U.S. patent application number 12/624443 was filed with the patent office on 2010-05-06 for method for predicting the response to a treatment.
Invention is credited to Joachim Moecks, Andreas Strauss, Gerhard Zugmaier.
Application Number | 20100112603 12/624443 |
Document ID | / |
Family ID | 35511140 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100112603 |
Kind Code |
A1 |
Moecks; Joachim ; et
al. |
May 6, 2010 |
METHOD FOR PREDICTING THE RESPONSE TO A TREATMENT
Abstract
The invention is related to a method of predicting the response
to a treatment with a HER inhibitor in a patient comprising the
steps of assessing a biomarker or a combination of biomarkers
selected from the group consisting of amphiregulin, an epidermal
growth factor, a transforming growth factor alpha, and a HER2
biomarker in a biological sample from the patient and predicting
the response to the treatment with the HER inhibitor in the patient
by evaluating the results of the first step. Further uses and
methods wherein these markers are used are disclosed.
Inventors: |
Moecks; Joachim; (Mannheim,
DE) ; Strauss; Andreas; (Penzberg, DE) ;
Zugmaier; Gerhard; (Stuttgart, DE) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.;PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
US
|
Family ID: |
35511140 |
Appl. No.: |
12/624443 |
Filed: |
November 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11438033 |
May 19, 2006 |
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12624443 |
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Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
G01N 2333/485 20130101;
A61P 35/00 20180101; C12Q 2600/158 20130101; C12Q 2600/106
20130101; G01N 33/57415 20130101; G01N 2333/495 20130101; C12Q
1/6886 20130101; G01N 2800/52 20130101; A61P 11/00 20180101; C12Q
2600/136 20130101; A61P 15/00 20180101; G01N 33/6872 20130101; A61P
13/08 20180101; G01N 2333/4756 20130101 |
Class at
Publication: |
435/7.21 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2005 |
EP |
05017663.5 |
Claims
1. A method of predicting the response of a patient to treatment
with a HER dimerization inhibitor comprising the steps of: (a)
determining the expression level one or more biomarker proteins
with one such biomarker protein being transforming growth factor
alpha; (b) determining whether the expression level assessed in
step (a) is above or below a certain quantity that is associated
with an increased or decreased clinical benefit to a patient; and
(c) predicting the response to the treatment with the HER inhibitor
in the patient by evaluating the results of step (b).
2. A method according to claim 1 wherein, in step (a), in addition
to the expression level of transforming growth factor alpha, the
level of expression of one or more biomarker proteins, selected
from the group consisting of epidermal growth factor, amphiregulin,
and HER2, is also determined.
3. A method according to claim 2 wherein, in step (a), the level of
expression of transforming growth factor alpha is determined in
combination with the level of expression of: epidermal growth
factor; HER2; or both amphiregulin and HER2.
4. A method according to claim 1 wherein the HER dimerization
inhibitor inhibits heterodimerization of HER2 with EGFR or
HER3.
5. A method according to claim 1 wherein the HER dimerization
inhibitor is an antibody.
6. A method according to claim 1 wherein the HER dimerization
inhibitor is the antibody 2C4.
7. A method according to claim 1 wherein the patient is a cancer
patient.
8. A method according to claim 1 wherein the patient is a breast
cancer, ovarian cancer, lung cancer or prostate cancer patient.
9. A method according to claim 1 wherein the level of expression of
said biomarker protein or proteins is assessed by detecting the
level of expression of fragments thereof.
10. A method according to claim 1 wherein the level of expression
said biomarker or biomarkers is determined using a reagent which
specifically binds with the biomarker protein or the fragment
thereof or the combination of biomarker proteins or the fragments
thereof.
11. A method according to claim 10 wherein said reagent is selected
from the group consisting of an antibody, a fragment of an
antibody, and an antibody derivative.
12. A method according to claim 1 wherein a biomarker protein is
the extracellular domain of HER2.
13. A method according to claim 12 wherein said extracellular
domain extracellular domain of HER2 has a molecular mass of
approximately 105,000 Daltons.
14. A method according to claim 1 wherein the amino acid sequence
of the transforming growth factor alpha biomarker protein is SEQ ID
NO: 3
15. A method according to claim 2 wherein the amino acid sequence
of the amphiregulin biomarker protein is SEQ ID NO: 1, the amino
acid sequence of the epidermal growth factor biomarker protein is
SEQ ID NO: 2, the amino acid sequence of the transforming growth
factor alpha biomarker protein is SEQ ID NO: 3, and the amino acid
sequence of the HER2 biomarker protein is SEQ ID NO: 4.
16. A method according to claim 1 wherein the amount of
transforming growth factor alpha is determined in a biological
sample of blood serum and the quantity that is associated with an
increased clinical benefit to a patient is within the range between
2.0 to 10.0 pg/ml of transforming growth factor alpha.
17. A method according to claim 2 wherein the amounts of
transforming growth factor alpha, amphiregulin, epidermal growth
factor, and HER2 marker proteins are determined in a biological
sample of blood serum and the quantities that are associated with
an increased clinical benefit to a patient are within the range of
between 2.0 to 10.0 pg/ml of transforming growth factor alpha,
between 6 to 15 pg/ml of amphiregulin, between 100 to 250 pg/ml of
epidermal growth factor, and between 12 to 22 ng/ml of HER2 marker
proteins.
18. A method according to claim 2 wherein the amounts of
transforming growth factor alpha, amphiregulin, epidermal growth
factor, and HER2 marker proteins are determined in a biological
sample of blood serum and the quantities that are associated with
an increased clinical benefit to a patient are about 3.5 pg/ml of
transforming growth factor alpha, about 12 pg/ml of amphiregulin,
about 150 pg/ml of epidermal growth factor, and about 18 ng/ml of
HER2 marker proteins.
Description
PRIORITY TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/438,033, filed May 19, 2006, now pending; which claims the
benefit of European Application No. 05017663.5, filed Aug. 12,
2005. The entire contents of the above-identified applications are
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention is related to a method of predicting the
response to a treatment with a HER inhibitor, preferably a HER
dimerization inhibitor, in a patient comprising the steps of
assessing a marker gene or a combination of marker genes selected
from the group consisting of an epidermal growth factor, a
transforming growth factor alpha and a HER2 marker gene or a
combination of marker genes comprising an amphiregulin marker gene
and a marker gene selected from an epidermal growth factor, a
transforming growth factor alpha and a HER2 marker gene in a
biological sample from the patient and predicting the response to
the treatment with the HER inhibitor in the patient by evaluating
the results of the first step. Further uses and methods wherein
these markers are used are disclosed.
BACKGROUND OF THE INVENTION
[0003] The human epidermal growth factor receptor (ErbB or HER)
family comprises four members (HER1-4) that, through the activation
of a complex signal cascade, are important mediators of cell
growth, survival and differentiation. At least 11 different gene
products from the epidermal growth factor (EGF) superfamily bind to
three of these receptors, EGFR (also called ErbB1 or HER1), HER3
(ErbB3) and HER4 (ErbB4). Although no ligand has been identified
that binds and activates HER2 (ErbB2 or neu), the prevailing
understanding is that HER2 is a co-receptor that acts in concert
with other HER receptors to amplify and in some cases initiate
receptor-ligand signaling. Dimerization with the same receptor type
(homodimerization) or another member of the HER family
(heterodimerization) is essential for their activity. HER2 is the
preferred dimerization partner for other HER family members. The
role of the HER family in many epithelial tumor types is well
documented and has led to the rational development of novel cancer
agents directed specifically to HER receptors. The recombinant
humanized anti-HER2 monoclonal antibody (MAb) trastuzumab is a
standard of care in patients with HER2-positive metastatic breast
cancer (MBC). Overexpression/amplification of the HER2
protein/gene, which occurs in 20-30% of breast cancer cases, is a
prerequisite for treatment with trastuzumab.
[0004] Pertuzumab (Omnitarg.TM.; formerly 2C4) is the first of a
new class of agents known as HER dimerization inhibitors (HDIs).
Pertuzumab binds to HER2 at its dimerization domain, thereby
inhibiting its ability to form active dimer receptor complexes and
thus blocking the downstream signal cascade that ultimately results
in cell growth and division. Pertuzumab is a fully humanized
recombinant monoclonal antibody directed against the extracellular
domain of HER2. Binding of Pertuzumab to the HER2 on human
epithelial cells prevents HER2 from forming complexes with other
members of the HER family (including EGFR, HER3, HER4) and probably
also HER2 homodimerization. By blocking complex formation,
Pertuzumab prevents the growth-stimulatory effects and cell
survival signals activated by ligands of HER1, HER3 and HER4 (e.g.
EGF, TGF.alpha., amphiregulin, and the heregulins). Other names for
Pertuzumab are 2C4 or Pertuzumab. Pertuzumab is a fully humanized
recombinant monoclonal antibody based on the human IgG1(.kappa.)
framework sequences. The structure of Pertuzumab consists of two
heavy chains (449 residues) and two light chains (214 residues).
Compared to Trastuzumab (Herceptin.RTM.), Pertuzumab has 12 amino
acid differences in the light chain and 29 amino acid differences
in the IgG1 heavy chain. WO 2004/092353 and WO 2004/091384 present
investigations that the formation of heterodimers of HER2 with
other receptors should be linked to the effectiveness or
suitability of Pertuzumab.
[0005] Zabrecky, J. R. et al., J. Biol. Chem. 266 (1991) 1716-1720
disclose that the release of the extracellular domain of HER2 may
have implications in oncogenesis and its detection could be useful
as a cancer diagnostic. Colomer, R. et al., Clin. Cancer Res. 6
(2000) 2356-2362 disclose circulating HER2 extracellular domain and
resistance to chemotherapy in advanced breast cancer. The
prognostic and predictive values of the extracellular domain of
HER2 is reviewed by Hait, W. N., Clin. Cancer Res. 7 (2001)
2601-2604.
SUMMARY OF THE INVENTION
[0006] There is still a need to provide further methods for
determining the progression of disease in a cancer patient treated
with a HER dimerization inhibitor.
[0007] Therefore, in an embodiment of the invention, a method of
predicting the response to a treatment with a HER inhibitor,
preferably a HER dimerization inhibitor, in a patient is provided
comprising the steps of: [0008] (a) determining the expression
level or amount of one or more biomarker in a biological sample
from a patient wherein the biomarker or biomarkers are selected
from the group consisting of: [0009] (1) transforming growth factor
alpha; [0010] (2) HER2; [0011] (3) amphiregulin; and [0012] (4)
epidermal growth factor; [0013] (b) determining whether the
expression level or amount assessed in step (a) is above or below a
certain quantity that is associated with an increased or decreased
clinical benefit to a patient; and [0014] (c) predicting the
response to the treatment with the HER inhibitor in the patient by
evaluating the results of step (b).
[0015] In another embodiment of the invention, a probe that
hybridizes with the polynucleotides of the above biomarkers under
stringent conditions or an antibody that binds to the proteins of
the above biomarkers is used for predicting the response to
treatment with a HER inhibitor in a patient or used for selecting a
composition for inhibiting the progression of disease in a
patient.
[0016] In still another embodiment of the invention, a kit is
provided comprising a probe that anneals with a biomarker
polynucleotide under stringent conditions or an antibody that binds
to the biomarker protein.
[0017] In still another embodiment of the invention, a method of
selecting a composition for inhibiting the progression of disease
in a patient is provided, the method comprising: [0018] (a)
separately exposing aliquots of a biological sample from a cancer
patient in the presence of a plurality of test compositions; [0019]
(b) comparing the level of expression of one or more biomarkers
selected from the group consisting of amphiregulin, epidermal
growth factor, transforming growth factor alpha and HER2 in the
aliquots of the biological sample contacted with the test
compositions and the level of expression of such biomarkers in an
aliquot of the biological sample not contacted with the test
compositions; and [0020] (c) selecting one of the test compositions
which alters the level of expression of a particular biomarker or
biomarkers in the aliquot of the biological sample contacted with
the test composition and the level of expression of the
corresponding biomarker or biomarkers in the aliquot of the
biological sample not contacted with the test composition is an
indication for the selection of the test composition.
[0021] In yet another embodiment of the invention, a method of
identifying a candidate agent is provided said method comprising:
[0022] (a) contacting an aliquot of a biological sample from a
cancer patient with the candidate agent and determining the level
of expression of one or more biomarkers selected from the group
consisting of amphiregulin, epidermal growth factor, transforming
growth factor alpha and HER2 in the aliquot; [0023] (b) determining
the level of expression of a corresponding biomarker or of a
corresponding combination of biomarkers in an aliquot of the
biological sample not contacted with the candidate agent; [0024]
(c) observing the effect of the candidate agent by comparing the
level of expression of the biomarker or biomarkers in the aliquot
of the biological sample contacted with the candidate agent and the
level of expression of the corresponding biomarker or biomarkers in
the aliquot of the biological sample not contacted with the
candidate agent; and [0025] (d) identifying said agent from said
observed effect, wherein an at least 10% difference between the
level of expression of the biomarker or biomarkers in the aliquot
of the biological sample contacted with the candidate agent and the
level of expression of the corresponding biomarker or biomarkers in
the aliquot of the biological sample not contacted with the
candidate agent is an indication of an effect of the candidate
agent.
[0026] In yet another embodiment, a candidate agent identified by
the method according to the invention or a pharmaceutical
preparation comprising an agent according to the invention is
provided.
[0027] In yet another embodiment of the invention, an agent
according to the invention is provided for the preparation of a
composition for the treatment of cancer.
[0028] In still another embodiment of the invention, a method of
producing a drug is provided comprising: [0029] (i) synthesizing
the candidate agent identified as described above or an analog or
derivative thereof in an amount sufficient to provide said drug in
a therapeutically effective amount to a subject; and/or [0030] (ii)
combining the drug candidate or the candidate agent identified as
described above or an analog or derivative thereof with a
pharmaceutically acceptable carrier.
[0031] In yet another embodiment of the invention, a biomarker
protein or a biomarker polynucleotide selected from the group
consisting of an amphiregulin biomarker, and epidermal growth
factor biomarker, a transforming growth factor alpha biomarker and
a HER2 biomarker protein or polynucleotide is used for deriving a
candidate agent or for selecting a composition for inhibiting the
progression of a disease in a patient.
[0032] In another embodiment of the invention, a HER inhibitor,
preferably a HER dimerization inhibitor, is used for the
manufacture of a medicament for treating a human cancer patient
characterized in that said treating or treatment includes assessing
in a biological sample from the patient: one or more biomarkers
selected from the group consisting of amphiregulin biomarker,
epidermal growth factor biomarker, transforming growth factor alpha
biomarker, and HER2 biomarker. In a particular embodiment, one or
more biomarkers are assessed wherein the biomarkers are selected
from the group consisting of epidermal growth factor, transforming
growth factor alpha, and HER2. In another particular embodiment, a
transforming growth factor alpha biomarker is assessed in
combination with one or more biomarkers selected from the group
consisting of epidermal growth factor, amphiregulin, and HER2. In
another particular embodiment, a HER2 biomarker is assessed in
combination with one or more biomarkers selected from the group
consisting of epidermal growth factor, transforming growth factor
alpha, and amphiregulin.
[0033] In another particular embodiment, a epidermal growth factor
biomarker is assessed in combination with one or more biomarkers
selected from the group consisting of amphiregulin, transforming
growth factor alpha, and HER2.
[0034] In another particular embodiment, an amphiregulin biomarker
is assessed in combination with one or more biomarkers selected
from the group consisting of epidermal growth factor, transforming
growth factor alpha, and HER2.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Scatterplot TGF-alpha logarithmic transformation
versus categorized clinical benefit
[0036] FIG. 2: Scatterplot Amphiregulin logarithmic transformation
versus categorized clinical benefit
[0037] FIG. 3: Ordinal clinical benefit TGF-alpha
[0038] FIG. 4: Ordinal clinical benefit Amphiregulin
[0039] FIG. 5: Ordinal clinical benefit EGF
[0040] FIG. 6: Ordinal clinical benefit HER2-ECD
[0041] FIG. 7: Overview exploratory cut-points and log-rank
p-values for TTP and TTD for Amphiregulin, EGF, TGF-alpha,
HER2-ECD
[0042] FIG. 8: TGF-alpha Kaplan Meier plot for time to
progression/death based on exploratory single marker cut-point
[0043] FIG. 9: TGF-alpha Kaplan Meier plot for time to death based
on exploratory single marker cut-point
[0044] FIG. 10: Amphiregulin Kaplan Meier plot for time to
progression/death based on exploratory single marker cut-point
[0045] FIG. 11: Amphiregulin Kaplan Meier plot for time to death
based on exploratory single marker cut-point
[0046] FIG. 12: EGF Kaplan Meier plot for time to progression/death
based on exploratory single marker cut-point
[0047] FIG. 13: EGF Kaplan Meier plot for time to death based on
exploratory single marker cut-point
[0048] FIG. 14: HER2-ECD Kaplan Meier plot for time to
progression/death based on exploratory single marker cut-point
[0049] FIG. 15: HER2-ECD Kaplan Meier plot for time to death based
on exploratory single marker cut-point
[0050] FIG. 16: As example for a combination score, further
improving the separation between the greater clinical
benefit/lesser clinical benefit groups in TTP: Ordinal clinical
benefit HER2-ECD TGF alpha combination
[0051] FIG. 17: Overview exploratory cut-points and log-rank
p-values for TTP and TTD for a combination of TGF-alpha and
HER2-ECD
[0052] FIG. 18: HER2-ECD/TGF-alpha Kaplan Meier plot for time to
progression/death based on exploratory combination marker
cut-point
[0053] FIG. 19: HER2-ECD/TGF-alpha Kaplan Meier plot for time to
death based on exploratory combination marker cut-point
DETAILED DESCRIPTION OF THE INVENTION
[0054] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0055] The term "biological sample" shall generally mean any
biological sample obtained from an individual, body fluid, cell
line, tissue culture, or other source. Body fluids are e.g. lymph,
sera, plasma, urine, semen, synovial fluid and spinal fluid.
Methods for obtaining tissue biopsies and body fluids from mammals
are well known in the art. If the term "sample" is used alone, it
shall still mean that the "sample" is a "biological sample", i.e.
the terms are used interchangeably.
[0056] The term "response of a patient to treatment with a HER
inhibitor" or "response of a patient to treatment with a HER
dimerization inhibitor" refers to the clinical benefit imparted to
a patient suffering from a disease or condition (such as cancer)
from or as a result of the treatment with the HER inhibitor (e.g.,
a HER dimerization inhibitor). A clinical benefit includes a
complete remission, a partial remission, a stable disease (without
progression), progression-free survival, disease free survival,
improvement in the time-to-progression (of the disease),
improvement in the time-to-death, or improvement in the overall
survival time of the patient from or as a result of the treatment
with the HER dimerization inhibitor. There are criteria for
determining a response to therapy and those criteria allow
comparisons of the efficacy to alternative treatments (Slapak and
Kufe, Principles of Cancer Therapy, in Harrisons's Principles of
Internal Medicine, 13th edition, eds. Isselbacher et al.,
McGraw-Hill, Inc., 1994). For example, a complete response or
complete remission of cancer is the disappearance of all detectable
malignant disease. A partial response or partial remission of
cancer may be, for example, an approximately 50 percent decrease in
the product of the greatest perpendicular diameters of one or more
lesions or where there is not an increase in the size of any lesion
or the appearance of new lesions.
[0057] As used herein, the term "progression of cancer" includes
and may refer to metastasis; a recurrence of cancer, or an at least
approximately 25 percent increase in the product of the greatest
perpendicular diameter of one lesion or the appearance of new
lesions. The progression of cancer, preferably breast cancer, is
"inhibited" if recurrence or metastasis of the cancer is reduced,
slowed, delayed, or prevented.
[0058] As used herein, the term "Time To Progression/death" (also
referred to as "TPP") or Progression-Free Survival (also referred
to as "PFS") refers to a clinical endpoint frequently used in
oncology trials (that includes but is not limited to clinical
trials with reference to the present invention). The measurement
for each patient equals the time elapsed from onset of the
treatment of a patient in a trial (as defined in the protocol [i.e,
see the examples infra]) until the detection of a malignancy
progression (as defined in the protocol) or the occurrence of any
fatality (whatever is first). If the observation of the patient was
stopped (e.g. at study end) after a period and no event was
observed, then this observation time t is called "censored."
[0059] As used herein, the term "Time To Death" (also referred to
as "TTD") or "Overall Survival" (also referred to as "OS") refers
to a clinical endpoint frequently used in oncology trials (that
includes but is not limited to clinical trials with reference to
the present invention). The measurement for each patient equals the
time elapsed from onset of the treatment of a patient in a trial
(as defined in the protocol [i.e., see the examples infra]) until
the occurrence of any fatality. If the observation of the patient
is stopped (e.g. at study end) after a period t and the patient
survived to this time, then this observation time t is called
"censored."
[0060] As used herein, the term "covariate" refers to certain
variables or information relating to a patient. The clinical
endpoints are frequently considered in regression models, where the
endpoint represent the dependent variable and the biomarkers
represent the main or target independent variables (regressors). If
additional variables from the clinical data pool are considered
these are denoted as (clinical) covariates. The term "clinical
covariate" here is used to describe all clinical information about
the patient, which are in general available at baseline. These
clinical covariates comprise demographic information like sex, age
etc., other anamnestic information, concomitant diseases,
concomitant therapies, result of physical examinations, common
laboratory parameters obtained, known properties of the target
tumor, information quantifying the extent of malignant disease,
clinical performance scores like ECOG or Karnofsky index, clinical
disease staging, timing and result of pretreatments and disease
history as well as all similar information, which may be associated
with the clinical prognosis.
[0061] As used herein, the term "raw analysis" or "unadjusted
analysis" refers to regression analyses, where over the considered
biomarkers no additional clinical covariates were used in the
regression model, neither as independent factors nor as stratifying
covariate.
[0062] As used herein, the term "adjusted by covariates" refers to
regression analyses, where over the considered biomarkers
additional clinical covariates were used in the regression model,
either as independent factors or as stratifying covariate.
[0063] As used herein, the term "univariate" refers to regression
models or graphical approaches where as independent variable only
one of the target biomarkers is part of the model. These univariate
models can be considered with and without additional clinical
covariates.
[0064] As used herein, the term "multivariate" refers to regression
models or graphical approaches where as independent variables more
than one of the target biomarkers are part of the model.
[0065] These multivariate models can be considered with and without
additional clinical covariates.
[0066] "Nucleotides" are "nucleosides" that further include a
phosphate group covalently linked to the sugar portion of the
nucleoside. For those "nucleosides" that include a pentofuranosyl
sugar, the phosphate group can be linked to either the 2', 3' or 5'
hydroxyl moiety of the sugar. A "nucleotide" is the "monomeric
unit" of an "oligonucleotide", more generally denoted herein as an
"oligomeric compound", or a "polynucleotide", more generally
denoted as a "polymeric compound". Another general expression
therefor is desoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). As used herein the term "polynucleotide" is synonymous with
"nucleic acid."
[0067] As used herein, the term "probe" refers to synthetically or
biologically produced nucleic acids (DNA or RNA) which, by design
or selection, contain specific nucleotide sequences that allow them
to hybridize under defined predetermined stringencies specifically
(i.e., preferentially) to "nucleic acids". A "probe" can be
identified as a "capture probe" meaning that it "captures" the
nucleic acid so that it can be separated from undesirable materials
which might obscure its detection. Once separation is accomplished,
detection of the captured "target nucleic acid" can be achieved
using a suitable procedure. "Capture probes" are often already
attached to a solid phase. According to the present invention, the
term hybridization under "stringent conditions" is given the same
meaning as in Sambrook et al. (Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1989), paragraph
1.101-1.104). Preferably, a "stringent hybridization" is the case
when a hybridization signal is still detectable after washing for 1
h with 1.times.SSC and 0.1% SDS at 50.degree. C., preferably at
55.degree. C., more preferably at 62.degree. C., and most
preferably at 68.degree. C., and more preferably for 1 hour with
0.2.times.SSC and 0.1% SDS at 50.degree.., preferably at 55.degree.
C., more preferably at 62.degree., and most preferably at
68.degree. C. The composition of the SSC buffer is described in
Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1989)).
[0068] As used herein, a "transcribed polynucleotide" is a
polynucleotide (e.g an RNA, a cDNA, or an analog of one of an RNA
or cDNA) which is complementary to or homologous with all or a
portion of a mature RNA made by transcription of a gene, such as
the marker gene of the invention, and normal post-transcriptional
processing (e.g. splicing), if any, of the transcript. The term
"cDNA" is an abbreviation for complementary DNA, the
single-stranded or double-stranded DNA copy of a mRNA. The term
"mRNA" is an abbreviation for messenger RNA- the RNA that serves as
a template for protein synthesis.
[0069] As used herein, the term "marker gene" or "biomarker gene"
is meant to include a gene which is useful according to this
invention for determining the progression of cancer in a patient,
particularly in a breast cancer patient.
[0070] As used herein, the term "marker polynucleotide" or
"biomarker polynucleotide" is meant to include a nucleotide
transcript (hnRNA or mRNA) encoded by a marker gene according to
the invention, or cDNA derived from the nucleotide transcript, or a
segment of said transcript or cDNA.
[0071] As used herein, the term "marker protein," "marker
polypeptide," "biomarker protein," or "biomarker polypeptide" is
meant to include a protein or polypeptide encoded by a marker gene
according to the invention or to a fragment thereof.
[0072] As used herein, the term "marker" and "biomarker" are used
interchangeably and refer to a marker gene, marker polynucleotide,
or marker protein as defined above.
[0073] As used herein, the term "gene product" refers to a marker
polynucleotide or marker protein encoded by a marker gene.
[0074] The expression of a marker gene "significantly" differs from
the level of expression of the marker gene in a reference sample if
the level of expression of the marker gene in a sample from the
patient differs from the level in a sample from the reference
subject by an amount greater than the standard error of the assay
employed to assess expression, and preferably at least 10%, and
more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%,
400%, 500% or 1,000% of that amount. Alternatively, expression of
the marker gene in the patient can be considered "significantly"
lower than the level of expression in a control subject if the
level of expression in a sample from the patient is lower than the
level in a sample from the control subject by an amount greater
than the standard error of the assay employed to assess expression,
and preferably at least 10%, and more preferably 25%, 50%, 75%,
100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% that
amount.
[0075] A marker polynucleotide or a marker protein "corresponds to"
another marker polynucleotide or marker protein if it is related
thereto, and in preferred embodiments is identical thereto.
[0076] The terms "level of expression" or "expression level" are
used interchangeably and generally refer to the amount of a
polynucleotide or an amino acid product or protein in a biological
sample. "Expression" generally refers to the process by which gene
encoded information is converted into the structures present and
operating in the cell. Therefore, according to the invention
"expression" of a gene may refer to transcription into a
polynucleotide, translation into a protein or even
posttranslational modification of the protein. Fragments of the
transcribed polynucleotide, the translated protein or the
postranslationally modified protein shall also be regarded as
expressed whether they originate from a transcript generated by
alternative splicing, a degraded transcript or from a
posttranslational processing of the protein, e.g. by proteolysis.
"Expressed genes" include those that are transcribed into a
polynucleotide as mRNA and then translated into a protein; and also
include expressed genes that are transcribed into RNA but not
translated into a protein (for example, transfer and ribosomal
RNAs).
[0077] The term "overexpression" or "increased expression" refers
to an upward deviation in levels of expression as compared to the
baseline expression level in a sample used as a control.
[0078] The term "underexpression" or "decreased expression" refers
to a downward deviation in levels of expression as compared to the
baseline expression level in a sample used as a control.
[0079] The term "amphiregulin" relates to a gene that encodes a
protein and to the protein itself that is a member of the epidermal
growth factor family. It is an autocrine growth factor as well as a
mitogen for astrocytes, Schwann cells, and fibroblasts. It is
related to epidermal growth factor (EGF) and transforming growth
factor alpha (TGF-alpha). This protein interacts with the
EGF/TGF-alpha receptor to promote the growth of normal epithelial
cells and inhibits the growth of certain aggressive carcinoma cell
lines. According to the invention, the amino acid sequence of
amphiregulin is the amino acid sequence according to SEQ ID NO: 1.
According to the invention, the nucleic acid sequence of the
"amphiregulin" cDNA is the nucleic acid sequence according to SEQ
ID NO: 5 which is accessible at GenBank with the accession number
NM.sub.--001657.
[0080] The term "transforming growth factor alpha" relates to a
gene that encodes a protein and to the protein itself that is a
member of the family of transforming growth factors (TGFs). These
are biologically active polypeptides that reversibly confer the
transformed phenotype on cultured cells. "Transforming growth
factor-alpha" shows about 40% sequence homology with epidermal
growth factor and competes with EGF for binding to the EGF
receptor, stimulating its phosphorylation and producing a mitogenic
response. According to the invention, the amino acid sequence of
"Transforming growth factor-alpha" is the amino acid sequence
according to SEQ ID NO: 3. According to the invention, the nucleic
acid sequence of the "transforming growth factor-alpha" cDNA is the
nucleic acid sequence according to SEQ ID NO: 7 which is accessible
at GenBank with the accession number NM.sub.--003236.
[0081] The term "epidermal growth factor" relates to a gene that
encodes a protein and to the protein itself that is a member of the
family of growth factors. "Epidermal growth factor (EGF)" has a
profound effect on the differentiation of specific cells in vivo
and is a potent mitogenic factor for a variety of cultured cells of
both ectodermal and mesodermal origin. The EGF precursor is
believed to exist as a membrane-bound molecule which is
proteolytically cleaved to generate the 53-amino acid peptide
hormone that stimulates cells to divide. According to the
invention, the amino acid sequence of "Epidermal growth factor" is
the amino acid sequence according to SEQ ID NO: 2. According to the
invention, the nucleic acid sequence of the "Epidermal growth
factor (EGF)" cDNA is the nucleic acid sequence according to SEQ ID
NO: 6 which is accessible at GenBank with the accession number
NM.sub.--001963. The "Epidermal Growth Factor Receptor" abbreviated
as EGFR, a 170-kD glycoprotein, is composed of an N-terminus
extracellular domain, a hydrophobic transmembrane domain, and a
C-terminus intracellular region containing the kinase domain. The
mRNA has different variants translated into different receptor
proteins. According to the invention, the amino acid sequence of
the "Epidermal growth factor receptor" is the amino acid sequence
according to SEQ ID NO: 11 (transcript variant 1; GenBank accession
number NM.sub.--005228), SEQ ID NO: 12 (transcript variant 2;
GenBank accession number NM.sub.--201282), SEQ ID NO: 13
(transcript variant 3; GenBank accession number NM.sub.--201283),
or SEQ ID NO: 14 (transcript variant 4; GenBank accession number
NM.sub.--201284). EGFR, encoded by the erbB1 gene, has been
causally implicated in human malignancy. In particular, increased
expression of EGFR has been observed in breast, bladder, lung,
head, neck and stomach cancer as well as glioblastomas. EGFR
ligandinduced dimerization activates the intrinsic RTK domain (an
Src homology domain 1, SH1), resulting in autophosphorylation on
six specific EGFR tyrosine residues in the noncatalytic tail of the
cytoplasmic domain. The cellular effects of EGFR activation in a
cancer cell include increased proliferation, promotion of cell
motility, adhesion, invasion, angiogenesis, and enhanced cell
survival by inhibition of apoptosis. Activated EGFR induces tumor
cell proliferation through stimulation of the mitogen-activated
protein kinase (MAPK) cascade.
[0082] The terms "human neu", "c-erbB-2", "erbB2", "erbB-2",
"HER-2/neu", "HER-2" and "HER2" are used interchangeably herein.
These terms relate to a gene that encodes a protein and to the
protein itself that is a member of the family of the epidermal
growth factor (EGF) receptor family of receptor tyrosine kinases.
This protein has no ligand binding domain of its own and therefore
cannot bind growth factors. However, it does bind tightly to other
ligand-bound EGF receptor family members to form a heterodimer,
stabilizing ligand binding and enhancing kinase-mediated activation
of downstream signalling pathways, such as those involving
mitogen-activated protein kinase and phosphatidylinositol-3 kinase.
Allelic variations at amino acid positions 654 and 655 of isoform a
(positions 624 and 625 of isoform b) have been reported, with the
most common allele, Ile654/Ile655 being preferred according to the
invention. Amplification and/or overexpression of this gene has
been reported in numerous cancers, including breast and ovarian
tumors. Alternative splicing results in several additional
transcript variants, some encoding different isoforms and others
that have not been fully characterized. According to the invention,
the amino acid sequence of HER2 is the amino acid sequence
according to SEQ ID NO: 4. According to the invention, the nucleic
acid sequence of the "HER2" cDNA is the nucleic acid sequence
according to SEQ ID NO: 8 which is accessible at GenBank with the
accession number NM.sub.--004448.2.
[0083] The "extracellular domain of HER2" or "shed extracellular
domain of HER2" or "HER2-ECD" is a glycoprotein of between 97 and
115 kDa which corresponds substantially to the extracellular domain
of the human HER2 gene product. It can be referred to as p105
(Zabrecky, J. R. et al., J. Biol. Chem. 266 (1991) 1716-1720; U.S.
Pat. No. 5,401,638; U.S. Pat. No. 5,604,107). The quantitation and
detection of the extracellular domain of HER2 is described in U.S.
Pat. No. 5,401,638 and U.S. Pat. No. 5,604,107.
[0084] The term "HER3" stands for another member of the epidermal
growth factor receptor (EGFR) family of receptor tyrosine kinases.
This membrane-bound protein has not an active kinase domain. The
protein can bind ligands but not transmit a signal into the cell.
It forms heterodimers with other EGF receptor family members which
do have kinase activity which leads to cell proliferation or
differentiation. Amplification of this gene and/or overexpression
of its protein is found in numerous cancers. According to the
invention, the amino acid sequence of the "HER3" cDNA is the amino
acid sequence according to SEQ ID NO: 9 which is accessible at
GenBank from the translation of the nucleic acid sequence of HER3
with the accession number NM.sub.--001005915. According to the
invention, the nucleic acid sequence of the "HER3" cDNA is the
nucleic acid sequence according to SEQ ID NO: 10 which is
accessible at GenBank with the accession number
NM.sub.--001005915.
[0085] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, and multispecific antibodies (e.g., bispecific
antibodies) formed from at least two intact antibodies, and
antibody fragments, so long as they exhibit the desired biological
activity of an antibody.
[0086] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler, G. et
al., Nature 256 (1975) 495-497, or may be made by recombinant DNA
methods (see, e.g., U.S. Pat. No. 4,816,567). "Antibody fragments"
comprise a portion of an intact antibody.
[0087] An antibody "which binds" an antigen of interest according
to the invention is one capable of binding that antigen with
sufficient affinity such that the antibody is useful in detecting
the presence of the antigen. One antibody according to the
invention binds human HER2 and does not (significantly) cross-react
with other proteins. In such embodiments, the extent of binding of
the antibody to other proteins will be less than 10% as determined
by fluorescence activated cell sorting (FACS) analysis or
radioimmunoprecipitation (RIA).
[0088] Dimerization--the pairing of receptors--is essential to the
signaling activity of all HER receptors. According to the
invention, the term "HER dimerization inhibitor" or preferably
"HER2 heterodimerization inhibitor" refers to a therapeutic agent
that binds to HER2 and inhibits HER2 heterodimerization. These are
preferably antibodies, preferably monoclonal antibodies, more
preferably humanized antibodies that bind to HER2 and inhibit HER2
heterodimerization. Examples of antibodies that bind HER2 include
4D5, 7C2, 7F3 or 2C4 as well as humanized variants thereof,
including huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,
huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 as described in
Table 3 of U.S. Pat. No. 5,821,337; and humanized 2C4 mutant
numbers 560, 561, 562, 568, 569, 570, 571, 574, or 56869 as
described in WO 01/00245. 7C2 and 7F3 and humanized variants
thereof are described in WO 98/17797. The term "HER dimerization
inhibitor" or "HER2 heterodimerization inhibitor" shall not apply
to Trastuzumab monoclonal antibodies commercially available as
"Herceptin.RTM." as the mechanism of action is different and as
Trastuzumab does not inhibit HER dimerization.
[0089] Preferred throughout the application is the "antibody 2C4",
in particular the humanized variant thereof (WO 01/00245; produced
by the hybridoma cell line deposited with the American Type Culture
Collection, Manassass, Va., USA under ATCC HB-12697), which binds
to a region in the extracellular domain of HER2 (e.g., any one or
more residues in the region from about residue 22 to about residue
584 of HER2, inclusive). The "epitope 2C4" is the region in the
extracellular domain of ErbB2 to which the antibody 2C4 binds. The
expression "monoclonal antibody 2C4" refers to an antibody that has
antigen binding residues of, or derived from, the murine 2C4
antibody of the Examples in WO 01/00245. For example, the
monoclonal antibody 2C4 may be murine monoclonal antibody 2C4 or a
variant thereof, such as humanized antibody 2C4, possessing antigen
binding amino acid residues of murine monoclonal antibody 2C4.
Examples of humanized 2C4 antibodies are provided in Example 3 of
WO 01/00245. Unless indicated otherwise, the expression "rhuMAb
2C4" when used herein refers to an antibody comprising the variable
light (VL) and variable heavy (VH) sequences of SEQ ID Nos. 3 and 4
of WO 01/00245, respectively, fused to human light and heavy IgG1
(non-A allotype) constant region sequences optionally expressed by
a Chinese Hamster Ovary (CHO) cell. Preferred embodiments of WO
01/00245 are preferred herein as well. The humanized antibody 2C4
is also called Pertuzumab.
[0090] A "kit" is any manufacture (e.g a package or container)
comprising at least one reagent, e.g a probe, for specifically
detecting a marker gene or protein of the invention. The
manufacture is preferably promoted, distributed, or sold as a unit
for performing the methods of the present invention.
[0091] The verbs "determine" and "assess" shall have the same
meaning and are used interchangeably throughout the
application.
[0092] Conventional techniques of molecular biology and nucleic
acid chemistry, which are within the skill of the art, are
explained in the literature. See, for example, Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989; Gait, M. J.
(ed.), Oligonucleotide synthesis--a practical approach, IRL Press
Limited, 1984; Hames, B. D. and Higgins, S. J. (eds.), Nucleic acid
hybridisation--a practical approach, IRL Press Limited, 1985; and a
series, Methods in Enzymology, Academic Press, Inc., all of which
are incorporated herein by reference. All patents, patent
applications, and publications mentioned herein, both supra and
infra, are hereby incorporated by reference in their entirety.
[0093] As used herein, the general form of a prediction rule
consists in the specification of a function of one or multiple
biomarkers potentially including clinical covariates to predict
response or non-response, or more generally, predict benefit or
lack of benefit in terms of suitably defined clinical
endpoints.
[0094] The simplest form of a prediction rule consists of an
univariate model without covariates, where the prediction is
determined by means of a cutoff or threshold. This can be phrased
in terms of the Heaviside function for a specific cutoff c and a
biomarker measurement x, where the binary prediction A or B is to
be made, then
If H(x-c)=0 then predict A.
If H(x-c)=1 then predict B.
[0095] This is the simplest way of using univariate biomarker
measurements in prediction rules. If such a simple rule is
sufficient, it allows for a simple identification of the direction
of the effect, i.e. whether high or low expression levels are
beneficial for the patient.
[0096] The situation can be more complicated if clinical covariates
need to be considered and/or if multiple biomarkers are used in
multivariate prediction rules. In order to illustrate the issues
here are two hypothetical examples:
Covariate Adjustment (Hypothetical Example):
[0097] For a biomarker X it is found in a clinical trial population
that high expression levels are associated with a worse prognosis
(univariate analysis). A closer analysis shows that there are two
tumor types in the population, one of which possess a worse
prognosis than the other one and at the same time the biomarker
expression for this tumor group is generally higher. An adjusted
covariate analysis reveals that for each of the tumor types the
relation of clinical benefit and prognosis is reversed, i.e. within
the tumor types, lower expression levels are associated with better
prognosis. The overall opposite effect was masked by the covariate
tumor type--and the covariate adjusted analysis as part of the
prediction rule reversed the direction.
Multivariate Prediction (Hypothetical Example):
[0098] For a biomarker X it is found in a clinical trial population
that high expression levels are slightly associated with a worse
prognosis (univariate analysis). For a second biomarker Y a similar
observation was made by univariate analysis. The combination of X
and Y revealed that a good prognosis is seen if both biomarkers are
low. This makes the rule to predict benefit if both biomarkers are
below some cutoffs (AND- connection of a Heaviside prediction
function). For the combination rule there is no longer a simple
rule phraseable in an univariate sense. E.g. having low expression
levels in X will not automatically predict a better prognosis.
[0099] These simple examples show that prediction rules with and
without covariates cannot be judged on the univariate level of each
biomarker. The combination of multiple biomarkers plus a potential
adjustment by covariates does not allow to assign simple
relationships towards single biomarkers.
[0100] In one embodiment of the invention, a method of predicting
the response to a treatment with a HER inhibitor, preferably a HER
dimerization inhibitor, in a patient comprises the steps of: [0101]
(a) determining the expression level or amount of one or more
biomarkers in a biological sample from a patient wherein the
biomarker or biomarkers are selected from the group consisting of:
[0102] (1) transforming growth factor alpha; [0103] (2) HER2;
[0104] (3) amphiregulin; and [0105] (4) epidermal growth factor;
[0106] (b) determining whether the expression level or amount
assessed in step (a) is above or below a certain quantity that is
associated with an increased or decreased clinical benefit to a
patient; and [0107] (c) predicting the response to the treatment
with the HER inhibitor in the patient by evaluating the results of
step (b).
[0108] In a more particular embodiment of the above method, the
expression level of the transforming growth factor alpha biomarker
is determined in combination with one or more biomarkers selected
from the group consisting of epidermal growth factor, amphiregulin,
and HER2. In another more particular embodiment of the above
method, the expression level of the HER2 biomarker is determined in
combination with one or more biomarkers selected from the group
consisting of epidermal growth factor, transforming growth factor
alpha, and amphiregulin. In another more particular embodiment of
the above method, the expression level of the epidermal growth
factor biomarker is determined in combination with one or more
biomarkers selected from the group consisting of amphiregulin,
transforming growth factor alpha, and HER2. In another more
particular embodiment of the above method, an amphiregulin
biomarker is assessed in combination with one or more biomarkers
selected from the group consisting of epidermal growth factor,
transforming growth factor alpha, and HER2.
[0109] The "quantity that is associated with an increased or
decreased clinical benefit to a patient" of the above method is
preferably a value expressed in mass/volume for blood serum or
blood plasma or mass/mass for tumor tissue. It can be measured by
methods known to the expert skilled in the art and also disclosed
by this invention. If the expression level or amount determined in
step (a) is above or below a certain quantity or value, the
response to the treatment can be determined.
[0110] With respect to the quantity in blood serum for the
transforming growth factor alpha marker protein, a range between
2.0-10.0 pg/ml, preferably a range between 2.0-5.0 pg/ml, and more
preferably about 3.5 pg/ml may be favorable for progression free
survival and overall survival when treatment with a HER inhibitor
is considered. See FIG. 7. Thus, in a preferred embodiment, the
quantity of transforming growth factor alpha marker protein in the
blood serum of a patient is within one of the foregoing ranges for
predicting a good response to treatment with a HER inhibitor in the
patient.
[0111] With respect to the quantity in blood serum for the HER2
marker protein (preferably the soluble HER2 extracellular domain
(HER2-ECD)), a range between 12-22 ng/ml, preferably about 18
ng/ml, may be favorable for progression free survival and overall
survival when treatment with a HER inhibitor is considered. See
FIG. 7. Thus, in a preferred embodiment, the quantity of HER2
marker protein in the blood serum of a patient is within the
foregoing range for predicting a good response to treatment with a
HER inhibitor in the patient.
[0112] With respect to the quantity in blood serum for the
epidermal growth factor marker protein, a range between 100-250
pg/ml, preferably about 150 pg/ml, may be favorable for progression
free survival and overall survival when treatment with a HER
inhibitor is considered. See FIG. 7. Thus, in a preferred
embodiment, the quantity of epidermal growth factor marker protein
in the blood serum of a patient is within the foregoing range for
predicting a good response to treatment with a HER inhibitor in the
patient.
[0113] With respect to the quantity in blood serum for the
amphiregulin marker protein, a range between 6-15 pg/ml, preferably
about 12 pg/ml, may be favorable for progression free survival and
overall survival when treatment with a HER inhibitor is considered.
See FIG. 7. Thus, in a preferred embodiment, the quantity of
amphiregulin marker protein in the blood serum of a patient is
within the foregoing range for predicting a good response to
treatment with a HER inhibitor in the patient.
[0114] Since the marker genes, in particular in serum, may be used
in multiple-marker prediction models potentially including other
clinical covariates, the direction of a beneficial effect of a
single marker gene within such models cannot be determined in a
simple way, and may contradict the direction found in univariate
analyses, i.e. the situation as described for the single marker
gene.
[0115] More preferably, in the method according to the invention,
the quantity or value (below or above which is associated with an
increased or decreased clinical benefit) is determined by: [0116]
(1) determining the expression level or amount of a biomarker or
combination of biomarkers in a plurality of biological samples from
patients before treatment with the HER inhibitor, [0117] (2)
treating the patients with the HER inhibitor, [0118] (3)
determining the clinical benefit of each patient; and [0119] (4)
correlating the clinical benefit of the patients treated with the
HER inhibitor to the expression level or amount of the biomarker or
combination of biomarkers.
[0120] The "quantity" is preferably a value expressed in
mass/volume for blood serum or blood plasma or mass/mass for tumor
tissue.
[0121] The present invention also considers mutants or variants of
the marker genes according to the present invention and used in the
methods according to the invention. In those mutants or variants
the native sequence of the marker gene is changed by substitutions,
deletions or insertions. "Native sequence" refers to an amino acid
or nucleic acid sequence which is identical to a wild-type or
native form of a marker gene or protein.
[0122] The present invention also considers mutants or variants of
the proteins according to the present invention and used in the
methods according to the invention. "Mutant amino acid sequence,"
"mutant protein" or "mutant polypeptide" refers to a polypeptide
having an amino acid sequence which varies from a native sequence
or is encoded by a nucleotide sequence intentionally made variant
from a native sequence. "Mutant protein," "variant protein" or
"mutein" means a protein comprising a mutant amino acid sequence
and includes polypeptides which differ from the amino acid sequence
of the native protein according to the invention due to amino acid
deletions, substitutions, or both.
[0123] The present invention also considers a method of predicting
the response to a treatment with a combination of a HER inhibitor
and another substance or agent as a chemotherapeutic agent or a
therapeutic antibody used for treating cancer. The chemotherapeutic
agent may be e.g. gemcitabine (Gemzar.RTM.; chemical name:
2',2'-difluorodeoxycytidine (dFdC)), carboplatin
(diammine-(cyclobutane-1,1-dicarboxylato (2-)-O,O')-platinum), or
paclitaxel (Taxol.RTM., chemical name:
.beta.-(benzoylamino)-.alpha.-hydroxy-,6,12b-bis(acetyloxy)-12-(benzoylox-
y)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13--
tetramethyl-5-oxo-7,11-methano-1H-cyclodeca(3,4)benz(1,2-b)oxet-9-yl
ester,(2aR-(2a-.alpha.,4-.beta.,4a-.beta.,6-.beta.,
9-.alpha.(.alpha.-R*,.beta.-S*),11-.alpha.,12-.alpha.,12a-.alpha.,
2b-.alpha.))-benzenepropanoic acid); or transtuzumab; or
erlotinib.
[0124] In a preferred embodiment of the invention, the biological
sample is blood serum, blood plasma or tumor tissue. Tumor tissue
may be formalin-fixed paraffin embedded tumor tissue or fresh
frozen tumor tissue.
[0125] In another preferred embodiment of the invention, the HER
dimerization inhibitor inhibits heterodimerization of HER2 with
EGFR or HER3, or HER4. Preferably, the HER dimerization inhibitor
is an antibody, preferably the antibody 2C4. Preferred throughout
the application is the "antibody 2C4", in particular the humanized
variant thereof (WO 01/00245; produced by the hybridoma cell line
deposited with the American Type Culture Collection, Manassass,
Va., USA under ATCC HB-12697), which binds to a region in the
extracellular domain of HER2 (e.g., any one or more residues in the
region from about residue 22 to about residue 584 of HER2,
inclusive). Examples of humanized 2C4 antibodies are provided in
Example 3 of WO 01/00245. The humanized antibody 2C4 is also called
Pertuzumab.
[0126] In still another preferred embodiment of the invention, the
patient is a cancer patient, preferably a breast cancer, ovarian
cancer, lung cancer or prostate cancer patient. The breast cancer
patient is preferably a metastatic breast cancer patient or a HER2
low expressing breast or metastatic breast cancer patient, or a
HER2 high expressing breast or metastatic breast cancer patient.
The ovarian cancer patient is preferably a metastatic ovarian
cancer patient. The lung cancer patient is preferably a non-small
cell lung cancer (NSCLC) patient.
[0127] It is preferred that two, three or all four marker genes,
marker polynucleotides or marker proteins are used in combination,
i.e. used in all disclosed embodiments of the invention or methods,
uses or kits according to the invention. The following are
preferred combinations of biomarkers in which the level of
expression or amounts are determined in accordance with the
invention:
[0128] In one particular embodiment, a transforming growth factor
alpha biomarker is assessed in combination with one or more
biomarkers selected from the group consisting of epidermal growth
factor, amphiregulin, and HER2. In another particular embodiment, a
HER2 biomarker is assessed in combination with one or more
biomarkers selected from the group consisting of epidermal growth
factor, transforming growth factor alpha, and amphiregulin.
[0129] In another particular embodiment, a epidermal growth factor
biomarker is assessed in combination with one or more biomarkers
selected from the group consisting of amphiregulin, transforming
growth factor alpha, and HER2. In another particular embodiment, an
amphiregulin biomarker is assessed in combination with one or more
biomarkers selected from the group consisting of epidermal growth
factor, transforming growth factor alpha, and HER2.
[0130] In a particularly preferred embodiment of the invention, the
combination of biomarkers consists of: [0131] the transforming
growth factor alpha and the HER2 biomarkers, or [0132] the
transforming growth factor alpha and the EGF biomarkers, or [0133]
the amphiregulin, the epidermal growth factor, the transforming
growth factor alpha and the HER2 biomarkers,
[0134] In a preferred embodiment of the invention, the level of
expression of the marker gene or the combination of marker genes in
the sample is assessed by detecting the level of expression of a
marker protein or a fragment thereof or a combination of marker
proteins or fragments thereof encoded by the marker gene or the
combination of marker genes. Preferably, the level of expression of
the marker protein or the fragment thereof or the combination of
marker proteins or the fragments thereof is detected using a
reagent which specifically binds with the marker protein or the
fragment thereof or the combination of marker proteins or the
fragments thereof. Preferably, the reagent is selected from the
group consisting of an antibody, a fragment of an antibody, and an
antibody derivative.
[0135] There are many different types of immunoassays which may be
used in the method of the present invention, e.g. enzyme linked
immunoabsorbent assay (ELISA), fluorescent immunosorbent assay
(FIA), chemical linked immunosorbent assay (CLIA), radioimmuno
assay (RIA), and immunoblotting. For a review of the different
immunoassays which may be used, see: Lottspeich and Zorbas (eds.),
Bioanalytik, 1.sup.st edition 1998, Spektrum Akademischer Verlag,
Heidelberg, Berlin, Germany. Therefore, in yet another preferred
embodiment of the invention, the level of expression is determined
using a method selected from the group consisting of proteomics,
flow cytometry, immunocytochemistry, immunohistochemistry,
enzyme-linked immunosorbent assay, multi-channel enzyme-linked
immunosorbent assay, and variations of these methods. Therefore
more preferably, the level of expression is determined using a
method selected from the group consisting of proteomics, flow
cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked
immunosorbent assay, multi-channel enzyme-linked immunosorbent
assay, and variations of these methods.
[0136] In another preferred embodiment of the invention, the
fragment of the marker protein is the extracellular domain of the
HER2 marker protein (HER2-ECD). Preferably, the extracellular
domain of the HER2 marker protein has a molecular mass of
approximately 105,000 Dalton. "Dalton" stands for a mass unit that
is equal to the weight of a hydrogen atom, or
1.657.times.10.sup.-24 grams.
[0137] In another preferred embodiment of the invention [0138] the
amino acid sequence of the amphiregulin marker protein is the amino
acid sequence SEQ ID NO: 1, [0139] the amino acid sequence of the
epidermal growth factor marker protein is the amino acid sequence
SEQ ID NO: 2, [0140] the amino acid sequence of the transforming
growth factor alpha marker protein is the amino acid sequence SEQ
ID NO: 3, or [0141] the amino acid sequence of the HER2 marker
protein is the amino acid sequence SEQ ID NO: 4.
[0142] In another preferred embodiment of the invention, the
quantity in blood serum for [0143] the transforming growth factor
alpha marker protein is between 2.0 to 10.0 pg/ml, preferably about
3.5 pg/ml, [0144] the epidermal growth factor marker protein is
between 100 to 250 pg/ml, preferably about 150 pg/ml, or pg/ml.
[0145] the amphiregulin marker protein is between 6 to 15 pg/ml,
preferably about 12 the HER2 marker protein is between 12 to 22
ng/ml, preferably about 18 ng/ml.
[0146] In still another preferred embodiment of the invention, the
"quantity" in blood serum for the extracellular domain of the HER2
marker protein is between 12 to 22 ng/ml, preferably about 18
ng/ml.
[0147] In yet another preferred embodiment of the invention, the
level of expression of the marker gene or the combination of marker
genes in the biological sample is assessed by detecting the level
of expression of a transcribed marker polynucleotide encoded by the
marker gene or a fragment of the transcribed marker polynucleotide
or of transcribed marker polynucleotides encoded by the combination
of marker genes or fragments of the transcribed marker
polynucleotide. Preferably, the transcribed marker polynucleotide
is a cDNA, mRNA or hnRNA or wherein the transcribed marker
polynucleotides are cDNA, mRNA or hnRNA.
[0148] Preferably, the step of detecting further comprises
amplifying the transcribed polynucleotide. The amplification is
performed preferably with the polymerase chain reaction which
specifically amplifies nucleic acids to detectable amounts. Other
possible amplification reactions are the Ligase Chain Reaction
(LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-569; and
Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193); Polymerase
Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1 (1991)
5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182
A2), 3SR (Kwoh, D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989)
1173-1177; Guatelli, J. C. et al., Proc. Natl. Acad. Sci. USA 87
(1990) 1874-1878; WO 92/08808), and NASBA (U.S. Pat. No.
5,130,238). Further, there are strand displacement amplification
(SDA), transcription mediated amplification (TMA), and
Q(3-amplification (for a review see e.g. Whelen, A. C. and Persing,
D. H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson, R. D. and
Myers T. W., Curr. Opin. Biotechnol. 4 (1993) 41-47). More
preferably, the step of detecting is using the method of
quantitative reverse transcriptase polymerase chain reaction.
[0149] Other suitable polynucleotide detection methods are known to
the expert in the field and are described in standard textbooks as
Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and
Ausubel, F. et al., Current Protocols in Molecular Biology, 1987,
J. Wiley and Sons, NY. There may be also further purification steps
before the polynucleotide detection step is carried out as e.g. a
precipitation step. The detection methods may include but are not
limited to the binding or intercalating of specific dyes as
ethidiumbromide which intercalates into the double-stranded
polynucleotides and changes their fluorescence thereafter. The
purified polynucleotide may also be separated by electrophoretic
methods optionally after a restriction digest and visualized
thereafter. There are also probe-based assays which exploit the
oligonucleotide hybridisation to specific sequences and subsequent
detection of the hybrid. It is also possible to sequence the DNA
after further steps known to the expert in the field. The preferred
template-dependent DNA polymerase is Taq polymerase.
[0150] In yet another preferred embodiment of the invention, the
level of expression of the marker gene is assessed by detecting the
presence of the transcribed marker polynucleotide or the fragment
thereof in a sample with a probe which anneals with the transcribed
marker polynucleotide or the fragment thereof under stringent
hybridization conditions or the level of expression of the
combination of the marker genes in the samples is assessed by
detecting the presence of transcribed marker polynucleotides or the
fragments thereof in a sample with probes which anneal with the
transcribed marker polynucleotides or the fragments thereof under
stringent hybridization conditions. This method may be performed in
a homogeneous assay system. An example for a "homogeneous" assay
system is the TagMan.RTM. system that has been detailed in U.S.
Pat. No. 5,210,015, U.S. Pat. No. 5,804,375 and U.S. Pat. No.
5,487,972. Briefly, the method is based on a double-labelled probe
and the 5'-3' exonuclease activity of Taq DNA polymerase. The probe
is complementary to the target sequence to be amplified by the PCR
process and is located between the two PCR primers during each
polymerisation cycle step. The probe has two fluorescent labels
attached to it. One is a reporter dye, such as 6-carboxyfluorescein
(FAM), which has its emission spectra quenched by energy transfer
due to the spatial proximity of a second fluorescent dye,
6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of each
amplification cycle, the Taq DNA polymerase in the process of
elongating a primed DNA strand displaces and degrades the annealed
probe, the latter due to the intrinsic 5'-3' exonuclease activity
of the polymerase. The mechanism also frees the reporter dye from
the quenching activity of TAMRA. As a consequence, the fluorescent
activity increases with an increase in cleavage of the probe, which
is proportional to the amount of PCR product formed. Accordingly,
an amplified target sequence is measured by detecting the intensity
of released fluorescence label. Another example for "homogeneous"
assay systems are provided by the formats used in the
LightCycler.RTM. instrument (see e.g. U.S. Pat. No. 6,174,670),
some of them sometimes called "kissing probe" formats. Again, the
principle is based on two interacting dyes which, however, are
characterized in that the emission wavelength of a donor-dye
excites an acceptor-dye by fluorescence resonance energy transfer.
The COBAS.RTM. AmpliPrep instrument (Roche Diagnostics GmbH,
D-68305 Mannheim, Germany) was recently introduced to expand
automation by isolating target sequences using biotinylated
sequence-specific capture probes along with streptavidin-coated
magnetic particles (Jungkind, D., J. Clin. Virol. 20 (2001) 1-6;
Stelzl, E. et al., J. Clin. Microbiol. 40 (2002) 1447-1450). It has
lately been joined by an additional versatile tool, the Total
Nucleic Acid Isolation (TNAI) Kit (Roche Diagnostics). This
laboratory-use reagent allows the generic, not sequence-specific
isolation of all nucleic acids from plasma and serum on the
COBAS.RTM. AmpliPrep instrument based essentially on the method
developed by Boom, R. et al., J. Clin. Microbiol. 28 (1990)
495-503.
[0151] In another preferred embodiment of the invention, the
nucleic acid sequence of the amphiregulin marker polynucleotide is
the nucleic acid sequence SEQ ID NO: 5, the nucleic acid sequence
of the epidermal growth factor marker polynucleotide is the nucleic
acid sequence SEQ ID NO: 6, the nucleic acid sequence of the
transforming growth factor alpha marker polynucleotide is the
nucleic acid sequence SEQ ID NO: 7, or the nucleic acid sequence of
the HER2 marker polynucleotide is the nucleic acid sequence SEQ ID
NO: 8.
[0152] In another embodiment of the invention, a probe that
hybridizes with the epidermal growth factor, transforming growth
factor alpha or HER2 marker polynucleotide under stringent
conditions or an antibody that binds to the epidermal growth
factor, transforming growth factor alpha or HER2 marker protein is
used for predicting the response to treatment with a HER inhibitor
in a patient or a probe that hybridizes with the amphiregulin,
epidermal growth factor, transforming growth factor alpha or HER2
marker polynucleotide under stringent conditions or an antibody
that binds to the amphiregulin, epidermal growth factor,
transforming growth factor alpha or HER2 marker protein is used for
selecting a composition for inhibiting the progression of disease
in a patient. The disease is preferably cancer and the patient is
preferably a cancer patient as disclosed above.
[0153] In another embodiment of the invention, a kit comprising a
probe that anneals with the amphiregulin, epidermal growth factor,
transforming growth factor alpha or HER2 marker polynucleotide
under stringent conditions or an antibody that binds to the
amphiregulin, epidermal growth factor, transforming growth factor
alpha or HER2 marker protein is provided. Such kits known in the
art further comprise plastics ware which can be used during the
amplification procedure as e.g. microtitre plates in the 96 or 384
well format or just ordinary reaction tubes manufactured e.g. by
Eppendorf, Hamburg, Germany and all other reagents for carrying out
the method according to the invention, preferably an immunoassay,
e.g. enzyme linked immunoabsorbent assay (ELISA), fluorescent
immunosorbent assay (FIA), chemical linked immunosorbent assay
(CLIA), radioimmuno assay (RIA), and immunoblotting. For a review
of the different immunoassays and reagents which may be used, see:
Lottspeich and Zorbas (eds.), Bioanalytik, 1.sup.st edition, 1998,
Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany.
Preferably combinations of the probes or antibodies to the various
marker polynucleotides or marker proteins are provided in the form
of kit as the preferred combinations of the marker polynucleotides
or marker proteins as disclosed above.
[0154] In another embodiment of the invention, a method of
selecting a composition for inhibiting the progression of disease
in a patient is provided, the method comprising: [0155] (a)
separately exposing aliquots of a biological sample from a cancer
patient in the presence of a plurality of test compositions; [0156]
(b) comparing the level of expression of one or more biomarkers
selected from the group consisting of amphiregulin, epidermal
growth factor, transforming growth factor alpha and HER2 in the
aliquots of the biological sample contacted with the test
compositions and the level of expression of such biomarkers in an
aliquot of the biological sample not contacted with the test
compositions; and [0157] (c) selecting one of the test compositions
which alters the level of expression of the biomarker or biomarkers
in the aliquot containing that test composition relative to the
aliquot not contacted with the test composition wherein an at least
10% difference between the level of expression of the biomarker or
biomarkers in the aliquot of the biological sample contacted with
the test composition and the level of expression of the
corresponding biomarker or biomarkers in the aliquot of the
biological sample not contacted with the test composition is an
indication for the selection of the test composition. The disease
is preferably cancer and the patient is preferably a cancer patient
as disclosed above.
[0158] In another embodiment of the invention, a method of
selecting a composition for inhibiting the progression of disease
in a patient is provided, the method comprising: [0159] (a)
separately exposing aliquots of a biological sample from a cancer
patient in the presence of a plurality of test compositions; [0160]
(b) comparing the level of expression of one or more biomarkers
selected from the group consisting of the amphiregulin, epidermal
growth factor, transforming growth factor alpha and HER2 in the
aliquots of the biological sample contacted with the test
compositions and the level of expression of such biomarkers in an
aliquot of the biological sample not contacted with the test
compositions; and [0161] (c) selecting one of the test compositions
which alters the level of expression of the biomarker or biomarkers
in the aliquot containing that test composition relative to the
aliquot not contacted with the test composition wherein an at least
10% difference between the level of expression of the biomarker or
biomarkers in the aliquot of the biological sample contacted with
the test composition and the level of expression of the
corresponding biomarker or biomarkers in the aliquot of the
biological sample not contacted with the test composition is an
indication for the selection of the test composition. The disease
is preferably cancer and the patient is preferably a cancer patient
as disclosed above.
[0162] The expression of a marker gene "significantly" differs from
the level of expression of the marker gene in a reference sample if
the level of expression of the marker gene in a sample from the
patient differs from the level in a sample from the reference
subject by an amount greater than the standard error of the assay
employed to assess expression, and preferably at least 10%, and
more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%,
400%, 500% or 1,000% of that amount. Alternatively, expression of
the marker gene in the patient can be considered "significantly"
lower than the level of expression in a reference subject if the
level of expression in a sample from the patient is lower than the
level in a sample from the reference subject by an amount greater
than the standard error of the assay employed to assess expression,
and preferably at least 10%, and more preferably 25%, 50%, 75%,
100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% that
amount. The difference of the level of expression be up to 10,000
or 50,000%. The difference of the level of expression is preferably
between 10% to 10,000%, more preferably 25% to 10,000%, 50% to
10,000%, 100% to 10,000%, even more preferably 25% to 5,000%, 50%
to 5,000%, 100% to 5,000%.
[0163] In another embodiment of the invention, a method of
identifying a candidate agent is provided said method comprising:
[0164] (a) contacting an aliquot of a biological sample from a
cancer patient with the candidate agent and determining the level
of expression of one or more biomarkers selected from the group
consisting of amphiregulin, epidermal growth factor, transforming
growth factor alpha and HER2 in the aliquot; [0165] (b) determining
the level of expression of a corresponding biomarker or biomarkers
in an aliquot of the biological sample not contacted with the
candidate agent; [0166] (c) observing the effect of the candidate
agent by comparing the level of expression of the biomarker or
biomarkers in the aliquot of the biological sample contacted with
the candidate agent and the level of expression of the
corresponding biomarker or biomarkers in the aliquot of the
biological sample not contacted with the candidate agent; and
[0167] (d) identifying said agent from said observed effect,
wherein an at least 10% difference between the level of expression
of the biomarker gene or combination of biomarker genes in the
aliquot of the biological sample contacted with the candidate agent
and the level of expression of the corresponding biomarker gene or
combination of biomarker genes in the aliquot of the biological
sample not contacted with the candidate agent is an indication of
an effect of the candidate agent.
[0168] In still another embodiment of the invention, a method of
identifying a candidate agent is provided said method comprising:
[0169] (a) contacting an aliquot of a biological sample from a
cancer patient with the candidate agent and determining the level
of expression in the aliquot of: [0170] (1) a biomarker or a
combination of biomarkers selected from the group consisting of
epidermal growth factor, transforming growth factor alpha and HER2
or; [0171] (2) a combination of biomarkers comprising amphiregulin
and one or more biomarkers selected from the group consisting of an
epidermal growth factor, a transforming growth factor alpha, and
HER2, [0172] (b) determining the level of expression of a
corresponding biomarker or biomarkers in an aliquot of the
biological sample not contacted with the candidate agent, [0173]
(c) observing the effect of the candidate agent by comparing the
level of expression of the biomarker or biomarkers in the aliquot
of the biological sample contacted with the candidate agent and the
level of expression of the corresponding biomarker or biomarkers in
the aliquot of the biological sample not contacted with the
candidate agent, [0174] (d) identifying said agent from said
observed effect, wherein an at least 10% difference between the
level of expression of the biomarker or biomarkers in the aliquot
of the biological sample contacted with the candidate agent and the
level of expression of the corresponding biomarker or biomarkers in
the aliquot of the biological sample not contacted with the
candidate agent is an indication of an effect of the candidate
agent.
[0175] Preferably, the candidate agent is a candidate inhibitory
agent. Preferably, said candidate agent is a candidate enhancing
agent.
[0176] In another embodiment of the invention, a candidate agent
derived by the method according to the invention is provided.
[0177] In another embodiment of the invention, a pharmaceutical
preparation comprising an agent according to the invention is
provided.
[0178] In yet another embodiment of the invention, an agent
according to the invention is used for the preparation of a
composition for the treatment of cancer. Preferred forms of cancer
are disclosed above.
[0179] In another preferred embodiment of the invention, a method
of producing a drug comprising the steps of the method according to
the invention and [0180] (i) synthesizing the candidate agent
identified in step (c) above or an analog or derivative thereof in
an amount sufficient to provide said drug in a therapeutically
effective amount to a subject; and/or [0181] (ii) combining the
drug candidate the candidate agent identified in step (c) above or
an analog or derivative thereof with a pharmaceutically acceptable
carrier.
[0182] In another embodiment of the invention, a marker protein or
a marker polynucleotide selected from the group consisting of a
amphiregulin, epidermal growth factor, transforming growth factor
alpha and HER2 marker protein or marker polynucleotide is used for
identifying a candidate agent or for selecting a composition for
inhibiting the progression of a disease in a patient. The disease
is preferably cancer and the patient is preferably a cancer patient
as disclosed above.
[0183] In another embodiment of the invention, a HER inhibitor is
used for the manufacture of a pharmaceutical composition for
treating a human cancer patient characterized in that said treating
or treatment includes assessing in a biological sample from the
patient [0184] (a) a marker gene or a combination of marker genes
selected from the group consisting of an epidermal growth factor, a
transforming growth factor alpha and a HER2 marker gene or; [0185]
(b) a combination of marker genes comprising an amphiregulin marker
gene and a marker gene selected from the group consisting of an
epidermal growth factor, a transforming growth factor alpha and a
HER2 marker gene.
[0186] The manufacture of a pharmaceutical composition for treating
a human cancer patient and particularly the formulation is
described in WO 01/00245, incorporated herein by reference,
particularly for the antibody 2C4.
[0187] In an preferred embodiment of the invention, in the use of
the HER dimerization inhibitor for the manufacture of a
pharmaceutical composition for treating a human cancer patient, the
treatment includes assessing the marker gene or the combination of
marker genes at least one time or repeatedly during treatment.
Preferably, the level of expression of the marker gene or the level
of expression of the combination of marker genes is assessed.
Preferably, the HER inhibitor is an antibody, preferably the
antibody 2C4. Preferably, the patient is a breast cancer, ovarian
cancer, lung cancer or prostate cancer patient.
[0188] In all embodiments of the invention, combinations of the
marker genes, marker polynucleotides or marker proteins are used as
disclosed above. In all embodiments of the invention, preferred
values for the difference of the level of expression determined in
the respective steps are also as disclosed above.
[0189] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
EXAMPLES
Statistical Methods
[0190] The statistical tasks comprise the following steps:
1. Pre-selection of candidate biomarkers 2. Pre-selection of
relevant clinical prognostic covariates 3. Selection of biomarker
prediction functions at an univariate level 4. Selection of
biomarker prediction functions including clinical covariates at an
univariate level 5. Selection of biomarker prediction functions at
a multivariate level 6. Selection of biomarker prediction functions
including clinical covariates at a multivariate level The following
text details the different steps:
[0191] Ad1: Pre-selection of candidate biomarkers: The statistical
pre-selection of candidate biomarkers is oriented towards the
strength of association with measures of clinical benefit. For this
purpose the different clinical endpoints may be transformed in
derived surrogate scores, as e.g. an ordinal assignment of the
degree of clinical benefit or morbidity scores regarding TTP or TTD
which avoid censored observations. These surrogate transformed
measures can be easily used for simple correlation analysis, e.g.
by the non-parametric Spearman rank correlation approach. An
alternative here is to use the biomarker measurements as metric
covariates in Time-to-event regression models, as e.g. Cox
proportional hazard regression. Depending on the statistical
distribution of the biomarker values this step may require some
pre-processing, as e.g. variance stabilizing transformations and
the use of suitable scales or, alternatively, a standardization
step like e.g. using percentiles instead of raw measurements. A
further approach is inspection of bivariate scatter plots, e.g. by
displaying the scatter of (x-axis=biomarker value, y-axis=measure
of clinical benefit) on a single patient basis. Here also some
non-parametric regression line as e.g. achieved by smoothing
splines can be useful to visualize the association of biomarker and
clinical benefit.
[0192] The goal of these different approaches is the pre-selection
of biomarker candidates, which show some association with clinical
benefit in at least one of the benefit measures employed, while
results for other measures are not contradictory. When there are
available control groups, then differences in association of
biomarkers with clinical benefit in the different arms could be a
sign of differential prediction which makes the biomarker eligible
for further consideration.
[0193] Ad2: Pre-selection of relevant clinical prognostic
covariates: The term "clinical covariate" here is used to describe
all other information about the patient, which are in general
available at baseline. These clinical covariates comprise
demographic information like sex, age etc., other anamnestic
information, concomitant diseases, concomitant therapies, result of
physical examinations, common laboratory parameters obtained, known
properties of the target tumor, information quantifying the extent
of malignant disease, clinical performance scores like ECOG or
Karnofsky index, clinical disease staging, timing and result of
pretreatments and disease history as well as all similar
information, which may be associated with the clinical prognosis.
The statistical pre-selection of clinical covariates parallels the
approaches for pre-selecting biomarkers and is as well oriented
towards the strength of association with measures of clinical
benefit. So in principle the same methods apply as considered under
1. In addition to statistical criteria, also criteria from clinical
experience and theoretical knowledge may apply to pre-select
relevant clinical covariates.
[0194] The prognosis by clinical covariates could interact with the
prognosis of the biomarkers. They will be considered for refined
prediction rules if necessary.
[0195] Ad3: Selection of biomarker prediction functions at an
univariate level: The term "prediction function" will be used in a
general sense to mean a numerical function of a biomarker
measurement which results in a number which is scaled to imply the
target prediction.
[0196] A simple example is the choice of the Heaviside function for
a specific cutoff c and a biomarker measurement x, where the binary
prediction A or B is to be made, then
If H(x-c)=0 then predict A.
If H(x-c)=1 then predict B.
[0197] This is probably the most common way of using univariate
biomarker measurements in prediction rules. The definition of a
prediction function usually recurs to an existing training data set
which can be used to explore the prediction possibilities. In order
to achieve a suitable cutoff c from the training set different
routes can be taken. First the scatterplot with smoothing spline
mentioned under 1 can be used to define the cutoff. Alternatively
some percentile of the distribution could be chosen, e.g. the
median or a quartile. Cutoffs can also be systematically extracted
by investigating all possible cutoffs according to their prediction
potential with regard to the measures of clinical benefit. Then
these results can be plotted to allow for an either manual
selection or to employ some search algorithm for optimality. This
was realized based on the endpoints TTP and TTD using a Cox model,
where at each test cutoff the biomarker was used as a binary
covariate. Prediction criteria were the resulting Hazard ratios.
Then the results for TTP and TTD can be considered together in
order to chose a cutoff which shows prediction in line with both
endpoints
[0198] Another uncommon approach for choosing a prediction function
can be based on a fixed parameter Cox regression model obtained
from the training set with biomarker values (possibly transformed)
as covariate. Then the prediction could simply depend on whether
the computed Hazard ratio is smaller or greater than 1.
[0199] A further possibility is to base the decision on some
likelihood ratio (or monotonic transform of it), where the target
probability densities were pre-determined in the training set for
separation of the prediction states. Then the biomarker would be
plugged into some function of the density ratios.
[0200] Ad4: Selection of biomarker prediction functions including
clinical covariates at an univariate level: Univariate here refers
to using only one biomarker--with regard to clinical covariates
this can be a multivariate model. This approach parallels the
search without clinical covariates, only that the methods should
allow for incorporating the relevant covariate information. The
scatterplot method of choosing a cutoff allows only a limited use
of covariates, e.g. a binary covariate could be color coded within
the plot. If the analysis relies on some regression approach then
the use of covariates (also many of them at a time) is usually
facilitated. The cutoff search based on the Cox model described
under 3, allows for an easy incorporation of covariates and thereby
leads to a covariate adjusted univariate cutoff search. The
adjustment by covariates may be done as covariates in the model or
via the inclusion in a stratified analysis.
[0201] Also the other choices of prediction functions allow for the
incorporation of covariates.
[0202] This is straightforward for the Cox model choice as
prediction function. There is the option to estimate the influence
of covariates on an interaction level, which means that e.g. for
different age groups different Hazard ratios apply.
[0203] For the likelihood ratio type of prediction functions, the
prediction densities must be estimated including covariates. Here
the methodology of multivariate pattern recognition can be used or
the biomarker values can be adjusted by multiple regression on the
covariates (prior to density estimation).
[0204] The CART technology (Classification And Regression Trees;
Breiman L., Friedman J. H., Olshen R. A., Stone C. J., Chapman
& Hall (Wadsworth, Inc.), New York, 1984) can be used for a
biomarker (raw measurement level) plus clinical covariates
employing a clinical benefit measure as response. This way cutoffs
are searched and a decision tree type of functions will be found
involving the covariates for prediction. The cutoffs and algorithms
chosen by CART are frequently close to optimal and may be combined
and unified by considering different clinical benefit measures.
[0205] Ad5: Selection of biomarker prediction functions at a
multivariate level: When there are several biomarker candidates
which maintain their prediction potential within the different
univariate prediction function choices, then a further improvement
may be achieved by combinations of biomarkers, i.e. considering
multivariate prediction functions.
[0206] Based on the simple Heaviside function model combinations of
biomarkers may be evaluated, e.g. by considering bivariate
scatterplots of biomarker values where optimal cutoffs are
indicated. Then a combination of biomarkers can be achieved by
combining different Heaviside function by the logical AND and OR
operators in order to achieve an improved prediction.
[0207] The CART technology (Classification And Regression Trees)
can be used for multiple biomarkers (raw measurement level) and a
clinical benefit measure as response, in order to achieve cutoffs
for biomarkers and decision tree type of functions for prediction.
The cutoffs and algorithms chosen by CART are frequently close to
optimal and may be combined and unified by considering different
clinical benefit measures.
[0208] The Cox-regression can be employed on different levels. A
first way is to incorporate the multiple biomarkers in a binary way
(i.e. based on Heaviside functions with some cutoffs). The other
option is to employ biomarkers in a metric way (after suitable
transformations), or a mixture of the binary and metric approach.
The evolving multivariate prediction function is of the Cox type as
described under 3.
[0209] The multivariate likelihood ratio approach is difficult to
realize but presents as well as an option for multivariate
prediction functions.
[0210] Ad6: Selection of biomarker prediction functions including
clinical covariates at a multivariate level: When there are
relevant clinical covariates then a further improvement may be
achieved by combining multiple biomarkers with multiple clinical
covariates. The different prediction function choices will be
evaluated with respect to the possibilities to include clinical
covariates.
[0211] Based on the simple logical combinations of Heaviside
functions for the biomarkers, further covariates may be included to
the prediction function based on logistic regression model obtained
in the training set.
[0212] The CART technology and the evolving decision trees can be
easily used with additional covariates, which would include these
in the prediction algorithm.
[0213] All prediction functions based on the Cox-regression can use
further clinical covariates. There is the option to estimate the
influence of covariates on an interaction level, which means that
e.g. for different age groups different Hazard ratios apply.
[0214] The multivariate likelihood ratio approach is not directly
extendible to the use of additional covariates.
Example 1
Baseline Blood Sera from HER2 Low Expressing Metastatic Breast
Cancer Patients Treated with Pertuzumab were Assessed for Levels of
HER Ligands and Shedded HER2 (HER2 ECD), as Described Below
[0215] Kits used for assessment of the serum biomarkers:
TABLE-US-00001 Marker Assay Distribution HER2-ECD Bayer HER-2/neu
ELISA, DakoCytomation N.V./S.A., Cat. #: EL501 Interleuvenlaan 12B,
B-3001 Heverlee Amphiregulin DuoSet ELISA Development R&D
Systems Ltd., 19 Barton System Human Amphiregulin, Lane, Abingdon
OX14 3NB, UK Cat. #: DY262 EGF Quantikine human EGF R&D Systems
Ltd., 19 Barton ELISA kit, Cat. #: DEG00 Lane, Abingdon OX14 3NB,
UK TGF-alpha Quantikine .RTM. Human TGF-alpha R&D Systems Ltd.,
19 Barton Immunoassay, Cat. #: DTGA00 Lane, Abingdon OX14 3NB,
UK
Protocols:
HER2-ECD:
[0216] HER2-ECD ELISA was performed according to the
recommendations of the manufacturer.
[0217] Amphiregulin: [0218] Prepare all reagents (provided with the
kit), standard dilutions (provided with the kit) and samples
[0219] Provide EvenCoat Goat Anti-mouse IgG microplate strips
(R&D, Cat. # CP002; not provided with the kit) in the frame.
The frame is now termed ELISA plate.
[0220] Determine of the required number of wells (number of
standard dilutions+number of samples).
[0221] Determine the plate layout.
[0222] Add 100 .mu.l diluted capture antibody (provided with the
kit; 1:180 in PBS) to each well.
[0223] Incubate at r.t. for 1 hour.
[0224] Aspirate each well and wash, repeating the process three
times for a total of four washes. Wash by filling each well with
400 .mu.l Wash buffer (not provided with the kit; 0.05% Tween-20 in
PBS was used), using a manifold dispenser, and subsequent
aspiration. After the last wash, remove any remaining Wash buffer
by aspirating. Invert the plate and blot it against clean paper
towels.
[0225] Add 100 .mu.l standard dilution or diluted sample (see
below) per well. Change tip after every pipetting step.
[0226] Cover plate with the adhesive strip (provided with the
kit).
[0227] Incubate for 2 hours at r.t. on a rocking platform.
[0228] Repeat the aspiration/wash as described previously.
[0229] Aspirated samples and wash solutions are treated with
laboratory disinfectant.
[0230] Add 100 .mu.l Detection Antibody (provided with the kit)
diluted 1:180 in Reagent diluent (not provided with the kit; 1% BSA
(Roth; Albumin Fraction V, Cat. # T844.2) in PBS was used) per
well
[0231] Incubate for 2 hours at r.t.
[0232] Repeat the aspiration/wash as described previously.
[0233] Add 100 .mu.l working dilution of the Streptavidin-HRP to
each well (provided with the kit; 1:200 dilution in Reagent
diluent). Cover with a new adhesive strip.
[0234] Incubate for 20 min at r.t.
[0235] Repeat the aspiration/wash as described previously.
[0236] Add 100 .mu.l Substrate Solution (R&D, Cat. # DY999; not
provided with the kit) to each well.
[0237] Incubate for 20 min at r.t. Protect from light. Add 50 .mu.l
Stop Solution (1.5 M H2504 (Schwefelsaure reinst, Merck, Cat. #
713); not provided with the kit) to each well. Mix carefully.
[0238] Determine the optical density of each well immediately,
using a microplate reader set to 450 nm.
Amphiregulin Standard Curve:
[0239] A 40 ng/ml amphiregulin stock solution was prepared in 1%
BSA in PBS, aliquotted and stored at -80.degree. C. Amphiregulin
solutions in 20% BSA in PBS were not stable beyond 2 weeks and were
therefore not used. From the aliquotted amphiregulin stock
solution, the amphiregulin standard curve was prepared freshly in
20% BSA in PBS prior to each experiment. The highest concentration
was 1000 pg/ml (1:40 dilution of the amphiregulin stock solution).
The standards provided with the ELISA kit produced a linear
standard curve. Excel-based analysis of the curves allowed the
determination of curve equations for every ELISA.
Amphiregulin Samples:
[0240] When samples were diluted 1:1 in Reagent Diluent, all
samples were within the linear range of the ELISA. Each sample was
measured in duplicates. Dependent on the quality of the data, and
on sufficient amounts of serum, determinations were repeated in
subsequent experiments if necessary.
[0241] EGF:
[0242] Prepare all reagents (provided with the kit), standard
dilutions (provided with the kit) and samples
[0243] Remove excess antibody-coated microtiter plate strips
(provided with the kit) from the frame. The frame is now termed
ELISA plate.
[0244] Determine of the required number of wells: (Number of
standard dilutions+number of samples).times.2
[0245] Determine the plate layout.
[0246] Add 50 .mu.l Assay Diluent RD1 (provided with the kit) to
each well
[0247] Add 200 .mu.l standard dilution or diluted sample (e.g. 1:20
in Calibrator Diluent RD6H) per well. Change tip after every
pipetting step.
[0248] Cover plate with the adhesive strip (provided with the
kit).
[0249] Incubate for 2 hours at r.t. on a rocking platform.
[0250] Aspirate each well and wash, repeating the process three
times for a total of four washes. Wash by filling each well with
400 .mu.l Wash Buffer (provided with the kit), using a manifold
dispenser, and subsequent aspiration. After the last wash, remove
any remaining Wash buffer by aspirating. Invert the plate and blot
it against clean paper towels.
[0251] Aspirated samples and wash solutions are treated with
laboratory disinfectant. Add 200 .mu.l of Conjugate (provided with
the kit) to each well. Cover with a new adhesive strip.
[0252] Incubate for 2 hours at r.t.
[0253] Repeat the aspiration/wash as described previously.
[0254] Add 200 .mu.l Substrate Solution (provided with the kit) to
each well.
[0255] Incubate for 20 min at r.t. Protect from light.
[0256] Add 50 .mu.l Stop Solution (provided with the kit) to each
well. Mix carefully.
[0257] Determine the optical density of each well within 30
minutes, using a microplate reader set to 450 nm.
EGF Standard Curve:
[0258] The standards provided with the ELISA kit produced a linear
standard curve. Also very small concentrations showed detectable
results.
[0259] EGF samples:
[0260] A total of four assays with the samples was performed. Each
sample was measured 2-5 times, the number of determinations being
dependent on the quality of the results (mean+/-SD) and the
availability of sufficient amounts of serum. When samples were
diluted 1:20 in Calibrator Diluent RD6H, all samples were within
the linear range of the ELISA.
TGF-Alpha:
[0261] Prepare all reagents (provided with the kit), standard
dilutions (provided with the kit) and samples
[0262] Remove excess antibody-coated microtiter plate strips
(provided with the kit) from the frame. The frame is now termed
ELISA plate.
[0263] Determine of the required number of wells: (Number of
standard dilutions+number of samples).times.2
[0264] Determine the plate layout.
[0265] Add 100 .mu.l Assay Diluent RD1W (provided with the kit) to
each well
[0266] Add 50 .mu.l standard dilution or sample per well. Change
tip after every pipetting step.
[0267] Cover plate with the adhesive strip (provided with the
kit).
[0268] Incubate for 2 hours at r.t. on a rocking platform.
[0269] Aspirate each well and wash, repeating the process three
times for a total of four washes. Wash by filling each well with
400 .mu.l Wash Buffer (provided with the kit), using a manifold
dispenser, and subsequent aspiration. After the last wash, remove
any remaining Wash buffer by aspirating. Invert the plate and blot
it against clean paper towels.
[0270] Aspirated samples and wash solutions are treated with
laboratory disinfectant.
[0271] Add 200 .mu.l of TGF-alpha Cojugate (provided with the kit)
to each well. Cover with a new adhesive strip.
[0272] Incubate for 2 hours at r.t.
[0273] Repeat the aspiration/wash as described previously.
[0274] Add 200 .mu.l Substrate Solution (provided with the kit) to
each well.
[0275] Incubate for 30 min at r.t. Protect from light.
[0276] Add 50 .mu.l Stop Solution (provided with the kit) to each
well. Mix carefully.
[0277] Determine the optical density of each well within 30
minutes, using a microplate reader set to 450 nm.
TGF-Alpha Standard Curve:
[0278] The standards provided with the ELISA kit produced a linear
standard curve. Also very small concentrations showed detectable
results.
TGF-Alpha Samples:
[0279] A total of four assays with the samples was performed.
Samples were measured in 2-4 independent assays.
[0280] The serum data was analyzed to identify factors the baseline
serum levels of which would be associated with response to the
Pertuzumab treatment. For all factors a skewed pattern of the
distribution (mean, standard deviation, median, minimum, maximum)
was observed. A monotonic transform was used to reduce the skewness
based on the logarithm: Log(x+1). In a univariate analysis, it was
explored whether suitable cut-points for the factors could be
defined which would relate to the probability of response (in this
example defined as clinical benefit). Here, patients with clinical
benefit were defined as those who achieved a partial response (PR)
or maintained stable disease for at least 6 months. Scatterplots of
the factors versus the response categories were investigated. FIG.
1 and FIG. 2 show a plotting of the clinical response categories
versus the logarithmic transformation of the serum levels of
TGF-alpha and amphiregulin, respectively, to exemplify the
approach.
[0281] Based on the scatterplots, cut-points were selected for the
factors to define groups of patients, who have experienced greater
clinical benefit. FIG. 3 (TGF-alpha), FIG. 4 (Amphiregulin), FIG. 5
(EGF), and FIG. 6 (HER2-ECD) show the clinical benefit in relation
to the different factor groupings based on the exploratory
cut-points calculated to the original factor units. The cut-points
separate out some of the patients without clinical benefit, and
hence, elevate the response rate for the group with greater
clinical benefit.
Example 2
[0282] In this example the exploratory cut-points from Example 1
were used to assess the univariate effect of the factor groupings
on different measures of the clinical benefit of the Pertuzumab
treatment, using time to progression/or death (TTP) and time to
death (TTD) as alternative clinical endpoints. Significant effects
were observed for TGF-alpha, Amphiregulin, EGF and HER2-ECD in
Kaplan-Meier estimates and log-rank tests for TTP and/or TTD, as
shown in an overview in FIG. 7.
[0283] The Kaplan-Meier plots displaying the hazard ratio are given
for TTP and TTD (highest number of events observed) in FIG. 8 and
FIG. 9 (TGF-alpha), 10 and 11 (Amphiregulin), 12 and 13 (EGF), and
14 and 15 (HER2-ECD), showing the pronounced effect of a grouping
based on these factors on the clinical outcome of the patients
treated with Pertuzumab.
Example 3
[0284] In this example multivariate approaches were used to
identify combinations of factors that would further improve the
identification of patients with greater benefit from the Pertuzumab
treatment. Results, as derived from a CART approach (Classification
And Regression Trees), are reflected. The CART classification
approach made it necessary to specify as the benefit group all
values in clinical benefit above of 0. As variables serum levels of
HER2-ECD, TGF-alpha, Amphiregulin, and EGF were employed. A
combination of serum HER2-ECD and serum TGF-alpha levels were
selected to give best results. From the CART results optimized
cut-points for a combination of serum HER2-ECD and serum TGF-alpha
levels were derived, resulting in a rule for exploratory
categorization of clinical benefit in the study population--a
combination of low serum HER2-ECD values and low serum TGF-alpha
values capturing 2/2 PR and 2/3 SD>6 months in the study
population and excluding a reasonable number of fast progressing
patients. FIG. 16 shows the clinical benefit in relation to the
TGF-alpha/HER2-ECD combination groupings based on the exploratory
combination cut-point. FIG. 17 summarizes the effect of a
combination of TGF-alpha and HER2-ECD on TTP and TTD. The
Kaplan-Meier estimates and the hazard ratios given in FIG. 18 (TTP)
and FIG. 19 (TTD) demonstrate the significant effect of the
grouping based on a combination of these factors for on the
clinical outcome of the patients treated with Pertuzumab.
[0285] Unless stated to the contrary, all compounds in the examples
were prepared and characterized as described. All ranges recited
herein encompass all combinations and subcombinations included
within that range limit. All patents and publications cited herein
are hereby incorporated by reference in their entirety for any
purpose.
Sequence CWU 1
1
141252PRTHomo sapiens 1Met Arg Ala Pro Leu Leu Pro Pro Ala Pro Val
Val Leu Ser Leu Leu1 5 10 15Ile Leu Gly Ser Gly His Tyr Ala Ala Gly
Leu Asp Leu Asn Asp Thr 20 25 30Tyr Ser Gly Lys Arg Glu Pro Phe Ser
Gly Asp His Ser Ala Asp Gly 35 40 45Phe Glu Val Thr Ser Arg Ser Glu
Met Ser Ser Gly Ser Glu Ile Ser 50 55 60Pro Val Ser Glu Met Pro Ser
Ser Ser Glu Pro Ser Ser Gly Ala Asp65 70 75 80Tyr Asp Tyr Ser Glu
Glu Tyr Asp Asn Glu Pro Gln Ile Pro Gly Tyr 85 90 95Ile Val Asp Asp
Ser Val Arg Val Glu Gln Val Val Lys Pro Pro Gln 100 105 110Asn Lys
Thr Glu Ser Glu Asn Thr Ser Asp Lys Pro Lys Arg Lys Lys 115 120
125Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Lys Lys Lys Asn
130 135 140Pro Cys Asn Ala Glu Phe Gln Asn Phe Cys Ile His Gly Glu
Cys Lys145 150 155 160Tyr Ile Glu His Leu Glu Ala Val Thr Cys Lys
Cys Gln Gln Glu Tyr 165 170 175Phe Gly Glu Arg Cys Gly Glu Lys Ser
Met Lys Thr His Ser Met Ile 180 185 190Asp Ser Ser Leu Ser Lys Ile
Ala Leu Ala Ala Ile Ala Ala Phe Met 195 200 205Ser Ala Val Ile Leu
Thr Ala Val Ala Val Ile Thr Val Gln Leu Arg 210 215 220Arg Gln Tyr
Val Arg Lys Tyr Glu Gly Glu Ala Glu Glu Arg Lys Lys225 230 235
240Leu Arg Gln Glu Asn Gly Asn Val His Ala Ile Ala 245
25021207PRTHomo sapiens 2Met Leu Leu Thr Leu Ile Ile Leu Leu Pro
Val Val Ser Lys Phe Ser1 5 10 15Phe Val Ser Leu Ser Ala Pro Gln His
Trp Ser Cys Pro Glu Gly Thr 20 25 30Leu Ala Gly Asn Gly Asn Ser Thr
Cys Val Gly Pro Ala Pro Phe Leu 35 40 45Ile Phe Ser His Gly Asn Ser
Ile Phe Arg Ile Asp Thr Glu Gly Thr 50 55 60Asn Tyr Glu Gln Leu Val
Val Asp Ala Gly Val Ser Val Ile Met Asp65 70 75 80Phe His Tyr Asn
Glu Lys Arg Ile Tyr Trp Val Asp Leu Glu Arg Gln 85 90 95Leu Leu Gln
Arg Val Phe Leu Asn Gly Ser Arg Gln Glu Arg Val Cys 100 105 110Asn
Ile Glu Lys Asn Val Ser Gly Met Ala Ile Asn Trp Ile Asn Glu 115 120
125Glu Val Ile Trp Ser Asn Gln Gln Glu Gly Ile Ile Thr Val Thr Asp
130 135 140Met Lys Gly Asn Asn Ser His Ile Leu Leu Ser Ala Leu Lys
Tyr Pro145 150 155 160Ala Asn Val Ala Val Asp Pro Val Glu Arg Phe
Ile Phe Trp Ser Ser 165 170 175Glu Val Ala Gly Ser Leu Tyr Arg Ala
Asp Leu Asp Gly Val Gly Val 180 185 190Lys Ala Leu Leu Glu Thr Ser
Glu Lys Ile Thr Ala Val Ser Leu Asp 195 200 205Val Leu Asp Lys Arg
Leu Phe Trp Ile Gln Tyr Asn Arg Glu Gly Ser 210 215 220Asn Ser Leu
Ile Cys Ser Cys Asp Tyr Asp Gly Gly Ser Val His Ile225 230 235
240Ser Lys His Pro Thr Gln His Asn Leu Phe Ala Met Ser Leu Phe Gly
245 250 255Asp Arg Ile Phe Tyr Ser Thr Trp Lys Met Lys Thr Ile Trp
Ile Ala 260 265 270Asn Lys His Thr Gly Lys Asp Met Val Arg Ile Asn
Leu His Ser Ser 275 280 285Phe Val Pro Leu Gly Glu Leu Lys Val Val
His Pro Leu Ala Gln Pro 290 295 300Lys Ala Glu Asp Asp Thr Trp Glu
Pro Glu Gln Lys Leu Cys Lys Leu305 310 315 320Arg Lys Gly Asn Cys
Ser Ser Thr Val Cys Gly Gln Asp Leu Gln Ser 325 330 335His Leu Cys
Met Cys Ala Glu Gly Tyr Ala Leu Ser Arg Asp Arg Lys 340 345 350Tyr
Cys Glu Asp Val Asn Glu Cys Ala Phe Trp Asn His Gly Cys Thr 355 360
365Leu Gly Cys Lys Asn Thr Pro Gly Ser Tyr Tyr Cys Thr Cys Pro Val
370 375 380Gly Phe Val Leu Leu Pro Asp Gly Lys Arg Cys His Gln Leu
Val Ser385 390 395 400Cys Pro Arg Asn Val Ser Glu Cys Ser His Asp
Cys Val Leu Thr Ser 405 410 415Glu Gly Pro Leu Cys Phe Cys Pro Glu
Gly Ser Val Leu Glu Arg Asp 420 425 430Gly Lys Thr Cys Ser Gly Cys
Ser Ser Pro Asp Asn Gly Gly Cys Ser 435 440 445Gln Leu Cys Val Pro
Leu Ser Pro Val Ser Trp Glu Cys Asp Cys Phe 450 455 460Pro Gly Tyr
Asp Leu Gln Leu Asp Glu Lys Ser Cys Ala Ala Ser Gly465 470 475
480Pro Gln Pro Phe Leu Leu Phe Ala Asn Ser Gln Asp Ile Arg His Met
485 490 495His Phe Asp Gly Thr Asp Tyr Gly Thr Leu Leu Ser Gln Gln
Met Gly 500 505 510Met Val Tyr Ala Leu Asp His Asp Pro Val Glu Asn
Lys Ile Tyr Phe 515 520 525Ala His Thr Ala Leu Lys Trp Ile Glu Arg
Ala Asn Met Asp Gly Ser 530 535 540Gln Arg Glu Arg Leu Ile Glu Glu
Gly Val Asp Val Pro Glu Gly Leu545 550 555 560Ala Val Asp Trp Ile
Gly Arg Arg Phe Tyr Trp Thr Asp Arg Gly Lys 565 570 575Ser Leu Ile
Gly Arg Ser Asp Leu Asn Gly Lys Arg Ser Lys Ile Ile 580 585 590Thr
Lys Glu Asn Ile Ser Gln Pro Arg Gly Ile Ala Val His Pro Met 595 600
605Ala Lys Arg Leu Phe Trp Thr Asp Thr Gly Ile Asn Pro Arg Ile Glu
610 615 620Ser Ser Ser Leu Gln Gly Leu Gly Arg Leu Val Ile Ala Ser
Ser Asp625 630 635 640Leu Ile Trp Pro Ser Gly Ile Thr Ile Asp Phe
Leu Thr Asp Lys Leu 645 650 655Tyr Trp Cys Asp Ala Lys Gln Ser Val
Ile Glu Met Ala Asn Leu Asp 660 665 670Gly Ser Lys Arg Arg Arg Leu
Thr Gln Asn Asp Val Gly His Pro Phe 675 680 685Ala Val Ala Val Phe
Glu Asp Tyr Val Trp Phe Ser Asp Trp Ala Met 690 695 700Pro Ser Val
Ile Arg Val Asn Lys Arg Thr Gly Lys Asp Arg Val Arg705 710 715
720Leu Gln Gly Ser Met Leu Lys Pro Ser Ser Leu Val Val Val His Pro
725 730 735Leu Ala Lys Pro Gly Ala Asp Pro Cys Leu Tyr Gln Asn Gly
Gly Cys 740 745 750Glu His Ile Cys Lys Lys Arg Leu Gly Thr Ala Trp
Cys Ser Cys Arg 755 760 765Glu Gly Phe Met Lys Ala Ser Asp Gly Lys
Thr Cys Leu Ala Leu Asp 770 775 780Gly His Gln Leu Leu Ala Gly Gly
Glu Val Asp Leu Lys Asn Gln Val785 790 795 800Thr Pro Leu Asp Ile
Leu Ser Lys Thr Arg Val Ser Glu Asp Asn Ile 805 810 815Thr Glu Ser
Gln His Met Leu Val Ala Glu Ile Met Val Ser Asp Gln 820 825 830Asp
Asp Cys Ala Pro Val Gly Cys Ser Met Tyr Ala Arg Cys Ile Ser 835 840
845Glu Gly Glu Asp Ala Thr Cys Gln Cys Leu Lys Gly Phe Ala Gly Asp
850 855 860Gly Lys Leu Cys Ser Asp Ile Asp Glu Cys Glu Met Gly Val
Pro Val865 870 875 880Cys Pro Pro Ala Ser Ser Lys Cys Ile Asn Thr
Glu Gly Gly Tyr Val 885 890 895Cys Arg Cys Ser Glu Gly Tyr Gln Gly
Asp Gly Ile His Cys Leu Asp 900 905 910Ile Asp Glu Cys Gln Leu Gly
Val His Ser Cys Gly Glu Asn Ala Ser 915 920 925Cys Thr Asn Thr Glu
Gly Gly Tyr Thr Cys Met Cys Ala Gly Arg Leu 930 935 940Ser Glu Pro
Gly Leu Ile Cys Pro Asp Ser Thr Pro Pro Pro His Leu945 950 955
960Arg Glu Asp Asp His His Tyr Ser Val Arg Asn Ser Asp Ser Glu Cys
965 970 975Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys
Met Tyr 980 985 990Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val
Val Gly Tyr Ile 995 1000 1005Gly Glu Arg Cys Gln Tyr Arg Asp Leu
Lys Trp Trp Glu Leu Arg 1010 1015 1020His Ala Gly His Gly Gln Gln
Gln Lys Val Ile Val Val Ala Val 1025 1030 1035Cys Val Val Val Leu
Val Met Leu Leu Leu Leu Ser Leu Trp Gly 1040 1045 1050Ala His Tyr
Tyr Arg Thr Gln Lys Leu Leu Ser Lys Asn Pro Lys 1055 1060 1065Asn
Pro Tyr Glu Glu Ser Ser Arg Asp Val Arg Ser Arg Arg Pro 1070 1075
1080Ala Asp Thr Glu Asp Gly Met Ser Ser Cys Pro Gln Pro Trp Phe
1085 1090 1095Val Val Ile Lys Glu His Gln Asp Leu Lys Asn Gly Gly
Gln Pro 1100 1105 1110Val Ala Gly Glu Asp Gly Gln Ala Ala Asp Gly
Ser Met Gln Pro 1115 1120 1125Thr Ser Trp Arg Gln Glu Pro Gln Leu
Cys Gly Met Gly Thr Glu 1130 1135 1140Gln Gly Cys Trp Ile Pro Val
Ser Ser Asp Lys Gly Ser Cys Pro 1145 1150 1155Gln Val Met Glu Arg
Ser Phe His Met Pro Ser Tyr Gly Thr Gln 1160 1165 1170Thr Leu Glu
Gly Gly Val Glu Lys Pro His Ser Leu Leu Ser Ala 1175 1180 1185Asn
Pro Leu Trp Gln Gln Arg Ala Leu Asp Pro Pro His Gln Met 1190 1195
1200Glu Leu Thr Gln 12053160PRTHomo sapiens 3Met Val Pro Ser Ala
Gly Gln Leu Ala Leu Phe Ala Leu Gly Ile Val1 5 10 15Leu Ala Ala Cys
Gln Ala Leu Glu Asn Ser Thr Ser Pro Leu Ser Ala 20 25 30Asp Pro Pro
Val Ala Ala Ala Val Val Ser His Phe Asn Asp Cys Pro 35 40 45Asp Ser
His Thr Gln Phe Cys Phe His Gly Thr Cys Arg Phe Leu Val 50 55 60Gln
Glu Asp Lys Pro Ala Cys Val Cys His Ser Gly Tyr Val Gly Ala65 70 75
80Arg Cys Glu His Ala Asp Leu Leu Ala Val Val Ala Ala Ser Gln Lys
85 90 95Lys Gln Ala Ile Thr Ala Leu Val Val Val Ser Ile Val Ala Leu
Ala 100 105 110Val Leu Ile Ile Thr Cys Val Leu Ile His Cys Cys Gln
Val Arg Lys 115 120 125His Cys Glu Trp Cys Arg Ala Leu Ile Cys Arg
His Glu Lys Pro Ser 130 135 140Ala Leu Leu Lys Gly Arg Thr Ala Cys
Cys His Ser Glu Thr Val Val145 150 155 16041255PRTHomo sapiens 4Met
Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu1 5 10
15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
20 25 30Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg
His 35 40 45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu
Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile
Gln Glu Val65 70 75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val
Arg Gln Val Pro Leu 85 90 95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln
Leu Phe Glu Asp Asn Tyr 100 105 110Ala Leu Ala Val Leu Asp Asn Gly
Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125Val Thr Gly Ala Ser Pro
Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140Leu Thr Glu Ile
Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln145 150 155 160Leu
Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170
175Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys
180 185 190His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly
Glu Ser 195 200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys
Ala Gly Gly Cys 210 215 220Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp
Cys Cys His Glu Gln Cys225 230 235 240Ala Ala Gly Cys Thr Gly Pro
Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255His Phe Asn His Ser
Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270Thr Tyr Asn
Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285Tyr
Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295
300Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn
Gln305 310 315 320Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu
Lys Cys Ser Lys 325 330 335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly
Met Glu His Leu Arg Glu 340 345 350Val Arg Ala Val Thr Ser Ala Asn
Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365Lys Ile Phe Gly Ser Leu
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380Pro Ala Ser Asn
Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe385 390 395 400Glu
Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410
415Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
420 425 430Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu 435 440 445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu
Leu Gly Ser Gly 450 455 460Leu Ala Leu Ile His His Asn Thr His Leu
Cys Phe Val His Thr Val465 470 475 480Pro Trp Asp Gln Leu Phe Arg
Asn Pro His Gln Ala Leu Leu His Thr 485 490 495Ala Asn Arg Pro Glu
Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510Gln Leu Cys
Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525Val
Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535
540Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His
Cys545 550 555 560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly
Ser Val Thr Cys 565 570 575Phe Gly Pro Glu Ala Asp Gln Cys Val Ala
Cys Ala His Tyr Lys Asp 580 585 590Pro Pro Phe Cys Val Ala Arg Cys
Pro Ser Gly Val Lys Pro Asp Leu 595 600 605Ser Tyr Met Pro Ile Trp
Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620Pro Cys Pro Ile
Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys625 630 635 640Gly
Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650
655Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly
660 665 670Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr
Met Arg 675 680 685Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu
Thr Pro Ser Gly 690 695 700Ala Met Pro Asn Gln Ala Gln Met Arg Ile
Leu Lys Glu Thr Glu Leu705 710 715 720Arg Lys Val Lys Val Leu Gly
Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735Gly Ile Trp Ile Pro
Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750Lys Val Leu
Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765Asp
Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775
780Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln
Leu785 790 795 800Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu
Asn Arg Gly Arg 805 810 815Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys
Met Gln Ile Ala Lys Gly 820 825 830Met Ser Tyr Leu Glu Asp Val Arg
Leu Val His Arg Asp Leu Ala Ala 835
840 845Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp
Phe 850 855 860Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr
His Ala Asp865 870 875 880Gly Gly Lys Val Pro Ile Lys Trp Met Ala
Leu Glu Ser Ile Leu Arg 885 890 895Arg Arg Phe Thr His Gln Ser Asp
Val Trp Ser Tyr Gly Val Thr Val 900 905 910Trp Glu Leu Met Thr Phe
Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925Arg Glu Ile Pro
Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940Pro Ile
Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met945 950 955
960Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe
965 970 975Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln
Asn Glu 980 985 990Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe
Tyr Arg Ser Leu 995 1000 1005Leu Glu Asp Asp Asp Met Gly Asp Leu
Val Asp Ala Glu Glu Tyr 1010 1015 1020Leu Val Pro Gln Gln Gly Phe
Phe Cys Pro Asp Pro Ala Pro Gly 1025 1030 1035Ala Gly Gly Met Val
His His Arg His Arg Ser Ser Ser Thr Arg 1040 1045 1050Ser Gly Gly
Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065Glu
Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1070 1075
1080Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu
1085 1090 1095Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg
Tyr Ser 1100 1105 1110Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr
Asp Gly Tyr Val 1115 1120 1125Ala Pro Leu Thr Cys Ser Pro Gln Pro
Glu Tyr Val Asn Gln Pro 1130 1135 1140Asp Val Arg Pro Gln Pro Pro
Ser Pro Arg Glu Gly Pro Leu Pro 1145 1150 1155Ala Ala Arg Pro Ala
Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1160 1165 1170Ser Pro Gly
Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 1180 1185Gly
Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1190 1195
1200Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp
1205 1210 1215Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly
Ala Pro 1220 1225 1230Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu
Asn Pro Glu Tyr 1235 1240 1245Leu Gly Leu Asp Val Pro Val 1250
125551270DNAHomo sapiens 5agacgttcgc acacctgggt gccagcgccc
cagaggtccc gggacagccc gaggcgccgc 60gcccgccgcc ccgagctccc caagccttcg
agagcggcgc acactcccgg tctccactcg 120ctcttccaac acccgctcgt
tttggcggca gctcgtgtcc cagagaccga gttgccccag 180agaccgagac
gccgccgctg cgaaggacca atgagagccc cgctgctacc gccggcgccg
240gtggtgctgt cgctcttgat actcggctca ggccattatg ctgctggatt
ggacctcaat 300gacacctact ctgggaagcg tgaaccattt tctggggacc
acagtgctga tggatttgag 360gttacctcaa gaagtgagat gtcttcaggg
agtgagattt cccctgtgag tgaaatgcct 420tctagtagtg aaccgtcctc
gggagccgac tatgactact cagaagagta tgataacgaa 480ccacaaatac
ctggctatat tgtcgatgat tcagtcagag ttgaacaggt agttaagccc
540ccccaaaaca agacggaaag tgaaaatact tcagataaac ccaaaagaaa
gaaaaaggga 600ggcaaaaatg gaaaaaatag aagaaacaga aagaagaaaa
atccatgtaa tgcagaattt 660caaaatttct gcattcacgg agaatgcaaa
tatatagagc acctggaagc agtaacatgc 720aaatgtcagc aagaatattt
cggtgaacgg tgtggggaaa agtccatgaa aactcacagc 780atgattgaca
gtagtttatc aaaaattgca ttagcagcca tagctgcctt tatgtctgct
840gtgatcctca cagctgttgc tgttattaca gtccagctta gaagacaata
cgtcaggaaa 900tatgaaggag aagctgagga acgaaagaaa cttcgacaag
agaatggaaa tgtacatgct 960atagcataac tgaagataaa attacaggat
atcacattgg agtcactgcc aagtcatagc 1020cataaatgat gagtcggtcc
tctttccagt ggatcataag acaatggacc ctttttgtta 1080tgatggtttt
aaactttcaa ttgtcacttt ttatgctatt tctgtatata aaggtgcacg
1140aaggtaaaaa gtattttttc aagttgtaaa taatttattt aatatttaat
ggaagtgtat 1200ttattttaca gctcattaaa cttttttaac caaacagaaa
aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa 127064877DNAHomo sapiens
6actgttggga gaggaatcgt atctccatat ttcttctttc agccccaatc caagggttgt
60agctggaact ttccatcagt tcttcctttc tttttcctct ctaagccttt gccttgctct
120gtcacagtga agtcagccag agcagggctg ttaaactctg tgaaatttgt
cataagggtg 180tcaggtattt cttactggct tccaaagaaa catagataaa
gaaatctttc ctgtggcttc 240ccttggcagg ctgcattcag aaggtctctc
agttgaagaa agagcttgga ggacaacagc 300acaacaggag agtaaaagat
gccccagggc tgaggcctcc gctcaggcag ccgcatctgg 360ggtcaatcat
actcaccttg cccgggccat gctccagcaa aatcaagctg ttttcttttg
420aaagttcaaa ctcatcaaga ttatgctgct cactcttatc attctgttgc
cagtagtttc 480aaaatttagt tttgttagtc tctcagcacc gcagcactgg
agctgtcctg aaggtactct 540cgcaggaaat gggaattcta cttgtgtggg
tcctgcaccc ttcttaattt tctcccatgg 600aaatagtatc tttaggattg
acacagaagg aaccaattat gagcaattgg tggtggatgc 660tggtgtctca
gtgatcatgg attttcatta taatgagaaa agaatctatt gggtggattt
720agaaagacaa cttttgcaaa gagtttttct gaatgggtca aggcaagaga
gagtatgtaa 780tatagagaaa aatgtttctg gaatggcaat aaattggata
aatgaagaag ttatttggtc 840aaatcaacag gaaggaatca ttacagtaac
agatatgaaa ggaaataatt cccacattct 900tttaagtgct ttaaaatatc
ctgcaaatgt agcagttgat ccagtagaaa ggtttatatt 960ttggtcttca
gaggtggctg gaagccttta tagagcagat ctcgatggtg tgggagtgaa
1020ggctctgttg gagacatcag agaaaataac agctgtgtca ttggatgtgc
ttgataagcg 1080gctgttttgg attcagtaca acagagaagg aagcaattct
cttatttgct cctgtgatta 1140tgatggaggt tctgtccaca ttagtaaaca
tccaacacag cataatttgt ttgcaatgtc 1200cctttttggt gaccgtatct
tctattcaac atggaaaatg aagacaattt ggatagccaa 1260caaacacact
ggaaaggaca tggttagaat taacctccat tcatcatttg taccacttgg
1320tgaactgaaa gtagtgcatc cacttgcaca acccaaggca gaagatgaca
cttgggagcc 1380tgagcagaaa ctttgcaaat tgaggaaagg aaactgcagc
agcactgtgt gtgggcaaga 1440cctccagtca cacttgtgca tgtgtgcaga
gggatacgcc ctaagtcgag accggaagta 1500ctgtgaagat gttaatgaat
gtgctttttg gaatcatggc tgtactcttg ggtgtaaaaa 1560cacccctgga
tcctattact gcacgtgccc tgtaggattt gttctgcttc ctgatgggaa
1620acgatgtcat caacttgttt cctgtccacg caatgtgtct gaatgcagcc
atgactgtgt 1680tctgacatca gaaggtccct tatgtttctg tcctgaaggc
tcagtgcttg agagagatgg 1740gaaaacatgt agcggttgtt cctcacccga
taatggtgga tgtagccagc tctgcgttcc 1800tcttagccca gtatcctggg
aatgtgattg ctttcctggg tatgacctac aactggatga 1860aaaaagctgt
gcagcttcag gaccacaacc atttttgctg tttgccaatt ctcaagatat
1920tcgacacatg cattttgatg gaacagacta tggaactctg ctcagccagc
agatgggaat 1980ggtttatgcc ctagatcatg accctgtgga aaataagata
tactttgccc atacagccct 2040gaagtggata gagagagcta atatggatgg
ttcccagcga gaaaggctta ttgaggaagg 2100agtagatgtg ccagaaggtc
ttgctgtgga ctggattggc cgtagattct attggacaga 2160cagagggaaa
tctctgattg gaaggagtga tttaaatggg aaacgttcca aaataatcac
2220taaggagaac atctctcaac cacgaggaat tgctgttcat ccaatggcca
agagattatt 2280ctggactgat acagggatta atccacgaat tgaaagttct
tccctccaag gccttggccg 2340tctggttata gccagctctg atctaatctg
gcccagtgga ataacgattg acttcttaac 2400tgacaagttg tactggtgcg
atgccaagca gtctgtgatt gaaatggcca atctggatgg 2460ttcaaaacgc
cgaagactta cccagaatga tgtaggtcac ccatttgctg tagcagtgtt
2520tgaggattat gtgtggttct cagattgggc tatgccatca gtaataagag
taaacaagag 2580gactggcaaa gatagagtac gtctccaagg cagcatgctg
aagccctcat cactggttgt 2640ggttcatcca ttggcaaaac caggagcaga
tccctgctta tatcaaaacg gaggctgtga 2700acatatttgc aaaaagaggc
ttggaactgc ttggtgttcg tgtcgtgaag gttttatgaa 2760agcctcagat
gggaaaacgt gtctggctct ggatggtcat cagctgttgg caggtggtga
2820agttgatcta aagaaccaag taacaccatt ggacatcttg tccaagacta
gagtgtcaga 2880agataacatt acagaatctc aacacatgct agtggctgaa
atcatggtgt cagatcaaga 2940tgactgtgct cctgtgggat gcagcatgta
tgctcggtgt atttcagagg gagaggatgc 3000cacatgtcag tgtttgaaag
gatttgctgg ggatggaaaa ctatgttctg atatagatga 3060atgtgagatg
ggtgtcccag tgtgcccccc tgcctcctcc aagtgcatca acaccgaagg
3120tggttatgtc tgccggtgct cagaaggcta ccaaggagat gggattcact
gtcttgatat 3180tgatgagtgc caactggggg tgcacagctg tggagagaat
gccagctgca caaatacaga 3240gggaggctat acctgcatgt gtgctggacg
cctgtctgaa ccaggactga tttgccctga 3300ctctactcca ccccctcacc
tcagggaaga tgaccaccac tattccgtaa gaaatagtga 3360ctctgaatgt
cccctgtccc acgatgggta ctgcctccat gatggtgtgt gcatgtatat
3420tgaagcattg gacaagtatg catgcaactg tgttgttggc tacatcgggg
agcgatgtca 3480gtaccgagac ctgaagtggt gggaactgcg ccacgctggc
cacgggcagc agcagaaggt 3540catcgtggtg gctgtctgcg tggtggtgct
tgtcatgctg ctcctcctga gcctgtgggg 3600ggcccactac tacaggactc
agaagctgct atcgaaaaac ccaaagaatc cttatgagga 3660gtcgagcaga
gatgtgagga gtcgcaggcc tgctgacact gaggatggga tgtcctcttg
3720ccctcaacct tggtttgtgg ttataaaaga acaccaagac ctcaagaatg
ggggtcaacc 3780agtggctggt gaggatggcc aggcagcaga tgggtcaatg
caaccaactt catggaggca 3840ggagccccag ttatgtggaa tgggcacaga
gcaaggctgc tggattccag tatccagtga 3900taagggctcc tgtccccagg
taatggagcg aagctttcat atgccctcct atgggacaca 3960gacccttgaa
gggggtgtcg agaagcccca ttctctccta tcagctaacc cattatggca
4020acaaagggcc ctggacccac cacaccaaat ggagctgact cagtgaaaac
tggaattaaa 4080aggaaagtca agaagaatga actatgtcga tgcacagtat
cttttctttc aaaagtagag 4140caaaactata ggttttggtt ccacaatctc
tacgactaat cacctactca atgcctggag 4200acagatacgt agttgtgctt
ttgtttgctc ttttaagcag tctcactgca gtcttatttc 4260caagtaagag
tactgggaga atcactaggt aacttattag aaacccaaat tgggacaaca
4320gtgctttgta aattgtgttg tcttcagcag tcaatacaaa tagatttttg
tttttgttgt 4380tcctgcagcc ccagaagaaa ttaggggtta aagcagacag
tcacactggt ttggtcagtt 4440acaaagtaat ttctttgatc tggacagaac
atttatatca gtttcatgaa atgattggaa 4500tattacaata ccgttaagat
acagtgtagg catttaactc ctcattggcg tggtccatgc 4560tgatgatttt
gccaaaatga gttgtgatga atcaatgaaa aatgtaattt agaaactgat
4620ttcttcagaa ttagatggcc ttatttttta aaatatttga atgaaaacat
tttattttta 4680aaatattaca caggaggcct tcggagtttc ttagtcatta
ctgtcctttt cccctacaga 4740attttccctc ttggtgtgat tgcacagaat
ttgtatgtat tttcagttac aagattgtaa 4800gtaaattgcc tgatttgttt
tcattataga caacgatgaa tttcttctaa ttatttaaat 4860aaaatcacca aaaacat
487774119DNAHomo sapiens 7ctggagagcc tgctgcccgc ccgcccgtaa
aatggtcccc tcggctggac agctcgccct 60gttcgctctg ggtattgtgt tggctgcgtg
ccaggccttg gagaacagca cgtccccgct 120gagtgcagac ccgcccgtgg
ctgcagcagt ggtgtcccat tttaatgact gcccagattc 180ccacactcag
ttctgcttcc atggaacctg caggtttttg gtgcaggagg acaagccagc
240atgtgtctgc cattctgggt acgttggtgc acgctgtgag catgcggacc
tcctggccgt 300ggtggctgcc agccagaaga agcaggccat caccgccttg
gtggtggtct ccatcgtggc 360cctggctgtc cttatcatca catgtgtgct
gatacactgc tgccaggtcc gaaaacactg 420tgagtggtgc cgggccctca
tctgccggca cgagaagccc agcgccctcc tgaagggaag 480aaccgcttgc
tgccactcag aaacagtggt ctgaagagcc cagaggagga gtttggccag
540gtggactgtg gcagatcaat aaagaaaggc ttcttcagga cagcactgcc
agagatgcct 600gggtgtgcca cagaccttcc tacttggcct gtaatcacct
gtgcagcctt ttgtgggcct 660tcaaaactct gtcaagaact ccgtctgctt
ggggttattc agtgtgacct agagaagaaa 720tcagcggacc acgatttcaa
gacttgttaa aaaagaactg caaagagacg gactcctgtt 780cacctaggtg
aggtgtgtgc agcagttggt gtctgagtcc acatgtgtgc agttgtcttc
840tgccagccat ggattccagg ctatatattt ctttttaatg ggccacctcc
ccacaacaga 900attctgccca acacaggaga tttctatagt tattgttttc
tgtcatttgc ctactgggga 960agaaagtgaa ggaggggaaa ctgtttaata
tcacatgaag accctagctt taagagaagc 1020tgtatcctct aaccacgaga
ctctcaacca gcccaacatc ttccatggac acatgacatt 1080gaagaccatc
ccaagctatc gccacccttg gagatgatgt cttatttatt agatggataa
1140tggttttatt tttaatctct taagtcaatg taaaaagtat aaaacccctt
cagacttcta 1200cattaatgat gtatgtgttg ctgactgaaa agctatactg
attagaaatg tctggcctct 1260tcaagacagc taaggcttgg gaaaagtctt
ccagggtgcg gagatggaac cagaggctgg 1320gttactggta ggaataaagg
taggggttca gaaatggtgc cattgaagcc acaaagccgg 1380taaatgcctc
aatacgttct gggagaaaac ttagcaaatc catcagcagg gatctgtccc
1440ctctgttggg gagagaggaa gagtgtgtgt gtctacacag gataaaccca
atacatattg 1500tactgctcag tgattaaatg ggttcacttc ctcgtgagcc
ctcggtaagt atgtttagaa 1560atagaacatt agccacgagc cataggcatt
tcaggccaaa tccatgaaag ggggaccagt 1620catttatttt ccattttgtt
gcttggttgg tttgttgctt tatttttaaa aggagaagtt 1680taactttgct
atttattttc gagcactagg aaaactattc cagtaatttt tttttcctca
1740tttccattca ggatgccggc tttattaaca aaaactctaa caagtcacct
ccactatgtg 1800ggtcttcctt tcccctcaag agaaggagca attgttcccc
tgacatctgg gtccatctga 1860cccatggggc ctgcctgtga gaaacagtgg
gtcccttcaa atacatagtg gatagctcat 1920ccctaggaat tttcattaaa
atttggaaac agagtaatga agaaataata tataaactcc 1980ttatgtgagg
aaatgctact aatatctgaa aagtgaaaga tttctatgta ttaactctta
2040agtgcaccta gcttattaca tcgtgaaagg tacatttaaa atatgttaaa
ttggcttgaa 2100attttcagag aattttgtct tcccctaatt cttcttcctt
ggtctggaag aacaatttct 2160atgaattttc tctttatttt ttttttataa
ttcagacaat tctatgaccc gtgtcttcat 2220ttttggcact cttatttaac
aatgccacac ctgaagcact tggatctgtt cagagctgac 2280cccctagcaa
cgtagttgac acagctccag gtttttaaat tactaaaata agttcaagtt
2340tacatccctt gggccagata tgtgggttga ggcttgactg tagcatcctg
cttagagacc 2400aatcaatgga cactggtttt tagacctcta tcaatcagta
gttagcatcc aagagacttt 2460gcagaggcgt aggaatgagg ctggacagat
ggcggaacga gaggttccct gcgaagactt 2520gagatttagt gtctgtgaat
gttctagttc ctaggtccag caagtcacac ctgccagtgc 2580cctcatcctt
atgcctgtaa cacacatgca gtgagaggcc tcacatatac gcctccctag
2640aagtgccttc caagtcagtc ctttggaaac cagcaggtct gaaaaagagg
ctgcatcaat 2700gcaagcctgg ttggaccatt gtccatgcct caggatagaa
cagcctggct tatttgggga 2760tttttcttct agaaatcaaa tgactgataa
gcattggctc cctctgccat ttaatggcaa 2820tggtagtctt tggttagctg
caaaaatact ccatttcaag ttaaaaatgc atcttctaat 2880ccatctctgc
aagctccctg tgtttccttg ccctttagaa aatgaattgt tcactacaat
2940tagagaatca tttaacatcc tgacctggta agctgccaca cacctggcag
tggggagcat 3000cgctgtttcc aatggctcag gagacaatga aaagccccca
tttaaaaaaa taacaaacat 3060tttttaaaag gcctccaata ctcttatgga
gcctggattt ttcccactgc tctacaggct 3120gtgacttttt ttaagcatcc
tgacaggaaa tgttttcttc tacatggaaa gatagacagc 3180agccaaccct
gatctggaag acagggcccc ggctggacac acgtggaacc aagccaggga
3240tgggctggcc attgtgtccc cgcaggagag atgggcagaa tggccctaga
gttcttttcc 3300ctgagaaagg agaaaaagat gggattgcca ctcacccacc
cacactggta agggaggaga 3360atttgtgctt ctggagcttc tcaagggatt
gtgttttgca ggtacagaaa actgcctgtt 3420atcttcaagc caggttttcg
agggcacatg ggtcaccagt tgctttttca gtcaatttgg 3480ccgggatgga
ctaatgaggc tctaacactg ctcaggagac ccctgccctc tagttggttc
3540tgggctttga tctcttccaa cctgcccagt cacagaagga ggaatgactc
aaatgcccaa 3600aaccaagaac acattgcaga agtaagacaa acatgtatat
ttttaaatgt tctaacataa 3660gacctgttct ctctagccat tgatttacca
ggctttctga aagatctagt ggttcacaca 3720gagagagaga gagtactgaa
aaagcaactc ctcttcttag tcttaataat ttactaaaat 3780ggtcaacttt
tcattatctt tattataata aacctgatgc ttttttttag aactccttac
3840tctgatgtct gtatatgttg cactgaaaag gttaatattt aatgttttaa
tttattttgt 3900gtggtaagtt aattttgatt tctgtaatgt gttaatgtga
ttagcagtta ttttccttaa 3960tatctgaatt atacttaaag agtagtgagc
aatataagac gcaattgtgt ttttcagtaa 4020tgtgcattgt tattgagttg
tactgtacct tatttggaag gatgaaggaa tgaacctttt 4080tttcctaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 411984624DNAHomo sapiens
8ggaggaggtg gaggaggagg gctgcttgag gaagtataag aatgaagttg tgaagctgag
60attcccctcc attgggaccg gagaaaccag gggagccccc cgggcagccg cgcgcccctt
120cccacggggc cctttactgc gccgcgcgcc cggcccccac ccctcgcagc
accccgcgcc 180ccgcgccctc ccagccgggt ccagccggag ccatggggcc
ggagccgcag tgagcaccat 240ggagctggcg gccttgtgcc gctgggggct
cctcctcgcc ctcttgcccc ccggagccgc 300gagcacccaa gtgtgcaccg
gcacagacat gaagctgcgg ctccctgcca gtcccgagac 360ccacctggac
atgctccgcc acctctacca gggctgccag gtggtgcagg gaaacctgga
420actcacctac ctgcccacca atgccagcct gtccttcctg caggatatcc
aggaggtgca 480gggctacgtg ctcatcgctc acaaccaagt gaggcaggtc
ccactgcaga ggctgcggat 540tgtgcgaggc acccagctct ttgaggacaa
ctatgccctg gccgtgctag acaatggaga 600cccgctgaac aataccaccc
ctgtcacagg ggcctcccca ggaggcctgc gggagctgca 660gcttcgaagc
ctcacagaga tcttgaaagg aggggtcttg atccagcgga acccccagct
720ctgctaccag gacacgattt tgtggaagga catcttccac aagaacaacc
agctggctct 780cacactgata gacaccaacc gctctcgggc ctgccacccc
tgttctccga tgtgtaaggg 840ctcccgctgc tggggagaga gttctgagga
ttgtcagagc ctgacgcgca ctgtctgtgc 900cggtggctgt gcccgctgca
aggggccact gcccactgac tgctgccatg agcagtgtgc 960tgccggctgc
acgggcccca agcactctga ctgcctggcc tgcctccact tcaaccacag
1020tggcatctgt gagctgcact gcccagccct ggtcacctac aacacagaca
cgtttgagtc 1080catgcccaat cccgagggcc ggtatacatt cggcgccagc
tgtgtgactg cctgtcccta 1140caactacctt tctacggacg tgggatcctg
caccctcgtc tgccccctgc acaaccaaga 1200ggtgacagca gaggatggaa
cacagcggtg tgagaagtgc agcaagccct gtgcccgagt 1260gtgctatggt
ctgggcatgg agcacttgcg agaggtgagg gcagttacca gtgccaatat
1320ccaggagttt gctggctgca agaagatctt tgggagcctg gcatttctgc
cggagagctt 1380tgatggggac ccagcctcca acactgcccc gctccagcca
gagcagctcc aagtgtttga 1440gactctggaa gagatcacag gttacctata
catctcagca tggccggaca gcctgcctga 1500cctcagcgtc ttccagaacc
tgcaagtaat ccggggacga attctgcaca atggcgccta 1560ctcgctgacc
ctgcaagggc tgggcatcag ctggctgggg ctgcgctcac tgagggaact
1620gggcagtgga ctggccctca tccaccataa cacccacctc tgcttcgtgc
acacggtgcc 1680ctgggaccag ctctttcgga acccgcacca agctctgctc
cacactgcca accggccaga 1740ggacgagtgt gtgggcgagg gcctggcctg
ccaccagctg tgcgcccgag ggcactgctg 1800gggtccaggg cccacccagt
gtgtcaactg cagccagttc cttcggggcc aggagtgcgt 1860ggaggaatgc
cgagtactgc aggggctccc cagggagtat gtgaatgcca ggcactgttt
1920gccgtgccac cctgagtgtc agccccagaa tggctcagtg acctgttttg
gaccggaggc 1980tgaccagtgt gtggcctgtg cccactataa ggaccctccc
ttctgcgtgg cccgctgccc 2040cagcggtgtg aaacctgacc tctcctacat
gcccatctgg
aagtttccag atgaggaggg 2100cgcatgccag ccttgcccca tcaactgcac
ccactcctgt gtggacctgg atgacaaggg 2160ctgccccgcc gagcagagag
ccagccctct gacgtccatc atctctgcgg tggttggcat 2220tctgctggtc
gtggtcttgg gggtggtctt tgggatcctc atcaagcgac ggcagcagaa
2280gatccggaag tacacgatgc ggagactgct gcaggaaacg gagctggtgg
agccgctgac 2340acctagcgga gcgatgccca accaggcgca gatgcggatc
ctgaaagaga cggagctgag 2400gaaggtgaag gtgcttggat ctggcgcttt
tggcacagtc tacaagggca tctggatccc 2460tgatggggag aatgtgaaaa
ttccagtggc catcaaagtg ttgagggaaa acacatcccc 2520caaagccaac
aaagaaatct tagacgaagc atacgtgatg gctggtgtgg gctccccata
2580tgtctcccgc cttctgggca tctgcctgac atccacggtg cagctggtga
cacagcttat 2640gccctatggc tgcctcttag accatgtccg ggaaaaccgc
ggacgcctgg gctcccagga 2700cctgctgaac tggtgtatgc agattgccaa
ggggatgagc tacctggagg atgtgcggct 2760cgtacacagg gacttggccg
ctcggaacgt gctggtcaag agtcccaacc atgtcaaaat 2820tacagacttc
gggctggctc ggctgctgga cattgacgag acagagtacc atgcagatgg
2880gggcaaggtg cccatcaagt ggatggcgct ggagtccatt ctccgccggc
ggttcaccca 2940ccagagtgat gtgtggagtt atggtgtgac tgtgtgggag
ctgatgactt ttggggccaa 3000accttacgat gggatcccag cccgggagat
ccctgacctg ctggaaaagg gggagcggct 3060gccccagccc cccatctgca
ccattgatgt ctacatgatc atggtcaaat gttggatgat 3120tgactctgaa
tgtcggccaa gattccggga gttggtgtct gaattctccc gcatggccag
3180ggacccccag cgctttgtgg tcatccagaa tgaggacttg ggcccagcca
gtcccttgga 3240cagcaccttc taccgctcac tgctggagga cgatgacatg
ggggacctgg tggatgctga 3300ggagtatctg gtaccccagc agggcttctt
ctgtccagac cctgccccgg gcgctggggg 3360catggtccac cacaggcacc
gcagctcatc taccaggagt ggcggtgggg acctgacact 3420agggctggag
ccctctgaag aggaggcccc caggtctcca ctggcaccct ccgaaggggc
3480tggctccgat gtatttgatg gtgacctggg aatgggggca gccaaggggc
tgcaaagcct 3540ccccacacat gaccccagcc ctctacagcg gtacagtgag
gaccccacag tacccctgcc 3600ctctgagact gatggctacg ttgcccccct
gacctgcagc ccccagcctg aatatgtgaa 3660ccagccagat gttcggcccc
agcccccttc gccccgagag ggccctctgc ctgctgcccg 3720acctgctggt
gccactctgg aaaggcccaa gactctctcc ccagggaaga atggggtcgt
3780caaagacgtt tttgcctttg ggggtgccgt ggagaacccc gagtacttga
caccccaggg 3840aggagctgcc cctcagcccc accctcctcc tgccttcagc
ccagccttcg acaacctcta 3900ttactgggac caggacccac cagagcgggg
ggctccaccc agcaccttca aagggacacc 3960tacggcagag aacccagagt
acctgggtct ggacgtgcca gtgtgaacca gaaggccaag 4020tccgcagaag
ccctgatgtg tcctcaggga gcagggaagg cctgacttct gctggcatca
4080agaggtggga gggccctccg accacttcca ggggaacctg ccatgccagg
aacctgtcct 4140aaggaacctt ccttcctgct tgagttccca gatggctgga
aggggtccag cctcgttgga 4200agaggaacag cactggggag tctttgtgga
ttctgaggcc ctgcccaatg agactctagg 4260gtccagtgga tgccacagcc
cagcttggcc ctttccttcc agatcctggg tactgaaagc 4320cttagggaag
ctggcctgag aggggaagcg gccctaaggg agtgtctaag aacaaaagcg
4380acccattcag agactgtccc tgaaacctag tactgccccc catgaggaag
gaacagcaat 4440ggtgtcagta tccaggcttt gtacagagtg cttttctgtt
tagtttttac tttttttgtt 4500ttgttttttt aaagatgaaa taaagaccca
gggggagaat gggtgttgta tggggaggca 4560agtgtggggg gtccttctcc
acacccactt tgtccatttg caaatatatt ttggaaaaca 4620gcta
46249183PRTHomo sapiens 9Met Arg Ala Asn Asp Ala Leu Gln Val Leu
Gly Leu Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser
Gln Ala Val Cys Pro Gly Thr 20 25 30Leu Asn Gly Leu Ser Val Thr Gly
Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45Leu Tyr Lys Leu Tyr Glu Arg
Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60Ile Val Leu Thr Gly His
Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr
Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95Leu Pro Leu
Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110Gly
Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120
125His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Gly Gln Phe Pro
130 135 140Met Val Pro Ser Gly Leu Thr Pro Gln Pro Ala Gln Asp Trp
Tyr Leu145 150 155 160Leu Asp Asp Asp Pro Arg Leu Leu Thr Leu Ser
Ala Ser Ser Lys Val 165 170 175Pro Val Thr Leu Ala Ala Val
180101050DNAHomo sapiens 10acacacacac acccctcccc tgccatccct
ccccggactc cggctccggc tccgattgca 60atttgcaacc tccgctgccg tcgccgcagc
agccaccaat tcgccagcgg ttcaggtggc 120tcttgcctcg atgtcctagc
ctaggggccc ccgggccgga cttggctggg ctcccttcac 180cctctgcgga
gtcatgaggg cgaacgacgc tctgcaggtg ctgggcttgc ttttcagcct
240ggcccggggc tccgaggtgg gcaactctca ggcagtgtgt cctgggactc
tgaatggcct 300gagtgtgacc ggcgatgctg agaaccaata ccagacactg
tacaagctct acgagaggtg 360tgaggtggtg atggggaacc ttgagattgt
gctcacggga cacaatgccg acctctcctt 420cctgcagtgg attcgagaag
tgacaggcta tgtcctcgtg gccatgaatg aattctctac 480tctaccattg
cccaacctcc gcgtggtgcg agggacccag gtctacgatg ggaagtttgc
540catcttcgtc atgttgaact ataacaccaa ctccagccac gctctgcgcc
agctccgctt 600gactcagctc accggtcagt tcccgatggt tccttctggc
ctcacccctc agccagccca 660agactggtac ctccttgatg atgacccaag
actgctcact ctaagtgcct cttccaaggt 720gcctgtcacc ttggccgctg
tctaaaggtc cattgctccc taagcaatag agggccccca 780gtagggggag
ctaggggcat ctgctccagg gaaaggaacc ctgtgtcctt gtggggctgg
840agtcagagct ggatctgtta accgtttttc taatttcaaa gtacagtgta
ccggaggcca 900ggcctgatgg cttacacctg taatcccagc attttgggag
gccaaggagg gcagatcact 960tgagatcagg agtttgagac cagcctggcc
aacatggcga aaccctgtct ctactaaaaa 1020tacaaaaaaa taaaataaaa
taaaaaatta 1050111210PRTHomo sapiens 11Met Arg Pro Ser Gly Thr Ala
Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser
Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys
Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln
Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile
Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr
Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90
95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp
Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu
Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro
Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile
Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln
Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro
Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys
Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215
220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly
Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg
Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro
Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn
Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys
Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp
Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330
335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser
Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser
Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile
Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile
Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu
Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln
Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly
Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455
460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys
Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys
His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro
Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu
Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu
Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570
575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu
Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
His Pro Asn Cys 610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu
Gly Cys Pro Thr Asn Gly625 630 635 640Pro Lys Ile Pro Ser Ile Ala
Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655Leu Leu Val Val Ala
Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670Ile Val Arg
Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685Val
Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695
700Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly
Ser705 710 715 720Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile
Pro Glu Gly Glu 725 730 735Lys Val Lys Ile Pro Val Ala Ile Lys Glu
Leu Arg Glu Ala Thr Ser 740 745 750Pro Lys Ala Asn Lys Glu Ile Leu
Asp Glu Ala Tyr Val Met Ala Ser 755 760 765Val Asp Asn Pro His Val
Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780Thr Val Gln Leu
Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790 795 800Tyr
Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810
815Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg
820 825 830Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys
Thr Pro 835 840 845Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys
Leu Leu Gly Ala 850 855 860Glu Glu Lys Glu Tyr His Ala Glu Gly Gly
Lys Val Pro Ile Lys Trp865 870 875 880Met Ala Leu Glu Ser Ile Leu
His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895Val Trp Ser Tyr Gly
Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910Lys Pro Tyr
Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925Lys
Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935
940Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro
Lys945 950 955 960Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala
Arg Asp Pro Gln 965 970 975Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg
Met His Leu Pro Ser Pro 980 985 990Thr Asp Ser Asn Phe Tyr Arg Ala
Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005Asp Val Val Asp Ala
Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020Phe Ser Ser
Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035Ser
Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045
1050Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
1055 1060 1065Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser
Ile Asp 1070 1075 1080Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn
Gln Ser Val Pro 1085 1090 1095Lys Arg Pro Ala Gly Ser Val Gln Asn
Pro Val Tyr His Asn Gln 1100 1105 1110Pro Leu Asn Pro Ala Pro Ser
Arg Asp Pro His Tyr Gln Asp Pro 1115 1120 1125His Ser Thr Ala Val
Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140Pro Thr Cys
Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155Gln
Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160 1165
1170Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala
Pro Gln 1190 1195 1200Ser Ser Glu Phe Ile Gly Ala 1205
121012628PRTHomo sapiens 12Met Arg Pro Ser Gly Thr Ala Gly Ala Ala
Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu
Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys Leu Thr Gln
Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe
Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val
Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu
Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile
Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr
Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120
125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn
Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp
Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly
Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys
Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile
Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser
Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235
240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr
Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys
Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn
Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala Cys Gly
Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys
Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys Asn Gly
Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala
Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360
365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val
Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro
Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn Leu Glu Ile
Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln Phe Ser Leu Ala
Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu Arg Ser Leu
Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450
455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys
Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys
His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro
Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu
Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu
Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570
575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu
Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
His Pro Asn Cys 610 615 620Thr Tyr Gly Ser62513405PRTHomo sapiens
13Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1
5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys
Gln 20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp
His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val
Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu
Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu
Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln
Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu
Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys
Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala
Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155
160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys
Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu
Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln
Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys
His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu
Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr
Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr
Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280
285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met
Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly
Pro Cys Arg Lys Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe
Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe
Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro
Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu
Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395
400Ile Thr Gly Leu Ser 40514705PRTHomo sapiens 14 Met Arg Pro Ser
Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys
Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly
Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40
45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu
Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu
Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg
Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu
Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro
Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe
Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln
Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser
Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185
190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg
Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys
Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu
Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr
Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met
Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys
Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly
Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310
315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys
Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr
Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg
Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu
Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Phe
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His
Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425
430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile
Ile Ser 450 455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
Trp Lys Lys Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys
Ile Ile Ser Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly
Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly
Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg
Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu
Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550
555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly
Pro 565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
His Cys Val 580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn
Asn Thr Leu Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys
His Leu Cys His Pro Asn Cys 610 615 620Thr Tyr Gly Pro Gly Asn Glu
Ser Leu Lys Ala Met Leu Phe Cys Leu625 630 635 640Phe Lys Leu Ser
Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His 645 650 655Gln Ser
Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro 660 665
670Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His
675 680 685Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser
Ser Cys 690 695 700His705
* * * * *