U.S. patent application number 12/360770 was filed with the patent office on 2009-10-15 for methods for diagnosing and treating kidney and colorectal cancer.
Invention is credited to MATTHIAS WABL, BRUCE WANG.
Application Number | 20090258358 12/360770 |
Document ID | / |
Family ID | 38533919 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090258358 |
Kind Code |
A1 |
WANG; BRUCE ; et
al. |
October 15, 2009 |
Methods for Diagnosing and Treating Kidney and Colorectal
Cancer
Abstract
Methods, reagents and kits for diagnosing and treating cancer
such as kidney or colorectal cancer are disclosed. An immunoassay
for detecting kidney or colorectal cancer is based on the relative
change of the ADAMTSL4 protein in urine or blood compared with
normal tissue. An immunohistochemical assay for detecting kidney or
colorectal cancer is based on the relative absence of labeled
antibody binding to cancerous tissue, compared with normal
tissue.
Inventors: |
WANG; BRUCE; (MOUNTAIN VIEW,
CA) ; WABL; MATTHIAS; (SAN FRANCISCO, CA) |
Correspondence
Address: |
King & Spalding LLP
P.O. Box 889
Belmont
CA
94002-0889
US
|
Family ID: |
38533919 |
Appl. No.: |
12/360770 |
Filed: |
January 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11388550 |
Mar 24, 2006 |
7488584 |
|
|
12360770 |
|
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/287.2; 530/387.9 |
Current CPC
Class: |
G01N 33/57419 20130101;
G01N 33/57438 20130101; G01N 33/57492 20130101; G01N 33/5082
20130101 |
Class at
Publication: |
435/6 ;
530/387.9; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 16/18 20060101 C07K016/18; C12M 1/34 20060101
C12M001/34 |
Claims
1. An antibody which specifically binds an epitope contained within
a sequence selected from the group consisting of an ADAMTSL4
isoform 1 amino acid sequence defined by SEQ ID NO:11 and an
ADAMTSL4 isoform 2 amino acid sequence defined by SEQ ID NO:12.
2. The antibody of claim 1 which specifically binds an epitope
contained within a sequence selected from the group consisting of
amino acid residues 1009 to 1024 of human ADAMTSL4 isoform 1
defined by SEQ ID NO:1 and amino acid residues 693-710 of human
ADAMTSL4 defined by SEQ ID NO:2.
3. The antibody of claim 1, labeled with a detectable marker
suitable for immunohistochemical detection of cancerous kidney,
colorectal, or neuronal tissue, based on the relative absence of
histochemical staining of the tissue compared with normal kidney,
colorectal or neuronal issue, respectively.
4. The antibody of claim 1, wherein the antibody is a polyclonal
antibody.
5. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
6. A diagnostic device for use in for screening for kidney or
colorectal cancer in a subject, or staging treatment of kidney or
colorectal cancer in a subject, comprising: (a) a structure for
receiving a body-fluid sample from the subject, (b) an antibody
which specifically binds a selected domain or epitope of ADAMTSL4,
wherein the antibody is associated with the structure and capable
of reacting with the body-fluid received in the structure to
produce, in combination with other reagents associated with the
structure, a detectable reaction product indicative of the presence
and/or level of ADAMTSL4 containing that epitope or domain, and (c)
a known standard indicator which, in combination with the reagents,
produces a known standard reaction product indicative of kidney or
colorectal cancer, against which the level of detectable body-fluid
reaction product can be assessed.
7. The device of claim 6, wherein said structure includes a porous
pad having the antibody embedded therein, for reaction with the
body-fluid sample when the sample is applied to the pad, and said
detectable body-fluid reaction product is indicated by a
colorimetric or fluorimetric indicator.
8. The device of claim 6, which includes a spectrophotometric
detector for generating a signal related to the level of reaction
product, a microprocessor for comparing said signal with a signal
value from the known standard indicator, and a display for
displaying an output of the microprocessor.
9. The device of claim 6, wherein the antibody specifically binds
an epitope contained within SEQ ID NO:1 or SEQ ID NO:2.
10. A method of screening for colorectal cancer in a human subject,
or staging treatment of colorectal cancer in a subject, comprising:
reacting a body-fluid sample from the subject with an antibody
which specifically binds a selected domain or epitope of ADAMTSL4,
and determining, from the presence and/or amount of immunoassay
product, whether the subject has a reduced level of antibody
binding to the selected domain or epitope of ADAMTSL4, as compared
to a normal range of antibody binding to the selected domain or
epitope of ADAMTSL4 in human samples, wherein the reduced level of
antibody binding to the selected domain or epitope of ADAMTSL4
indicates the presence of and/or staging treatment of colorectal
cancer.
11. The method of claim 10, wherein the body-fluid sample is urine,
and the level of ADAMTSL4 indicative or colorectal cancer is less
than about 0.1 ng/ml.
12. The method of claim 10, wherein said reacting includes applying
the body fluid to a solid-phase immunoassay device, the level of
ADAMTSL4 in the sample is indicated qualitatively by a colorimetric
or fluorometric indicator, and said determining includes comparing
the indicator with a known standard.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of currently
pending U.S. application Ser. No. 11/388,550, filed 24 Mar. 2006,
the contents of which are expressly incorporated herein by
reference.
I. REFERENCES
[0002] The following references are cited below in support of the
background of the invention or methods employed in practicing the
invention. [0003] 1. Buchner and Meisler, "TSRC1, a widely
expressed gene containing seven thrombospondin type I repeats,"
Gene, 307:23-30 (2003). [0004] 2. Nusse, et al., "Mode of proviral
activation of a putative mammary oncogene (int-1) on mouse
chromosome 15," Nature, 307:131-136 (1984). [0005] 3. Nusse, et
al., "Many tumors induced by the mouse mammary tumor virus contain
a provirus integrated in the same region of the host genome," Cell,
31:99-109 (1982). [0006] 4. Sorensen, et al., "Sequence tags of
provirus integration sites in DNAs of tumors induced by the murine
retrovirus SL3-3," J Virol, 70:4063-4070 (1996). [0007] 5. Lund, et
al., "Genome-wide retroviral insertional tagging of genes involved
in cancer in Cdkn2a-deficient mice," Nat Genet, 32:160-165 (2002).
[0008] 6. Mikkers, et al., "High-throughput retroviral tagging to
identify components of specific signaling pathways in cancer," Nat
Genet, 32:153-159 (2002). [0009] 7. Collier, et al., "Cancer gene
discovery in solid tumours using transposon-based somatic
mutagenesis in the mouse," Nature, 436:272-276 (2005). [0010] 8.
Dupuy, et al., "Mammalian mutagenesis using a highly mobile somatic
Sleeping Beauty transposon system," Nature, 436:221-226 (2005).
II. BACKGROUND
[0011] Cancer is caused by genetic aberrations, i.e., mutations. In
mutant cells the normal balance between the factors that promote
and restrain growth is disrupted, and as a result, these mutant
cells proliferate continuously--the hallmark of tumor cells.
Mutations can arise spontaneously or by external factors such as
chemical mutagens, radiation, or viral integration, which inserts
extra-genomic DNA that may or may not contain an oncogene. A
cellular gene can be modified by point mutation, insertion and
frame shift (including truncation), (functional) deletion
(including silencing), or translocation, which sometimes can result
in gene fusion. In this way, proto-oncogenes become oncogenes,
which promote proliferation, and tumor suppressor genes become
inactivated, also inducing tumor growth. Any combination of the
above-mentioned changes in DNA can contribute to tumor formation.
There are two ways by which mutations result in transformation: the
expression level of the genes is changed, or their function is
altered. The consequences of these changes may or may not be held
in check by the immune system (immune surveillance).
[0012] Heretofore, there has been no demonstrated link between
changes in ADAMTSL4 levels and kidney and/or colorectal cancer.
Such a link could have a number of important diagnostic and
therapeutic applications. It has now been discovered that (i)
ADAMTSL4 levels change, e.g. decrease significantly in kidney
cancer and colon tumor cells, and (ii) this change can be measured
in blood-fluid and urine sample of patients.
III. SUMMARY
[0013] In one aspect, a histological method for examining human
tissue for the presence and extent of cancer is provided. This
method involves the steps of staining the human tissue with an
antibody specific against a selected domain or epitope of ADAMTSL4
and labeled with a detectable marker, to attach the marker to the
surface of tissue cells having surface bound ADAMTSL4 protein with
that epitope or domain, and determining, based on a reduced
distribution and extent of detectable marker with respect to the
distribution and extent of marker in normal cells, the presence and
extent of cancer in the tissue.
[0014] In one embodiment, the antibody is specific against an
epitope contained within at least one of SEQ ID NO:1 and SEQ ID
NO:2. In other embodiments, the antibody may be specific against a
thrombospondin repeat. In specific embodiments, the antibody is
specific against a thrombospondin repeat selected from the group
consisting of SEQ ID NOS:3-9. In another embodiment, the antibody
is specific against an ADAM-TS spacer represented by SEQ ID NO:10.
In an additional embodiment, the antibody is specific against an
epitope contained within the ADAMTSL4 isoform 1 amino acid defined
by SEQ ID NO:11 or the ADAMTSL4 isoform 2 amino acid defined by SEQ
ID NO:12.
[0015] The human tissue may be selected from the group consisting
of kidney tissue, colon tissue, and/or rectal tissue.
[0016] The antibody may further be labeled with a detectable marker
suitable for immunohistochemical detection of cancerous kidney,
colorectal, or neuronal tissue, based on the relative absence of
histochemical staining of the tissue compared with normal kidney
colorectal, or neuronal issue, respectively.
[0017] Also disclosed is a method for identifying genetic mutations
associated with an increased risk of kidney and/or colorectal
cancer. The method involves (a) extracting genomic DNA from cells
from cancerous tissue from human patients, (b) for the DNA
extracted from cells from each tissue, comparing the sequence of
the DNA in a region selected from at least one of (i) a plurality
of exons 1 to 17 of the ADAMTSL4 on chromosome 1q21, including
adjacent splice site acceptor and donor sequences of the exons,
(ii) a 5' UTR region within 10 kB or less of exon 1 of the gene,
and (iii) a 3' UTR region within 10 kB or less of exon 17, with a
homologous region of DNA from cells from normal, wildtype human
tissue, and (c), by said comparing, identifying one or more
mutations in said regions associated with an increased risk of
kidney or colorectal cancer. In one embodiment, the DNA that is
compared is a 5' UTR region within 10 kB or less of exon 1 of the
ADAMTSL4 gene.
[0018] The method may be used in constructing a gene chip designed
for genetic screening for risk of cancer. For each mutation
identified in step (c), a gene fragment capable of binding
selectively to genomic DNA fragments carrying that mutation, but
not to corresponding wildtype DNA fragments is produced, and the
different-sequence fragments are attached at known positions on a
gene-chip substrate.
[0019] In yet another aspect, there is provided a method for
screening for kidney or colorectal cancer in a human subject, or
staging treatment of kidney or colorectal cancer in a subject by
reacting a body-fluid sample from the subject with an antibody
specific against a selected domain or epitope of ADAMTSL4, and
determining from the presence and/or amount of immunoassay product,
whether the subject has a reduced level of ADAMTSL4 protein lacking
the specific domain or epitope, when compared with a normal range
of ADAMTSL4 in human samples, as an indicator of kidney and/or
colorectal cancer. The body-fluid sample may be urine, and the
assayed level of ADAMTSL4 indicative or kidney or colorectal cancer
may be a level less than about 0.1 ng/ml.
[0020] The method may be carried out by applying the body fluid to
a solid-phase immunoassay device, the level of ADAMTSL4 in the
sample may be indicated qualitatively by a colorimetric or
fluorometric indicator, and the determining step may include
comparing the indicator with a known standard.
[0021] In a related aspect, a diagnostic device for use in for
screening for kidney and/or colorectal cancer in a human subject,
or staging treatment of kidney and/or colorectal cancer in a
subject is provided. The device comprises (a) structure for
receiving a body-fluid sample from the subject, (b) an antibody
specific against a selected domain or epitope of ADAMTSL4, and
associated with the structure and capable of reacting with
body-fluid received in the structure, to produce, in combination
with other reagents associated with the structure, a detectable
reaction indicative of the presence of ADAMTSL4 sample protein
containing that epitope or domain, and (c) a known-standard
indicator against which the level of detectable reaction produced
can be assessed as a reduced level associated with kidney or
colorectal cancer.
[0022] The structure in the device may include a porous pad having
the antibody embedded therein, for reaction with the fluid sample
when the sample is applied to the pad, the detectable reaction may
be indicated by a colorimetric or fluorometric indicator, and the
known standard indicator may include an indicia that represents a
level of ADAMTSL4 containing the epitope or domain corresponding to
that associated with kidney and/or colorectal cancer.
[0023] The device may be employed in a kit which includes a
spectrophotometric detector for generating a signal related to the
level of ADAMTSL4 produced, a microprocessor for comparing the
signal with a known-standard signal value associated with kidney
and/or colorectal cancer, and a display for displaying an output of
the microprocessor. The anti-ADAMTSL4 binding protein may be
specific against an epitope contained within SEQ ID NO:11 and/or
SEQ ID NO:12. In another embodiment, the anti-ADAMTSL4 binding
protein may be specific against an epitope selected from SEQ ID
NO:1 and/or SEQ ID NO:2
[0024] Also provided is a method of treating kidney or colorectal
cancer in a subject by the steps of (a) reacting a body-fluid
sample from the subject with an antibody specific against a
selected domain or epitope of ADAMTSL4, (b) determining from the
presence and/or amount of immunoassay product, whether the subject
has a reduced level of ADAMTSL4 protein lacking the specific domain
or epitope, when compared with a normal range of ADAMTSL4 in human
samples, as an indicator of kidney and/or colorectal cancer, and
(c) if the subject has such a reduced ADAMTSL4 level, administering
a therapeutically effective amount of a ADAMTSL4 antibody
effective, when bound to the surface of kidney or colorectal cancer
cells, to inhibit growth or viability of the cells. One exemplary
antibody for use in the method is a human or humanized
anti-ADAMTSL4 antibody specific against an epitope contained within
SEQ ID NO:11 or SEQ ID NO:22. In a specific embodiment, the
anti-ADAMTSL4 antibody is specific against an epitope selected from
SEQ ID NO:1 and/or SEQ ID NO:2.
[0025] These and other aspects, objects, advantages, and features
of the invention will become apparent to those persons skilled in
the art upon reading the details of the invention as more fully
described below.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. The dimensions
of the various features are arbitrarily expanded or reduced for
clarity.
[0027] FIG. 1 shows the genomic organization of the human ADAMTSL4
gene on chromosome 1. The structures of isoform 1 and isoform 2 are
indicated.
[0028] FIG. 2 shows the genomic organization of the mouse ADAMTSL4
locus, as viewed by a customized screen print of the UCSC genome
web site browser (March 2005 version of the mm6 gene assembly).
Top, base position on chromosome 3. The vertical handlebars
represent the retroviral insertions into the locus found in three
independent tumors.
[0029] FIG. 3 shows an example of immunohistochemical stains of
human kidney tumor (renal cell carcinoma) (left), and matched
normal tissue from the same patient (right). The polyclonal rabbit
antibody serum used reacts to 2 epitopes: PPQLRPSRKRPCNSQP (amino
acid residues 1009 to 1024, SEQ ID NO:1), encoded by exon 16; this
epitope is found only in isoform 1, (GenBank Accession No.
NP.sub.--061905) and SRESGEELDERSCAAGAR (amino acid residues 693 to
710; SEQ ID NO:2), encoded by exon 11; this epitope is found in
both isoforms 1 (GenBank Accession No. NP.sub.--061905, SEQ ID NO:
11) and 2 (GenBank Accession No. NP.sub.--079284, SEQ ID
NO:12).
[0030] FIGS. 4A and 4B show a solid-phase diagnostic device for
determining CELSR1 levels in a human patient, at initial (4A) and
final stages (4B) of the assay.
[0031] FIG. 5 shows a portion of a gene chip useful for diagnosing
genetic predisposition to kidney cancer, constructed in accordance
with the present invention.
V. DETAILED DESCRIPTION
[0032] Before the present methods to diagnose and treat kidney and
colon and rectal, together colorectal, cancers are described, it is
to be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0033] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although any methods and materials
similar or equivalent to those described herein can be used in the
practice or testing of the present invention, some potential and
preferred methods and materials are now described. All publications
mentioned herein are incorporated herein by reference to disclose
and describe the methods and/or materials in connection with which
the publications are cited. It is understood that the present
disclosure supersedes any disclosure of an incorporated publication
to the extent there is a contradiction.
[0035] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the sequence" includes reference to one or more
sequences and equivalents thereof known to those skilled in the
art, and so forth.
[0036] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
A. DEFINITIONS
[0037] The following terms have the definitions given below, unless
otherwise indicated in the specification.
[0038] "Screening" for kidney or colorectal cancer, in accordance
with the present invention, means testing individuals for a level
of ADAMTSL4 that is indicative of kidney and/or colorectal cancer
or an elevated risk of kidney and/or colorectal cancer.
[0039] "Staging" treatment of kidney or colorectal cancer, in
accordance with the present invention, involves determining the
stage of kidney and/or colorectal cancer in an individual, based on
the level of ADAMTSL4 detected, and tailoring the treatment to that
stage. There are four recognized stages of cancer, which are
defined by the degree of localization of cancer cells. In addition,
kidney cancer may be defined as early stage at which the cancer is
responsive to a number of hormonal-based therapies, and a later,
more serious androgen-independent stage.
[0040] "A reduced level of ADAMTSL4 protein" may include, as an
indicator of cancer, a reduced level of wildtype ADAMTSL4 protein
or a reduced level of ADAMTSL4 protein having a specific epitope or
domain. That is, either the absence of any ADAMTSL4 protein or the
presence of a defective ADAMTSL4 protein may be indicative of
cancer, e.g., kidney and/or colorectal cancer.
[0041] "Colorectal" as used herein refers to colon and/or rectal
tissue. "Colorectal cancer" refers to cancer of the colon, rectum,
anus, and/or appendix.
B. ADAMTSL4 PROTEIN AND EXPRESSION
[0042] The thrombospondin type 1 repeat domain is found in many
proteins with diverse biological functions including cellular
adhesion, angiogenesis, and patterning of the developing nervous
system. Alternate transcriptional splice variants, encoding
different isoforms, have been characterized.
[0043] The thrombospondin type 1 repeat containing protein,
ADAMTSL4, (previously TSRC1) is a member of the ADAMTS-like gene
family (a disintegrin and metalloproteinase with thrombospondin
motifs). TSRs are extracellular protein domains that often contain
a protein binding site. The thrombospondin type 1 repeat domain is
found in many proteins with diverse biological functions including
cellular adhesion, angiogenesis, and patterning of the developing
nervous system (Buchner and Meisler). The human and the mouse genes
encode proteins of 1074 and 1036 amino acid residues (AA),
respectively (Buchner et al.). At least two alternatively spliced
transcript variants encoding isoforms 1 and 2 are known in humans.
Isoform 1 includes seven TSR (SEQ ID NOs:3-9) and an ADAM-TS spacer
(SEQ ID NO:10). Isoform 2 is a truncated version containing TSR 1-4
(SEQ ID NO:3-6) and the ADAM-TS spacer (SEQ ID NO:10). Isoform 2
has a unique 24 amino acid sequence at the C-terminus. The full
length isoform 1, GenBank Accession No. NM.sub.--019032, encodes a
1074 AA protein, GenBank Accession No. NP.sub.--061905, SEQ ID
NO:11. The truncated isoform 2, GenBank Accession No.
NM.sub.--025008, encodes an 877 AA protein, GenBank Accession No.
NP.sub.--079284, SEQ ID NO:12. Isoforms 1 and 2 have calculated
molecular weights of 116415 and 95001 Da, respectively. Mouse
ADAMTSL4 also has an isoform, GenBank Accession No. BC016215. The
4.4 kb mouse ADAMTSL4 transcript is expressed in a wide range of
fetal and adult tissues.sup.(1).
[0044] Specific binding sites contained in the thrombospondin type
1 repeat include WSXW, a heparin binding motif, CSVTCG, a CD36,
binding motif, and GGWSH, a fibronectin and TGF-.beta. binding
motif.sup.(1). The mouse ADAMTSL4 protein is widely expressed in
the brain, spinal cord, skin, muscle, liver, lung, spleen and
heart, etc..sup.(1).
C. IDENTIFICATION OF ADAMTSL4 AS A CANCER GENE
[0045] Cancer genes (oncogenes and tumor suppressor genes) were
defined in a high throughput manner by using proviral tagging.
Although viruses have not yet been implicated as a major cause of
cancers in humans, research using tumor viruses has led to the
discovery of many oncogenes and protooncogenes. In proviral
tagging, mice are infected with a retrovirus that does not contain
an oncogene (e.g., murine leukemia virus, MLV or murine mammary
tumor virus, MMTV).sup.(2-6). Recently, the host range of this
approach has been broadened by the use of a transposon.sup.(7,
8).
[0046] During retroviral infection, the virus integrates into the
cellular genome and inserts its DNA near or within genes, which
leads to various outcomes: (i) the insertion site is too far away
from a protooncogene and thus does not activate it. In this case,
there will be no selection for that cell. (ii) The provirus inserts
within 200 kb of a protooncogene, but not within the gene (type 1).
Here, either the viral promoter or the viral enhancer increases the
expression level of the protooncogene. (iii) The provirus inserts
within a gene, destroying or altering its function (type 2). There
will be no selection for a cell that contains either type 1 or type
2 insertion events in a gene that is not a protooncogene or tumor
suppressor gene. If integration results in the formation of a
tumor, genes adjacent to the integration site can be identified,
and classified as either protooncogenes or tumor suppressor genes.
This method has been used to identify many new protooncogenes as
well as to confirm already known protooncogenes discovered by
virtue of their homology to viral oncogenes.sup.(2, 3, 5, 6). A
tumor suppressor may be scored if a retrovirus lands within a gene
and truncates or destroys it. In these cases, the suppressor may be
haplo-insufficient, or alternatively, the mutation on the other
allele is provided spontaneously by the mouse. The integration
event may also lead to more complex consequences, such as a
dominant negative effect of the truncated gene product or the
transcription of anti-sense or microRNA.
In a screen with T lymphotropic virus SL3-3, two independent tumors
were recovered that contained proviral integrations within 5 kb of
the 3' end of the ADAMTSL4 gene (FIG. 2, handlebars at base
positions 223S-15-1-3 and 2102S-117-31-1). In a second screen with
B lymphotropic virus Akv1-99, one tumor was recovered that also
contained a proviral integration in the same region, 5 kb of the 3'
end of the ADAMTSL4 gene (FIG. 2, handlebar at base position
02-1157S-1). These integrations affect the transcription of the
canonical ADAMTSL4 gene and of the truncated form, as present in
the transcript for the mouse ADAMTS-like 4 mRNA (GenBank Accession
No. BC016215), which may represent a functionally different version
of ADAMTSL4.
D. EXPRESSION OF ADAMTSL4 IN HUMAN TUMORS AND IN NORMAL TISSUE
[0047] The mutations identified as causal in mouse tumor formation
affected both transcripts of the mouse. ADAMTSL4 protein expresses
in a wide variety of normal human tissues (www.t1dbase.org)
including the brain, ovaries, placenta, spinal cord, testis, and
thalamus. Additionally, ADAMTSL4 was found to be strongly expressed
in kidney and colon cells. However, the antigenic epitopes to the
ADAMTSL4 antibody were missing in colorectal carcinoma and in
tumors of the kidney (FIG. 3), whereas the normal counterpart of
these tumor cells strongly expressed the ADAMTSL4 protein (FIG. 3).
Clearly, the absence of part or all of these epitopes is a marker
for these tumors.
[0048] More generally, the invention provides a histological method
for examining tissue that normally expressed ADAMTSL4 for the
presence and extent of cancer. The method is especially useful for
examining kidney, colon, and/or rectal tissue. In the method
directed to examining kidney tissue, kidney tissue is stained with
a labeled antibody specific against a selected domain or epitope of
ADAMTSL4, e.g., fluorescence-labeled antibody (see Section E
below), to attach the marker to the surface of tissue cells. The
presence and extent of kidney cancer in the tissue is then
determined based on a reduced distribution and extent of detectable
marker with respect to the distribution and extent of marker in
normal kidney cells. It will be appreciated that a similar method
may be used for other tissues including colorectal tissue.
E. PREPARATION OF ANTI-ADAMTSL4 ANTIBODY
[0049] This section describes production of anti-ADAMTSL4
antibodies useful for diagnostic and therapeutic purposes, as
described further in the sections below. The anti-ADAMTSL4 antibody
used in the present invention can be obtained by any variety of
conventional methods to produce a monoclonal, polyclonal, and/or
recombinant antibody. One preferred antibody, particularly for
diagnostic use, is a mouse monoclonal antibody, prepared according
to well-known hybridoma methodology. Briefly, human ADAMTSL4 may be
first obtained, for example, by expressing the ADAMTSL4 gene. The
purified ADAMTSL4 protein acts as an immunogen. Alternatively, a
partial peptide of ADAMTSL4 can be used as a sensitization antigen.
In particular, for generating antibodies specific against a
selected epitope or domain of ADAMTSL4, a peptide defining that
domain or epitope may be used as the immunogen. These peptides can
be defined by the sequences given in the Sequence Listing below.
For example, to generate an antibody specific against an epitope
contained in SEQ ID NO:1, the peptide defined by this sequence is
employed as the immunogen.
[0050] Anti-ADAMTSL4 antibodies useful in diagnostic applications
may be labeled with a variety of detectable labels, including
detectable reporters, such as enzymes for enzyme-linked
immunosorbent assays (ELISA), detectable particles, such as gold
particles and reporter-carrying liposomes, colorimetric or
fluorescent reporters, labels such as quantum dot nanocrystal
particles, radiolabels, and labels such as a biotin label by which
secondary detectable labels, such as a reporter-labeled
streptavidin label can be attached. In some assay formats, an
unlabeled anti-ADAMTSL4 antibody, for example, a mouse IgG
antibody, is detected by reaction with a labeled antibody, e.g., a
labeled anti-mouse IgG antibody.
[0051] For therapeutic uses, human monoclonal antibodies having
binding activity to ADAMTSL4 can be produced by sensitizing in
vitro human lymphocytes with ADAMTSL4, and causing the sensitized
lymphocytes to fuse with the human-derived myeloma cells having a
permanent division potential. Alternatively, ADAMTSL4 as an antigen
can be administered to a transgenic animal having all the
repertories of a human antibody gene to obtain anti-ADAMTSL4
antibody-producing cells, and then human antibodies for ADAMTSL4
may be obtained from the immortalized anti-ADAMTSL4
antibody-producing cells.
[0052] In still other methods, human or humanized antibodies
specific against ADAMTSL4 antigen can be prepared by recombinant
techniques, such as have been reported (see, for example, U.S. Pat.
Nos. 6,090,382 and 6,258,562).
F. DIAGNOSTIC METHODS AND REAGENTS
[0053] In one aspect, the invention includes a method of screening
for kidney and/or colorectal cancer in a human subject. In another
aspect, the invention includes a method of staging treatment of
kidney and/or colorectal cancer in a subject. This is done, in
accordance with the invention, by reacting a body-fluid sample from
the subject with an antibody specific against a selected domain or
epitope of ADAMTSL4, and determining from the presence and/or
amount of immunoassay product, whether the subject has a reduced
level of ADAMTSL4 protein lacking the specific domain or epitope,
when compared with a normal range of ADAMTSL4 in human samples.
Reduced levels are an indicator of kidney or colorectal cancer.
[0054] Preferred body-fluid samples are blood and urine. Where
urine is assayed, the assayed level of ADAMTSL4 indicative of
kidney or colorectal cancer is typically in the range of less than
about 0.1 ng/ml sample fluid.
[0055] The assay may be carried out by any of a variety of assay
methods used for detecting body-fluid antigens, including ELISA
techniques, homogeneous assays, for example, involving fluorescence
quenching, and a variety of solid-phase sandwich assays in which
the ADAMTSL4 antigen is captured by an anti-ADAMTSL4 antibody
carried on a solid support, and the immobilized antigen-antibody
complex is labeled with a second anti-ADAMTSL4 antibody, e.g., a
second antibody carrying a colorimetric or gold-particle
reporter.
[0056] FIGS. 4A and 4B illustrate a solid-phase assay strip
constructed in accordance with an embodiment of the invention,
suitable for carrying out a sandwich immunoassay of the type just
mentioned, and shown in initial and final assay states,
respectively. The strip, indicated generally at 10, includes a
porous support or pad 12 having a sample-application zone 14 in an
upstream region of the support and a sample-detection zone 16 in a
downstream region. The sample-application zone includes a
detectable anti-ADAMTSL4 antibody reagent, e.g., anti-ADAMTSL4
antibodies labeled with gold particles, and carried in the zone in
an unbound, i.e., non-immobilized form. This reagent is indicated
by solid circles, such as at 18. Anti-ADAMTSL4 antibodies, which
may be the same or different from those in the labeled antibody
reagent, are immobilized to the solid support within the detection
zone, and are indicated by the "Y" shapes, such as at 20.
[0057] Also shown is a reference zone 22 which is located adjacent
the detection zone and has one or more colored or shaded regions
corresponding to different assay levels of ADAMTSL4 in a body-fluid
sample. In the embodiment shown, zone 22 includes three regions
22a, 22b, and 22c, corresponding to an assayed level of ADAMTSL4
(a) below that associated with cancer, (b) corresponding to a lower
threshold level associated with cancer, and (c) a level that is
substantially higher, e.g., 2-3 times, higher than the threshold
layer in region 22b, respectively. These three regions provide a
known standard indicator against which the level of detectable
reaction produced can be assessed as a level associated with kidney
or colorectal cancer. Together, the assay strip and reference zone
constitute an assay device for use in screening for kidney and/or
colorectal cancer in a human subject, or for staging treatment of
kidney and/or colorectal cancer in a human subject.
[0058] In operation, a known volume of a body-fluid sample to be
tested is added to the sample-application zone of the strip, where
it diffuses into the zone, allowing the antibody reagent to react
with ADAMTSL4 antigen in the sample to form an antigen-antibody
complex. This complex and unbound antibody reagent then migrate
downstream by capillarity toward the detection zone, where the
antigen-antibody complex is captured by the immobilize antibody and
the unbound reagent is carried to the end of the support, as
indicated at 24. As can be appreciated, the higher the
concentration of antigen in the body fluid, the higher the density
of captured reagent in the detection zone and the greater the color
or intensity in this zone. This color or intensity produced in the
detection zone is compared with the standards in the reference zone
to determine a qualitative level of ADAMTSL4 associated with the
presence or absence of kidney or colorectal cancer. If a
sub-threshold level or threshold level of ADAMTSL4 is observed in
the assay, the subject can be classified in a higher-probability
category for the presence of cancer and the subject may be
recommended for additional testing and/or more frequent
testing.
[0059] In another embodiment, the assay device includes an assay
strip like that described above, but where the known-reference
indicator is provided by a strip-reader instrument reader having
(i) a reader slot for receiving the assay strip, (ii) a light
source and an optical detection, e.g., a spectrophotometric
detector, for detecting an assay-related optical condition at the
detection zone of the assay strip, (iii) an electronics or
processor unit which records and processes a signal from the
optical detector, and converts the signal to an assayed level of
ADAMTSL4, and (iv) a user display screen or window. The instrument
may report the actual ADAMTSL4 body-fluid sample detected, allowing
the operator to compare the displayed value with known standard
indicator levels provided with the assay strip or instrument, to
assess whether the subject has a reduced ADAMTSL4 level associated
with kidney cancer and/or colorectal cancer, or to assess the
possible stage of the cancer, for purposes of treatment design.
Alternatively, the instrument itself may contain stored
known-standard indicator levels which can be compared internally
with an assayed level to generate an output that indicates whether
an reduced ADAMTSL4 level associated with kidney or colorectal
cancer has been detected, or to indicate the stage of the
cancer.
G. IDENTIFYING GENETIC MUTATION ASSOCIATED WITH CANCER
[0060] In another aspect, the invention provides a method for
identifying mutations associated with increased risk of cancer,
such as kidney and/or colorectal cancer, in a human subject. The
section below is described in relation to kidney cancer; however,
it will be appreciated that the method may be practiced for other
cancers involving decreased expression of ADAMTSL4 such as
colorectal cancer. In practicing the method, genomic DNA is
extracted from human patients having kidney cancer, preferably
including patients from men or women representing different racial
and age groups. The DNA sequences that are examined, in particular;
are (i) one or more of exons 1 to 17 of the ADAMTSL4 on chromosome
1q21, including adjacent splice site acceptor and donor sequences
of the exons, (ii) a 5' UTR region within 10 kB or less of exon 1
of the gene, and (iii) a 3' UTR region within 10 kB or less of exon
17.
[0061] Mutations at one or more sites along the region are
identified by comparing each of the sequences with sequences from
the same region derived from normal (wildtype) kidney tissue.
Preferably sequences from a number of wildtype individuals are
determined to ensure a true wildtype sequence. For each extracted
DNA, the patient and wildtype sequences are compared to identify
mutations in the patient sequences, and thus mutations that are
likely associated with increased risk of kidney cancer.
[0062] Once a large number of these mutations are identified, e.g.,
at least 50-200 or more, they may be used in constructing a genetic
screening device, e.g., a gene chip, useful for screening
individuals for genetic predisposition to kidney cancer. In one
embodiment, the device includes a gene chip, such as shown at 30 in
FIG. 5, having an array of regions, such as regions 34, 36, each
containing bound known-sequence fragments, such as fragment 37 in
region 34. The fragments or probes are preferably 25-70 bases in
lengths, and each includes one of the above-identified mutations
upstream of the ADAMTSL4 gene that is associated with kidney
cancer. Gene-chip construction and detection of mutant sequences
with such chips are well known.
[0063] In a typical genetic-screening procedure, patient cells are
obtained, genomic DNA is extracted, and sequence regions of
interest are amplified by standard PCR, employing fluoresceinated
probes. The amplified material is then reacted with the chip-array
sequences, under suitable hybridization conditions, and the array
surface is washed to remove unbound material, and then scanned with
a suitable chip reader to identify any mutated sequences associated
with kidney cancer. The figure shows binding of a labeled genomic
DNA fragment, indicated at 42, to an array region 38 having bound
probe molecules 40. Detection of a fluorescent signal in this array
region is diagnostic of a known genetic mutation in the critical
upstream ADAMTSL4 region may be diagnostic of a genetic
predisposition to kidney cancer.
[0064] In an alternative embodiment, the mutations identified as
above are used to construct a set of molecular inversion probes
(MIPs) capable of identifying the presence of genomic mutations.
The construction and use of MIPs for identifying genetic mutations
have been described (see, for example, Wang, et al., Nucleic Acids
Research, (England) 2005, Vol. 33, p. 21).
H. TREATMENT METHODS AND PHARMACEUTICAL PREPARATIONS
[0065] The invention also includes methods for treating, e.g.,
reducing the tumor burden in a human subject with a cancer
characterized by a reduced expression of ADAMTSL4 in the cancer
cells. The section below is described in relation to kidney cancer;
however, it will be appreciated that the method may be practiced
for other cancers involving decreased expression of ADAMTSL4 such
as colorectal cancer.
[0066] In one general immunotherapy approach, a patient diagnosed
with kidney cancer is first confirmed as having reduced levels of
ADAMTSL4, according to assay methods described above. If the
subject tests positive in this assay, he is treated by
administration of anti-ADAMTSL4 antibody. Preferably the antibody
is a human or humanized antibody, prepared as described above, and
is administered by IV or subcutaneous injection in a suitable
physiological carrier. The antibody dose is preferably 1 to 10
mg/injection, and the patient is treated at intervals of every 14
days or so. During treatment, the patient is monitored for change
in status of the cancer, typically by a combination of a
tumor-visualization procedure and levels of kidney-related
antigens, as above. The treatment may also be carried out in
combination with other kidney-cancer treatments, including drug or
radioisotope therapy, and may be continued until a desired
diminution in tumor size is observed.
[0067] While the invention has been described with respect to
particular embodiments and applications, it will be appreciated
that various changes and modification may be made without departing
from the invention as claimed.
TABLE-US-00001 Sequences: SEQ ID NO: 1: Antibody epitope sequence
(residues 1009 to 1024 of isoform 1) PPQLRPSRKRPCNSQP SEQ ID NO: 2:
Antibody epitope sequence (residues 693-710 of isoform 1 and 2)
SRESGEELDERSCAAGAR SEQ ID NO: 3: Human Thrombospondin Repeat 1
(TSR1) from isoform 1 (residues 47-102)
WGPWVQWASCSQPCGVGVQRRSRTCQLPTVQLHPSLPLPPRPPRHPEALL PRGQGP SEQ ID
NO: 4: Human Thrombospondin Repeat 2 (TSR2) from isoform 1
(residues 668-724)
WKRVGHSACSASCGKGVWRPIFLCISRESGEELDERSCAAGARPPASPEP CHGTPCP SEQ ID
NO: 5: Human Thrombospondin Repeat 3 (TSR3) from isoform 1
(residues 726-784)
YWEAGEWTSCSRSCGPGTQHRQLQCRQEFGGGGSSVPPERCGHLPRPNIT QSCQLRLCG SEQ ID
NO: 6: Human Thrombospondin Repeat 4 (TSR4) from isoform 1
(residues 786-842)
WEVGSPWSQCSVRCGRGQRSRQVRCVGNNGDEVSEQECASGPPQPPSREA CDMGPCT SEQ ID
NO: 7: Human Thrombospondin Repeat 5 (TSR5) from isoform 1
(residues 847-909)
HSDWSSKCSAECGTGIQRRSVVCLGSGAALGPGQGEAGAGTGQSCPTGSR PPDMRACSLGPCE
SEQ ID NO: 8: Human Thrombospondin Repeat 6 (TSR6) from isoform 1
(residues 914-971)
WYTGPWGECSSECGSGTQRRDIICVSKLGTEFNVTSPSNCSHLPRPPALQ PCQGQACQ SEQ ID
NO: 9: Human Thrombospondin Repeat 7 (TSR7) from isoform 1
(residues 973-1026)
RWFSTPWSPCSRSCQGGTQTREVQCLSTNQTLSTRCPPQLRPSRKRPCNS QPCS SEQ ID NO:
10: Human ADAM-TS Spacer from isoform 1 (residues 485-599)
RLVSGNLTDRGGPLGYQKILWIPAGALRLQIAQLRPSSNYLALRGPGGRS
IINGNWAVDPPGSYRAGGTVFRYNRPPREEGKGESLSAEGPTTQPVDVYM IFQEENPGVFYQYVI
SEQ ID NO: 11: Human thrombospondin repeat containing 1, isoform 1
(Gen Bank Accession No. NP_061905)
MENWTGRPWLYLLLLLSLPQLCLDQEVLSGHSLQTPTEEGQGPEGVWGPW
VQWASCSQPCGVGVQRRSRTCQLPTVQLHPSLPLPPRPPRHPEALLPRGQ
GPRPQTSPETLPLYRTQSRGRGGPLRGPASHLGREETQEIRAARRSRLRD
PIKPGMFGYGRVPFALPLHRNRRHPRSPPRSELSLISSRGEEAIPSPTPR
AEPFSANGSPQTELPPTELSVHTPSPQAEPLSPETAQTEVAPRTRPAPLR
HHPRAQASGTEPPSPTHSLGEGGFFRASPQPRRPSSQGWASPQVAGRRPD
PFPSVPRGRGQQGQGPWGTGGTPHGPRLEPDPQHPGAWLPLLSNGPHASS
LWSLFAPSSPIPRCSGESEQLRACSQAPCPPEQPDPRALQCAAFNSQEFM
GQLYQWEPFTEVQGSQRCELNCRPRGFRFYVRHTEKVQDGTLCQPGAPDI
CVAGRCLSPGCDGILGSGRRPDGCGVCGGDDSTCRLVSGNLTDRGGPLGY
QKILWIPAGALRLQIAQLRPSSNYLALRGPGGRSIINGNWAVDPPGSYRA
GGTVFRYNRPPREEGKGESLSAEGPTTQPVDVYMIFQEENPGVFYQYVIS
SPPPILENPTPEPPVPQLQPEILRVEPPLAPAPRPARTPGTLQRQVRIPQ
MPAPPHPRTPLGSPAAYWKRVGHSACSASCGKGVWRPIFLCISRESGEEL
DERSCAAGARPPASPEPCHGTPCPPYWEAGEWTSCSRSCGPGTQHRQLQC
RQEFGGGGSSVPPERCGHLPRPNITQSCQLRLCGHWEVGSPWSQCSVRCG
RGQRSRQVRCVGNNGDEVSEQECASGPPQPPSREACDMGPCTTAWFHSDW
SSKCSAECGTGIQRRSWCLGSGAALGPGQGEAGAGTGQSCPTGSRPPDMR
ACSLGPCERTWRWYTGPWGECSSECGSGTQRRDIICVSKLGTEFNVTSPS
NCSHLPRPPALQPCQGQACQDRWFSTPWSPCSRSCQGGTQTREVQCLSTN
QTLSTRCPPQLRPSRKRPCNSQPCSQRPDDQCKDSSPHCPLWQARLCVYP
YYTATCCRSCAHVLERSPQDPS SEQ ID NO: 12: Human thrombospondin repeat
containing 1, isoform 2 (Gen Bank Accession No. NP_079284)
MENWTGRPWLYLLLLLSLPQLCLDQEVLSGHSLQTPTEEGQGPEGVWGPW
VQWASCSQPCGVGVQRRSRTCQLPTVQLHPSLPLPPRPPRHPEALLPRGQ
GPRPQTSPETLPLYRTQSRGRGGPLRGPASHLGREETQEIRAARRSRLRD
PIKPGMFGYGRVPFALPLHRNRRHPRSPPRSELSLISSRGEEAIPSPTPR
AEPFSANGSPQTELPPTELSVHTPSPQAEPLSPETAQTEVAPRTRPAPLR
HHPRAQASGTEPPSPTHSLGEGGFFRASPQPRRPSSQGWASPQVAGRRPD
PFPSVPRGRGQQGQGPWGTGGTPHGPRLEPDPQHPGAWLPLLSNGPHASS
LWSLFAPSSPIPRCSGESEQLRACSQAPCPPEQPDPRALQCAAFNSQEFM
GQLYQWEPFTEVQGSQRCELNCRPRGFRFYVRHTEKVQDGTLCQPGAPDI
CVAGRCLSPGCDGILGSGRRPDGCGVCGGDDSTCRLVSGNLTDRGGPLGY
QKILWIPAGALRLQIAQLRPSSNYLALRGPGGRSIINGNWAVDPPGSYRA
GGTVFRYNRPPREEGKGESLSAEGPTTQPVDVYMIFQEENPGVFYQYVIS
SPPPILENPTPEPPVPQLQPEILRVEPPLAPAPRPARTPGTLQRQVRIPQ
MPAPPHPRTPLGSPAAYWKRVGHSACSASCGKGVWRPIFLCISRESGEEL
DERSCAAGARPPASPEPCHGTPCPPYWEAGEWTSCSRSCGPGTQHRQLQC
RQEFGGGGSSVPPERCGHLPRPNITQSCQLRLCGHWEVGSPWSQCSVRCG
RGQRSRQVRCVGNNGDEVSEQECASGPPQPPSREACDMGPCTTAWFHSDW
SSKVSPEPPAISCILGNHAQDTSAFPA
Sequence CWU 1
1
12116PRTHomo sapiens 1Pro Pro Gln Leu Arg Pro Ser Arg Lys Arg Pro
Cys Asn Ser Gln Pro1 5 10 15218PRTHomo sapiens 2Ser Arg Glu Ser Gly
Glu Glu Leu Asp Glu Arg Ser Cys Ala Ala Gly1 5 10 15Ala
Arg356PRTHomo sapiens 3Trp Gly Pro Trp Val Gln Trp Ala Ser Cys Ser
Gln Pro Cys Gly Val1 5 10 15Gly Val Gln Arg Arg Ser Arg Thr Cys Gln
Leu Pro Thr Val Gln Leu 20 25 30His Pro Ser Leu Pro Leu Pro Pro Arg
Pro Pro Arg His Pro Glu Ala 35 40 45Leu Leu Pro Arg Gly Gln Gly Pro
50 55457PRTHomo sapiens 4Trp Lys Arg Val Gly His Ser Ala Cys Ser
Ala Ser Cys Gly Lys Gly1 5 10 15Val Trp Arg Pro Ile Phe Leu Cys Ile
Ser Arg Glu Ser Gly Glu Glu 20 25 30Leu Asp Glu Arg Ser Cys Ala Ala
Gly Ala Arg Pro Pro Ala Ser Pro 35 40 45Glu Pro Cys His Gly Thr Pro
Cys Pro 50 55559PRTHomo sapiens 5Tyr Trp Glu Ala Gly Glu Trp Thr
Ser Cys Ser Arg Ser Cys Gly Pro1 5 10 15Gly Thr Gln His Arg Gln Leu
Gln Cys Arg Gln Glu Phe Gly Gly Gly 20 25 30Gly Ser Ser Val Pro Pro
Glu Arg Cys Gly His Leu Pro Arg Pro Asn 35 40 45Ile Thr Gln Ser Cys
Gln Leu Arg Leu Cys Gly 50 55657PRTHomo sapiens 6Trp Glu Val Gly
Ser Pro Trp Ser Gln Cys Ser Val Arg Cys Gly Arg1 5 10 15Gly Gln Arg
Ser Arg Gln Val Arg Cys Val Gly Asn Asn Gly Asp Glu 20 25 30Val Ser
Glu Gln Glu Cys Ala Ser Gly Pro Pro Gln Pro Pro Ser Arg 35 40 45Glu
Ala Cys Asp Met Gly Pro Cys Thr 50 55763PRTHomo sapiens 7His Ser
Asp Trp Ser Ser Lys Cys Ser Ala Glu Cys Gly Thr Gly Ile1 5 10 15Gln
Arg Arg Ser Val Val Cys Leu Gly Ser Gly Ala Ala Leu Gly Pro 20 25
30Gly Gln Gly Glu Ala Gly Ala Gly Thr Gly Gln Ser Cys Pro Thr Gly
35 40 45Ser Arg Pro Pro Asp Met Arg Ala Cys Ser Leu Gly Pro Cys Glu
50 55 60858PRTHomo sapiens 8Trp Tyr Thr Gly Pro Trp Gly Glu Cys Ser
Ser Glu Cys Gly Ser Gly1 5 10 15Thr Gln Arg Arg Asp Ile Ile Cys Val
Ser Lys Leu Gly Thr Glu Phe 20 25 30Asn Val Thr Ser Pro Ser Asn Cys
Ser His Leu Pro Arg Pro Pro Ala 35 40 45Leu Gln Pro Cys Gln Gly Gln
Ala Cys Gln 50 55954PRTHomo sapiens 9Arg Trp Phe Ser Thr Pro Trp
Ser Pro Cys Ser Arg Ser Cys Gln Gly1 5 10 15Gly Thr Gln Thr Arg Glu
Val Gln Cys Leu Ser Thr Asn Gln Thr Leu 20 25 30Ser Thr Arg Cys Pro
Pro Gln Leu Arg Pro Ser Arg Lys Arg Pro Cys 35 40 45Asn Ser Gln Pro
Cys Ser 5010115PRTHomo sapiens 10Arg Leu Val Ser Gly Asn Leu Thr
Asp Arg Gly Gly Pro Leu Gly Tyr1 5 10 15Gln Lys Ile Leu Trp Ile Pro
Ala Gly Ala Leu Arg Leu Gln Ile Ala 20 25 30Gln Leu Arg Pro Ser Ser
Asn Tyr Leu Ala Leu Arg Gly Pro Gly Gly 35 40 45Arg Ser Ile Ile Asn
Gly Asn Trp Ala Val Asp Pro Pro Gly Ser Tyr 50 55 60Arg Ala Gly Gly
Thr Val Phe Arg Tyr Asn Arg Pro Pro Arg Glu Glu65 70 75 80Gly Lys
Gly Glu Ser Leu Ser Ala Glu Gly Pro Thr Thr Gln Pro Val 85 90 95Asp
Val Tyr Met Ile Phe Gln Glu Glu Asn Pro Gly Val Phe Tyr Gln 100 105
110Tyr Val Ile 115111074PRTHomo sapiens 11Met Glu Asn Trp Thr Gly
Arg Pro Trp Leu Tyr Leu Leu Leu Leu Leu1 5 10 15Ser Leu Pro Gln Leu
Cys Leu Asp Gln Glu Val Leu Ser Gly His Ser 20 25 30Leu Gln Thr Pro
Thr Glu Glu Gly Gln Gly Pro Glu Gly Val Trp Gly 35 40 45Pro Trp Val
Gln Trp Ala Ser Cys Ser Gln Pro Cys Gly Val Gly Val 50 55 60Gln Arg
Arg Ser Arg Thr Cys Gln Leu Pro Thr Val Gln Leu His Pro65 70 75
80Ser Leu Pro Leu Pro Pro Arg Pro Pro Arg His Pro Glu Ala Leu Leu
85 90 95Pro Arg Gly Gln Gly Pro Arg Pro Gln Thr Ser Pro Glu Thr Leu
Pro 100 105 110Leu Tyr Arg Thr Gln Ser Arg Gly Arg Gly Gly Pro Leu
Arg Gly Pro 115 120 125Ala Ser His Leu Gly Arg Glu Glu Thr Gln Glu
Ile Arg Ala Ala Arg 130 135 140Arg Ser Arg Leu Arg Asp Pro Ile Lys
Pro Gly Met Phe Gly Tyr Gly145 150 155 160Arg Val Pro Phe Ala Leu
Pro Leu His Arg Asn Arg Arg His Pro Arg 165 170 175Ser Pro Pro Arg
Ser Glu Leu Ser Leu Ile Ser Ser Arg Gly Glu Glu 180 185 190Ala Ile
Pro Ser Pro Thr Pro Arg Ala Glu Pro Phe Ser Ala Asn Gly 195 200
205Ser Pro Gln Thr Glu Leu Pro Pro Thr Glu Leu Ser Val His Thr Pro
210 215 220Ser Pro Gln Ala Glu Pro Leu Ser Pro Glu Thr Ala Gln Thr
Glu Val225 230 235 240Ala Pro Arg Thr Arg Pro Ala Pro Leu Arg His
His Pro Arg Ala Gln 245 250 255Ala Ser Gly Thr Glu Pro Pro Ser Pro
Thr His Ser Leu Gly Glu Gly 260 265 270Gly Phe Phe Arg Ala Ser Pro
Gln Pro Arg Arg Pro Ser Ser Gln Gly 275 280 285Trp Ala Ser Pro Gln
Val Ala Gly Arg Arg Pro Asp Pro Phe Pro Ser 290 295 300Val Pro Arg
Gly Arg Gly Gln Gln Gly Gln Gly Pro Trp Gly Thr Gly305 310 315
320Gly Thr Pro His Gly Pro Arg Leu Glu Pro Asp Pro Gln His Pro Gly
325 330 335Ala Trp Leu Pro Leu Leu Ser Asn Gly Pro His Ala Ser Ser
Leu Trp 340 345 350Ser Leu Phe Ala Pro Ser Ser Pro Ile Pro Arg Cys
Ser Gly Glu Ser 355 360 365Glu Gln Leu Arg Ala Cys Ser Gln Ala Pro
Cys Pro Pro Glu Gln Pro 370 375 380Asp Pro Arg Ala Leu Gln Cys Ala
Ala Phe Asn Ser Gln Glu Phe Met385 390 395 400Gly Gln Leu Tyr Gln
Trp Glu Pro Phe Thr Glu Val Gln Gly Ser Gln 405 410 415Arg Cys Glu
Leu Asn Cys Arg Pro Arg Gly Phe Arg Phe Tyr Val Arg 420 425 430His
Thr Glu Lys Val Gln Asp Gly Thr Leu Cys Gln Pro Gly Ala Pro 435 440
445Asp Ile Cys Val Ala Gly Arg Cys Leu Ser Pro Gly Cys Asp Gly Ile
450 455 460Leu Gly Ser Gly Arg Arg Pro Asp Gly Cys Gly Val Cys Gly
Gly Asp465 470 475 480Asp Ser Thr Cys Arg Leu Val Ser Gly Asn Leu
Thr Asp Arg Gly Gly 485 490 495Pro Leu Gly Tyr Gln Lys Ile Leu Trp
Ile Pro Ala Gly Ala Leu Arg 500 505 510Leu Gln Ile Ala Gln Leu Arg
Pro Ser Ser Asn Tyr Leu Ala Leu Arg 515 520 525Gly Pro Gly Gly Arg
Ser Ile Ile Asn Gly Asn Trp Ala Val Asp Pro 530 535 540Pro Gly Ser
Tyr Arg Ala Gly Gly Thr Val Phe Arg Tyr Asn Arg Pro545 550 555
560Pro Arg Glu Glu Gly Lys Gly Glu Ser Leu Ser Ala Glu Gly Pro Thr
565 570 575Thr Gln Pro Val Asp Val Tyr Met Ile Phe Gln Glu Glu Asn
Pro Gly 580 585 590Val Phe Tyr Gln Tyr Val Ile Ser Ser Pro Pro Pro
Ile Leu Glu Asn 595 600 605Pro Thr Pro Glu Pro Pro Val Pro Gln Leu
Gln Pro Glu Ile Leu Arg 610 615 620Val Glu Pro Pro Leu Ala Pro Ala
Pro Arg Pro Ala Arg Thr Pro Gly625 630 635 640Thr Leu Gln Arg Gln
Val Arg Ile Pro Gln Met Pro Ala Pro Pro His 645 650 655Pro Arg Thr
Pro Leu Gly Ser Pro Ala Ala Tyr Trp Lys Arg Val Gly 660 665 670His
Ser Ala Cys Ser Ala Ser Cys Gly Lys Gly Val Trp Arg Pro Ile 675 680
685Phe Leu Cys Ile Ser Arg Glu Ser Gly Glu Glu Leu Asp Glu Arg Ser
690 695 700Cys Ala Ala Gly Ala Arg Pro Pro Ala Ser Pro Glu Pro Cys
His Gly705 710 715 720Thr Pro Cys Pro Pro Tyr Trp Glu Ala Gly Glu
Trp Thr Ser Cys Ser 725 730 735Arg Ser Cys Gly Pro Gly Thr Gln His
Arg Gln Leu Gln Cys Arg Gln 740 745 750Glu Phe Gly Gly Gly Gly Ser
Ser Val Pro Pro Glu Arg Cys Gly His 755 760 765Leu Pro Arg Pro Asn
Ile Thr Gln Ser Cys Gln Leu Arg Leu Cys Gly 770 775 780His Trp Glu
Val Gly Ser Pro Trp Ser Gln Cys Ser Val Arg Cys Gly785 790 795
800Arg Gly Gln Arg Ser Arg Gln Val Arg Cys Val Gly Asn Asn Gly Asp
805 810 815Glu Val Ser Glu Gln Glu Cys Ala Ser Gly Pro Pro Gln Pro
Pro Ser 820 825 830Arg Glu Ala Cys Asp Met Gly Pro Cys Thr Thr Ala
Trp Phe His Ser 835 840 845Asp Trp Ser Ser Lys Cys Ser Ala Glu Cys
Gly Thr Gly Ile Gln Arg 850 855 860Arg Ser Val Val Cys Leu Gly Ser
Gly Ala Ala Leu Gly Pro Gly Gln865 870 875 880Gly Glu Ala Gly Ala
Gly Thr Gly Gln Ser Cys Pro Thr Gly Ser Arg 885 890 895Pro Pro Asp
Met Arg Ala Cys Ser Leu Gly Pro Cys Glu Arg Thr Trp 900 905 910Arg
Trp Tyr Thr Gly Pro Trp Gly Glu Cys Ser Ser Glu Cys Gly Ser 915 920
925Gly Thr Gln Arg Arg Asp Ile Ile Cys Val Ser Lys Leu Gly Thr Glu
930 935 940Phe Asn Val Thr Ser Pro Ser Asn Cys Ser His Leu Pro Arg
Pro Pro945 950 955 960Ala Leu Gln Pro Cys Gln Gly Gln Ala Cys Gln
Asp Arg Trp Phe Ser 965 970 975Thr Pro Trp Ser Pro Cys Ser Arg Ser
Cys Gln Gly Gly Thr Gln Thr 980 985 990Arg Glu Val Gln Cys Leu Ser
Thr Asn Gln Thr Leu Ser Thr Arg Cys 995 1000 1005Pro Pro Gln Leu
Arg Pro Ser Arg Lys Arg Pro Cys Asn Ser Gln 1010 1015 1020Pro Cys
Ser Gln Arg Pro Asp Asp Gln Cys Lys Asp Ser Ser Pro 1025 1030
1035His Cys Pro Leu Val Val Gln Ala Arg Leu Cys Val Tyr Pro Tyr
1040 1045 1050Tyr Thr Ala Thr Cys Cys Arg Ser Cys Ala His Val Leu
Glu Arg 1055 1060 1065Ser Pro Gln Asp Pro Ser 107012877PRTHomo
sapiens 12Met Glu Asn Trp Thr Gly Arg Pro Trp Leu Tyr Leu Leu Leu
Leu Leu1 5 10 15Ser Leu Pro Gln Leu Cys Leu Asp Gln Glu Val Leu Ser
Gly His Ser 20 25 30Leu Gln Thr Pro Thr Glu Glu Gly Gln Gly Pro Glu
Gly Val Trp Gly 35 40 45Pro Trp Val Gln Trp Ala Ser Cys Ser Gln Pro
Cys Gly Val Gly Val 50 55 60Gln Arg Arg Ser Arg Thr Cys Gln Leu Pro
Thr Val Gln Leu His Pro65 70 75 80Ser Leu Pro Leu Pro Pro Arg Pro
Pro Arg His Pro Glu Ala Leu Leu 85 90 95Pro Arg Gly Gln Gly Pro Arg
Pro Gln Thr Ser Pro Glu Thr Leu Pro 100 105 110Leu Tyr Arg Thr Gln
Ser Arg Gly Arg Gly Gly Pro Leu Arg Gly Pro 115 120 125Ala Ser His
Leu Gly Arg Glu Glu Thr Gln Glu Ile Arg Ala Ala Arg 130 135 140Arg
Ser Arg Leu Arg Asp Pro Ile Lys Pro Gly Met Phe Gly Tyr Gly145 150
155 160Arg Val Pro Phe Ala Leu Pro Leu His Arg Asn Arg Arg His Pro
Arg 165 170 175Ser Pro Pro Arg Ser Glu Leu Ser Leu Ile Ser Ser Arg
Gly Glu Glu 180 185 190Ala Ile Pro Ser Pro Thr Pro Arg Ala Glu Pro
Phe Ser Ala Asn Gly 195 200 205Ser Pro Gln Thr Glu Leu Pro Pro Thr
Glu Leu Ser Val His Thr Pro 210 215 220Ser Pro Gln Ala Glu Pro Leu
Ser Pro Glu Thr Ala Gln Thr Glu Val225 230 235 240Ala Pro Arg Thr
Arg Pro Ala Pro Leu Arg His His Pro Arg Ala Gln 245 250 255Ala Ser
Gly Thr Glu Pro Pro Ser Pro Thr His Ser Leu Gly Glu Gly 260 265
270Gly Phe Phe Arg Ala Ser Pro Gln Pro Arg Arg Pro Ser Ser Gln Gly
275 280 285Trp Ala Ser Pro Gln Val Ala Gly Arg Arg Pro Asp Pro Phe
Pro Ser 290 295 300Val Pro Arg Gly Arg Gly Gln Gln Gly Gln Gly Pro
Trp Gly Thr Gly305 310 315 320Gly Thr Pro His Gly Pro Arg Leu Glu
Pro Asp Pro Gln His Pro Gly 325 330 335Ala Trp Leu Pro Leu Leu Ser
Asn Gly Pro His Ala Ser Ser Leu Trp 340 345 350Ser Leu Phe Ala Pro
Ser Ser Pro Ile Pro Arg Cys Ser Gly Glu Ser 355 360 365Glu Gln Leu
Arg Ala Cys Ser Gln Ala Pro Cys Pro Pro Glu Gln Pro 370 375 380Asp
Pro Arg Ala Leu Gln Cys Ala Ala Phe Asn Ser Gln Glu Phe Met385 390
395 400Gly Gln Leu Tyr Gln Trp Glu Pro Phe Thr Glu Val Gln Gly Ser
Gln 405 410 415Arg Cys Glu Leu Asn Cys Arg Pro Arg Gly Phe Arg Phe
Tyr Val Arg 420 425 430His Thr Glu Lys Val Gln Asp Gly Thr Leu Cys
Gln Pro Gly Ala Pro 435 440 445Asp Ile Cys Val Ala Gly Arg Cys Leu
Ser Pro Gly Cys Asp Gly Ile 450 455 460Leu Gly Ser Gly Arg Arg Pro
Asp Gly Cys Gly Val Cys Gly Gly Asp465 470 475 480Asp Ser Thr Cys
Arg Leu Val Ser Gly Asn Leu Thr Asp Arg Gly Gly 485 490 495Pro Leu
Gly Tyr Gln Lys Ile Leu Trp Ile Pro Ala Gly Ala Leu Arg 500 505
510Leu Gln Ile Ala Gln Leu Arg Pro Ser Ser Asn Tyr Leu Ala Leu Arg
515 520 525Gly Pro Gly Gly Arg Ser Ile Ile Asn Gly Asn Trp Ala Val
Asp Pro 530 535 540Pro Gly Ser Tyr Arg Ala Gly Gly Thr Val Phe Arg
Tyr Asn Arg Pro545 550 555 560Pro Arg Glu Glu Gly Lys Gly Glu Ser
Leu Ser Ala Glu Gly Pro Thr 565 570 575Thr Gln Pro Val Asp Val Tyr
Met Ile Phe Gln Glu Glu Asn Pro Gly 580 585 590Val Phe Tyr Gln Tyr
Val Ile Ser Ser Pro Pro Pro Ile Leu Glu Asn 595 600 605Pro Thr Pro
Glu Pro Pro Val Pro Gln Leu Gln Pro Glu Ile Leu Arg 610 615 620Val
Glu Pro Pro Leu Ala Pro Ala Pro Arg Pro Ala Arg Thr Pro Gly625 630
635 640Thr Leu Gln Arg Gln Val Arg Ile Pro Gln Met Pro Ala Pro Pro
His 645 650 655Pro Arg Thr Pro Leu Gly Ser Pro Ala Ala Tyr Trp Lys
Arg Val Gly 660 665 670His Ser Ala Cys Ser Ala Ser Cys Gly Lys Gly
Val Trp Arg Pro Ile 675 680 685Phe Leu Cys Ile Ser Arg Glu Ser Gly
Glu Glu Leu Asp Glu Arg Ser 690 695 700Cys Ala Ala Gly Ala Arg Pro
Pro Ala Ser Pro Glu Pro Cys His Gly705 710 715 720Thr Pro Cys Pro
Pro Tyr Trp Glu Ala Gly Glu Trp Thr Ser Cys Ser 725 730 735Arg Ser
Cys Gly Pro Gly Thr Gln His Arg Gln Leu Gln Cys Arg Gln 740 745
750Glu Phe Gly Gly Gly Gly Ser Ser Val Pro Pro Glu Arg Cys Gly His
755 760 765Leu Pro Arg Pro Asn Ile Thr Gln Ser Cys Gln Leu Arg Leu
Cys Gly 770 775 780His Trp Glu Val Gly Ser Pro Trp Ser Gln Cys Ser
Val Arg Cys Gly785 790 795 800Arg Gly Gln Arg Ser Arg Gln Val Arg
Cys Val Gly Asn Asn Gly Asp 805 810 815Glu Val Ser Glu Gln Glu Cys
Ala Ser Gly Pro Pro
Gln Pro Pro Ser 820 825 830Arg Glu Ala Cys Asp Met Gly Pro Cys Thr
Thr Ala Trp Phe His Ser 835 840 845Asp Trp Ser Ser Lys Val Ser Pro
Glu Pro Pro Ala Ile Ser Cys Ile 850 855 860Leu Gly Asn His Ala Gln
Asp Thr Ser Ala Phe Pro Ala865 870 875
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