U.S. patent application number 14/502769 was filed with the patent office on 2015-05-28 for protein biomarker and uses thereof.
This patent application is currently assigned to Daiichi Sankyo Company, Limited. The applicant listed for this patent is Daiichi Sankyo Company, Limited, Kinki University, U3 Pharma GmbH. Invention is credited to Robert Allen BECKMAN, Sabine BLUM, Daniel J. FREEMAN, Xiaoping JIN, Renee Jeanne MENDELL-HARARY, Kazuhiko NAKAGAWA, Matthias SCHNEIDER, Kimio YONESAKA.
Application Number | 20150147326 14/502769 |
Document ID | / |
Family ID | 52744738 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150147326 |
Kind Code |
A1 |
SCHNEIDER; Matthias ; et
al. |
May 28, 2015 |
PROTEIN BIOMARKER AND USES THEREOF
Abstract
The present invention is directed to methods of identifying and
treating a human subject harboring a tumor or other disease
comprising assessing HRG gene expression at a protein level in the
human subject and administering a treatment comprising an anti-HER3
antibody to the human subject whose HRG gene expression at a
protein level is assessed as high. The present invention is also
directed to methods of identifying a human subject harboring a
tumor or other disease comprising assessing HRG gene expression at
a protein level in the human subject and withholding a treatment
comprising an anti-HER3 antibody to the human subject whose HRG
gene expression at a protein level is assessed as low. The
invention is also directed to methods of performing an ELISA,
including sequential steps of contacting a solid surface with a
plurality of solutions each comprising in turn a capture antibody,
a blocking agent, a sample suspected of containing an analyte, a
detection antibody and an enzyme conjugate, in which the solid
surface is subjected to a wash process after each sequential
step.
Inventors: |
SCHNEIDER; Matthias;
(Neufarn, DE) ; BLUM; Sabine; (Munchen, DE)
; MENDELL-HARARY; Renee Jeanne; (Skillman, NJ) ;
FREEMAN; Daniel J.; (Holmdel, NJ) ; BECKMAN; Robert
Allen; (Blue Bell, PA) ; JIN; Xiaoping;
(Hillsborough, NJ) ; YONESAKA; Kimio; (Osakasayama
City, JP) ; NAKAGAWA; Kazuhiko; (Osakasayama City,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Daiichi Sankyo Company, Limited
U3 Pharma GmbH
Kinki University |
Tokyo
Martinsried
Osaka |
|
JP
DE
JP |
|
|
Assignee: |
Daiichi Sankyo Company,
Limited
Tokyo
JP
U3 Pharma GmbH
Martinsried
DE
Kinki University
Osaka
JP
|
Family ID: |
52744738 |
Appl. No.: |
14/502769 |
Filed: |
September 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61884983 |
Sep 30, 2013 |
|
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Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/142.1; 424/158.1; 435/29; 435/7.1; 435/7.72;
435/7.92; 435/7.94; 436/501; 600/1 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/57423 20130101; A61P 11/00 20180101; A61K 39/39558
20130101; C07K 16/2863 20130101; C07K 16/40 20130101; C07K 16/32
20130101; A61K 2039/505 20130101; C07K 2317/76 20130101; A61K
31/517 20130101; C07K 2317/21 20130101; G01N 33/5695 20130101; G01N
2333/4756 20130101; A61K 45/06 20130101; A61P 43/00 20180101; A61N
5/10 20130101 |
Class at
Publication: |
424/136.1 ;
424/158.1; 424/142.1; 424/133.1; 435/29; 436/501; 435/7.1;
435/7.92; 435/7.94; 435/7.72; 600/1 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 16/40 20060101 C07K016/40; A61N 5/10 20060101
A61N005/10; A61K 31/517 20060101 A61K031/517; A61K 45/06 20060101
A61K045/06; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395 |
Claims
1-74. (canceled)
75. A method of treating a human subject harboring a locally
advanced or metastatic non-small cell lung cancer (NSCLC) tumor
comprising administering a treatment comprising an anti-HER3
antibody to a human subject diagnosed with a locally advanced or
metastatic NSCLC whose HRG gene expression at a protein level is
assessed as high.
76. The method of claim 75 in which the HRG gene expression at a
protein level is assessed as high if a protein concentration value
is observed, which is above a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
77. The method of claim 76 in which the predetermined threshold is
statistically determined, refined, adjusted and/or confirmed
through, on, or based on, randomized clinical data and optionally
non-clinical data.
78. The method of claim 76 in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about
4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL,
about 8000 pg/mL, about 9000 pg/mL, about 10000 pg/mL, about 11000
pg/mL, about 12000 pg/mL, about 13000 pg/mL, about 14000 pg/mL,
about 15000 pg/mL, about 16000 pg/mL, about 17000 pg/mL, about
18000 pg/mL, about 19000 pg/mL, about 20000 pg/mL, about 22000
pg/mL, about 24000 pg/mL, about 26000 pg/mL, about 28000 pg/mL,
about 30000 pg/mL, about 35000 pg/mL, about 40000 pg/mL, about
50000 pg/mL, about 60000 pg/mL, about 70000 pg/mL, about 80000
pg/Lm, about 90000 pg/mL and about 100000 pg/mL.
79. The method of claim 75, wherein the subject harbors wild-type
EGFR.
80. The method of claim 75, wherein the tumor has progressed on at
least one prior systemic therapy.
81. The method of claim 75, further comprising assessing gene
expression at a protein level in the human subject diagnosed with
the locally advanced or metastatic NSCLC, wherein the HRG gene
expression at a protein level is assessed using ELISA or
immunohistochemistry techniques.
82. The method of claim 76 in which the biological sample comprises
a whole blood or serum sample.
83. The method of claim 75 in which the anti-HER3 antibody is
selected from the group consisting of patritumab, duligotumab
(MEHD-7945A), seribantumab (MM-121), MM-111, LJM716, RG-7116,
tri-specific anti-EGFR/ERBB3 zybody, huHER3-8, and a derivative or
fragment of any of these.
84. The method of claim 75 in which the treatment comprises an
anti-HER3 antibody in combination with one or more of (i) a HER
inhibitor, (ii) a chemotherapy, (iii) radiation and (iv) an other
targeted agent.
85. The method of claim 84, wherein the HER inhibitor is selected
from the group consisting of trastuzumab, T-DM1, lapatinib,
pertuzumab, cetuximab, panitumumab gefitinib, afatinib,
dacomitinib, KD-019 and erlotinib.
86. The method of claim 84, wherein the chemotherapy is selected
from the group consisting of cisplatin, carboplatin, gemcitabine,
pemetrexed, irinotecan, 5-fluoruracil, paclitaxel, docetaxel, and
capecitabine.
87. The method of claim 75, wherein the HRG expression is assessed
using an FDA-approved test.
88. A method of treating a human subject harboring a locally
advanced or metastatic non-small cell lung cancer (NSCLC) tumor
comprising withholding a treatment comprising an anti-HER3 antibody
from a human subject diagnosed with a locally advanced or
metastatic NSCLC whose HRG gene expression at a protein level is
assessed as low.
89. The method of claim 88 in which the HRG gene expression at a
protein level is assessed as low if a protein concentration value
is observed, which is at or below a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
90. The method of claim 89 in which the predetermined threshold is
statistically determined, refined, adjusted and/or confirmed
through, on, or based on, randomized clinical data and optionally
non-clinical data.
91. The method of claim 90 in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about
3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL,
about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL, about 10000
pg/mL, about 11000 pg/mL, about 12000 pg/mL, about 13000 pg/mL,
about 14000 pg/mL, about 15000 pg/mL, about 16000 pg/mL, about
17000 pg/mL, about 18000 pg/mL, about 19000 pg/mL, about 20000
pg/mL, about 22000 pg/mL, about 24000 pg/mL, about 26000 pg/mL,
about 28000 pg/mL, about 30000 pg/mL, about 35000 pg/mL, about
40000 pg/mL, about 50000 pg/mL, about 60000 pg/mL, about 70000
pg/mL, about 80000 pg/mL, about 90000 pg/mL and about 100000
pg/mL.
92. The method of claim 88, wherein the subject harbors wild-type
EGFR.
93. The method of claim 88, wherein the tumor has progressed on at
least one prior systemic therapy.
94. The method of claim 89 in which the biological sample comprises
a whole blood or serum sample.
95. The method of claim 88 in which the treatment withheld
comprises an anti-HER3 antibody in combination with one or more of
(i) a HER inhibitor, (ii) a chemotherapy, (iii) radiation and (iv)
an other targeted agent.
96. A method of receiving or undergoing a treatment for a locally
advanced or metastatic non-small cell lung cancer (NSCLC) tumor or
abstaining therefrom comprising: providing an autologous tissue
sample or consenting to a taking of same to facilitate an
assessment of HRG gene expression at a protein level in a human
subject diagnosed with a locally advanced or metastatic NSCLC; and
receiving or undergoing a treatment comprising an anti-HER3
antibody if the HRG gene expression at a protein level is assessed
as high, or abstaining from a treatment comprising an anti-HER3
antibody if the HRG gene expression at a protein level is assessed
as low.
97. The method of claim 96 in which (i) the HRG gene expression at
a protein level is assessed as high if a protein concentration
value is observed, which is above a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC or (ii) the HRG gene expression at a
protein level is assessed as low if a protein concentration value
is observed, which is at or below the predetermined threshold, from
the biological sample taken from the subject diagnosed with a
locally advanced or metastatic NSCLC.
98. The method of claim 97 in which the predetermined threshold is
statistically determined, refined, adjusted and/or confirmed
through, on, or based on, randomized clinical data and optionally
non-clinical data.
99. The method of claim 98 in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about
4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL,
about 8000 pg/mL, about 9000 pg/mL, about 10000 pg/mL, about 11000
pg/mL, about 12000 pg/mL, about 13000 pg/mL, about 14000 pg/mL,
about 15000 pg/mL, about 16000 pg/mL, about 17000 pg/mL, about
18000 pg/mL, about 19000 pg/mL, about 20000 pg/mL, about 22000
pg/mL, about 24000 pg/mL, about 26000 pg/mL, about 28000 pg/mL,
about 30000 pg/mL, about 35000 pg/mL, about 40000 pg/mL, about
50000 pg/mL, about 60000 pg/mL, about 70000 pg/mL, about 80000
pg/Lm, about 90000 pg/mL and about 100000 pg/mL.
100. A method of electing a treatment for a locally advanced or
metastatic non-small cell lung cancer (NSCLC) tumor comprising:
receiving an assessment of HRG gene expression at a protein level
in a human subject diagnosed with a locally advanced or metastatic
NSCLC; and electing to withhold or abstain from a treatment
comprising an anti-HER3 antibody if HRG gene expression at a
protein level is assessed as low, or electing to receive or undergo
a treatment comprising an anti-HER3 antibody if HRG gene expression
at a protein level is assessed as high.
101. The method of claim 100 in which (i) the HRG gene expression
at a protein level is assessed as high if a protein concentration
value is observed, which is above a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC or (ii) the HRG gene expression at a
protein level is assessed as low if a protein concentration value
is observed, which is at or below the predetermined threshold, from
the biological sample taken from the subject diagnosed with a
locally advanced or metastatic NSCLC.
102. The method of claim 101 in which the predetermined threshold
is statistically determined, refined, adjusted and/or confirmed
through, on, or based on, randomized clinical data and optionally
non-clinical data.
103. The method of claim 102 in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about
4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL,
about 8000 pg/mL, about 9000 pg/mL, about 10000 pg/mL, about 11000
pg/mL, about 12000 pg/mL, about 13000 pg/mL, about 14000 pg/mL,
about 15000 pg/mL, about 16000 pg/mL, about 17000 pg/mL, about
18000 pg/mL, about 19000 pg/mL, about 20000 pg/mL, about 22000
pg/mL, about 24000 pg/mL, about 26000 pg/mL, about 28000 pg/mL,
about 30000 pg/mL, about 35000 pg/mL, about 40000 pg/mL, about
50000 pg/mL, about 60000 pg/mL, about 70000 pg/mL, about 80000
pg/Lm, about 90000 pg/mL and about 100000 pg/mL.
Description
FIELD OF THE INVENTION
[0001] The field of the invention is molecular biology, oncology,
clinical diagnostics, clinical treatment, and methods of performing
an enzyme-linked immunosorbent assay (ELISA) are also described
herein.
BACKGROUND
[0002] Most cancer drugs are effective in some patients, but not in
others. This results from genetic variation among tumors, and can
be observed even among tumors within the same patient. Variable
patient response is particularly pronounced with respect to
targeted therapeutics. Therefore, the full potential of targeted
therapies cannot be realized without suitable tests for determining
which patients will benefit from which drugs. According to the
National Institutes of Health (NIH), the term "biomarker" is
defined as "a characteristic that is objectively measured and
evaluated as an indicator of normal biologic or pathogenic
processes or pharmacological response to a therapeutic
intervention." (Biomarkers Definitions Working Group, 2001, Clin.
Pharmacol. Ther. 69:89-95)
[0003] The development of improved diagnostics based on the
discovery of biomarkers has the potential to accelerate new drug
development by identifying, in advance, those patients most likely
to show a clinical response to a given drug. This would
significantly reduce the size, length and cost of clinical trials.
Technologies such as genomics, proteomics and molecular imaging
currently enable rapid, sensitive and reliable detection of
specific gene mutations, expression levels of particular genes, and
other molecular biomarkers. In spite of the availability of various
technologies for molecular characterization of tumors, the clinical
utilization of cancer biomarkers remains largely unrealized because
few cancer biomarkers have been discovered. For example, a recent
review article states: "There is a critical need for expedited
development of biomarkers and their use to improve diagnosis and
treatment of cancer." (Cho, 2007, Molecular Cancer 6:25) Another
recent review article on cancer biomarkers contains the following
comments: "The challenge is discovering cancer biomarkers. Although
there have been clinical successes in targeting molecularly defined
subsets of several tumor types--such as chronic myeloid leukemia,
gastrointestinal stromal tumor, lung cancer and glioblastoma
multiforme--using molecularly targeted agents, the ability to apply
such successes in a broader context is severely limited by the lack
of an efficient strategy to evaluate targeted agents in patients.
The problem mainly lies in the inability to select patients with
molecularly defined cancers for clinical trials to evaluate these
exciting new drugs. The solution requires biomarkers that reliably
identify those patients who are most likely to benefit from a
particular agent. (Sawyers, 2008, Nature 452:548-552, at 548)
Comments such as the foregoing illustrate the recognition of a need
for the discovery of clinically useful biomarkers and diagnostic
methods based on such biomarkers.
[0004] There are three distinct types of cancer biomarkers: (1)
prognostic biomarkers, (2) predictive biomarkers, and (3)
pharmacodynamic biomarkers. A prognostic biomarker is used to
classify a cancer, e.g., a solid tumor, according to
aggressiveness, i.e., rate of growth and/or metastasis, and
refractiveness to treatment. This is sometimes called
distinguishing "good outcome" tumors from "poor outcome" tumors. A
predictive biomarker is used to assess the probability that a
particular patient will benefit from treatment with a particular
drug. For example, patients with breast cancer in which the ERBB2
(HER2) gene is amplified are likely to benefit from treatment with
trastuzumab (HERCEPTIN.RTM.), whereas patients without ERBB2 gene
amplification are unlikely to benefit from treatment with
trastuzumab. A pharmacodynamic biomarker is an indication of the
effect(s) of a drug on its molecular target while the patient is
taking the drug. Accordingly, pharmacodynamic biomarkers often are
used to guide dosage level and dosing frequency, during the early
stages of clinical development of a new drug. For a discussion of
cancer biomarkers, see, e.g., Sawyers, 2008, Nature
452:548-552.
[0005] Tumors driven by EGFR or HER2 often respond to treatment
with inhibitors of EGFR or HER2, but these tumors invariably
develop resistance to these inhibitors. At least one mechanism of
acquired resistance to anti-EGFR or anti-HER2 treatment is
activation of HER3 (also known as ERBB3) signaling. See, e.g.,
Engelman et al, 2006, Clin. Cancer Res. 12:4372; Ritter et al,
2007, Clin. Cancer Res. 13:4909; Sergina et al, 2007, Nature
445:437. HER3 plays an important role in development of drug
resistance, as well as being involved in tumor initiation and
maintenance, through its heterodimerization with EGFR and HER2.
Consequently, there has been interest in development of HER3
inhibitors, especially anti-HER3 antibodies, since HER3 lacks
kinase activity.
[0006] As with other types of targeted therapy, some, but not all,
tumors respond to anti-HER3 therapy. Therefore, there is a need for
diagnostic methods based on predictive biomarkers that can be used
to identify patients with tumors that are likely (or unlikely) to
respond to treatment with a HER3 inhibitor such as an anti-HER3
antibody.
SUMMARY
[0007] The present invention is directed to methods of treating a
human subject harboring a locally advanced or metastatic non-small
cell lung cancer (NSCLC) tumor comprising administering a treatment
comprising an anti-HER3 antibody to a human subject diagnosed with
a locally advanced or metastatic NSCLC whose HRG gene expression at
a protein level is assessed as high.
[0008] Some embodiments comprise assessing HRG gene expression at a
protein level in a human subject diagnosed with a locally advanced
or metastatic NSCLC and administering a treatment comprising an
anti-HER3 antibody to a human subject whose HRG gene expression at
a protein level is assessed as high.
[0009] Some embodiments comprise ordering an assessment of HRG gene
expression at a protein level in a human subject diagnosed with a
locally advanced or metastatic NSCLC and administering a treatment
comprising an anti-HER3 antibody to the human subject whose HRG
gene expression at a protein level is assessed as high.
[0010] In a particular embodiment of the invention, the HRG gene
expression at a protein level is assessed as high if a protein
concentration value is observed, which is above a predetermined
threshold, from a biological sample taken from the subject
diagnosed with a locally advanced or metastatic NSCLC.
[0011] In some embodiments, the predetermined threshold is chosen
statistically to minimize undesirable effects of false positives
and false negatives. In a preferred embodiment, the predetermined
threshold value is selected from the group consisting of about 0
pg/mL, about 980 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about
3000 pg/mL, about 4000 pg/mL, and about 5000 pg/mL.
[0012] In some embodiments, the subject harbors wild-type EGFR. In
preferred embodiments, the tumor has also progressed on at least
one prior systemic therapy. In more preferred embodiments, a tumor
tissue or fragment thereof for or with which the HRG gene
expression is assessed had been removed from the subject prior to
any (systemic) therapy.
[0013] Some embodiments comprise assessing HRG gene expression at a
protein level in a human subject diagnosed with a locally advanced
or metastatic NSCLC, where HRG gene expression at a protein level
is assessed using ELISA or immunohistochemistry techniques.
[0014] In some embodiments, the biological sample comprises a whole
blood or serum sample.
[0015] In some embodiments, the anti-HER3 antibody is selected from
the group consisting of patritumab, duligotumab (MEHD-7945A),
seribantumab (MM-121), MM-111, LJM716, RG-7116, tri-specific
anti-EGFR/ERBB3 zybody, huHER3-8, or a derivative or fragment of
any of these.
[0016] In some embodiments, the treatment comprises an anti-HER3
antibody in combination with one or more of (i) a HER inhibitor,
(ii) a chemotherapy, (iii) radiation, and (iv) an other targeted
agent.
[0017] For example, In some embodiments the HER inhibitor is
selected from the group consisting of trastuzumab, T-DM1,
lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib,
dacomitinib, KD-019 and erlotinib.
[0018] In some embodiments, the chemotherapy is selected from the
group consisting of cisplatin, carboplatin, gemcitabine,
pemetrexed, irinotecan, 5-fluoruracil, paclitaxel, docetaxel, and
capecitabine. However, other chemotherapies can be applied.
[0019] The present invention is also directed to methods of
treating a human subject harboring a locally advanced or metastatic
non-small cell lung cancer (NSCLC) tumor comprising assessing HRG
gene expression at a protein level in a human subject diagnosed
with a locally advanced or metastatic NSCLC, and withholding a
treatment comprising an anti-HER3 antibody to a human subject whose
HRG gene expression at a protein level is assessed as low.
[0020] Some embodiments comprise ordering an assessment of an HRG
gene expression at a protein level in a human subject diagnosed
with a locally advanced or metastatic NSCLC and withholding a
treatment comprising an anti-HER3 antibody to the human subject
whose HRG gene expression at a protein level is assessed as
low.
[0021] In some embodiments, the HRG gene expression at a protein
level is assessed as low if a protein concentration value is
observed, which is at or below a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
[0022] In some embodiments, the predetermined threshold is chosen
statistically to minimize undesirable effects of false positives
and false negatives. In some embodiments, the predetermined
threshold value is selected from the group consisting of 0 pg/mL,
about 980 pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000
pg/mL, about 3000 pg/mL, about 4000 pg/mL, and about 5000
pg/mL.
[0023] In some embodiments, the subject harbors wild-type EGFR. In
preferred embodiments, the tumor has progressed on at least one
prior systemic therapy. In more preferred embodiments, a tumor
tissue or fragment thereof for or with which the HRG gene
expression is assessed has been removed from the subject prior to
any (systemic) therapy.
[0024] In some embodiments, HRG gene expression at a protein level
is assessed using ELISA or immunohistochemistry techniques.
[0025] In some embodiments, the biological sample comprises a whole
blood or serum sample.
[0026] In some embodiments, the treatment withheld comprises an
anti-HER3 antibody in combination with one or more of (i) a HER
inhibitor, (ii) a chemotherapy, (iii) radiation, and (iv) an other
targeted agent.
[0027] Some embodiments comprise treating a human subject whose HRG
gene expression at a protein level is assessed as low with a HER
inhibitor selected from the group consisting of trastuzumab, T-DM1,
lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib,
dacomitinib, KD-019 and erlotinib.
[0028] Some embodiments comprise treating a human subject whose HRG
gene expression at a protein level is assessed as low with a
chemotherapy selected from the group consisting of cisplatin,
carboplatin, gemcitabine, pemetrexed, irinotecan, 5-fluoruracil,
paclitaxel, docetaxel, and capecitabine. However, other
chemotherapies can be applied.
[0029] Some embodiments comprise treating a human subject whose HRG
gene expression at an mRNA level is assessed as low or high with
crizotinib. In some embodiments, the subject treated with
crizotinib has an ALK gene rearrangement or fusion.
[0030] The invention is also directed to kits for facilitating an
assessment of HRG gene expression at a protein level.
[0031] The invention is also directed to methods of identifying a
human patient diagnosed with a locally advanced or metastatic
non-small cell lung cancer (NSCLC) who is likely to benefit from a
treatment comprising an anti-HER3 antibody comprising obtaining a
biological sample from a human patient diagnosed with a locally
advanced or metastatic NSCLC, using the sample, determining a value
for HRG gene expression at a protein level in the human patient,
and, optionally, recording the value determined.
[0032] Some embodiments comprise receiving a biological sample from
a human patient diagnosed with a locally advanced or metastatic
NSCLC; using the sample, determining a value for HRG gene
expression at a protein level in the human subject; and,
optionally, recording the value determined.
[0033] Some embodiments comprise assessing if the value determined
is below, at, or above a predetermined threshold value. In some
embodiments, the predetermined threshold value is selected from the
group consisting of 0 pg/mL, about 980 pg/mL, about 1000 pg/mL,
about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about 4000
pg/mL, and about 5000 pg/mL.
[0034] Some embodiments involve characterizing the HRG gene
expression at a protein level as high if the value determined is
above the predetermined threshold value.
[0035] Some embodiments involve characterizing the HRG gene
expression at a protein level as low if the value determined is at
or below the predetermined threshold value.
[0036] Some embodiments comprise reporting the value determined to
an attending physician or other medical practitioner.
[0037] In some embodiments, the sample comprises a whole blood or
serum sample.
[0038] In some embodiments, the subject does not harbor an
epidermal growth factor receptor (EGFR) sensitizing mutation. In
preferred embodiments, the subject harbors wild-type EGFR. In even
more preferred embodiments, the tumor has progressed on at least
one prior systemic therapy. In more preferred embodiments, a tumor
tissue or fragment thereof for or with which the HRG gene
expression is assessed has been removed from the subject prior to
any (systemic) therapy.
[0039] In some embodiments, the treatment comprises an anti-HER3
antibody in combination with one or more of (i) a HER inhibitor,
(ii) a chemotherapy, (iii) radiation, and (iv) an other targeted
agent.
[0040] The invention is also directed to methods where HRG gene
expression is assessed as high based on randomized clinical
data.
[0041] The invention is also directed to methods of receiving or
undergoing a treatment for a locally advanced or metastatic
non-small cell lung cancer (NSCLC) tumor or abstaining therefrom.
In some embodiments, the methods comprise providing an autologous
tissue sample or consenting to a taking of same to facilitate an
assessment of HRG gene expression at a protein level in a human
subject diagnosed with a locally advanced or metastatic NSCLC; and
receiving a treatment comprising an anti-HER3 antibody if HRG gene
expression at a protein level is assessed as high, or abstaining
from a treatment comprising an anti-HER3 antibody if HRG gene
expression at a protein level is assessed as low.
[0042] The invention is also directed to methods of electing a
treatment for a locally advanced or metastatic non-small cell lung
cancer (NSCLC) tumor. In some embodiments, the methods comprise
receiving an assessment of HRG gene expression at a protein level
in a human subject diagnosed with a locally advanced or metastatic
NSCLC; and electing to withhold a treatment comprising an anti-HER3
antibody if HRG gene expression at a protein level is assessed as
low, or electing to administer a treatment comprising an anti-HER3
antibody if HRG gene expression at a protein level is assessed as
high.
[0043] The invention is also directed to methods of performing an
ELISA, including sequential steps of contacting a solid surface
with a plurality of solutions each comprising in turn a capture
antibody, a blocking agent, a sample suspected of containing an
analyte, a detection antibody and an enzyme conjugate, in which the
solid surface is subjected to a wash process after each sequential
step, the wash process comprising (a) cycling wash buffer on and
off the solid surface at a rapid rate until bubbles are observed at
which point the cycled wash buffer is removed, (b) optionally
repeating step (a) using fresh wash buffer; and (c) rinsing the
solid surface with fresh wash buffer, provided that after
completing the wash process following the enzyme conjugate
sequential step, the solid surface is contacted with a solution
comprising an enzyme substrate.
[0044] Some embodiments comprise adding to the solid surface a stop
solution.
[0045] Some embodiments comprise obtaining a spectrophotometric
reading to obtain a measure of an amount of analyte present in the
sample, if any.
[0046] In some embodiments, the wash buffer comprises a
surfactant.
[0047] In some embodiments, the wash buffer comprises phosphate
buffered saline (PBS).
[0048] In preferred embodiments, the wash buffer has a pH ranging
from 7.2-7.4.
[0049] In some embodiments, the cycling step comprises applying and
aspirating the wash buffer on and off the solid surface using a
"piston" action.
[0050] In some embodiments, the wash buffer is cycled 20 or more
times on and off the solid surface.
[0051] In some embodiments, the rinsing step of the wash process
removes substantially all bubbles on the solid surface.
[0052] In some embodiments, the solution comprising the detection
antibody excludes normal goat serum.
[0053] In some embodiments, the "piston" action is facilitated by
use of a pipette.
[0054] In some embodiments, the capture antibody and the detection
antibody specifically recognize human heregulin (HRG).
[0055] In some embodiments, the solution comprising the sample
comprises undiluted serum or undiluted plasma.
[0056] In some embodiments, the capture antibody is mouse
anti-human HRG.
[0057] In some embodiments, the detection antibody is biotinylated
goat anti-human HRG.
[0058] In some embodiments, the enzyme conjugate is
streptavidin-horse radish peroxidase.
[0059] In some embodiments, the enzyme substrate is a mixture of
hydrogen peroxide and tetramethylbenzidine.
[0060] In some embodiments, the solid surface is contacted with the
solution comprising the capture antibody for at least 8 hours at a
temperate in the range of about 65.degree. to 80.degree. F.
[0061] In some embodiments, the solution comprising the blocking
agent comprises 1% bovine serum albumin in phosphate buffered
saline at a pH in the range of 7.2 to 7.4.
[0062] In some embodiments, the solid surface is contacted with the
solution comprising the blocking agent for at least one hour at a
temperate in the range of about 65.degree. to 80.degree. F.
[0063] In some embodiments, the solid surface is contacted with the
solution comprising the sample for about 2 hours at a temperate in
the range of about 65.degree. to 80.degree. F.
[0064] In some embodiments, the solid surface is contacted with the
solution comprising the detection antibody for about 2 hours at a
temperate in the range of about 65.degree. to 80.degree. F.
[0065] In some embodiments, the solid surface is contacted with the
solution comprising the enzyme conjugate for about 20 minutes at a
temperate in the range of about 65.degree. to 80.degree. F.
[0066] In some embodiments, the solid surface is contacted with the
solution comprising the enzyme substrate for about 20 minutes at a
temperate in the range of about 65.degree. to 80.degree. F.
[0067] In some embodiments, the solid surface is a microplate
comprising one or more wells.
[0068] In some embodiments, the cycling step (a) is performed two
times before performing the rinsing step (c).
[0069] In some embodiments, the surfactant is polyethylene glycol
sorbitan monolaurate.
[0070] The present invention includes the following (1) to (114),
but is not limited thereto.
[0071] (1) A method of treating a human subject harboring a locally
advanced or metastatic non-small cell lung cancer (NSCLC) tumor
comprising:
[0072] assessing HRG gene expression at a protein level in a human
subject diagnosed with a locally advanced or metastatic NSCLC;
and
[0073] administering a treatment comprising an anti-HER3 antibody
to a human subject whose HRG gene expression at a protein level is
assessed as high.
[0074] (2) The method of (1) in which the HRG gene expression at a
protein level is assessed as high if a protein concentration value
is observed, which is above a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
[0075] (3) The method of (2) in which the predetermined threshold
is chosen statistically to minimize undesirable effects of false
positives and false negatives.
[0076] (4) The method of (1) in which the predetermined threshold
value is selected from the group consisting of about 0 pg/mL, about
980 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL,
about 4000 pg/mL, and about 5000 pg/mL.
[0077] (5) The method of (1), wherein the subject harbors wild-type
EGFR.
[0078] (6) The method of (5), wherein the tumor has progressed on
at least one prior systemic therapy.
[0079] (7) The method of (1) in which HRG gene expression at a
protein level is assessed using ELISA or immunohistochemistry
techniques.
[0080] (8) The method of (2) in which the biological sample
comprises a whole blood or serum sample.
[0081] (9) The method of (1) in which the anti-HER3 antibody is
selected from the group consisting of patritumab, duligotumab
(MEHD-7945A), seribantumab (MM-121), MM-111, LJM716, RG-7116,
tri-specific anti-EGFR/ERBB3 zybody, huHER3-8, or a derivative or
fragment of any of these.
[0082] (10) The method of (1) in which the treatment comprises an
anti-HER3 antibody in combination with one or more of (i) a HER
inhibitor, (ii) a chemotherapy, (iii) radiation, and (iv) an other
targeted agent.
[0083] (11) The method of (10), wherein the HER inhibitor is
selected from the group consisting of trastuzumab, T-DM1,
lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib,
dacomitinib, KD-019 and erlotinib.
[0084] (12) The method of (10), wherein the chemotherapy is
selected from the group consisting of cisplatin, carboplatin,
gemcitabine, pemetrexed, irinotecan, 5-fluoruracil, paclitaxel,
docetaxel, and capecitabine.
[0085] (13) A method of treating a human subject harboring a
locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor comprising:
[0086] assessing HRG gene expression at a protein level in a human
subject diagnosed with a locally advanced or metastatic NSCLC;
and
[0087] withholding a treatment comprising an anti-HER3 antibody to
a human subject whose HRG gene expression at a protein level is
assessed as low.
[0088] (14) The method of (13) in which the HRG gene expression at
a protein level is assessed as low if a protein concentration value
is observed, which is at or below a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
[0089] (15) The method of (14) in which the predetermined threshold
is chosen statistically to minimize undesirable effects of false
positives and false negatives.
[0090] (16) The method of (14) in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about
4000 pg/mL, and about 5000 pg/mL.
[0091] (17) The method of (13), wherein the subject harbors
wild-type EGFR.
[0092] (18) The method of (17), wherein the tumor has progressed on
at least one prior systemic therapy.
[0093] (19) The method of (13) in which HRG gene expression at a
protein level is assessed using ELISA or immunohistochemistry
techniques.
[0094] (20) The method of (14) in which the biological sample
comprises a whole blood or serum sample.
[0095] (21) The method of (13) in which the treatment withheld
comprises an anti-HER3 antibody in combination with one or more of
(i) a HER inhibitor, (ii) a chemotherapy, (iii) radiation, and (iv)
an other targeted agent.
[0096] (22) The method of (13) further comprising treating a human
subject whose HRG gene expression at a protein level is assessed as
low with a HER inhibitor selected from the group consisting of
trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab
gefitinib, afatinib, dacomitinib, KD-019 and erlotinib.
[0097] (23) The method of (13), further comprising treating a human
subject whose HRG gene expression at a protein level is assessed as
low with a chemotherapy selected from the group consisting of
cisplatin, carboplatin, gemcitabine, pemetrexed, irinotecan,
5-fluoruracil, paclitaxel, docetaxel, and capecitabine.
[0098] (24) A kit for facilitating an assessment of HRG gene
expression at a protein level.
[0099] (25) A method of identifying a human patient diagnosed with
a locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor who is likely to benefit from a treatment comprising an
anti-HER3 antibody comprising:
[0100] obtaining a biological sample from a human patient diagnosed
with a locally advanced or metastatic NSCLC;
[0101] using the sample, determining a value for HRG gene
expression at a protein level in the human patient; and
[0102] recording the value determined.
[0103] (26) The method of (25), further comprising assessing if the
value determined is below, at, or above a predetermined threshold
value.
[0104] (27) The method of (26) in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about
4000 pg/mL, and about 5000 pg/mL.
[0105] (28) The method of (26), further comprising characterizing
the HRG gene expression at a protein level as high if the value
determined is above the predetermined threshold value.
[0106] (29) The method of (26), further comprising characterizing
the HRG gene expression at a protein level as low if the value
determined is at or below the predetermined threshold value.
[0107] (30) The method of (25), further comprising reporting the
value determined to an attending physician or other medical
practitioner.
[0108] (31) The method of (25) in which the sample comprises a
whole blood or serum sample.
[0109] (32) The method of (25), wherein the subject does not harbor
an EGFR sensitizing mutation.
[0110] (33) The method of (25), wherein the subject harbors
wild-type EGFR.
[0111] (34) The method of (33), wherein the tumor has progressed on
at least one prior systemic therapy.
[0112] (35) The method of (25), wherein the treatment comprises an
anti-HER3 antibody in combination with one or more of (i) a HER
inhibitor, (ii) a chemotherapy, (iii) radiation, and (iv) an other
targeted agent.
[0113] (36) A method of performing an ELISA, including sequential
steps of contacting a solid surface with a plurality of solutions
each comprising in turn a capture antibody, a blocking agent, a
sample suspected of containing an analyte, a detection antibody and
an enzyme conjugate, in which the solid surface is subjected to a
wash process after each sequential step, the wash process
comprising:
[0114] (a) cycling wash buffer on and off the solid surface at a
rapid rate until bubbles are observed at which point the cycled
wash buffer is removed;
[0115] (b) optionally repeating step (a) using fresh wash buffer;
and
[0116] (c) rinsing the solid surface with fresh wash buffer;
provided that after completing the wash process following the
enzyme conjugate sequential step, the solid surface is contacted
with a solution comprising an enzyme substrate.
[0117] (37) The method of (36) further comprising adding to the
solid surface a stop solution.
[0118] (38) The method of (37) further comprising obtaining a
spectrophotometric reading to obtain a measure of an amount of
analyte present in the sample, if any.
[0119] (39) The method of (36) in which the wash buffer comprises a
surfactant.
[0120] (40) The method of (39) in which the wash buffer comprises
phosphate buffered saline (PBS).
[0121] (41) The method of (40) in which the wash buffer has a pH
ranging from 7.2-7.4.
[0122] (42) The method of (36) in which the cycling step comprises
applying and aspirating the wash buffer on and off the solid
surface using a "piston" action.
[0123] (43) The method of (42) in which the wash buffer is cycled
20 or more times on and off the solid surface.
[0124] (44) The method of (36) in which the rinsing step of the
wash process removes substantially all bubbles on the solid
surface.
[0125] (45) The method of (36) in which the solution comprising the
detection antibody excludes normal goat serum.
[0126] (46) The method of (42) in which the "piston" action is
facilitated by use of a pipette.
[0127] (47) The method of (36) in which the capture antibody and
the detection antibody specifically recognize human heregulin
(HRG).
[0128] (48) The method of (36) in which the solution comprising the
sample comprises undiluted serum or undiluted plasma.
[0129] (49) The method of (47) in which the capture antibody is a
mouse anti-human HRG antibody.
[0130] (50) The method of (49) in which the detection antibody is a
biotinylated goat anti-human HRG antibody.
[0131] (51) The method of (50) in which the enzyme conjugate is
streptavidin-horse radish peroxidase.
[0132] (52) The method of (51) in which the enzyme substrate is a
mixture of hydrogen peroxide and tetramethylbenzidine.
[0133] (53) The method of (49) in which the solid surface is
contacted with the solution comprising the capture antibody for at
least 8 hours at a temperate in the range of about 65.degree. to
80.degree. F.
[0134] (54) The method of (36) in which the solution comprising the
blocking agent comprises 1% bovine serum albumin in phosphate
buffered saline at a pH in the range of 7.2 to 7.4.
[0135] (55) The method of (54) in which the solid surface is
contacted with the solution comprising the blocking agent for at
least one hour at a temperate in the range of about 65.degree. to
80.degree. F.
[0136] (56) The method of (48) in which the solid surface is
contacted with the solution comprising the sample for about 2 hours
at a temperate in the range of about 65.degree. to 80.degree.
F.
[0137] (57) The method of (50) in which the solid surface is
contacted with the solution comprising the detection antibody for
about 2 hours at a temperate in the range of about 65.degree. to
80.degree. F.
[0138] (58) The method of (51) in which the solid surface is
contacted with the solution comprising the enzyme conjugate for
about 20 minutes at a temperate in the range of about 65.degree. to
80.degree. F.
[0139] (59) The method of (52) in which the solid surface is
contacted with the solution comprising the enzyme substrate for
about 20 minutes at a temperate in the range of about 65.degree. to
80.degree. F.
[0140] (60) The method of (36) in which the solid surface is a
microplate comprising one or more wells.
[0141] (61) The method of (43) in which the cycling step (a) is
performed two times before performing the rinsing step (c).
[0142] (62) The method of (40) in which the surfactant is
polyethylene glycol sorbitan monolaurate.
[0143] (63) The method of any one of (1)-(35), wherein HRG gene
expression is assessed as high based on randomized clinical
data.
[0144] (64) A method of receiving or undergoing a treatment for a
locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor or abstaining therefrom comprising:
[0145] providing an autologous tissue sample or consenting to a
taking of same to facilitate an assessment of HRG gene expression
at a protein level in a human subject diagnosed with a locally
advanced or metastatic NSCLC; and
[0146] receiving or undergoing a treatment comprising an anti-HER3
antibody if HRG gene expression at a protein level is assessed as
high, or
[0147] abstaining from a treatment comprising an anti-HER3 antibody
if HRG gene expression at a protein level is assessed as low.
[0148] (65) A method of electing a treatment for a locally advanced
or metastatic non-small cell lung cancer (NSCLC) tumor
comprising:
[0149] receiving an assessment of HRG gene expression at a protein
level in a human subject diagnosed with a locally advanced or
metastatic NSCLC; and
[0150] electing to withhold or abstain from a treatment comprising
an anti-HER3 antibody if HRG gene expression at a protein level is
assessed as low, or
[0151] electing to receive or undergo a treatment comprising an
anti-HER3 antibody if HRG gene expression at a protein level is
assessed as high.
[0152] (66) A method of identifying a human patient diagnosed with
a locally advanced or metastatic non-small cell lung cancer (NSCLC)
who is likely to benefit from a treatment comprising an anti-HER3
antibody comprising:
[0153] receiving a biological sample from a human patient diagnosed
with a locally advanced or metastatic NSCLC;
[0154] using the sample, determining a value for HRG gene
expression at a protein level in the human patient; and
[0155] optionally, recording the value determined.
[0156] (67) A method of treating a human subject harboring a
locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor comprising:
[0157] ordering an assessment of HRG gene expression at a protein
level in a human subject diagnosed with a locally advanced or
metastatic NSCLC; and
[0158] administering a treatment comprising an anti-HER3 antibody
to the human subject whose HRG gene expression at a protein level
is assessed as high.
[0159] (68) A method of withholding treatment of a human subject
harboring a locally advanced or metastatic non-small cell lung
cancer (NSCLC) tumor comprising:
[0160] ordering an assessment of HRG gene expression at a protein
level in a human subject diagnosed with a locally advanced or
metastatic NSCLC; and
[0161] withholding a treatment comprising an anti-HER3 antibody to
the human subject whose HRG gene expression at a protein level is
assessed as low.
[0162] (69) A method of treating a human subject harboring a
locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor comprising administering a treatment comprising an anti-HER3
antibody to a human subject diagnosed with a locally advanced or
metastatic NSCLC whose HRG gene expression at a protein level is
assessed as high.
[0163] (70) The method of (69) in which the HRG gene expression at
a protein level is assessed as high if a protein concentration
value is observed, which is above a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
[0164] (71) The method of (70) in which the predetermined threshold
is chosen statistically to minimize undesirable effects of false
positives and false negatives.
[0165] (72) The method of (69) in which the predetermined threshold
value is selected from the group consisting of about 0 pg/mL, about
980 pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL,
about 3000 pg/mL, about 4000 pg/mL, and about 5000 pg/mL.
[0166] (73) The method of (69), wherein the subject harbors
wild-type EGFR.
[0167] (74) The method of (73), wherein the tumor has progressed on
at least one prior systemic therapy.
[0168] (75) The method of (69), further comprising assessing gene
expression at a protein level in the human subject diagnosed with
the locally advanced or metastatic NSCLC, wherein HRG gene
expression at a protein level is assessed using ELISA or
immunohistochemistry techniques.
[0169] (76) The method of (70) in which the biological sample
comprises a whole blood or serum sample.
[0170] (77) The method of (69) in which the anti-HER3 antibody is
selected from the group consisting of patritumab, duligotumab
(MEHD-7945A), seribantumab (MM-121), MM-111, LJM716, RG-7116,
tri-specific anti-EGFR/ERBB3 zybody, huHER3-8, or a derivative or
fragment of any of these.
[0171] (78) The method of (69) in which the treatment comprises
administering an anti-HER3 antibody in combination with one or more
of (i) a HER inhibitor, (ii) a chemotherapy, (iii) radiation, and
(iv) an other targeted agent.
[0172] (79) The method of (78), wherein the HER inhibitor is
selected from the group consisting of trastuzumab, T-DM1,
lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib,
dacomitinib, KD-019 and erlotinib.
[0173] (80) The method of (78), wherein the chemotherapy is
selected from the group consisting of cisplatin, carboplatin,
gemcitabine, pemetrexed, irinotecan, 5-fluoruracil, paclitaxel,
docetaxel, and capecitabine.
[0174] (81) A method of treating a human subject harboring a
locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor comprising:
[0175] withholding a treatment comprising an anti-HER3 antibody
from a human subject diagnosed with a locally advanced or
metastatic NSCLC whose HRG gene expression at a protein level is
assessed as low.
[0176] (82) The method of (81) in which the HRG gene expression at
a protein level is assessed as low if a protein concentration value
is observed, which is at or below a predetermined threshold, from a
biological sample taken from the subject diagnosed with a locally
advanced or metastatic NSCLC.
[0177] (83) The method of (82) in which the predetermined threshold
is chosen statistically to minimize undesirable effects of false
positives and false negatives.
[0178] (84) The method of (82) in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about
3000 pg/mL, about 4000 pg/mL, and about 5000 pg/mL.
[0179] (85) The method of (81), wherein the subject harbors
wild-type EGFR.
[0180] (86) The method of (85), wherein the tumor has progressed on
at least one prior systemic therapy.
[0181] (87) The method of (81), further comprising assessing HRG
gene expression at a protein level in the human subject diagnosed
with the locally advanced or metastatic NSCLC, wherein HRG gene
expression at a protein level is assessed using ELISA or
immunohistochemistry techniques.
[0182] (88) The method of (82) in which the biological sample
comprises a whole blood or serum sample.
[0183] (89) The method of (81) in which the treatment withheld
comprises administering an anti-HER3 antibody in combination with
one or more of (i) a HER inhibitor, (ii) a chemotherapy, (iii)
radiation, and (iv) an other targeted agent.
[0184] (90) The method of (81) further comprising treating a human
subject whose HRG gene expression at a protein level is assessed as
low with a HER inhibitor selected from the group consisting of
trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab
gefitinib, afatinib, dacomitinib, KD-019 and erlotinib.
[0185] (91) The method of (81), further comprising treating a human
subject whose HRG gene expression at a protein level is assessed as
low with a chemotherapy selected from the group consisting of
cisplatin, carboplatin, gemcitabine, pemetrexed, irinotecan,
5-fluoruracil, paclitaxel, docetaxel, and capecitabine.
[0186] (92) A method of identifying a human patient diagnosed with
a locally advanced or metastatic non-small cell lung cancer (NSCLC)
tumor who is likely to benefit from a treatment comprising
administering an anti-HER3 antibody to the patient comprising:
[0187] obtaining a biological sample taken from a human patient
diagnosed with a locally advanced or metastatic NSCLC;
[0188] using the sample, determining a value for HRG gene
expression at a protein level in the human patient; and
[0189] optionally, recording the value determined.
[0190] (93) The method of (92), further comprising assessing if the
value determined is below, at, or above a predetermined threshold
value.
[0191] (94) The method of (93) in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about
3000 pg/mL, about 4000 pg/mL, and about 5000 pg/mL.
[0192] (95) The method of (93), further comprising characterizing
the HRG gene expression at a protein level as high if the value
determined is above the predetermined threshold value.
[0193] (96) The method of (93), further comprising characterizing
the HRG gene expression at a protein level as low if the value
determined is at or below the predetermined threshold value.
[0194] (97) The method of (92), further comprising reporting the
value determined to an attending physician or other medical
practitioner.
[0195] (98) The method of (92) in which the sample comprises a
whole blood or serum sample.
[0196] (99) The method of (92), wherein the subject does not harbor
an EGFR sensitizing mutation.
[0197] (100) The method of (92), wherein the subject harbors
wild-type EGFR.
[0198] (101) The method of (100), wherein the tumor has progressed
on at least one prior systemic therapy.
[0199] (102) The method of (92), wherein the treatment comprises an
anti-HER3 antibody in combination with one or more of (i) a HER
inhibitor, (ii) a chemotherapy, (iii) radiation, and (iv) an other
targeted agent.
[0200] (103) The method of any one of (69)-(102), wherein HRG gene
expression is assessed as high based on randomized clinical
data.
[0201] (104) A method of any of the preceding claims, wherein HRG
gene expression is assessed using an regulatory authority-approved
test.
[0202] (105) The method of (104), wherein the regulatory
authority-approved test is an FDA (Food and Drug Administration,
the United States)-approved, EMA (European Medicines agency,
European Union)-approved or PMDA (Pharmaceuticals and Medical
Devices Agency, Japan)-approved test.
[0203] (106) The method of (2) in which the predetermined threshold
value is selected from the group consisting of 0 pg/mL, about 980
pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about
3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL,
about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL, about 10000
pg/mL, about 11000 pg/mL, about 12000 pg/mL, about 13000 pg/mL,
about 14000 pg/mL, about 15000 pg/mL, about 16000 pg/mL, about
17000 pg/mL, about 18000 pg/mL, about 19000 pg/mL, about 20000
pg/mL, about 22000 pg/mL, about 24000 pg/mL, about 26000 pg/mL,
about 28000 pg/mL, about 30000 pg/mL, about 35000 pg/mL, about
40000 pg/mL, about 50000 pg/mL, about 60000 pg/mL, about 70000
pg/mL, about 80000 pg/mL, about 90000 pg/mL and about 100000
pg/mL.
[0204] (107) The method of (14) in which the predetermined
threshold value is selected from the group consisting of 0 pg/mL,
about 980 pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000
pg/mL, about 3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about
6000 pg/mL, about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL,
about 10000 pg/mL, about 11000 pg/mL, about 12000 pg/mL, about
13000 pg/mL, about 14000 pg/mL, about 15000 pg/mL, about 16000
pg/mL, about 17000 pg/mL, about 18000 pg/mL, about 19000 pg/mL,
about 20000 pg/mL, about 22000 pg/mL, about 24000 pg/mL, about
26000 pg/mL, about 28000 pg/mL, about 30000 pg/mL, about 35000
pg/mL, about 40000 pg/mL, about 50000 pg/mL, about 60000 pg/mL,
about 70000 pg/mL, about 80000 pg/mL, about 90000 pg/mL and about
100000 pg/mL.
[0205] (108) The method of (26) in which the predetermined
threshold value is selected from the group consisting of 0 pg/mL,
about 980 pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000
pg/mL, about 3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about
6000 pg/mL, about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL,
about 10000 pg/mL, about 11000 pg/mL, about 12000 pg/mL, about
13000 pg/mL, about 14000 pg/mL, about 15000 pg/mL, about 16000
pg/mL, about 17000 pg/mL, about 18000 pg/mL, about 19000 pg/mL,
about 20000 pg/mL, about 22000 pg/mL, about 24000 pg/mL, about
26000 pg/mL, about 28000 pg/mL, about 30000 pg/mL, about 35000
pg/mL, about 40000 pg/mL, about 50000 pg/mL, about 60000 pg/mL,
about 70000 pg/mL, about 80000 pg/mL, about 90000 pg/mL and about
100000 pg/mL.
[0206] (109) The method of (6) in which a tumor tissue or fragment
thereof for or with which the HRG gene expression is assessed has
been removed from the subject prior to any therapy.
[0207] (110) The method of (18) in which a tumor tissue or fragment
thereof for or with which the HRG gene expression is assessed has
been removed from the subject prior to any therapy.
[0208] (111) The method of (34) in which a tumor tissue or fragment
thereof for or with which the HRG gene expression is assessed has
been removed from the subject prior to any therapy.
[0209] (112) The method of (74) in which a tumor tissue or fragment
thereof for or with which the HRG gene expression is assessed has
been removed from the subject prior to any therapy.
[0210] (113) The method of (86) in which a tumor tissue or fragment
thereof for or with which the HRG gene expression is assessed has
been removed from the subject prior to any therapy.
[0211] (114) The method of (101) in which a tumor tissue or
fragment thereof for or with which the HRG gene expression is
assessed has been removed from the subject prior to any
therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0212] FIG. 1 depicts progression-free survival (showing high- and
low-dose patritumab+erlotinib vs. placebo+erlitonib) for all
subjects from the study in Example 2.
[0213] FIG. 2 depicts overall survival (showing high- and low-dose
patritumab+erlotinib vs. placebo+erlitonib) for all subjects from
the study in Example 2.
[0214] FIG. 3 depicts progression free survival (showing high- and
low-dose patritumab+erlotinib vs. placebo+erlitonib) for subjects
from the study in Example 3 assessed as having high HRG gene
expression at a protein level.
[0215] FIG. 4 depicts progression free survival (showing pooled
patritumab+erlotinib vs. placebo+erlitonib) for subjects from the
study in Example 3 assessed as having high HRG gene expression at a
protein level.
[0216] FIG. 5 depicts overall survival (showing high- and low-dose
patritumab+erlotinib vs. placebo+erlitonib) for subjects from the
study in Example 4 assessed as having high HRG gene expression at a
protein level.
[0217] FIG. 6 depicts overall survival (showing pooled
patritumab+erlotinib vs. placebo+erlitonib) for subjects from the
study in Example 4 assessed as having high HRG gene expression at a
protein level.
[0218] FIG. 7 depicts progression free survival (showing pooled
patritumab+erlotinib vs. placebo+erlitonib) for subjects from the
study in Example 5 assessed as having low HRG gene expression at a
protein level.
[0219] FIG. 8 depicts overall survival (showing pooled
patritumab+erlotinib vs. placebo+erlitonib) for subjects from the
study in Example 5 assessed as having low HRG gene expression at a
protein level.
[0220] FIG. 9 depicts efficacy determined in vitro by measuring
reduction of phospho-HER3 and phospho-AKT levels by Western
blotting.
[0221] FIG. 10 depicts Western blots showing that U3-1287 can block
ligand-dependent basal HER3 phosphorylation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0222] As used herein, unless indicated otherwise, when referring
to a numerical value, the term "about" means plus or minus 10% of
the enumerated value.
[0223] The term "antibody" refers to intact antibody, monoclonal or
polyclonal antibodies. The antibody may be generated using
recombinant DNA technologies. The antibody may originate for
example, from a mouse, rat, rabbit or any other mammal. The
antibody may also be a human antibody which may be obtained, for
example, from a transgenic non-human mammal capable of expressing
human Ig genes. The antibody may also be a humanised antibody which
may comprise, for example, one or more complementarity determining
regions of non-human origin. It may also comprise a surface residue
of a human antibody and/or framework regions of a human antibody.
The antibody may also be a chimeric antibody which may comprise,
for example, variable domains of a non-human antibody and constant
domains of a human antibody.
[0224] As used herein, "cancer" and "tumor" are
interchangeable.
[0225] As used herein, "treatment" means a medical care given to a
subject or patient, or administration of a dose of a medicine. In
some embodiments, "treatment" could be "phamaceutical composition",
"medicament" or "agent" that could comprise a HER inhibitor such as
anti-HER3 antibody. In some embodiments, "treatment" could be a
"chemotherapy, "immune therapy, "immunotherapy" or
"radiotherapy".
[0226] As used herein, "EGFR mutation" means any mutation in an
EGFR gene. "EGFR mutation" can be, for example, an EGFR exon 19
deletion and/or an exon 21 (L858R) substitution mutation. However,
"EGFR mutation" is not limited thereto.
[0227] As used herein, "HER" is one selected from the group
consisting of HER1 (EGFR), HER2, HER3 and HER4.
[0228] As used herein, "HER3" means the human protein encoded by
the gene identified by Entrez Gene ID No. 2065, and allelic
variants thereof
[0229] As used herein, "HER inhibitor" means a molecule (small
molecule or macromolecule, e.g., an antibody or antigen binding
fragment thereof) that inhibits, neutralizes, prevents or
eliminates at least a portion of the biological activity of a HER.
Preferably, a HER inhibitor binds to the HER. However, "HER
inhibitor" can be a molecule that does not directly bind to the
HER, as long as said molecule inhibits, neutralizes, prevents or
eliminates at least a portion of the biological activity of the
HER. Examples of HER1 inhibitors (EGFR inhibitor) include
lapatinib, erlotinib, cetuximab, gefitinib, afatinib, dacomitinib,
panitumumab and KD-019. Examples of HER2 inhibitors include
trastuzumab, pertuzumab and trastuzumab emtansine (T-DM1).
[0230] As used herein, "HER3 inhibitor" means a molecule (small
molecule or macromolecule, e.g., an antibody or antigen binding
fragment thereof) that inhibits, neutralizes, prevents or
eliminates at least a portion of the biological activity of HER3.
Preferably, the HER3 inhibitor binds to HER3. However, "HER3
inhibitor" can be a molecule that does not directly bind to HER3,
as long as said molecule inhibits, neutralizes, prevents or
eliminates at least a portion of the biological activity of HER3.
The effect on "biological activity" can be direct or indirect, such
as downstream signal transduction and heterodimerization with other
HER family molecules such as EGFR, HER2 and HER4. For example, the
HER3 inhibitor can be an inhibitor of EGFR/HER3, HER2/HER3 or
HER4/HER3 heterodimerization, or an inhibitor of a signal
transduction derived from any of these heterodimerizations. In this
context, "HER3 inhibitor" can include, for example pertuzumab,
nimotuzumab, MM-111 and cetuximab. Further, without being bound by
theory it is believed that HER3 forms a heterodimer with non-HER
receptors, such as MET (c-MET). Thus, in some embodiments "HER3
inhibitor" can include, for example, a MET inhibitor such as
onartuzumab and/or tivantinive.
[0231] As used herein, "HRG" (also known as neuregulin-1, NRG1,
heregulin, and HRG1) means the human protein encoded by the gene
identified by Entrez Gene ID No. 3084, and allelic variants
thereof.
[0232] As used herein, "non-small cell lung cancer" and "non-small
cell lung carcinoma" are interchangeable.
[0233] As used herein, "predetermined threshold (value)" means the
threshold numeric value at which a classifier gives the desirable
balance between (the cost of) false negatives and false
positives.
[0234] Preferably, "predetermined threshold (value)" means the
potential threshold numeric value to divide the entire population
(of patients or subjects) into two (or more) subgroups so that it
can bring clinical benefit to patients with the threshold or higher
(HRG) gene expression (used herein as "high HRG" subgroup),
compared to patients with the lower (HRG) gene expression than the
threshold (used herein as "low HRG" subgroup).
[0235] In case a threshold value is a dCt, preferably,
"predetermined threshold (value)" means the potential threshold
numeric value to divide the entire population (of patients or
subjects) into two (or more) subgroups so that it can bring
clinical benefit to patients with the threshold or lower value
(used herein as "high HRG" subgroup), compared to patients with the
higher value than the threshold (used herein as "low HRG"
subgroup).
[0236] In some embodiments, "predetermined threshold" is
statistically (and clinically) determined, refined, adjusted and/or
confirmed through, on, or based on, a clinical study and analyses
of outcome thereof (collectively, "clinical data"), and/or a
preclinical or non-clinical study (collectively, "non-clinical
data"), in order to minimize undesirable effects of false positives
and false negatives.
[0237] In some embodiments, "predetermined threshold" is
statistically (and clinically) determined, refined, adjusted and/or
confirmed on, or based on, clinical data (and optionally
non-clinical data), further more preferably randomized clinical
data (and optionally non-clinical data), to ensure all patients
that benefit from treatment are included in the HRG high
subgroup.
[0238] More preferably, "predetermined threshold" is determined,
refined, adjusted and/or confirmed through, on, or based on
pharmacological characteristics (i.e., mechanism of action),
preclinical or non-clinical study data, clinical study data, and
commercial sample data purchased from external companies or the
like, in order to maximize clinical benefit from "high HRG"
subgroup compared with "low HRG" subgroup. Some statistical method
such as Adaptive Biomarker Threshold Design (i.e., maximum
likelihood approach), Jiang W, Freidlin B, Simon R.
Biomarker-Adaptive Threshold Design: A Procedure for Evaluating
Treatment With Possible Biomarker-Defined Subset Effect, J Natl
Cancer Inst. 2007; 99(13):1036-43, and the like is used to
determine, refine, adjust and/or confirm the threshold using the
all available data of pre/non-clinical studies, clinical studies,
commercial sample, etc. (to ensure all patients that benefit from
treatment are included in the HRG high subgroup). In some
embodiments, "predetermined threshold" is determined so that high
HRG subgroup can be larger or can include all patients that drive
benefit from treatment.
[0239] As used herein, "subject," "human subject," and "patient"
are interchangeable.
[0240] As used herein, "subject suffering from a cancer" and
"subject harboring a cancer" are interchangeable.
[0241] In some preferred embodiments, when a group of patients
suffering from a cancer are treated by administering a HER3
inhibitor or placebo with or without a further medicament, and said
group is divided into "high HRG" subgroup and "low HRG" subgroup
using the predetermined threshold, average anti-cancer efficacy of
the administered HER3 inhibitor is better than that of control
(e.g. placebo) with clinical(ly) (meaningful) benefit in the "high
HRG" subgroup, while average anti-cancer efficacy of the
administered HER3 inhibitor is slightly better or not better than
that of control (e.g. placebo) with no clinical(ly) (meaningful)
benefit in the "low HRG" subgroup. In more preferred embodiments,
average anti-cancer efficacy of the administered HER3 inhibitor is
statistically significantly better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit in "high HRG"
subgroup, while average anti-cancer efficacy of the administered
HER3 inhibitor is not statistically significantly better than that
of control (e.g. placebo) with no clinical(ly) (meaningful) benefit
in the "low HRG" subgroup.
[0242] In other preferred embodiments, when a group of patients
suffering from a cancer is divided into a "high HRG" subgroup and a
"low HRG" subgroup using the predetermined threshold, and each
group is treated by administering a HER3 inhibitor or placebo with
or without a further medicament, average anti-cancer efficacy of
the administered HER3 inhibitor is better than that of a control
(e.g. placebo) with clinical(ly) (meaningful) benefit in the "high
HRG" subgroup, while average anti-cancer efficacy of the
administered HER3 inhibitor is slightly better or not better than
that of control (e.g. placebo) with no clinical(ly) (meaningful)
benefit in the "low HRG" subgroup. In more preferred embodiments,
average anti-cancer efficacy of the administered HER3 inhibitor is
statistically significantly better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit in the "high HRG"
subgroup, while average anti-cancer efficacy of the administered
HER3 inhibitor is not statistically significantly better than that
of control (e.g. placebo) with no clinical(ly) (meaningful) benefit
in the "low HRG" subgroup.
[0243] In other embodiments, "predetermined threshold" can be the
median of HRG levels which are measured in pre-/non-clinical study,
clinical study and/or commercial sample, for example with a group
of patients suffering from a cancer whose HRG levels are
measureable (can be measured) or detectable. In other preferred
embodiments, when a group of patients suffering from a cancer, such
as non-small cell lung cancer (NSCLC), are treated by administering
a HER3 inhibitor or placebo with or without a further medicament,
and the group is divided into a high HRG subgroup and low HRG
subgroup using the median HRG level of the patients as the
predetermined threshold, average anti-cancer efficacy of the
administered HER3 inhibitor is better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit in the "high HRG"
subgroup, while average anti-cancer efficacy of the administered
HER3 inhibitor is slightly bettr or not better than that of control
(e.g. placebo) with no clinical(ly) (meaningful) benefi tin the
"low HRG" subgroup. In more preferred embodiments, average
anti-cancer efficacy of the administered HER3 inhibitor is
statistically significantly better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit in the "high HRG"
subgroup, while average anti-cancer efficacy of the administered
HER3 inhibitor is not statistically significantly better than that
of control (e.g. placebo) with no clinical(ly) (meaningful) benefit
in the "low HRG" subgroup. In some embodiments, the predetermined
threshold is the median of HRG level of a group of patients
suffering from a cancer, and said threshold can be refined or
adjusted (to ensure all patients that benefit from treatment are
included in the HRG high sub group).
[0244] In other preferred embodiments, when a group of patients
suffering from a cancer is divided into a "high HRG" subgroup and
"low HRG" subgroup using the predetermined threshold, and the "high
HRG" subgroup is treated by administering a HER3 inhibitor or
placebo with or without a further medicament, average anti-cancer
efficacy of the administered HER3 inhibitor is better than that of
a control (e.g. placebo) with clinical(ly) (meaningful) benefit in
the "high HRG" subgroup. In more preferred embodiments, average
anti-cancer efficacy of the administered HER3 inhibitor is
statistically significantly better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit in the "high HRG"
subgroup.
[0245] In other preferred embodiments, when "high HRG" patients
suffering from a cancer are identified using the predetermined
threshold, and the patients are treated by administering a HER3
inhibitor or placebo with or without a further medicament, average
anti-cancer efficacy of the administered HER3 inhibitor is better
than that of a control (e.g. placebo) with clinical(ly)
(meaningful) benefit. In more preferred embodiments, average
anti-cancer efficacy of the administered HER3 inhibitor is
statistically significantly better than that of control (e.g.
placebo) with clinical(ly) (meaningful) benefit.
[0246] As used herein, "further medicament" means any therapeutic
or prophylactic molecule other than the HER3 inhibitor which is to
be used in combination with said molecule. In some embodiments,
"further medicament" is one or more of a HER inhibitor, a
chemotherapy, or a radiation therapy.
[0247] In some embodiments, an indicator (index) of "anti-cancer
efficacy" can be progression-free survival (PFS) or overall
survival (OS), but is not limited thereto. The indicator can be any
surrogate marker of anti-cancer efficacy of a HER3 inhibitor.
[0248] As used herein, "high HRG" is a numerical value
representing, or represents, a level of HRG gene expression at or
above a predetermined threshold. In the present invention, "high
HRG", "high HRG (sub)group" and "high HRG patient (or subject)"
mean a level of HRG gene expression at or above a (predetermined)
threshold, (sub)group having level(s) of HRG gene expression at or
above a (predetermined) threshold, and, patient (or subject) having
a level of HRG gene expression at or above a (predetermined)
threshold, respectively. The HRG classification can be based on HRG
gene expression at a protein level, for example.
[0249] As used herein, "low HRG" is a numerical value representing,
or represents, a level of HRG gene expression at or below a
predetermined threshold. In the present invention, "low HRG", "low
HRG (sub)group" and "low HRG patient (or subject)" mean a level of
HRG gene expression at or below a (predetermined) threshold,
(sub)group having level(s) of HRG gene expression at or below a
(predetermined) threshold, and, patient (or subject) having a level
of HRG gene expression at or below a (predetermined) threshold,
respectively. The HRG classification can be based on HRG gene
expression at a protein level, for example.
[0250] As used herein, "response" or "responding" to treatment
means, with regard to a treated tumor, that the tumor displays: (a)
slowing of growth, (b) cessation of growth, or (c) regression.
[0251] The methods disclosed herein can be used for identifying a
subject, for example a human subject, harboring or diagnosed with a
tumor or cancer cells. In some embodiments, the subject harbors
solid or liquid tumors that may be driven by the HER3 pathway, or
that may have resistance to other therapies mediated by the HER3
pathway. In some embodiments, the subject harbors lung cancer,
colorectal cancer, head and neck cancer, breast cancer,
gastrointestinal cancer, pancreatic cancer, prostate cancer,
ovarian cancer, endometrial cancer, salivary gland cancer, renal
cancer, colon cancer, gastric cancer (stomach cancer), thyroid
cancer, bladder cancer, glioma, melanoma, metastatic breast cancer,
epidermal carcinoma, esophageal cancer, cervical cancer, squamous
cell carcinoma, small-cell lung cancer, or non-small cell lung
cancer. In some embodiments, the methods disclosed herein can be
used to identify a subject harboring a locally advanced or
metastatic tumor, such as a locally advanced or metastatic NSCLC
(tumor) or locally advanced or metastatic head and neck cancer. In
some embodiments, methods disclosed herein can be used to identify
a subject, such as a subject harboring a locally advanced or
metastatic NSCLC (tumor), that is likely to benefit from a
treatment comprising an anti-HER3 antibody or HER3 inhibitor having
a low molecular weight. In some embodiments, the subject is
harboring a Stage III, e.g., Stage Mb, or Stage IV tumor. Methods
of identifying a subject can comprise, for example, assessing HRG
gene expression at a protein level in a human subject diagnosed
with a tumor or cancer.
[0252] In some embodiments, methods disclosed herein can be used to
identify a subject harboring a locally advanced or metastatic NSCLC
(tumor), that is likely to benefit from a treatment comprising
(administering) an anti-HER3 antibody or HER3 inhibitor having a
low molecular weight, provided that, any subject who having an ALK
gene fusion or rearrangement is excluded from those to whom the
methods are applied.
[0253] In some embodiments, the methods disclosed herein can be
used to treat a subject identified as harboring a tumor or cancer
cells. In some embodiments, methods of identifying or treating a
human subject harboring a locally advanced or metastatic NSCLC
(tumor) can comprise assessing HRG gene expression at a protein
level in a human subject diagnosed with a locally advanced or
metastatic NSCLC. In some embodiments, the subject does not harbor
an epidermal growth factor receptor (EGFR) sensitizing mutation. In
some embodiments, the subject harbors wild-type EGFR. In some
embodiments, the subject does not harbor an ALK gene fusion or
rearrangement. In some embodiments, the disease or tumor has
progressed on at least one prior systemic therapy, such as
chemotherapy. Some embodiments comprise administering a treatment
comprising an anti-HER3 antibody to a human subject whose HRG gene
expression at a protein level is assessed as high. In some
embodiments, treatment comprises (administering) an anti-HER3
antibody in combination with at least one agent that inhibits a HER
family receptor other than HER3. In some embodiments, treatment
comprises administering an anti-HER3 antibody in combination with
at least one agent that inhibits a non-HER family tyrosine kinase
receptor. In some embodiments, an anti-HER3 antibody is
administered in combination with non-specific chemotherapy.
[0254] In some preferred embodiments, patients to whom the methods
disclosed herein can be applied are heregulin high, EGFR wild-type
subjects with locally advanced or metastatic non-small cell lung
cancer who have progressed on at least one prior systemic therapy.
In some embodiments, the patients are HER inhibitor naive. In
preferred embodiments, a tumor tissue or fragment thereof for or
with which the HRG gene expression is assessed has been removed
from the subject or patient prior to any (systemic) therapy.
[0255] In some preferred embodiments, patients to whom the methods
disclosed herein can be applied include a subject with a first-line
metastatic or locally advanced head and neck cancer that will be
concurrently treated with one or more of cetuximab, cisplatin,
panitumumab, 5-fluoruracil, radiotherapy, and radiation therapy
(locally advanced only).
[0256] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with a second-line
metastatic NSCLC or other cancer that will be concurrently treated
with docataxel.
[0257] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with a NSCLC or other
cancer that will be concurrently treated with an immune
therapy.
[0258] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with a third line, HER2
positive, (metastatic) breast cancer that will be concurently
treated with a PI3K pathway inhibitor.
[0259] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with HER2 negative
(metastatic) breast cancer that will be concurrently treated with a
hormone therapy or PI3K pathway inhibitor.
[0260] In the present invention, PI3K pathway inhibitors include
PI3K inhibitors, mTOR inhibitors and AKT inhibitors.
[0261] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with a first-line
metastatic EGFR-sensitizing mutant positive for NSCLC or other
cancer that will be concurrently treated with one or more of
erlotinib, gefitini, and afitinib.
[0262] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with a first-line
metastatic NSCLC or other cancer that will be concurrently treated
with platinum-based chemotherapy.
[0263] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject with RAS wild-type
colorectal cancer that will be concurrently treated with one or
more of cetuximab, panitumumab, and chemotherapy.
[0264] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject that with HER2 positive
first line metastatic breast cancer or other cancer that will be
concurrently treated with one or more of trastuzumab, paclitaxel,
docataxel, T-DM1 and pertuzumab.
[0265] In some embodiments, patients to whom the methods disclosed
herein can be applied include a subject that with HER2 positive
second or later line metastatic breast cancer or other cancer that
will be concurrently treated with one or more of lapatinib,
capecitabine, trastuzumab, and paclitaxel.
[0266] In some embodiments, patients to whom the methods disclosed
herein can be applied have not failed with an earlier line of
therapy. In some embodiments, patients to whom the methods
disclosed herein can be applied have not failed with an earlier
line of therapy and the patients have been classified as "high
HRG."
[0267] In some embodiments, the methods disclosed herein can be
used to identify and/or treat HRG high, EGFR wild-type subjects
with locally advanced or metastatic NSCLC who will benefit from
treatment of patritumab in combination with a HER inhibitor.
[0268] In some embodiments, the methods disclosed herein can be
used to identify and/or treat HRG high, EGFR wild-type subjects
with locally advanced or metastatic NSCLC who will benefit from
treatment of patritumab in combination with chemotherapy.
[0269] In some embodiments, the methods disclosed herein can be
used to identify and/or treat HRG high, EGFR mutated subjects, for
example with locally advanced or metastatic NSCLC who will benefit
from treatment of patritumab in combination with a HER
inhibitor.
[0270] In some embodiments, the methods disclosed herein can be
used to identify and/or treat HRG high, EGFR mutated subjects with
locally advanced or metastatic NSCLC who will benefit from
treatment of patritumab in combination with chemotherapy.
[0271] In some embodiments, the methods disclosed herein can be
used to identify and/or treat a "HRG high" patient suffering from a
cancer who will benefit from treatment of patritumab in combination
with an immune therapy or immunotherapy. Such cancers include
NSCLC.
[0272] In some embodiments, the methods disclosed herein can be
used to identify and/or treat a "HRG high" patient suffering from a
cancer who will benefit from treatment of patritumab in combination
with a hormone therapy or PI3K (phosphoinositide 3-kinase) pathway
inhibitor. Such cancers include breast cancer, preferably,
HER2-negative breast cancer. Such PI3K pathway inhibitors include
PI3K inhibitors, AKT inhibitors and mTOR (mammallian Target Of
Rapamycin) inhibitors.
[0273] In some embodiments, the methods disclosed herein can be
used to identify and/or treat a "HRG high" patient suffering from a
cancer who will benefit from treatment of patritumab in combination
with a PI3K inhibitor. Such cancers include breast cancer,
preferably, HER2-positive breast cancer.
[0274] In some embodiments, the methods disclosed herein can be
used to identify and/or treat a "HRG high" patient suffering from a
cancer who will benefit from treatment of patritumab in combination
with a ALK inhibitor. Such cancers include NSCLC. Such ALK
(anaplastic lymphoma kinase) inhibitor includes crizotinib
(Xalkori).
[0275] In some embodiments, the methods disclosed herein can be
used to identify and/or treat acute respiratory distress syndrome,
pulmonary fibrosis, schizophrenia, heart disease, atherosclerosis,
and Duchenne's muscular dystrophy.
HER3 Antibodies
[0276] Antibodies suitable for treatment are not particularly
limited, and can be any protein or ligand that can bind to HER3. In
some embodiments, the antibodies can be binding proteins or
fragments thereof that bind to HER3. In some preferred embodiments,
the antibodies can inhibit, neutralize, prevent or eliminate at
least a portion of the biological activity of HER3.
[0277] HER3 antibodies can be, for example, one or more of
patritumab, duligotumab (MEHD-7945A), seribantumab (MM-121),
MM-111, LJM716, RG-7116 (glyco engineered anti-HER3 monoclonal
antibody), tri-specific anti-EGFR/ERBB3 zybody, huHER3-8 or a
derivative or fragment of any of these that can bind to HER3.
[0278] Antibody fragments include, for example, Fab fragments, Fab'
fragments, F(ab').sub.2 fragments, Fv fragments, diabodies
(Hollinger et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:6444-6448), single chain antibody molecules (Pluckthun in: The
Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore,
eds., Springer Verlag, N.Y. (1994), 269-315), scFv fragments, and
other fragments that can inhibit HER3.
[0279] Derivatives of antibodies or antibody fragments can include,
for example, a bispecific antibody, a multispecific antibody, a
biscFv fragment, a diabody, a nanobody, an antibody-drug conjugate,
an immunotoxin, and/or an immunocytokine, but are not limited
thereto.
[0280] Further examples of suitable antibodies can be found, for
example, in U.S. Pat. No. 7,705,130, which is herein incorporated
by reference in its entirety.
[0281] According to the present invention, an isolated binding
protein that is capable of binding to HER3 interacts with at least
one epitope in the extracellular part of HER3. The epitopes are
preferably located in domain L1, which is the amino terminal
domain, in domain S1 and S2, which are the two Cysteine-rich
domains, or in domain L2, which is flanked by the two Cysteine-rich
domains. The epitopes may also be located in combinations of
domains such as but not limited to an epitope comprised by parts of
L1 and S1.
Biological Sample
[0282] A biological sample taken from a subject, such as a subject
diagnosed with a locally advanced or metastatic NSCLC, can be used
as a source of protein or a source of thin sections for
immunohistochemistry (IHC), so the level of HRG gene expression in
the sample can be determined. The biological sample can comprise,
for example, blood, e.g., whole blood, or blood derivatives
including exosomes, serum, plasma, tissue, cells, and/or
circulating tumor cells. In some embodiments, the biological sample
can be taken from a tumor.
[0283] The biological sample can be obtained by any known methods,
such as venipuncture or with conventional tumor biopsy instruments
and procedures. Endoscopic biopsy, excisional biopsy, incisional
biopsy, fine needle biopsy, punch biopsy, shave biopsy and skin
biopsy are examples of recognized medical procedures that can be
used by one of skill in the art to obtain tumor samples. The
biological sample should be large enough to provide sufficient
protein or thin sections for measuring HRG gene expression.
[0284] In some embodiments, the methods described herein comprise
providing an autologous tissue sample or consenting to the taking
of an autologous tissue sample, e.g., to facilitate an assessment
of HRG gene expression at a protein level in a human subject
diagnosed with a locally advanced or metastatic NSCLC.
[0285] The biological sample can be in any form that allows
measurement of HRG expression or content. In other words, the
sample must be sufficient for protein extraction or preparation of
thin sections. Accordingly, the sample can be fresh, preserved
through suitable cryogenic techniques, or preserved through
non-cryogenic techniques. For example, a standard process for
handling clinical biopsy specimens is to fix the tissue sample in
formalin and then embed it in paraffin. Samples in this form are
commonly known as formalin-fixed, paraffin-embedded (FFPE) tissue.
Suitable techniques of tissue preparation for subsequent analysis
are well-known to those of skill in the art.
HRG Gene Expression
[0286] As described herein, determining or measuring the level of
HRG gene expression in a biological sample can be performed by any
suitable method. Several such methods are known in the art. For
example, determining HRG gene expression can be done by measuring
the level or amount of HRG protein in a sample.
[0287] HRG is produced in numerous isoforms upon expression of the
HRG gene. Without being bound by theory, it is believed that the
EGF-like domain of HRG is essential for binding to HER3. Therefore,
when determining the level of HRG gene expression as described
herein, the HRG assay preferably can be, for example designed to
detect at least the EGF-like domain of HRG, in order to detect
most, if not all, of the HRG isoforms present. If an anti-HRG
antibody is used to detect HRG protein, the antibody can recognize
the EGF-like domain. In some embodiments, however, the anti-HRG
antibody used to detect HRG protein does not recognize the EGF-like
domain.
[0288] HRG gene expression can be detected by any known methods.
Non-limiting examples of suitable detection methods for measuring
the level of HRG gene expression at the protein level include
enzyme linked immunosorbent assay (ELISA) and IHC analysis.
ELISA
[0289] Performing an HRG ELISA requires at least one antibody
against HRG, e.g., a detection antibody. HRG protein from a sample
to be analyzed can be immobilized on a solid support such as a
polystyrene microtiter plate. This immobilization can be by
non-specific binding, e.g., through adsorption to the surface.
Alternatively, immobilization can be by specific binding, e.g.,
through binding of HRG from the sample by a capture antibody in a
"sandwich" ELISA. After the HRG is immobilized, the detection
antibody can be added, and the detection antibody can form a
complex with the bound HRG. The detection antibody can be linked to
an enzyme, either directly or indirectly, e.g., through a secondary
antibody that specifically recognizes the detection antibody.
Between each step, the plate, with bound HRG, can be washed with a
mild detergent solution. Typical ELISA protocols can include one or
more blocking steps, which involve use of a
non-specifically-binding protein such as bovine serum albumin to
block unwanted non-specific binding of protein reagents to the
plate. After a final wash step, the plate can be developed by
addition of an appropriate enzyme substrate to produce a visible
signal that indicates the quantity of HRG in the sample. The
substrate can be, e.g., a chromogenic substrate or a fluorogenic
substrate. ELISA methods, reagents and equipment are well-known in
the art and commercially available.
[0290] Some embodiments of performing an ELISA are described below.
The disclosed methods provide for accurate results when performing
an ELISA on a variety of sample types, including biological samples
that contain high amounts of multiple different proteins (e.g.,
samples such as plasma and serum). In one aspect, the disclosed
ELISA method may be performed to detect natural and/or recombinant
human Heregulin (HRG).
[0291] The ELISA methods disclosed herein comprise sequential steps
of contacting a solid surface with a plurality of solutions each
comprising in turn a capture antibody, a blocking agent, a sample
suspected of containing an analyte, a detection antibody and an
enzyme conjugate, in which the solid surface is subjected to a wash
process after each sequential step, the wash process comprising:
[0292] (a) cycling wash buffer on and off the solid surface at a
rapid rate until bubbles are observed at which point the cycled
wash buffer is removed; [0293] (b) optionally repeating step (a)
using fresh wash buffer; and [0294] (c) rinsing the solid surface
with fresh wash buffer; provided that after completing the wash
process following the enzyme conjugate sequential step, the solid
surface is contacted with a solution comprising an enzyme
substrate.
[0295] The cycling of wash buffer may comprise applying and
aspirating the wash buffer on and off the solid surface quickly and
repeatedly using a "piston" action. The "piston" action may be
performed by repeatedly pumping the plunger end of a pipette (such
as, for example, an air displacement pipette, a positive
displacement pipette or a multichannel pipettor) to move the wash
buffer in and out of an associated pipette tip. Alternatively, the
bulbous end of a transfer pipette or a Pasteur pipette can be
repeatedly squeezed and released to carry out the piston action.
Other comparable means of dispensing and removing wash buffer may
be employed.
[0296] Not to be bound by theory, it is believed that such a piston
action effectively exposes non-specifically adsorbed proteins to
the wash buffer, effectively removing the proteins. The wash buffer
is cycled on an off the solid surface until bubbles are observed,
indicating sufficient washing of the solid surface has occurred. In
some embodiments, the wash buffer is cycled 10 to 30 times on and
off the solid surface. In some embodiments the wash buffer is
cycled about 20 times on and off the solid surface. In some
embodiments, the wash buffer is cycled at least 10, at least 20, or
at least 30 times on and off of the solid surface.
[0297] In some embodiments, the cycling step (a) is performed one
to two, three or four times before performing the rinsing step (c).
In a specific embodiment, the cycling step (a) is performed two
times before performing the rinsing step (c). In some embodiments,
the cycling step (a) is performed at least two times before
performing the rinse step (c).
[0298] The rinsing step involves applying and removing wash buffer
to and from the solid surface. The rinsing step may involve a
standard "fill and aspirate" methodology whereby the solid surface
is contacted one time with wash buffer which is then immediately
removed via aspiration. Alternatively, the wash buffer may be
removed by decantation. The rinsing step is conducted to ensure
that no bubbles remain on the solid surface. Accordingly, in some
embodiments the rinsing step does not involve performing multiples
cycles or a pistoning action. If the solid surface contains wells
or raised edges, the surface may be inverted and blotted on a dry,
flat, absorbent surface after removing the wash buffer to ensure
all wash buffer is removed.
ELISA Solid Surfaces
[0299] In some embodiments, the ELISA method disclosed herein is
performed on a solid surface that is a cell culture plate
comprising one or more wells. An exemplary solid surface for use in
the disclosed ELISA methods is a microtitre plate (also known as a
microplate or microwell plate). Suitable plates may be made of a
polystyrene, polycarbonate or polypropylene. In a specific
embodiment, the ELISA is performed using a 96-well microplate.
ELISA Solutions Comprising a Capture Antibody
[0300] The disclosed ELISA methods involve contacting a solid
surface with a solution comprising a capture antibody. The capture
antibody may be a monoclonal or a polyclonal antibody and
recognizes an antigen of interest that is the analyte to be
detected in the ELISA. In some embodiments, the capture antibody is
mouse antibody raised against a specific human protein or peptide.
In a specific embodiment the capture antibody is a mouse anti-human
HRG antibody.
[0301] The solution comprising a capture antibody may comprise the
antibody reconstituted with phosphate buffered saline (PBS). In
some embodiments, the solution may contain about 1.5 to about 5.0
ug/mL antibody. In a specific embodiment the solution contains
about 4.0 ug/mL antibody in PBS.
[0302] In some embodiments, the solid surface may be incubated with
the solution comprising a capture antibody for at least 4 hours at
a temperature of about 65.degree. to about 80.degree. F. In some
embodiments the solid surface is incubated with the solution for
about 8 hours or overnight at about 70.degree. F. In a specific
embodiment, the solid surface is covered and/or sealed while in
contact with the capture antibody solution.
ELISA Solutions Containing a Blocking Agent
[0303] The solid surface is blocked by adding a solution containing
a blocking agent. In some embodiments, the blocking agent solution
comprises about 1% bovine serum albumin (BSA) in PBS at a pH
between about 7.2 and about 7.4. In some embodiments the blocking
agent solution contains less than less than about 0.5%
proteases.
[0304] The solid surface may be incubated with the blocking agent
solution for at least one hour. In some embodiments, the solid
surface is incubated with the blocking agent solution at about
70.degree. F.
ELISA Sample Solutions
[0305] The solution comprising a sample suspected of containing an
analyte may consist of an undiluted biological sample or it may
comprise a biological sample diluted with a solution such as, for
example, PBS. In a specific embodiment, the sample solution
consists of undiluted serum or plasma and the serum or plasma is
contacted directly with the solid surface.
[0306] The analyte may be, for example, an antigen such as a
protein or peptide. In a specific embodiment, the analyte is a
growth factor such as HRG or, specifically, soluble HRG.
[0307] The solid surface may be incubated with the sample solution
for about 2 hours or longer at about 70.degree. F. In a specific
embodiment, the solid surface is covered and/or sealed during
incubation with the sample solution.
ELISA Solutions Containing a Detection Antibody
[0308] A detection antibody is an antibody that recognizes an
antigen of interest that is the analyte being detected in the
ELISA. As such, the capture antibody and the detection antibody
both recognize the same antigen. However, these two antibodies may
not recognize the same region or epitope of the antigen. The
detection antibody may a monoclonal antibody or a polyclonal
antibody. In some embodiments, the detection antibody is goat
antibody raised against a specific human protein or peptide. In a
specific embodiment the capture antibody is a goat anti-human HRG
antibody.
[0309] The solution comprising a detection antibody may comprise
the antibody reconstituted with phosphate buffered saline (PBS) or
BSA solution. In some embodiments, the solution may contain from
about 100 to about 200 ng/mL antibody in a solution of 1% BSA in
PBS, pH 7.2-7.4. In a specific embodiment the solution contains
about 150 ng/mL antibody in a solution of 1% BSA in PBS, pH
7.2-7.4. In some embodiments, the detection antibody solution does
not contain normal goat serum.
[0310] The detection antibody is conjugated with a detection agent
such as, for example, biotin. Such a detection agent permits
binding of the detection antibody to an enzyme/substrate detection
system such as, for example, an avidin or streptavidin linked
enzyme. In a specific embodiment, the detection antibody is
biotinylated anti-human HRG antibody.
[0311] In some embodiments, the solid surface is incubated with the
detection antibody solution for at least 1.5, 2 or 3 hours at a
temperature of about 70.degree. F. In some embodiments, the solid
surface is covered and/or sealed while in contact with the
detection antibody solution.
ELISA Solutions Containing an Enzyme Conjugate
[0312] The disclosed ELISA methods involve contacting the solid
surface with an enzyme conjugate comprising a binding agent coupled
to a reporter enzyme such as alkaline phosphatase (AP) or
horseradish peroxidase (HRP). The binding agent binds to the
detection agent that is conjugated to the detection antibody.
Streptavidin is an exemplary binding agent that binds to a
biotinylated antibody. The reporter enzyme converts a colorless
substrate to a detectable, colored product. An exemplary enzyme is
horse radish peroxidase (HRP), which converts a hydrogen
peroxide/tetramethylbenzidine substrate mixture to a detectable
chromogen. In some embodiments, the enzyme conjugate employed in
the ELISA is streptavidin-horse radish peroxidase (HRP).
[0313] In some embodiments, the enzyme conjugate solution comprises
the enzyme conjugate diluted in a solution of 1% BSA in PBS, pH
7.2-7.4. In some embodiments the solid surface may be incubated
with the enzyme conjugate solution for about 20 minutes at about
70.degree. F. and away from direct light. The solid surface may be
covered and/or sealed during incubation.
ELISA Solutions Comprising an Enzyme Substrate
[0314] After completing the wash process following the enzyme
conjugate sequential step, the solid surface is contacted with a
solution comprising an enzyme substrate. Suitable substrates
include colorless products that are converted to colored products
upon exposure to the enzyme linked to the binding agent of the
enzyme conjugate. Upon conversion from a colorless product to a
colored product, the optical density of the enzyme substrate
solution on the solid surface can be measured on an ELISA plate
reader at target wavelengths.
[0315] Exemplary, suitable enzyme substrates include Nitroblue
Tetrazolium (NBT) with the 5-Bromo-4-Chloro-3-Indolyl Phosphate
(BCIP) (alkaline phosphatase substrate), Fast Red TR/Naphthol AS-MX
with 4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid
2,4-dimethylanilide phosphate (alkalai phosphate substrate) and a
1:1 mixture of hydrogen peroxide (H.sub.2O.sub.2) and
tetramethylbenzidine (HRP substrate mixture).
[0316] In some embodiments, the enzyme substrate solution is
incubated with the solid surface for at least 20 minutes at about
70.degree. F., away from direct light.
ELISA Wash Buffer
[0317] The solid surface is washed after contacting with each of
the capture antibody, blocking agent, sample, detection antibody
and enzyme conjugate solutions. Wash buffer contains a surfactant,
such as a polysorbate surfactant. A specific suitable surfactant is
polyethylene glycol sorbitan monolaurate (e.g., TWEEN.RTM. 20,
Sigma Aldrich, Missouri, USA). The surfactant may be present in,
for example, PBS pH 7.2-7.4 or Tris Buffered Saline, pH 8.0. In a
specific embodiment, the wash buffer comprises 0.05% polyethylene
glycol sorbitan monolaurate in PBS, pH 7.2-7.4.
[0318] The solid surface of the ELISA may be contacted with a stop
solution after incubation with the enzyme substrate solution is
complete. In some embodiments, the stop solution comprises
2-NH.sub.2SO.sub.4. The stop solution may be thoroughly mixed with
the existing solution on the plate after added.
[0319] The optical density of the ELISA solid surface may be
determined by a spectrophotometric reading after the solid surface
is contacted with the stop solution. In some embodiments, the
optical density is determined immediately upon addition of the stop
solution.
[0320] In one embodiment of the disclosed ELISA methods, the method
is performed with a DuoELISA Development kit or detecting human
NRG1-b1/HRG1-b1 (R&D Systems, Inc., Minneapolis, USA; catalog
number DY377).
Immunohistochemistry
[0321] The presence and level of HRG in a sample can be determined
by immunohistochemistry (IHC) or immunofluorescence (IF). Assaying
HRG by IHC or IF requires at least one antibody against HRG.
Anti-HRG antibodies suitable for IHC and IF are commercially
available. For example, suitable antibodies can be purchased from
R&D Systems (Minneapolis, Minn.), abeam (Cambridge, Mass.),
Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), or Novus
Biologicals (Littleton, Colo.). Using standard techniques, the
anti-HRG antibody can be used to detect the presence of HRG protein
in thin sections, e.g., 5 micron sections, obtained from tumors,
including FFPE sections and frozen tumor sections. Typically, the
tumor sections are initially treated in such a way as to retrieve
the antigenic structure of proteins that were fixed in the initial
process of collecting and preserving the tumor material. Slides are
then blocked to prevent non-specific binding by the anti-HRG
detection antibody. The presence of HRG protein is then detected by
binding of the anti-HRG antibody (primary antibody) to the HRG
protein. The detection antibody (secondary antibody), which
recognizes and binds to the primary antibody, is linked to a
detectable enzyme or fluorophore. Typically, the tumor sections are
washed and blocked with non-specific protein such as bovine serum
albumin between steps. If the detection antibody is linked to a
detectable enzyme, the slide is developed using an appropriate
enzyme substrate to produce a visible signal. If the detection
antibody is linked to a fluorophore, the slide is viewed by using a
fluorescence microscope. The samples can be counterstained with,
for example, hematoxylin.
Assessing HRG Gene Expression
[0322] HRG gene expression can be assessed in a biological sample
from a human patient, such as a biological sample obtained from,
taken from, or received from a human patient. Some embodiments
comprise ordering or receiving an assessment of HRG gene expression
at a protein level. Some embodiments comprise determining a value
for HRG gene expression at a protein level and, optionally,
recording the value determined.
[0323] In some embodiments, HRG expression at a protein level is
assessed using a regulatory authority-approved test. In some
embodiments, the regulatory authority-approved test is an
FDA-approved test, an EMA-approved test, or a JPMA-approved
test.
[0324] HRG Gene Expression levels can be interpreted with respect
to a predetermined threshold. An HRG gene expression level that is
equal to or higher than the threshold score can be interpreted as
predictive of the likelihood that a subject would respond to
treatment with a HER3 inhibitor, e.g., an anti-HER3 antibody. In
some embodiments, HRG gene expression levels lower than the
threshold score can be interpreted as predictive of a tumor being
resistant (non-responsive) to treatment with a HER3 inhibitor.
[0325] In some embodiments, HRG gene expression can be assessed as
"high HRG" or "low HRG" based on a numerical value representing the
level of HRG gene expression in a biological sample. A subject can
be assessed as high HRG or low HRG based on, for example, HRG
expression at a protein level. The expression level can be assessed
by any known methods, such as those described above.
[0326] For example, HRG gene expression at a protein level can be
assessed as "high HRG" if a protein concentration value is
observed, which is at or above a predetermined threshold, from a
biological sample. In some embodiments, the predetermined threshold
is chosen statistically to minimize undesirable effects of false
positives and false negatives and can be, for example, 0 pg/mL,
about 980 pg/mL, about 1000 pg/mL, about 1622 pg/mL, about 2000
pg/mL, about 3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about
5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL, about 8000 pg/mL,
about 9000 pg/mL, about 10000 pg/mL, about 11000 pg/mL, about 12000
pg/mL, about 13000 pg/mL, about 14000 pg/mL, about 15000 pg/mL,
about 16000 pg/mL, about 17000 pg/mL, about 18000 pg/mL, about
19000 pg/mL, about 20000 pg/mL, about 22000 pg/mL, about 24000
pg/mL, about 26000 pg/mL, about 28000 pg/mL, about 30000 pg/mL,
about 35000 pg/mL, about 40000 pg/mL, about 50000 pg/mL, about
60000 pg/mL, about 70000 pg/mL, about 80000 pg/mL, about 90000
pg/mL or about 100000 pg/mL. In some embodiments, HRG expression
can be assessed as "high HRG" if the protein concentration value in
the biological sample is more than: about 0 pg/mL, about 980 pg/mL,
about 1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000
pg/mL, about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about
7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL, about 10000 pg/mL,
about 11000 pg/mL, about 12000 pg/mL, about 13000 pg/mL, about
14000 pg/mL, about 15000 pg/mL, about 16000 pg/mL, about 17000
pg/mL, about 18000 pg/mL, about 19000 pg/mL, about 20000 pg/mL,
about 22000 pg/mL, about 24000 pg/mL, about 26000 pg/mL, about
28000 pg/mL, about 30000 pg/mL, about 35000 pg/mL, about 40000
pg/mL, about 50000 pg/mL, about 60000 pg/mL, about 70000 pg/mL,
about 80000 pg/mL, about 90000 pg/mL or about 100000 pg/mL. In some
embodiments, the HRG expression is assessed as "low HRG" if the
protein concentration value is less than: about 980 pg/mL, about
1000 pg/mL, about 1622 pg/mL, about 2000 pg/mL, about 3000 pg/mL,
about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000
pg/mL, about 8000 pg/mL, about 9000 pg/mL, about 10000 pg/mL, about
11000 pg/mL, about 12000 pg/mL, about 13000 pg/mL, about 14000
pg/mL, about 15000 pg/mL, about 16000 pg/mL, about 17000 pg/mL,
about 18000 pg/mL, about 19000 pg/mL, about 20000 pg/mL, about
22000 pg/mL, about 24000 pg/mL, about 26000 pg/mL, about 28000
pg/mL, about 30000 pg/mL, about 35000 pg/mL, about 40000 pg/mL,
about 50000 pg/mL, about 60000 pg/mL, about 70000 pg/mL, about
80000 pg/mL, about 90000 pg/mL or about 100000 pg/mL.
[0327] In some embodiments, the HRG gene expression at a protein
level can be assessed based on soluble HRG that can originate from
one or more sources. For example, in some embodiments HRG gene
expression at a protein level can originate from both normal and
tumor cells.
[0328] In some embodiments, higher HRG gene expression is
correlated with better hazard ratios and p-values.
Treatment
[0329] In some embodiments, the subject can be treated by
administering a treatment comprising an anti-HER3 antibody to a
subject suffering from a cancer or other disease with HRG gene
expression assessed as high. In some embodiments, the subject can
be treated by withholding a treatment comprising an anti-HER3
antibody from a subject suffering from a cancer or other disease
with HRG gene expression assessed as low.
[0330] In some embodiments, the subject can be treated by receiving
or undergoing a treatment comprising an anti-HER3 antibody if HRG
gene expression at a protein level is assessed as high or
abstaining from a treatment comprising an anti-HER3 antibody if HRG
gene expression at a protein level is assessed as low.
[0331] In some embodiments, the subject can be treated by electing
to withhold or abstain from a treatment comprising an anti-HER3
antibody if HRG gene expression at a protein level is assessed as
low or electing to administer a treatment comprising an anti-HER3
antibody if HRG gene expression at a protein level is assessed as
high.
[0332] The anti-HER3 antibody can be any protein or ligand that can
bind to HER3, such as those discussed above. In some embodiments,
the anti-HER3 antibody is one or more of patritumab (U3-1287),
duligotumab (MEHD-7945A), MM-111, LJM716, RG-7116, tri-specific
anti-EGFR/ERBB3 zybody, huHER3-8 and seribantumab (MM-121).
[0333] The anti-HER3 antibody can be administered at any suitable
dose. For example, the antibody can be administered at about 9
mg/kg or more, about 12 mg/kg or more, about 15 mg/kg or more, or
about 18 mg/kg or more. In some embodiments, the antibody can be
administered at about 9 mg/kg or less, about 12 mg/kg or less,
about 15 mg/kg or less, or about 18 mg/kg or less.
[0334] The anti-HER3 antibody can be administered by any suitable
method. For example, in some embodiments the antibody is
administered intravenously. However, the administration route is
not limited to the intravenous one, but can be any other suitable
one as well.
[0335] In some embodiments, the anti-HER3 antibody is administered
one or more times every week or more frequently, or, every two
weeks, or every three weeks, or less frequently.
[0336] In some embodiments, the treatment comprises administering
an anti-HER3 antibody in combination with a tyrosine kinase
inhibitor or HER inhibitor, such as an epidermal growth factor
receptor inhibitor. The treatment can comprise administering an
anti-HER3 antibody in combination with, for example, one or more of
trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab
gefitinib, afatinib, dacomitinib, KD-019 and erlotinib.
[0337] In some embodiments, the treatment comprises administering
an anti-HER3 antibody in combination with a chemotherapy. The
treatment can comprise administering an anti-HER3 antibody in
combination with, for example, one or more of such as cisplatin,
5-fluoruracil, paclitaxel, capecitabine, and other
chemotherapies.
[0338] In some embodiments, the treatment comprises administering
an anti-HER3 antibody in combination with both a tyrosine kinase
inhibitor or HER inhibitor and chemotherapy. The treatment can
comprise administering an anti-HER3 antibody in combination with,
for example, one or more of trastuzumab, T-DM1, lapatinib,
pertuzumab, cetuximab, panitumumab, gefitinib, dacomitinib, KD-019,
afatinib, dacomitinib, KD-019 and erlotinib, and one or more of
cisplatin, carboplatin, gemcitabine, permetrexed, irinotecan,
5-fluoruracil, paclitaxel, docetaxel, capecitabine, and other
chemotherapies.
[0339] In some embodiments, the treatment comprises administering
an anti-HER3 antibody in combination with radiotherapy. In some
embodiments, treatment comprises administering an anti-HER3
antibody in combination with radiotherapy and one or more of a
tyrosine kinase inhibitor, HER inhibitor, and chemotherapy.
[0340] In some embodiments, anti-HER3 antibodies can be
administered in combination with first-line treatments for
metastatic or locally advanced head and neck cancer, such as
radiotherapy or radiation therapy, cetuximab, cisplatin, and/or
5-fluoruracil.
[0341] In some embodiments, anti-HER3 antibodies can be
administered in combination with first-line treatments for
metastatic or locally advanced head and neck cancer, such as
cetuximab, cisplatin, and/or 5-fluoruracil.
[0342] In some embodiments, anti-HER3 antibodies can be
administered in combination with first-line treatments for NSCLC,
such as erlotinib or platinum-based chemotherapy.
[0343] In some embodiments, anti-HER3 antibodies can be
administered in combination with second-line treatments for NSCLC,
such as docetaxel.
[0344] In some embodiments, anti-HER3 antibodies can be
administered in combination with treatments for RAS wild-type
cancer colorectal cancer and other cancer, such as cetuximab,
panitumumab, and/or chemotherapy.
[0345] In some embodiments, anti-HER3 antibodies can be
administered in combination with radiation, cisplatin, cetuximab,
5-fluoruracil, and/or other HER inhibitors or chemotherapies.
[0346] In some embodiments, anti-HER3 antibodies can be
administered in combination with one or more of trastuzumab,
paclitaxel, lapatinib, capecitabine, and/or other HER inhibitors or
chemotherapies.
Test Kits
[0347] In some embodiments, the test kit contains materials for
determining HRG content by IHC. An IHC kit, for example, may
contain a primary antibody against HRG, and a secondary antibody
conjugated to a reporter enzyme, e.g., horseradish peroxidase. In
some embodiments, the secondary antibody is replaced with a
conjugated polymer that specifically recognizes the primary
antibody.
EXAMPLES
[0348] The invention is further illustrated by the following
examples. The examples are provided for illustrative purposes only,
and are not to be construed as limiting the scope or content of the
invention in any way.
[0349] Abbreviations: AE--adverse event; CI--confidence interval;
CR--complete response; DLT--dose limiting toxicity; FAS--full
analysis set; FFPE--formalin-fixed, paraffin-embedded; HR--hazard
ratio; IN--intravenous; ITT--intent to treat; MTD--maximum
tolerated dose; OS--overall survival; PD--progressive disease;
PFS--progression-free survival; PH--proportional hazards; PO--oral;
PR--partial response; SD--stable disease.
Example 1
Phase 1b/2 Clinical Trial
[0350] This and other examples provide the results of a randomized,
placebo-controlled, double-blind Phase 1b/2 study designed to
evaluate the safety and efficacy of patritumab in combination with
erlotinib in EGFR-inhibitor treatment-naive subjects with Stage
IIIb/IV NSCLC who had progressed after at least 1 prior
chemotherapy regimen.
[0351] The study comprised a Phase 1b open-label, single-arm
portion to assess safety and tolerability of patritumab in
combination with erlotinib, and to determine the dosage for the
Phase 2 portion, followed by a randomized, placebo-controlled Phase
2 portion to assess efficacy and safety of the combination therapy
relative to erlotinib plus placebo. Based on Phase 1 study results
in which a maximum tolerated dose was not reached, the preliminary
human pharmacokinetic profile supported intravenous patritumab
administration at or above 9 mg/kg once every 3 weeks to achieve
circulating levels exceeding those showing maximal efficacy and
pharmacodynamics in experimental animal models. A higher
maintenance dose level of 18 mg/kg was also included to accommodate
the possible effect of reduced tumor tissue penetration in the
clinical setting relative to animal models. Due to the lack of dose
limiting toxicity in a monotherapy Phase 1 study, the Phase 1b was
designed as a dose-de-escalation study, with once daily oral
administration of 150 mg of erlotinib and IV administration of 18
mg/kg patritumab every three weeks, with a provision for dose
de-escalation from this maximal dose if it exceeded the MTD. As no
DLTs were seen in this Phase 1b cohort, doses at this level and
below were allowed in the Phase 2 portion.
[0352] In both portions of the study, subjects received 150 mg of
erlotinib orally once daily. At the beginning of every 3 weeks
treatment cycle, subjects received an IV infusion of patritumab or
placebo (in Phase 2 portion). Three treatment regimens were
evaluated: the combination of 150 mg erlotinib daily and 18 mg/kg
patritumab every 3 weeks ("high dose"); the combination of 150 mg
erlotinib daily and 18 mg/kg patritumab loading with 9 mg/kg
patritumab maintenance every 3 weeks ("low dose"); and the
combination of 150 mg erlotinib daily and placebo every 3 weeks
("placebo"). Tumors were to be assessed every 6 weeks (.+-.3 days)
up to the first 24 weeks of the study, then every 12 weeks (.+-.7
days) independent of treatment cycle.
[0353] An "HRG high" subject was defined as a subject with observed
soluble HRG concentration of higher than 980 pg/mL, the Quartile 3
of the sample set. Soluble HRG concentration was measured by the
improved ELISA method disclosed herein.
[0354] In the study, 202 serum samples were collected from 215
randomized subjects. Concentration of soluble HRG in the samples
were measured in duplicate in 5 batches, and mean values of the
soluble HRG concentration were obtained for each subject. During
the measurement, assay reproducibility was confirmed by repeat
measurement of a control sample, which was recombinant heregulin in
fetal bovine serum at a concentration of 4000 pg/mL. HRG data of
greater than 0 pg/mL were obtained for 70 subjects. The Quartile 3
was determined using all the 202 samples.
[0355] The sample size for the Phase 2 portion was calculated based
on a one-sided log-rank test with 80% power to detect a 50%
improvement (that is, HR of 0.667) in median PFS of 3.3 vs 2.2
months between any patritumab arm compared to the control at a
significance level of one-sided alpha=0.1.
[0356] The primary analyses for this study occurred when 162 PFS
events (and 110 PFS events per comparison of patritumab 18
mg/kg+erlotinib and control arms, and of patritumab 9
mg/kg+erlotinib and control arms) had been observed. At the point
of primary analysis, the treatment assignment for all subjects was
unblinded to designated study personnel for analysis after data
were reconciled and cleaned, and a snapshot of the clean database
was created. To minimize potential bias, individual treatment
assignment was not divulged to subjects or Investigators until
study closure.
[0357] All efficacy analyses were performed on the full analysis
set, which includes all subjects in the randomized analysis set who
received at least one dose of randomized study medication. The
primary efficacy endpoint was PFS. PFS is defined as the time from
the date of randomization to the earlier of the dates of the first
objective documentation of radiographic disease progression or
death due to any cause. A subject who was alive with no objective
documentation of (radiographic) disease progression by the data
cut-off date was to be censored at the date of the last evaluable
tumor assessment. The key secondary efficacy endpoint, overall
survival, was defined as the time from the date of randomization to
death due to any cause and was analyzed in the same manner as the
primary efficacy endpoint PFS.
[0358] The primary analysis for PFS used a stratified log-rank
linear trend test for the dose-response relationship, followed by
pair-wise comparisons of each patritumab arm and the control using
the stratified log-rank test, accounting for the stratification
factors at randomization: histology (Adenocarcinoma vs
Non-Adenocarcinoma) and best response to prior therapy (CR/PR vs SD
vs PD). Kaplan-Meier curves were generated for PFS and used to
calculate medians and 95% CIs for each treatment group. Estimates
of the HR between each patritumab arm and the control along with
95% CIs were calculated using a stratified Cox's proportional
hazards model.
[0359] The primary analysis for PFS in HRG-high group on the FAS
used a stratified log-rank test for the comparisons of each
patritumab arm and the control and the comparison of the combined
patritumab arm and the control. The stratification factors included
histology (Adenocarcinoma vs Non-Adenocarcinoma) and best response
to prior therapy (CR/PR/SD vs PD). Estimates of the HR between each
patritumab arm and the control and between the combined patritumab
arm and the control along with 95% CIs were calculated using a
stratified Cox's proportional hazards model with the same
stratification factors used for the stratified log-rank test.
[0360] Unless otherwise indicated, log-rank p-values and HRs for
PFS and OS were based on the primary analysis adjusted for the
stratification factors at randomization as described above.
[0361] The Phase 1b portion of the trial enrolled 7 subjects (4
male; median age [range], 68 years [48-78]) all of whom received
the combination of 150 mg erlotinib daily and 18 mg/kg patritumab
every 3 weeks. AEs grade .gtoreq.3 occurred in 2 subjects: one
grade 3 case each of pain, fatigue, headache, dehydration,
diarrhea, and blood creatinine increase; none were related to
patritumab. Three subjects had four serious AEs: grade 3 pain
(unrelated to study treatment), grade 3 dehydration
(erlotinib-related), and grade 1 decreased appetite (erlotinib- and
patritumab-related) and grade 1 pyrexia (unrelated) in one subject.
Most reported AEs were grade 1 or 2 and were considered
erlotinib-related. The only patritumab-related AE reported in
.gtoreq.2 subjects was decreased appetite (2 subjects).
[0362] No response was recorded and stable disease was noted in
four subjects (83, 87, 90, and 117 days). All 7 subjects
discontinued from study treatment due to disease progression; 6
subjects were followed until death, and 1 subject withdrew consent
for follow-up.
[0363] No DLTs were reported during the phase 1b study. Therefore,
the Phase 2 dose regimens were a patritumab 18 mg/kg loading dose,
with subsequent administration of either a 9 mg/kg patritumab or 18
mg/kg patritumab maintenance dose every 3 weeks. Subjects were also
administered 150 mg/day erlotinib during the phase 2 trial.
[0364] For the Phase 2 portion, 3 subjects were randomized but not
treated, thus there were 212 subjects in the FAS and safety
analysis set. The analysis results presented below are based on
primary analyses of efficacy data (except for OS) from the locked
database (as of data cut-off date Oct. 30, 2012). OS data was not
mature yet at the time of primary analysis, and the preliminary
results from updated OS analysis based on a data cut-off date of
Apr. 19, 2013 are presented below.
[0365] Dispositions of the 215 subjects enrolled into the
randomized Phase 2 portion of the study are summarized in Table 1.
Demographic information for the full analysis set is summarized in
Table 2. There was no meaningful difference among treatment groups
with respect to demographic characteristics.
TABLE-US-00001 TABLE 1 Phase 2 Subject Disposition Placebo + 18
mg/kg + 9 mg/kg + Total erlotinib erlotinib erlotinib Phase 2
Subject Accounting (N = 71) (N = 72) (N = 72) (N = 215)
Enrolled/Randomized but Not Dosed 0 2 (2.8%) 1 (1.4%) 3 (1.4%)
Treatment Status Ongoing on the Study Treatment 5 (7.0%) 5 (6.9%) 6
(8.3%) 16 (7.4%) Discontinued from Study Treatment 66 (93.0%) 65
(90.3%) 65 (90.3%) 196 (91.2%) Primary Reason for Adverse Event 5
(7.0%) 7 (9.7%) 6 (8.3%) 18 (8.4%) Discontinuing Study Lost to
Follow-up 0 0 0 0 Treatment Death 4 (5.6%) 11 (15.3%) 2 (2.8%) 17
(7.9%) Protocol Violation 0 0 0 0 Subject Withdrew Consent 3 (4.2%)
2 (2.8%) 4 (5.6%) 9 (4.2%) Study Terminated by Sponsor 0 0 0 0
Progressive Disease (Radiographic Progression) 50 (70.4%) 42
(58.3%) 45 (62.5%) 137 (63.7%) Other 4 (5.6%) 3 (4.2%) 8 (11.1%) 15
(7.0%) On-Study Death.sup.[a] 13 (18.3%) 20 (27.8%) 9 (12.5%) 42
(19.5%) Primary Cause of Adverse Event 5 (7.0%) 11 (15.3%) 4 (5.6%)
20 (9.3%) On-Study Death Disease Progression 8 (11.3%) 8 (11.1%) 4
(5.6%) 20 (9.3%) Unknown 0 0 1 (1.4%) 1 (0.5%) Other 0 1 (1.4%) 0 1
(0.5%) Notes: Percentages are based on the number of subjects in
the Enrolled/Randomized Analysis Set. .sup.[a]On-Study Death = Y if
the date of death occurred on or after the date of first drug
administration and within the AE collection period (up to 53 days
after the last dose of patritumab or more than 30 days after the
last dose of erlotinib, whichever is later).
TABLE-US-00002 TABLE 2 Demographic and Baseline Characteristics
(Full Analysis Set) Placebo + 18 mg/kg + 9 mg/kg + erlotinib
erlotinib erlotinib Total (N = 71) (N = 70) (N = 71) (N = 212) Age
(yrs) [a] Median 60.0 62.0 65.0 62.5 Minimum 35 41 44 35 Maximum 88
82 84 88 <60 33 (46.5%) 28 (40.0%) 24 (33.8%) 85 (40.1%) >=60
38 (53.5%) 42 (60.0%) 47 (66.2%) 127 (59.9%) Gender Male 43 (60.6%)
38 (54.3%) 48 (67.6%) 129 (60.8%) Female 28 (39.4%) 32 (45.7%) 23
(32.4%) 83 (39.2%) Race White 69 (97.2%) 68 (97.1%) 71 (100.0%) 208
(98.1%) Black or African American 1 (1.4%) 1 (1.4%) 0 2 (0.9%)
Asian 0 1 (1.4%) 0 1 (0.5%) Other/Specify 1 (1.4%) 0 0 1 (0.5%)
Weight (kg) n 71 70 71 212 Mean 74.68 73.59 72.34 73.53 SD 14.337
17.506 14.369 15.422 Median 74.00 72.00 72.30 72.55 Minimum 42.6
44.0 42.0 42.0 Maximum 108.6 121.0 114.0 121.0 Smoking Status Never
5 (7.0%) 10 (14.3%) 11 (15.5%) 26 (12.3%) Current 13 (18.3%) 12
(17.1%) 9 (12.7%) 34 (16.0%) Former 53 (74.6%) 48 (68.6%) 51
(71.8%) 152 (71.7%) Pack Years (PY) <=15 PY 11 (15.5%) 9 (12.9%)
7 (9.9%) 27 (12.7%) >15 PY 50 (70.4%) 43 (61.4%) 47 (66.2%) 140
(66.0%) Missing 10 (14.1%) 18 (25.7%) 17 (23.9%) 45 (21.2%) Notes:
Denominator for percentages is the number of subjects in the FAS.
[a]: Age in years is calculated using the informed consent date and
the birth date
[0366] Subject baseline characteristics with regard to NSCLC
history and prior therapy are shown in Table 3. Subjects generally
appeared to be well balanced among treatment groups.
TABLE-US-00003 TABLE 3 Baseline Prognostic and Disease
Characteristics (Full Analysis Set) Placebo + 18 mg/kg + 9 mg/kg +
erlotinib erlotinib erlotinib Total (N = 71) (N = 70) (N = 71) (N =
212) Baseline ECOG Performance Status 0 Fully Active 25 (35.2%) 33
(47.1%) 30 (42.3%) 88 (41.5%) 1 Restricted in Physically Strenuous
Activity 46 (64.8%) 37 (52.9%) 41 (57.7%) 124 (58.5%) Histology
Adenocarcinoma 42 (59.2%) 46 (65.7%) 44 (62.0%) 132 (62.3%)
Squamous 21 (29.6%) 19 (27.1%) 23 (32.4%) 63 (29.7%) Other 8
(11.3%) 5 (7.1%) 4 (5.6%) 17 (8.0%) NSCLC Tumor Staging at Study
Entry (CRF) IIIB 7 (9.9%) 5 (7.1%) 9 (12.7%) 21 (9.9%) IV 64
(90.1%) 65 (92.9%) 62 (87.3%) 191 (90.1%) Time from Initial
Diagnosis of NSCLC to Study Treatment (months) <6 months 10
(14.1%) 16 (22.9%) 14 (19.7%) 40 (18.9%) 6-12 months 37 (52.1%) 33
(47.1%) 35 (49.3%) 105 (49.5%) >12 months 24 (33.8%) 21 (30.0%)
22 (31.0%) 67 (31.6%) Number of Prior NSCLC Therapies 0 0 0 0 0 1
47 (66.2%) 50 (71.4%) 48 (67.6%) 145 (68.4%) 2 24 (33.8%) 19
(27.1%) 22 (31.0%) 65 (30.7%) 3 0 1 (1.4%) 1 (1.4%) 2 (0.9%) Best
Response to Prior Chemotherapy.sup.[a] CR/PR 23 (32.4%) 19 (27.1%)
17 (23.9%) 59 (27.8%) SD 29 (40.8%) 28 (40.0%) 34 (47.9%) 91
(42.9%) PD 19 (26.8%) 23 (32.9%) 20 (28.2%) 62 (29.2%) Exposure to
Prior Platinum Therapy Yes 70 (98.6%) 70 (100.0%) 70 (98.6%) 210
(99.1%) No 1 (1.4%) 0 1 (1.4%) 2 (0.9%) Prior Radiation Therapy Yes
24 (33.8%) 25 (35.7%) 22 (31.0%) 71 (33.5%) No 47 (66.2%) 45
(64.3%) 49 (69.0%) 141 (66.5%) Notes: Percentages reflect
proportion of subjects in Full Analysis Set (FAS). Baseline = last
non-missing value before initial administration of study treatment.
.sup.[a]If a subject has two lines of prior chemotherapy regimens,
the best response to the most recent chemotherapy regimen
(excluding `Not Applicable`) was used.
Example 2
Progression Free Survival and Overall Survival in Full Analysis
Set
[0367] Primary analysis of PFS for the FAS is presented in Table 4.
Kaplan-Meier estimates of progression-free survival in the FAS are
presented in FIG. 1 (showing high- and low-dose
patritumab+erlotinib vs. placebo+erlitonib). Overall Survival (OS)
results in the unselected FAS are presented in Table 5 and in FIG.
2 (showing high- and low-dose patritumab+erlotinib vs.
placebo+erlitonib). There was no significant improvement in PFS or
OS for the combination of patritumab with erlotinib as compared to
erlotinib plus placebo in the full analysis set, and the study was
considered as negative for the unselected ITT population.
[0368] The number of subjects with the response being CR/PR in low-
and high-dose patritumab treatment groups were respectively 9
(12.9%) and 5 (7.1%) vs placebo 4 (5.6%).
TABLE-US-00004 TABLE 4 Analysis of Progression-Free Survival in
Full Analysis Set Placebo + 18 mg/kg + 9 mg/kg + erlotinib
erlotinib erlotinib (N = 71) (N = 70) (N = 71) Subjects (%) with
events 59 (83.1%) 58 (82.9%) 52 (73.2%) Subjects (%) without events
(censored) 12 (16.9%) 12 (17.1%) 19 (26.8%) Time to event
(months).sup.[a] Median 1.6 1.4 2.5 95% CI for Median [1.4; 2.6]
[1.3; 2.7] [1.5; 3.0] Stratified Logrank P-Value.sup.[b] 0.9735
0.1512 Hazard Ratio (relative to Placebo).sup.[b] 0.978 0.770 95%
CI [0.674; 1.420] [0.523; 1.131] 80% CI [0.767; 1.248] [0.598;
0.990] P-value for Hazard Ratio 0.9075 0.1828 Notes: PFS is defined
as the time from the randomization date to the date of the first
objective documentation of disease progression or death resulting
from any cause, whichever comes first. .sup.[a]Kaplan-Meier
Estimate. CI for median was computed using the Brookmeyer-Crowley
method. .sup.[b]Stratified log-rank and stratified Cox PH were
stratified by best response to prior therapy and histology subtype
(Adenocarcinoma vs. Non-Adenocarcinoma).
TABLE-US-00005 TABLE 5 Analysis of Overall Survival in Full
Analysis Set Placebo + 18 mg/kg + 9 mg/kg + erlotinib erlotinib
erlotinib (N = 71) (N = 70) (N = 71) Subjects (%) with events 48
(67.6%) 54 (77.1%) 46 (64.8%) Subjects (%) without 23 (32.4%) 16
(22.9%) 25 (35.2%) events (censored) Time to event (months).sup.[a]
Median 7.2 5.3 6.3 95% CI for Median [5.4; 10.6] [4.0; 6.9] [5.4;
9.3] Stratified Logrank 0.3823 0.3673 P-Value.sup.[b] Hazard Ratio
1.208 0.858 (relative to Placebo).sup.[b] 95% CI [0.807; 1.808]
[0.566; 1.301] 80% CI [0.928; 1.572] [0.653; 1.127] P-value for
Hazard Ratio 0.3585 0.4712 Notes: OS is defined as the time from
the randomization date to the date of death. .sup.[a]Kaplan-Meier
Estimate. CI for median was computed using the Brookmeyer-Crowley
method. .sup.[b]Stratified log-rank and stratified Cox PH were
stratified by best response to prior therapy and histology subtype
(adenocarcinoma vs. non-adenocarcinoma).
Example 3
Progression Free Survival in Subjects with Tumors Expressing High
Soluble HRG Levels
[0369] Kaplan-Meier estimates of progression-free survival in
subjects with tumors expressing high HRG at a protein level,
defined as protein concentration >980 pg/mL, are presented in
FIG. 3 (showing high- and low-dose patritumab+erlotinib vs.
placebo+erlitonib) and FIG. 4 (showing pooled patritumab+erlotinib
vs. placebo+erlotinib).
Example 4
Overall Survival in Subjects with Tumors Expressing High Soluble
HRG Levels
[0370] Preliminary OS results in the subset of subjects with tumors
expressing high levels of HRG at a protein level, defined as
protein concentration >980 pg/mL, are presented in FIG. 5
(showing high- and low-dose patritumab+erlotinib vs.
placebo+erlitonib) and FIG. 6 (showing pooled patritumab+erlotinib
vs. placebo+erlitonib).
Example 5
Efficacy in Subjects with Tumors Expressing Low Soluble HRG
Levels
[0371] In contrast with subjects whose tumors expressed high levels
of HRG, subjects with tumors expressing low levels of HRG showed no
clear treatment difference in PFS and OS. Kaplan-Meier estimates of
PFS in subjects with tumors expressing low HRG at a protein level,
defined as protein concentration <980 pg/mL, are presented in
FIG. 7 (showing pooled patritumab+erlotinib vs. placebo+erlitonib).
Kaplan-Meier estimates of OS in subjects with tumors expressing low
HRG at a protein level, defined as protein concentration <980
pg/mL, are presented in FIG. 8 (showing pooled patritumab+erlotinib
vs. placebo+erlitonib).
Example 6
Potential Cut-Off Values for Soluble HRG Protein Concentration Vs.
Clinical Benefit
[0372] Data was used to calculate hazard ratios between the pooled
dose of patritumab and placebo based on several potential cut-off
values for HRG protein concentration. These hazard ratios are shown
in Table 6. It appears that higher HRG expression is generally
correlated with a greater clinical benefit in terms of PFS. Raising
the cutoff from 980 pg/mL to, for example, 3000 pg/mL results in
additional improvement in the average benefit as judged by the
hazard ratio.
TABLE-US-00006 TABLE 6 Hazard ratios and p-values for PFS in the
soluble HRG high group as a function of cutoff HRG protein
concentration cut-off HR for PFS (pooled Log-rank p-value for
(pg/mL) n dose vs placebo) PFS 0 (median) 66 0.58 0.08 980 (Q3) 46
0.41 0.02 1622.5 39 0.31 0.01 2000 34 0.28 0.008 3000 30 0.24 0.006
4000 27 0.26 0.01 5000 23 0.40 0.13
Example 7
Improved ELISA Method
[0373] ELISAs were performed to detect soluble HRG in serum samples
using the human NRG1-b1/HRG1-b1 DuoSet ELISA Development kit
(R&D Systems, Inc., Minneapolis, Minn.; catalog #DY377). To
perform the assays, 100 .mu.l of capture antibody solution
containing 4.0 .mu.g/ml mouse anti-human NRG1-b1 in PBS was added
to each well of a 96-well plate. The capture antibody solution was
prepared by diluting 180-fold with PBS (e.g., 5 .mu.l of the
original solution in the kit was added to 895 .mu.l of PBS for 8
wells). The plates were then covered and kept at room temperature
overnight.
[0374] The capture antibody solution was then aspirated and the
wells were washed by either (i) a "control wash" method in which
each well was filled with 400 .mu.l wash buffer using a squirt
bottle after which the wash buffer was removed, and the process was
repeated two additional times for a total of three washes, or (ii)
an "improved wash" method. For the "improved wash" method, 180
.mu.l of wash buffer was added to each well using a multichannel
pipettor and the wash buffer was cycled by performing a piston
action 20 times until the wash buffer was bubbly. The wash buffer
was then aspirated and the cycling was repeated with another 180
.mu.l of wash buffer using a piston action 20 times until the wash
buffer was bubbly. The wash buffer was then aspirated and a rinse
step was performed in which 200 .mu.l of wash buffer was added to
each well and then immediately aspirated, ensuring that all bubbles
were removed. Thus, the improved wash method involved two wash
steps using a piston action and one rinse step. The wash buffer
contained 0.05% TWEEN.RTM. 20 detergent in PBS, pH 7.2-7.4 and was
prepared by diluting the provided solution 25-fold with distilled
water (i.e., 4 ml of original solution+96 ml of distilled
water).
[0375] 300 .mu.l of a reagent diluent containing 1% BSA in PBS, pH
7.2-7.4, 0.2 um filtered was then added to each well and the plate
was covered and kept at room temperature for one hour. During this
time, a heregulin standard was prepared (described below).
Following the 1 hour incubation, the reagent diluent was aspirated
and the wells were washed using either the control wash method or
the improved wash method described above.
[0376] 100 .mu.l of undiluted serum or plasma (or heregulin
standard) was then added to each well, after which the plate was
covered and kept at room temperature for two hours. The sample (or
standard) was aspirated and the wells were washed using either the
control wash method or the improved wash method.
[0377] 100 .mu.l of a detection antibody solution containing 150
ng/ml biotinylated goat anti-human NRG1-b1 antibody in reagent
diluent was then added to each well and the plates were covered and
kept at room temperature for two hours. The detection antibody
solution was prepared by diluting the original solution 180-fold
with reagent diluent (e.g. 5 .mu.l of original solution+895 .mu.l
of Reagent Diluent for 8 wells). Although the kit instructions call
for the addition of 2% heat inactivated normal goat serum (NGS),
NGS was not included in the detection antibody solution for these
experiments.
[0378] The detection antibody solution was aspirated and the wells
were washed using either the control wash method or the improved
wash method. 100 .mu.l of a working solution of streptavidin-HRP
solution was then added to each well and allowed to incubate at
room temperature for 20 minutes under shaded conditions. The vial
of streptavidin-HRP solution provided with the kit contained 1.0 mL
of streptavidin conjugated to HRP and the working concentration was
prepared by diluting the contents of the vial with reagent diluent
as directed on the vial label. Following the 20 minute incubation,
the streptavidin-HRP solution was aspirated and the wells were
washed using either the control wash method or the improved wash
method.
[0379] 100 .mu.l of substrate solution containing a 1:1 mixture of
H.sub.2O.sub.2 (Color reagent A) and tetramethylbenzidine (Color
reagent B) was then added to each well and allowed to incubate at
room temperature for 20 minutes under shaded conditions after which
50 .mu.L of stop solution was added to each well. The stop solution
contained 2-NH.sub.2SO.sub.4. The optical density of each well was
then measured at OD.sub.450 (correction by OD.sub.570).
[0380] Preparation of the heregulin Standard was as follows:
TABLE-US-00007 TABLE 7 Preparation of heregulin Standard Final
concentration Stock 275 ng/ml Compound + Reagent Diluent 0.5 ml
Solution A 4000 pg/ml Stock 5 .mu.l + Reagent Diluent 337 ml B 2000
pg/ml A 200 .mu.l + Reagent Diluent 200 .mu.l C 1000 pg/ml B 200
.mu.l + Reagent Diluent 200 .mu.l D 500 pg/ml C 200 .mu.l + Reagent
Diluent 200 .mu.l E 250 pg/ml D 200 .mu.l + Reagent Diluent 200
.mu.l F 125 pg/ml E 200 .mu.l + Reagent Diluent 200 .mu.l G 62.5
pg/ml F 200 .mu.l + Reagent Diluent 200 .mu.l H 0 pg/ml Reagent
Diluent 200 .mu.l
[0381] The attempts to detect soluble HRG using the published kit
instructions (with the control wash method) failed consistently.
However, a varied range of soluble HRG in serum was be able to be
measured with high reproducibility using the process with the
improved wash method.
Example 8
Biomarker Identification
[0382] The HRG biomarker was identified by analysing the anti-tumor
activity of the anti-HER3 antibody U3-1287 on various human cancer
xenografts in vivo and analysis of the expression of HRG of these
cell lines in vitro. Human tumor cell lines of various indications
were grown as xenografts in mice and treated with the anti-HER3
antibody U3-1287 for several weeks. Inhibition of tumor growth was
analysed by comparing the tumor volumes of control mice and mice
treated with U3-1287. Human tumor cell lines were grown in vitro
and analysed for HRG protein expression by Western blotting. The
results of this analysis are shown in Table 8. Basal activity of
HER3 as measured by Western blotting did not correlate with in vivo
efficacy of U3-1287, predominantly in FISH positive breast cancer
models. In contrast, expression of HRG correlated very well with in
vivo efficacy of U3-1287, as seen for 15 of the 17 models
analyzed.
TABLE-US-00008 TABLE 8 Retrospective in vitro analysis of cell
lines used for in vivo xenografts Phospho In vivo HER HER3 HRG
efficacy Corre- Cell Line Indication (WB) (WB) (WB) (SA) lation
Sum225 BC FISH+ve + + - No Yes MDA- BC FISH+ve + + - No Yes MB453
BT474 BC FISH+ve + + - No Yes HCC 1569 BC FISH+ve + + - No Yes
ZR75-1 BC FISH+ve + - - No Yes MCF-7 BC FISH+ve + - - No Yes T47D
BC FISH+ve + + - No Yes NCI-H441 NSCLC + + - No Yes A549 NSCLC + +
+ Yes Yes Calu-3 NSCLC + + + Yes Yes NC-H1975 NSCLC + + + Yes Yes
A375 Melanoma + - - No Yes HT-144 Melanoma + - - No Yes HT-29 Colon
+ + - Yes No MKN-45 Gastric + + - Yes No BxPCS Pancreas + + + Yes
Yes FaDu Head&Neck + + + Yes Yes
[0383] U3-1287 efficacy was determined in vitro by measuring
reduction of phospho-HER3 and phospho-AKT levels. Basal HER3
phosphorylation could be blocked in cell lines that endogenously
express heregulin (A549) as well as in cells that do not have basal
HER3 activation but were stimulated with exogenous heregulin
(CaOV3). U3-1287 efficacy results are shown in FIG. 9.
[0384] Unexpectedly cells that have basal HER3 phosphorylation but
do not express heregulin showed no efficacy upon U3-1287 treatment
(BT474 basal) and even more surprisingly, this could be overcome by
exogenously added heregulin (BT474+HRG), as shown in FIG. 10.
INCORPORATION BY REFERENCE
[0385] The entire disclosure of each of the patent documents and
scientific articles cited herein is incorporated by reference for
all purposes.
* * * * *