttgt 203720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 37gtgcaggaag gagagaatgg
203820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38gcacaccaag gagattgacc 203920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39cctgcaatgg ttcttggttt 204020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 40gggaaaatcg atgttggaga
204120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41gggctgttgg ctatctcgta 204220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42agagaccgag ctcagaggtg 20438PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 43Gly Tyr Ser Phe Thr Gly Tyr
Asn 1 5 448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gly Tyr Ser Phe Thr Gly Tyr Asn 1 5
458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5
468PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5
478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Ile Asp Pro Tyr Asn Gly Asp Thr 1 5
488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Ile Asp Pro Tyr Tyr Gly Thr Pro 1 5
498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Ile Asn Thr Tyr Thr Gly Glu Pro 1 5
508PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Ile Asn Thr Glu Thr Gly Glu Pro 1 5
5111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5
10 5211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5
10 5326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser
Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser
20 25 5426PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser
20 25 5526PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Asp Ile Val Leu Thr Leu Ser Pro Ala Ser Leu Ala
Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
20 25 5626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala
Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser
20 25 5726PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Pro Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
20 25 5826PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
20 25 5926PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala
Met Ser Val Gly 1 5 10 15 Gln Arg Val Thr Met Ser Cys Lys Ser Ser
20 25 6026PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
20 25 615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Ser Ser Val Ser Tyr 1 5 626PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 62Gln
Asp Ile Asn Lys Tyr 1 5 6310PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 63Gln Ser Val Asn Ser Ser Asn
Tyr Ser Tyr 1 5 10 6412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Gln Ser Leu Leu Tyr Ser Ser
Asn Gln Lys Asn Tyr 1 5 10 6512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 65Gln Ser Leu Leu Asn Ser Arg
Thr Arg Lys Asn Tyr 1 5 10 6611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 66Gln Ser Ile Val His Ser Asn
Gly Asn Thr Tyr 1 5 10 6711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Gln Ser Leu Val His Ser Asn
Gly Asn Thr Tyr 1 5 10 6812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Gln Ser Leu Leu Asn Ser Asn
Asn Gln Lys Asn Tyr 1 5 10 6911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Gln Ser Leu Val His Ser Asn
Gly Asp Thr Tyr 1 5 10 7026PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Asp Val Leu Leu Thr Gln Thr
Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile
Ser Cys Arg Ser Ser 20 25 7126PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Asp Ile Val Met Thr Gln Ser
Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu
Ser Cys Arg Ala Ser 20 25 7226PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 72Glu Ile Val Leu Thr Gln Ser
Pro Thr Thr Met Thr Ala Ser Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile
Thr Cys Ser Ala Ser 20 25 7311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 73Gln Ser Leu Val His Ser Asn
Gly Asn Thr Tyr 1 5 10 746PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 74Gln Ser Ile Ser Asp Tyr 1 5
757PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Ser Ser Ile Ser Ser Asn Phe 1 5
7625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25
7725PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys
Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser 20 25
7825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys
Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser 20 25
7925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Thr Thr Ser 20 25
8025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25
8125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 25
8225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val
Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25
8325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Thr Cys Lys Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 25
8425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val
Arg Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25 858PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Gly
Tyr Ser Phe Thr Gly Tyr Asn 1 5 868PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Gly
Tyr Thr Phe Thr Asp Tyr Ser 1 5 878PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 87Gly
Tyr Thr Phe Thr Asn Tyr Gly 1 5 888PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Gly
Phe Asn Ile Lys Asp Ile Tyr 1 5 898PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 89Gly
Tyr Ser Phe Thr Gly Tyr Asn 1 5 908PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 90Gly
Phe Thr Phe Ser Ser Tyr Ala 1 5 918PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 91Asp
Tyr Ser Phe Thr Thr Tyr Trp 1 5 928PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Gly
Phe Thr Phe Ser Ser Tyr Ser 1 5 938PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Asp
Tyr Ser Phe Thr Thr Tyr Trp 1 5 9425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Lys Phe Ser Cys Ala Ala Ser 20 25 9525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Gln
Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10
15 Thr Val Lys Ile Ser Cys Lys Ala Ser 20 25 968PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 96Gly
Phe Thr Phe Ile Asn Tyr Ala 1 5 978PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Gly
Tyr Thr Leu Thr Asp Tyr Ser 1 5 9817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 98Met
His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile 1 5 10
15 Tyr 9917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro
Arg Leu Leu Ile 1 5 10 15 His 10017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Met
His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 1 5 10
15 Lys 10117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 101Leu Ala Trp Tyr Gln Gln Arg Pro Gly
Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr 10217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10
15 Tyr 10317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 103Leu Glu Trp Tyr Leu Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr 10417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Leu
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10
15 Tyr 10517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 105Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Val 1 5 10 15 Tyr 10617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Leu
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10
15 Tyr 10717PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 107Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr 10817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 108Leu
His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 1 5 10
15 Lys 10917PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 109Leu His Trp Tyr Gln Gln Lys Pro Gly
Phe Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr 11017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Met
Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile Gly 1 5 10
15 Asn 11117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 111Met His Trp Val Lys Gln Ala Pro Gly
Lys Gly Leu Lys Trp Met Gly 1 5 10 15 Trp 11217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 112Met
His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly 1 5 10
15 Trp 11317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 113Met His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Arg 11417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Met
Tyr Trp Val Lys Gln Ser His Arg Lys Ser Leu Glu Trp Ile Gly 1 5 10
15 Tyr 11517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 115Met Ser Trp Val Arg Gln Ser Pro Glu
Lys Arg Leu Glu Trp Val Ala 1 5 10 15 Glu 11617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 116Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10
15 Met 11717PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 117Met Ser Trp Val Arg Gln Thr Pro Glu
Lys Arg Leu Glu Trp Val Ala 1 5 10 15 Thr 11817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10
15 Met 1198PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Ile Asp Pro Tyr Tyr Gly Thr Pro 1 5
1208PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Ile Asn Thr Glu Thr Gly Glu Pro 1 5
1218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 121Ile Asn Thr Tyr Thr Gly Glu Pro 1 5
1228PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Ile Asp Pro Ala Tyr Gly Asn Thr 1 5
1238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Ile Asp Pro Tyr Asn Gly Asp Thr 1 5
1248PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Ile Ser Ser Gly Gly Thr Tyr Thr 1 5
1258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Ile Asp Pro Ser Asp Ser Glu Thr 1 5
1268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Ile Ser Thr Ile Gly Ser Tyr Thr 1 5
1278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Ile Asp Pro Ser Asp Ser Glu Thr 1 5
12817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg
Leu Glu Trp Ile Ala 1 5 10 15 Glu 12917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 129Met
His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly 1 5 10
15 Trp 1308PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Ile Ser Ser Gly Gly Ser Asp Thr 1 5
1318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Ile Asn Thr Glu Thr Gly Glu Pro 1 5
13236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Asn Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu Ala Glu Asp Val Ala 20 25 30 Thr Tyr Tyr Cys 35
13336PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 133Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Arg Asp Tyr Ser Phe Ser Ile Ser Asn
Leu Glu Pro Glu Asp Ile Ala 20 25 30 Thr Tyr Tyr Cys 35
13436PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr His Phe Thr Leu Asn Ile His Pro
Val Glu Glu Glu Asp Thr Ala 20 25 30 Thr Tyr Tyr Cys 35
13536PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser
Val Lys Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35
13636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser
Val Gln Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35
13736PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Tyr Cys 35
13836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Phe Cys 35
13936PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ile
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser
Val Gln Ala Glu Asp Leu Ala 20 25 30 Asp Tyr Phe Cys 35
14036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Phe Cys 35
1419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Phe Gln Gly Ser Gly Tyr Pro Phe Thr 1 5
1429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Leu Gln Tyr Asp Asn Leu Leu Arg Thr 1 5
1439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Gln His Ser Trp Glu Ile Pro Trp Thr 1 5
1449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Gln Gln Tyr Tyr Ile Tyr Pro Arg Thr 1 5
1458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Lys Gln Ser Tyr Asn Leu Trp Thr 1 5
1469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Phe Gln Gly Ser His Val Pro Tyr Thr 1 5
1479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Ser Gln Ser Thr His Ile Pro Arg Thr 1 5
1489PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Gln Gln His Tyr Asn Thr Pro Leu Thr 1 5
1499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Ser Gln Ser Thr His Val Pro Arg Thr 1 5
15036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Asn Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Phe Cys 35
15136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 151Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Ser Asp Phe Thr Leu Ser Ile Asn Ser
Val Glu Pro Glu Asp Val Gly 20 25 30 Val Tyr Tyr Cys 35
15236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 152Asn Leu Pro Ser Gly Val Pro Pro Arg Phe Ser
Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu Thr Ile Gly Thr
Met Glu Ala Glu Asp Val Ala 20 25 30 Thr Tyr Tyr Cys 35
1539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 153Ser Gln Ser Thr His Val Pro Arg Thr 1 5
1549PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 154Gln Asn Gly His Ser Phe Pro Tyr Thr 1 5
1559PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 155Gln Gln Gly Ser Ser Leu Pro Arg Thr 1 5
15638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 156Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr
Leu Thr Val Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu
Lys Ser Leu Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35
15738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala
Phe Ser Leu Glu Thr 1 5 10 15 Ser Ala Ser Thr Ala Tyr Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp 20 25 30 Thr Ala Thr Tyr Phe Cys 35
15838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 158Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala
Phe Ser Leu Glu Thr 1 5 10 15 Ser Ala Ser Thr Ala Tyr Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp 20 25 30 Thr Ala Thr Tyr Phe Cys 35
15938PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 159Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr
Ile Thr Ala Asp Thr 1 5 10 15 Ser Ser Asn Thr Ala Tyr Leu Gln Leu
Ser Ser Leu Thr Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35
16038PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Ser Tyr Asn Gln Lys Ser Lys Gly Lys Ala Thr
Leu Thr Ala Asp Arg 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met His Leu
Asn Ser Leu Thr Ser Glu Asp 20 25 30 Ser Gly Ile Tyr Tyr Cys 35
16138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 161Tyr Tyr Pro Asp Thr Val Thr Gly Arg Phe Thr
Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Tyr Leu Glu Met
Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35
16238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 162Arg
Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 1 5 10
15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp
20 25 30 Ser Ala Val Tyr Tyr Cys 35 16338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 163Asp
Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10
15 Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp
20 25 30 Thr Ala Met Tyr Cys Cys 35 16438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 164Arg
Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 1 5 10
15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp
20 25 30 Ser Ala Val Tyr Tyr Cys 35 16513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 165Ala
Leu Asn Ser Leu Leu Arg Leu Asn Ala Met Asp Tyr 1 5 10
16611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 166Ala Arg Thr Gly Ser Ser Gly Tyr Phe Asp Cys 1
5 10 16711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 167Ala Arg Asp Gly Phe Leu Tyr Phe Phe Asp Tyr 1
5 10 16810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 168Ala Arg Arg Tyr Asp Thr Ala Met Asp Tyr 1 5 10
16911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 169Ala Arg Gly Ala Tyr Gly Ser Ser Tyr Ala Tyr 1
5 10 17015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 170Thr Arg Ile Tyr Tyr Asp Tyr Gly Ser Trp Asp
Gly Phe Ala Tyr 1 5 10 15 17113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 171Ser Arg Asp Gly Asn Tyr
Arg Tyr Tyr Thr Leu Asp Phe 1 5 10 17212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 172Thr
Arg Glu Asp Tyr Arg Tyr Ala Trp Phe Ala Tyr 1 5 10
17313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 173Ser Arg Asp Gly Asn Tyr Arg Ser Tyr Thr Met
Asp Tyr 1 5 10 17437PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 174Tyr Tyr Pro Asp Thr Val Thr Gly Arg
Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Leu Glu
Met Ser Ser Leu Arg Ser Glu Asp Thr 20 25 30 Ala Met Tyr Tyr Cys 35
17538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Thr Tyr Val Asp Asp Phe Lys Arg Arg Phe Ala
Phe Ser Leu Glu Thr 1 5 10 15 Ser Ala Ser Ala Ala Tyr Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp 20 25 30 Thr Ala Thr Tyr Phe Cys 35
17612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 176Ala Arg Asp Gly Asn Trp Asp Gly Gly Ser Leu
Thr 1 5 10 17711PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 177Thr Arg Asp Gly Ser Ser Thr Trp Phe
Ser Tyr 1 5 10 1789PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 178Gly Ser Gly Thr Lys Leu Glu Ile Lys 1
5 17910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 179Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 180Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 181Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 182Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys 1 5 10
18310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 185Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10
18610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 186Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 187Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
18910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 189Phe Gly Ala Gly Thr Lys Leu Ala Leu Lys 1 5 10
19011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1
5 10 19111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 191Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1
5 10 19211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 192Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1
5 10 19311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 193Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1
5 10 19411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 194Trp Gly Gln Gly Thr Leu Val Ala Val Ser Ala 1
5 10 19511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 195Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1
5 10 19611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 196Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1
5 10 19711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 197Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1
5 10 19811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 198Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1
5 10 19911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 199Gly Ala Lys Ala Pro Leu Ser Gln Ser Pro Gln 1
5 10 20015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 200Leu Trp Thr Thr Gly Val Lys Glu Pro Gln Ser
Pro Ser Pro Gln 1 5 10 15 20112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 201Leu Leu Gly Pro Arg Asp
Ser Gly Arg Cys Leu Cys 1 5 10 20216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 202Ile
Leu Trp Thr Thr Gly Val Lys Glu Pro Gln Ser Pro Ser Pro Gln 1 5 10
15 20314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 203Leu Thr Thr Gly Ala Lys Ala Pro Leu Ser Gln
Ser Pro Gln 1 5 10
* * * * *