U.S. patent application number 15/386723 was filed with the patent office on 2017-07-27 for biomarker profiles for predicting outcomes of cancer therapy with erbb3 inhibitors and/or chemotherapies.
The applicant listed for this patent is Merrimack Pharmaceuticals, Inc.. Invention is credited to Bambang ADIWIJAYA, Akos CZIBERE, William KUBASEK, Gavin MACBEATH, Sharon MOULIS, Rachel C. NERING, Lin NIE, Defne YARAR.
Application Number | 20170210810 15/386723 |
Document ID | / |
Family ID | 52424111 |
Filed Date | 2017-07-27 |
United States Patent
Application |
20170210810 |
Kind Code |
A1 |
ADIWIJAYA; Bambang ; et
al. |
July 27, 2017 |
BIOMARKER PROFILES FOR PREDICTING OUTCOMES OF CANCER THERAPY WITH
ERBB3 INHIBITORS AND/OR CHEMOTHERAPIES
Abstract
Provided are methods for optimizing therapy of, treating a
patient having, or selecting (identifying) patients who will
benefit from treatment for, a cancer (e.g., a non-hematological
cancer; e.g., a gynecological cancer). The methods comprise
determining whether the patient will benefit from treatment with an
ErbB3 inhibitor (e.g., an anti-ErbB3 antibody), with or without
either a taxane or an aromatase inhibitor, or with a taxane or an
aromatase inhibitor in the absence of an ErbB3 inhibitor, based on
levels of particular biomarkers and combinations of biomarkers
measured in a biological sample obtained from the patient. The
methods further comprise optimizing the patient's therapy,
selecting the patient for treatment, or treating the patient
accordingly. In various aspects the biological samples are sections
of a biopsy (e.g., a formalin fixed paraffin embedded biopsy). In
other aspects the biomarkers are proteins and/or nucleic acids. In
other aspects the biomarkers function in ErbB-mediated signal
transduction.
Inventors: |
ADIWIJAYA; Bambang;
(Belmont, MA) ; CZIBERE; Akos; (Medford, MA)
; KUBASEK; William; (Belmont, MA) ; MACBEATH;
Gavin; (Wakefield, MA) ; MOULIS; Sharon;
(Manchester, NH) ; NERING; Rachel C.; (Stoneham,
MA) ; NIE; Lin; (Needham Heights, MA) ; YARAR;
Defne; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merrimack Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
52424111 |
Appl. No.: |
15/386723 |
Filed: |
December 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15156262 |
May 16, 2016 |
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15386723 |
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PCT/US2014/072594 |
Dec 29, 2014 |
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15156262 |
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61921185 |
Dec 27, 2013 |
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62005683 |
May 30, 2014 |
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62027042 |
Jul 21, 2014 |
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62055382 |
Sep 25, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/112 20130101;
G01N 2333/71 20130101; C07K 16/3023 20130101; C07K 2317/34
20130101; C07K 16/3069 20130101; C07K 2317/565 20130101; C12Q
2600/158 20130101; G01N 33/574 20130101; C07K 16/3015 20130101;
A61K 39/3955 20130101; A61K 31/337 20130101; C07K 2317/56 20130101;
C07K 16/2863 20130101; C12Q 2600/106 20130101; C12Q 1/6886
20130101; G01N 2800/52 20130101; C07K 2317/21 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; C12Q 1/68 20060101
C12Q001/68; A61K 31/337 20060101 A61K031/337 |
Claims
1-56. (canceled)
57. A method of treating a human patient having lung cancer with a
heregulin RNA in situ hybridization (RNA-ISH) score of 1+ or higher
measured in a biological sample from the patient, the method
comprising administering to the patient an effective amount of: 1)
a first agent that is an anti-ErbB3 antibody, wherein the
anti-ErbB3 antibody comprises CDRH1, CDRH2, and CDRH3 sequences
comprising the amino acid sequences set forth in SEQ ID NO: 3
(CDRH1), SEQ ID NO: 4 (CDRH2), and SEQ ID NO: 5 (CDRH3),
respectively, and CDRL1, CDRL2, and CDRL3 sequences comprising the
amino acid sequences set forth in SEQ ID NO: 6 (CDRL1), SEQ ID NO:
7 (CDRL2), and SEQ ID NO: 8 (CDRL3), respectively; and 2) a second
agent that is an anticancer agent, wherein the RNA-ISH uses one or
more probes comprising: a) an 18- to 25-nucleotide region that is
complementary to the target RNA, b) a spacer sequence; and c) a
14-nucleotide tail sequence.
58. The method of claim 57, wherein the anti-ErbB3 antibody
comprises V.sub.H and V.sub.L amino acid sequences set for the in
SEQ ID NO:1 and SEQ ID NO:2, respectively.
59. The method of claim 57, wherein the second anticancer agent is
a taxane.
60. The method of claim 59, wherein the second anticancer agent is
paclitaxel.
61. The method of claim 59, wherein the second anticancer agent is
docetaxel.
62. The method of claim 57, wherein the one or more probes comprise
a pool of oligonucleotides that hybridize to a select region of the
heregulin mRNA.
63. The method of claim 57, wherein the probes specifically
hybridize to heregulin mRNAs that encode each of heregulin isoforms
.alpha., .beta.1, .beta.1b, .beta.1c, .beta.1d, .beta.2, .beta.2b,
.beta.3, .beta.3b, .gamma., .gamma.2, .gamma.3, ndf43, ndf43b, and
GGF2.
64. The method of claim 57, wherein the probes hybridize in pairs
to multiple adjacent sequences on the heregulin mRNA.
65. The method of claim 64, wherein the pairs of probes have
different tail sequences which form a 28-base hybridization site
for a preamplifier oligo.
66. The method of claim 57, wherein the probes span about 1 kb of
the heregulin mRNA and comprise about 20 oligonucleotide
hybridization pairs.
67. The method of claim 57, wherein the sample is a microtome
section of a biopsy.
68. The method of claim 67, wherein the biopsy is a formalin fixed
and paraffin embedded biopsy.
69. The method of claim 57, wherein the sample was taken from the
patient within 90 days prior to treating the patient.
70. The method of claim 57, wherein the treatment produces at least
one therapeutic effect selected from the group consisting of
reduction in size of a tumor, reduction in number of metastatic
lesions over time, complete response, partial response, stable
disease, or a pathologic complete response.
71. The method of claim 57, wherein the lung cancer is NSCLC.
72. A method of treating a human patient having lung cancer
comprising: 1) selecting a human lung cancer patient having a
heregulin RNA in situ hybridization (RNA-ISH) score of 1+ or higher
measured in a biological sample from the patient, and 2)
administering to the patient (a) an effective amount of a first
agent that is an anti-ErbB3 antibody, wherein the anti-ErbB3
antibody comprises CDRH1, CDRH2, and CDRH3 sequences comprising the
amino acid sequences set forth in SEQ ID NO: 3 (CDRH1), SEQ ID NO:
4 (CDRH2), and SEQ ID NO: 5 (CDRH3), respectively, and CDRL1,
CDRL2, and CDRL3 sequences comprising the amino acid sequences set
forth in SEQ ID NO: 6 (CDRL1), SEQ ID NO: 7 (CDRL2), and SEQ ID NO:
8 (CDRL3), respectively; and (b) a second agent that is an
anticancer agent, wherein the RNA-ISH uses one or more probes
comprising: a) an 18- to 25-nucleotide region that is complementary
to the target RNA, b) a spacer sequence; and c) a 14-nucleotide
tail sequence.
73. The method of claim 72, wherein the anti-ErbB3 antibody
comprises V.sub.H and V.sub.L amino acid sequences set for the in
SEQ ID NO:1 and SEQ ID NO:2, respectively.
74. The method of claim 72, wherein the second anticancer agent is
a taxane.
75. The method of claim 74, wherein the second anticancer agent is
paclitaxel.
76. The method of claim 74, wherein the second anticancer agent is
docetaxel.
77. The method of claim 72, wherein the treatment produces at least
one therapeutic effect selected from the group consisting of
reduction in size of a tumor, reduction in number of metastatic
lesions over time, complete response, partial response, stable
disease, or a pathologic complete response.
78. The method of claim 72, wherein the lung cancer is NSCLC.
79. A method of treating a human patient having lung cancer with a
positive heregulin RNA in situ hybridization (RNA-ISH) score
measured in a biological sample from the patient, wherein the
positive score is at least 1 dot/cell in at least 10% of tumor
cells from the biological sample, and wherein the method comprises
administering to the patient an effective amount of: 1) a first
agent that is an anti-ErbB3 antibody, wherein the anti-ErbB3
antibody comprises CDRH1, CDRH2, and CDRH3 sequences comprising the
amino acid sequences set forth in SEQ ID NO: 3 (CDRH1), SEQ ID NO:
4 (CDRH2), and SEQ ID NO: 5 (CDRH3), respectively, and CDRL1,
CDRL2, and CDRL3 sequences comprising the amino acid sequences set
forth in SEQ ID NO: 6 (CDRL1), SEQ ID NO: 7 (CDRL2), and SEQ ID NO:
8 (CDRL3), respectively; and 2) a second agent that is an
anticancer agent.
80. The method of claim 79, wherein the anti-ErbB3 antibody
comprises V.sub.H and V.sub.L amino acid sequences set for the in
SEQ ID NO:1 and SEQ ID NO:2, respectively.
81. The method of claim 79, wherein the second anticancer agent is
a taxane.
82. The method of claim 81, wherein the second anticancer agent is
paclitaxel.
83. The method of claim 81, wherein the second anticancer agent is
docetaxel.
84. The method of claim 79, wherein the positive score is at least
1-3 dots/cell in at least 10% of tumor cells.
85. The method of claim 79, wherein the positive score is at least
4 dots/cell in at least 10% of tumor cells.
86. The method of claim 79, wherein the biological sample comprises
at least 50 tumor cells.
87. The method of claim 79, wherein the sample is a microtome
section of a biopsy.
88. The method of claim 87, wherein the biopsy is a formalin fixed
and paraffin embedded biopsy.
89. The method of claim 79, wherein the sample was taken from the
patient within 90 days prior to treating the patient.
90. The method of claim 79, wherein the treatment produces at least
one therapeutic effect selected from the group consisting of
reduction in size of a tumor, reduction in number of metastatic
lesions over time, complete response, partial response, stable
disease, or a pathologic complete response.
91. The method of claim 79, wherein the lung cancer is NSCLC.
92. A method of treating a human patient having lung cancer
comprising: 1) selecting a human lung cancer patient having a
positive RNA in situ hybridization (RNA-ISH) score measured in a
biological sample from the patient, wherein the positive score is
at least 1 dot/cell in at least 10% of tumor cells from the
biological sample, and 2) administering to the patient (a) an
effective amount of a first agent that is an anti-ErbB3 antibody,
wherein the anti-ErbB3 antibody comprises CDRH1, CDRH2, and CDRH3
sequences comprising the amino acid sequences set forth in SEQ ID
NO: 3 (CDRH1), SEQ ID NO: 4 (CDRH2), and SEQ ID NO: 5 (CDRH3),
respectively, and CDRL1, CDRL2, and CDRL3 sequences comprising the
amino acid sequences set forth in SEQ ID NO: 6 (CDRL1), SEQ ID NO:
7 (CDRL2), and SEQ ID NO: 8 (CDRL3), respectively; and (b) a second
agent that is an anticancer agent.
93. The method of claim 92, wherein the anti-ErbB3 antibody
comprises V.sub.H and V.sub.L amino acid sequences set for the in
SEQ ID NO:1 and SEQ ID NO:2, respectively.
94. The method of claim 92, wherein the second anticancer agent is
a taxane.
95. The method of claim 92, wherein the second anticancer agent is
paclitaxel.
96. The method of claim 92, wherein the second anticancer agent is
docetaxel.
97. The method of claim 92, wherein the treatment produces at least
one therapeutic effect selected from the group consisting of
reduction in size of a tumor, reduction in number of metastatic
lesions over time, complete response, partial response, stable
disease, or a pathologic complete response.
98. The method of claim 92, wherein the lung cancer is NSCLC.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/156,262, filed on May 16, 2016, which is a
continuation-in-part of International Application No.
PCT/US2014/072594, filed on Dec. 29, 2014, which claims priority
to, and the benefit of, the following U.S. Provisional
Applications: No. 61/921,185, filed on Dec. 27, 2013; No.
62/005,683, filed on May 30, 2014; No. 62/027,042, filed on Jul.
21, 2014; and No. 62/055,382, filed on Sep. 25, 2014. The contents
of the aforementioned applications are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 20,
2016, is named Sequence_Listing_MMJ-044PCCPCN.txt and is 42,104
bytes in size.
BACKGROUND
[0003] Targeted therapies for the treatment of cancer include
monoclonal antibodies that bind to antigens that are expressed on
tumor cells. For example, cetuximab (Erbitux.RTM.) is a monoclonal
antibody that targets the epidermal growth factor receptor (EGFR,
also known as ErbB1 or HER1). While such monoclonal antibodies have
been shown to be effective in some patients, the response rate for
targeted therapies (and for untargeted chemotherapeutics) is never
100%. For example, only about 15-20% of patients whose tumors
express EGFR respond to cetuximab monotherapy. Thus, expression by
a tumor of an antigen that is targeted by a therapeutic monoclonal
antibody does not necessarily predict responsiveness to treatment
with the antibody.
[0004] In addition to EGFR, the ErbB/HER subfamily of polypeptide
growth factor receptors include the neu oncogene product ErbB2
(HER2), and the more recently identified ErbB3 (HER3) and ErbB4
(HER4) proteins. Experiments in vitro have indicated that the
protein tyrosine kinase activity of the ErbB3 protein is
significantly attenuated relative to that of other ErbB/HER family
members. Despite its deficient kinase activity, the ErbB3 protein
has been shown to be phosphorylated in a variety of cellular
contexts and to play an important role in ErbB signal transduction,
e.g., in cancer cells. For example, ErbB3 is constitutively
phosphorylated on tyrosine residues in a subset of human breast
cancer cell lines that highly express this protein.
[0005] An explanation for the phosphorylation of ErbB3 and its
oncogenic impact is that, in addition to forming various active
homodimers, ErbBs are cell surface receptor proteins that form
heterodimeric receptor complexes that mediate ligand-dependent (and
in some cases ligand-independent) activation of multiple signal
transduction pathways. ErbB3, upon binding to heregulin (HRG), its
primary physiological ligand, heterodimerizes more efficiently with
other ErbB family members than it does in the absence of ligand. It
is the fully kinase-active ErbB hetero-partner of ErbB3 in such
heterodimers that is believed to phosphorylate ErbB3, promoting
high levels of (potentially oncogenic) signal transduction by the
heterodimer. ErbB3-containing heterodimers (such as ErbB2/ErbB3) in
tumor cells have been shown to be the most mitogenic and oncogenic
ErbB receptor complexes. Accordingly, ErbB3 inhibitors, including
anti-ErbB3 monoclonal antibodies, are in development for use in the
treatment of various cancers.
[0006] The variable response rates of patients to monoclonal
antibody therapies and chemotherapies indicates that methods are
needed for accurately predicting which patients are likely to
respond to therapeutic treatment with such targeted and untargeted
agents so that the treatment can be administered to only those
patients who are likely to receive benefits that outweigh the
financial costs and potential deleterious effects of treatment
(including the damage to the patient due to tumor growth over time
during the administration of the ineffective treatments).
Particular biomarkers or sets of biomarkers (e.g., gene products
such as proteins or RNAs) in tumors may be found for which a
particular concentration range for each biomarker (e.g., in the
set) correlates with tumor responsiveness to a particular
therapy.
[0007] Accordingly, considerable efforts are being made to discover
and identify characteristic biomarkers whose levels are indicative
of the probability of a particular individual tumor being
responsive to particular therapies. The following disclosure
provides novel biomarker criteria that allow for optimization of
tumor therapy using ErbB3 inhibitors, chemotherapeutic agents (such
as taxanes, anti-estrogens, topoisomerase inhibitors, and
nucleoside analogs) or combinations thereof, and provides
additional benefits.
SUMMARY
[0008] Provided herein are methods for a) optimizing therapy for,
or b) selecting for ErbB3-targeted or other heregulin-inhibitory
treatment and/or chemotherapeutic treatment or other
heregulin-neutral treatment, or c) for treating; a patient having a
cancer (e.g., a non-hematological cancer). These methods comprise
determining whether the patient is likely to benefit from treatment
with an ErbB3 inhibitor (i.e., an anti-ErbB3 antibody or another
agent that inhibits the activation of ErbB3 by heregulin) or any
other heregulin-inhibitory treatment with or without treatment with
a heregulin-neutral treatment such as a taxane or estrogen
inhibitor and whether or not the patient is likely to benefit from
treatment with a chemotherapeutic agent without co-administration
of an ErbB3 inhibitor. The determination is based on the measuring
or scoring of levels of one or more of four particular biomarkers;
ErbB2, ErbB3, ErbB4, and HRG. Each of the four particular
biomarkers may be detected and measured as a protein, and may also
or alternatively be detected and measured as an RNA (e.g., a gene
transcript) that encodes the protein or specifically hybridizes
with sequences encoding the protein. These levels are measured in
at least one biological sample (biopsy) obtained from the patient.
With regard to ErbB2 (HER2), certain cancer types typically express
this marker at low levels and many rarely express ErbB2 at levels
high enough to be scored as 2+ or 3+. As the frequency of cancers
of these types that overexpress ErbB2 is low (e.g., less than 10%,
or less than 5% of such cancers overexpress ErbB2, or in some cases
less than 30% or less than 20%), it is possible in such cases to
score levels of ErbB2 the practice of the disclosed methods by
reference to cancer type (with cancer types that infrequently
overexpress ErbB2 being scored as having fewer than 126,000 ErbB2
receptors per cell or as ErbB2 1+), rather than by measuring ErbB2
in a biological sample. Such cancer types may be defined in terms
of the organ or tissue of origin as well as by the ethnicity of the
patient.
[0009] Accordingly, in one aspect, the invention provides, a method
of I) selecting therapy for a patient having a cancer, II)
improving or optimizing therapeutic efficacy of treatment of a
cancer in a patient, III) treating a cancer in a patient, or IV)
ordering treatment of a cancer in a patient; the method comprising:
[0010] (a) obtaining one or more biomarker scores obtained from a
biological sample from the patient, wherein the scored biomarker is
one or more of ErbB2, ErbB3, ErbB4, or HRG, or any combination
thereof; and [0011] (b) if the one or more scores meet a threshold,
then [0012] 1) selecting, and/or [0013] 2) administering, and/or
[0014] 3) ordering the administration of, [0015] an effective
amount of an ErbB3 inhibitor to the patient, and optionally thereby
improving or optimizing therapeutic efficacy of treatment of the
cancer in the patient, wherein the threshold is one or more of the
following: [0016] (i) an immunohistochemistry (IHC) score for ErbB2
of less than 2+, [0017] (ii) fewer than 126,000 ErbB2 receptors per
tumor cell; [0018] (iii) an ErbB3 IHC score of 2+ or higher; [0019]
(iv) an ErbB4 IHC score of less than 1+; [0020] (v) a HRG RNA-ISH
score of 1+ or higher; [0021] (vi) a HRG RT-PCR score of greater
than or equal to -5; [0022] (vii) fewer than 126,000 ErbB2
receptors per tumor cell and a HRG RT-PCR score of greater than or
equal to -5; [0023] (viii) an IHC score for ErbB2 of less than 2+
with a HRG RNA-ISH score of 1+ or higher; [0024] (ix) an IHC score
for ErbB2 of less than 2+ with a HRG RNA-ISH score of 2+ or higher;
[0025] (x) an IHC score for ErbB2 of less than 3+ with a HRG
RNA-ISH score of 2+ or higher; or [0026] (xi) fewer than 200,000
ErbB2 receptors per tumor cell with a HRG RNA-ISH score of 2+ or
higher.
[0027] In one variation of the above aspect, the scored biomarkers
comprise ErbB3 and HRG or comprise ErbB3 and HRG and ErbB2, and the
biological sample comprises tumor cells and the tumor cells
comprise human phosphoinisitide-3-kinase catalytic subunit
(PI3KCA)-encoding sequences comprising an activating mutation of
PI3KCA and the one or more scores include (iii) and either a)
either or both of (v) and (vi) or b) any one of (vii), (viii),
(ix), (x), or (xi).
[0028] In an alternative embodiment, one or more scores for ErbB2,
ErbB3, ErbB4, or HRG are measured in a patient biopsy of a cancer,
and if the one or more scores indicate any one of the following
conditions, a therapy for the patient is selected and/or ordered
and/or administered that comprises ErbB3 inhibitor therapy and
taxane therapy or ErbB3 inhibitor therapy and an anti-estrogen
therapy: [0029] a) ErbB2 low; [0030] b) HRG positive; [0031] c)
ErbB2 low AND HRG positive; [0032] d) ErbB3 medium/high; [0033] e)
ErbB2 low AND ErbB3 medium/high; [0034] f) HRG positive AND ErbB3
medium/high; [0035] g) ErbB2 low AND HRG positive AND ErbB3
medium/high; [0036] h) ErbB4 negative; [0037] i) ErbB2 low AND
ErbB4 negative; [0038] j) HRG positive AND ErbB4 negative; [0039]
k) ErbB2 low AND HRG positive AND ErbB4 negative; [0040] l) ErbB3
medium/high AND ErbB4 negative; [0041] m) ErbB2 low AND ErbB3
medium/high AND ErbB4 negative; [0042] n) HRG positive AND ErbB3
medium/high AND ErbB4 negative; or [0043] o) ErbB2 low AND HRG
positive AND ErbB3 medium/high AND ErbB4 negative; where, in a)-o)
immediately above, ErbB2 low is defined as ErbB2.ltoreq.(about)
126,000 receptors per cell; HRG positive is defined as HRG
score.gtoreq.1; ErbB3 medium/high is defined as ErbB3
score.gtoreq.2; and ErbB4 negative is defined as ErbB4 score=0.
[0044] In another aspect, the invention provides, a method of I)
selecting therapy for a patient having a cancer, II) optimizing
therapeutic efficacy of treatment of cancer in a patient, III)
treating cancer in a patient, or IV ordering the treatment of a
cancer in a patient; the method comprising: [0045] (a) obtaining
one or more biomarker scores obtained from a biological sample from
the patient, wherein the scored biomarker is one or more of ErbB2,
ErbB3, ErbB4, or HRG, or any combination thereof; and [0046] (b) if
the one or more scores meet a threshold, then administering to the
patient (or ordering the administration to the patient of) an
effective amount of an estrogen inhibitor (e.g., an aromatase
inhibitor, e.g., exemestane), [0047] wherein the threshold is one
or more of the following: [0048] (i) an immunohistochemistry (IHC)
score for ErbB2 of less than 2+, [0049] (ii) fewer than 126,000
ErbB2 receptors per tumor cell; [0050] (iii) an ErbB3 IHC score of
2+ or higher; [0051] (iv) an ErbB4 IHC score of less than 1+;
[0052] (v) a HRG RNA-ISH score of 1+ or higher; [0053] (vi) a HRG
RT-PCR score of greater than or equal to -5; [0054] (vii) fewer
than 126,000 ErbB2 receptors per tumor cell with a HRG RT-PCR score
of greater than or equal to -5; [0055] (viii) an IHC score for
ErbB2 of less than 2+ with a HRG RNA-ISH score of 1+ or higher;
[0056] (ix) an IHC score for ErbB2 of less than 2+ with a HRG
RNA-ISH score of 2+ or higher; [0057] (x) an IHC score for ErbB2 of
less than 3+ with a HRG RNA-ISH score of 2+ or higher; or [0058]
(xi) fewer than 200,000 ErbB2 receptors per tumor cell with a HRG
RNA-ISH score of 2+ or higher.
[0059] In one variation of the above aspect, the scored biomarkers
comprise ErbB3 and HRG or comprise ErbB3 and HRG and ErbB2, and the
biological sample comprises tumor cells and the tumor cells
comprise human phosphoinisitide-3-kinase catalytic subunit
(PI3KCA)-encoding sequences comprising an activating mutation of
PI3KCA and the one or more scores include (iii) and either a)
either or both of (v) and (vi) or b) any one of (vii), (viii),
(ix), (x), or (xi).
[0060] The estrogen inhibitor may be an estrogen receptor blocker
such as tamoxifen, a selective estrogen receptor modulator such as
raloxifene or an aromatase inhibitor such as exemestane.
[0061] For use in the described methods, exemplary assays include
immunohistochemistry assays, immunofluorescence assays, and in situ
hybridization assays, such as those provided below.
[0062] An exemplary assay by which a HRG score can be determined is
an RNA-in situ hybridization (RNA-ISH) assay. In one embodiment,
the RNA-ISH is read out via a chromogenic signal as set forth
below. In a particular embodiment, the probes used to detect HRG by
RNA-ISH hybridize specifically to a nucleic acid that comprises
nucleotides 442-2977 of the nucleotide sequence set forth in
GenBank accession number NM-013956 (SEQ ID NO:42). In certain
embodiments the probes hybridize specifically to RNAs encoding each
of the HRG isoforms .alpha., .beta.1, .beta.1b, .beta.1c, .beta.1d,
.beta.2, .beta.2b, .beta.3, .beta.3b, .gamma., .gamma.2, .gamma.3,
ndf43, ndf34b, and GGF2.
[0063] In another embodiment, the HRG score is determined by RT-PCR
using probes specific for HRG.
[0064] In the case of ErbB2, ErbB3, and ErbB4, the score can be
determined using an immunohistochemistry (IHC) assay. In one
embodiment, the IHC assay is a read out quantitatively via a
chromogenic signal (qIHC). In other embodiments, the IHC assay is
an immunofluorescence assay.
[0065] ErbB3 inhibitors for use in the methods described herein
include antibodies, nucleic acids (such an RNA that inhibits the
expression of ErbB3 or of heregulin), or proteins (e.g., an
anti-heregulin antibody, a soluble form of the ErbB3 receptor that
inhibits signaling by trapping ErbB3 ligands) or small molecules
(e.g., a sheddase protease inhibitor that blocks the cleavage of
active heregulin from its larger precursor protein). In each of the
foregoing methods, the ErbB3 inhibitor may be formulated with a
pharmaceutically acceptable carrier.
[0066] In a particular embodiment, the ErbB3 inhibitor is an
anti-ErbB3 antibody. An exemplary anti-ErbB3 antibody is MM-121
(SAR256212), comprising V.sub.H and V.sub.L sequences as shown in
SEQ ID NOs: 1 and 2, respectively. Another exemplary anti-ErbB3
antibody is an antibody comprising in amino-terminal to
carboxy-terminal order, V.sub.H CDR1, 2 and 3 sequences as shown in
SEQ ID NOs: 3-5, respectively, and V.sub.L CDR1, 2 and 3 sequences
as shown in SEQ ID NOs: 6-8, respectively. In other embodiments,
the anti-ErbB3 antibody is Ab #3 (comprising V.sub.H and V.sub.L
sequences as shown in SEQ ID NOs: 9 and 10, respectively), Ab #14
(comprising V.sub.H and V.sub.L sequences as shown in SEQ ID NOs:
17 and 18, respectively), Ab #17 (comprising V.sub.H and V.sub.L
sequences as shown in SEQ ID NOs: 25 and 26, respectively) or Ab
#19 (comprising V.sub.H and V.sub.L sequences as shown in SEQ ID
NOs: 33 and 34, respectively). In still other embodiments, the
anti-ErbB3 antibody is mAb 1B4C3, mAb 2D1D12, AMG-888, humanized
mAb 8B8, AV-203, MM-141 or MEHD7945A. In another embodiment,
administration of the anti-ErbB3 antibody inhibits growth or
invasiveness or metastasis of the tumor.
[0067] The methods provided herein can be used to determine
whether: I) a cancer in a patient is likely to respond to treatment
with ErbB3 inhibitors, II) to select treatment for a cancer in a
patient, III) to order treatment for a cancer in a patient, or IV
to treat a cancer in a patient. In one embodiment, the cancer is a
non-hematological cancer (e.g., a solid tumor). In a particular
embodiment, the cancer is an ovarian cancer. In another embodiment,
the cancer is a platinum resistant ovarian cancer. In another
embodiment, the cancer is a breast cancer. In a further embodiment,
the cancer is either or both of ER+ and PR+. In yet a further
embodiment, the cancer is either or both of ER+ and PR+ and is a
HER2 negative breast cancer. In an additional embodiment the cancer
is a lung cancer, e.g. a non-small cell lung cancer (NSCLC).
[0068] Any suitable tumor biopsy sample can be used in the methods
described herein. In one embodiment, the sample is a microtome
section of a biopsy (e.g., which was formalin fixed and paraffin
embedded prior to microtome sectioning). In another embodiment the
biopsy is a blood draw comprising circulating tumor cells. In a
further embodiment, the biopsy is obtained within 30, 60, or 90
days prior to treating the patient.
[0069] In another aspect, the treatment methods provided herein
further comprise administering to the patient at least one
additional anti-cancer agent that is not an ErbB3 inhibitor. In one
embodiment, the at least one additional anti-cancer agent comprises
at least one chemotherapeutic drug, such as a drug(s) selected from
the group consisting of platinum-based chemotherapy drugs, taxanes,
tyrosine kinase inhibitors, and combinations thereof.
[0070] In another aspect, the treatment further comprises
administering paclitaxel in combination with the ErbB3 inhibitor.
In a particular embodiment, the method comprises at least one
cycle, wherein the cycle is a period of 4 weeks, wherein for each
cycle the anti-ErbB3 antibody is administered every other week at a
dose of 20 mg/kg (except for the first cycle, in which the initial
administration of antibody may be at 40 mg/kg) and paclitaxel is
administered once per week at a dose of 80 mg/m.sup.2.
[0071] In another aspect, the treatment further comprises
administering exemestane in combination with the ErbB3 inhibitor.
In a particular embodiment, the ErbB3 inhibitor is an anti-ErbB3
antibody that is administered at an initial loading dose of 40
mg/kg and a weekly dose of 20 mg/kg thereafter together with daily
administration of 25 mg of exemestane (e.g., in the form of a
single tablet).
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] In the figure descriptions below, indications such as
"left," "right," "top," or "bottom" refer to the orientation of the
figure that agrees with the orientation of the text annotation.
[0073] FIG. 1 shows an exemplary heregulin (HRG) RNA staining
pattern in a micrograph of an ovarian cancer biopsy. Inset on upper
right shows a low magnification image of the same biopsy
sample.
[0074] FIG. 2 shows reference tissue microarray (TMA) analysis
using the HRG RNA-ISH assay. The four upper panes show
representative high magnification images for each cell line TMA
(cell line indicated by heading). Lower panes show TMAs, with the
three cell plugs along the top of each lower pane being internal
reference plugs for sample orientation. Rectangles drawn over
individual plugs indicate the locations of the corresponding high
magnification images in the panels above each array.
[0075] FIG. 3 shows the comparison of HRG RNA levels in four
control cell lines measured by (Y axis) RNA-ISH scoring of average
spots per cell area and (X-axis) qPCR.
[0076] FIG. 4 is a schematic exemplification of the steps of a qIHC
assay.
[0077] FIGS. 5A and 5B summarize biomarker analysis data from the
clinical trial described in Example 5. FIG. 5A is a graph showing
the number of patients that scored positive for any one or more of
six biomarkers out of the 220 patients in the safety population.
For each assay type (y-axis), the numbers of patients (x-axis) in
the control arm and the number of patients who received MM-121
therapy are indicated. FIG. 5B shows the ranking of biomarkers by
biomarker-treatment interactions. The x-axis shows biomarkers and
variables and the y-axis shows P-values. A horizontal line has been
drawn at a P-value of 0.4 as an indicator of very high predictive
value--this is an arbitrary cutoff--in this context P-values of
greater than 0.4 may still correspond to highly predictive
outcomes.
[0078] FIGS. 6A to 6D summarize hazard ratio (HR) data for the
various biomarkers from the clinical trial described in Example 5.
In these results, HR of less than one indicates that patients
receiving the study treatment are surviving longer than patients
receiving control treatment. FIG. 6A shows the relationship between
local HR and ErbB2 levels. The speckled dots in this figure are
observed HRs, whereas the thick solid line provides a smoothed
rendering (Smoothed HR) of these data accounting for noise in the
ErbB2 measurements. The thinner solid lines above and below the
dots represent the 95% Confidence Interval (CI). The dashed line
shows the cumulative percentage of patients by ErbB2 level. FIG. 6B
is three adjacent graphs showing the relationships between local
HRs (treatment vs. control) and biomarker levels for HRG, ErbB4,
and ErbB3 as indicated by the headers. The solid dots indicate
local HR per the Y axis labeling on the left, whereas the speckled
dots show the prevalence, i.e., percentage of patients with the
given biomarker value, per the Y axis labeling on the right. X axis
labeling is discrete for each biomarker. The black dashes above
and/or below each solid dot represent the 95% CI of the HR
estimates. FIG. 6C shows local HR (Y-axis) as a function of HRG
levels (X axis) for RT-PCR of archived tissue (FFPE) from ovarian
cancers. FIG. 6D shows local HR (Y-axis) as a function of HRG
levels (X axis) for RT-PCR of samples from archived tissue (FFPE)
from breast cancers.
[0079] FIGS. 7A to 7G show hazard ratio (HR) analysis data from the
clinical trial described in Example 5. FIGS. 7A-7F show bivariate
HR scans for six pairwise biomarker comparisons set forth in
Example 1: Headings indicate biomarker pairs. Cumulative HR scans
were performed by selecting a subpopulation of patients based on
their status regarding the 2.sup.nd biomarker of each indicated
pair, and then plotting cumulative HR across the prevalence levels
of the first biomarker of each pair. The dashed lines represent
patients with a score above zero for the biomarker; the thickest
solid lines represent patients with a 2.sup.nd biomarker score of 1
or higher (i.e. detectable levels of the 2.sup.nd biomarker), and
the thinnest solid lines represent patients with a 2.sup.nd
biomarker score of greater than or equal to 2. FIG. 7G shows local
HR as a function of ErbB2 levels for all subjects ("Unselected")
and for subjects with a HRG score of 1+ or greater ("HRG+"). Dots
and dashes indicate individual data points, heavy and light
continuous lines indicate smoothed data plots.
[0080] FIGS. 8A to 8I illustrate responses of biomarker profile
positive and negative subpopulations from the clinical trial
described in Example 5. Data collected at 60 weeks are summarized
in FIGS. 8A-8F, whereas data collected at about 16 months is
summarized in FIGS. 8G-8I. For each of the six plots in FIGS.
8A-8E, the treatment arm (paclitaxel+MM-121) is a solid line and
the control arm (paclitaxel without MM-121) is a dashed line. FIG.
8A shows a Kaplan-Meier progression-free survival (PFS) plot for
the overall (unselected) safety population. FIGS. 8B-8E are
Kaplan-Meier plots for the pairwise biomarker combination ErbB2 low
(ErbB2<2+) and HRG positive (HRG.gtoreq.1 "HRG+"). Data for
patients positive for this biomarker profile (BM+) are shown in
FIG. 8B and FIG. 8D, and data for patients negative for this
biomarker profile (BM-) are shown in FIG. 8C and FIG. 8E. FIG. 8B
and FIG. 8C are progression free survival (PFS) plots, while FIG.
8D and FIG. 8E are overall survival (OS) plots. FIG. 8F sets forth
best response rates in the treatment and control arms for the same
biomarker profile positive and negative subpopulations. Percentages
of Progressive Disease (PD), Stable Disease (SD), and Partial
Response (PR) outcomes are shown. FIG. 8G: PFS for entire study
population (unstratified). FIG. 8H: OS for entire study population
(unstratified). FIG. 8I: PFS for entire study population
(stratified). The data set forth in FIG. 8I demonstrate (inter
alia) that of the patients treated with paclitaxel alone, BM-
patients (light dashed line, in this case, heregulin-) achieved
much longer PFS than did BM+ patients (light solid line, in this
case, heregulin+), indicating that heregulin is a predictive
biomarker for this standard of care therapy.
[0081] FIGS. 9A to 9D show calculated observed local HRs
("local.hr.obs" Y axis--left scale) from the clinical trial
described in Example 6. These HRs are plotted as a series of often
overlapping points or dots, with the diameter of each point
indicating the 95% confidence interval for that data point, with HR
values plotted against regressed HER2 receptors per cell from
cytokeratin (CK) positive cells (X-axis, log 10 values shown).
Prevalence for each HER2 amount is indicated by a plot line
descending from left to right and read off the Y axis--right scale.
FIG. 9A--Median CK+cells, all patients; FIG. 9B--Median CK+ cells,
HRG+ patients; FIG. 9C--Top 10% of CK+cells, all patients; FIG.
9D--Top 10% of CK+ cells, HRG+ patients. These results demonstrate
that patients with tumors with detectable HRG levels and low HER2
levels (of log 10 5.1 or less, or 5.2 or less, or 5.3 or less) are
more likely to benefit from MM-121 therapy than are patients with
tumors exhibiting the same HER2 levels but that have not been
further selected for having detectable levels of HRG.
[0082] FIGS. 10A to 10I provide Kaplan Meier survival curves
generated from data obtained from the breast cancer clinical trial
described in Example 6. FIGS. 10A and 10B summarize data collected
at about 60 weeks, whereas FIGS. 10C-10E summarize data collected
at between 4 and 20 months. FIG. 10A: Progression-free survival
(PFS) for subpopulation with biomarker profile negative tumors (NOT
log 10 ErbB2.ltoreq.5.1 and detectable HRG). These results show
that there was little if any benefit from adding MM-121 to control
(exemestane) therapy in the biomarker negative patient population.
FIG. 10B: PFS for subpopulation with biomarker profile positive
(log 10 ErbB2.ltoreq.5.1 and detectable HRG) tumors. These results
show that there was a dramatic benefit from adding MM-121 to
exemestane therapy in the biomarker positive population, as all
control (exemestane alone) treated patients exhibited disease
progression within 20 weeks, while over half of the MM-121 plus
exemestane treated patients had not exhibited disease progression
at this time point, and over 35% still had not progressed within 60
weeks. FIG. 10C: PFS for entire study population (unstratified).
FIG. 10D: OS for entire unstratified study population. FIG. 10E:
PFS for entire study population (stratified), with BM+ patients
treated with erlotinib alone, BM+ patients treated with MM-121
+erlotinib, BM- patients treated with erlotinib alone, and BM-
patients treated with MM-121 +erlotinib. The data set forth in FIG.
10E demonstrate (inter alia) that of the patients treated with
exemestane alone, BM- patients (light dashed line, in this case,
heregulin-) achieved much longer PFS than did BM+ patients (light
solid line, in this case, heregulin+), indicating that heregulin is
a predictive biomarker for this standard of care therapy. FIG. 10F
depicts overall survival in patients who progressed in the adjuvant
setting prior to entering study. FIG. 10G depicts overall survival
in patients who progressed in the metastatic setting prior to
entering study. FIG. 10H depicts progression free survival in
patients who progressed in the adjuvant setting prior to entering
study. FIG. 10I depicts progression free survival in patients who
progressed in the metastatic setting prior to entering study.
[0083] FIG. 11 is a graph showing data indicating the best overall
response in the BM+ (heregulin+) study population from a Phase 2
trial of MM-121+erlotinib in EGFR-wild-type non-small cell lung
cancer (NSCLC) patients. Patients were treated with erlotinib alone
(N=30) or MM-121+erlotinib (n=70). Of the BM+ patients (in this
case, heregulin+) treated with erlotinib alone, none had a complete
response (CR) by the end of the study; 6.7% of patients had a
partial response (PR), 36.7% had stable disease (SD), and 53.3% had
progressive disease (PD). Of the patients treated with the
combination therapy of MM-121+erlotinib, 1 patient (1.4%) had a CR,
4.3% had a PR, 30% had SD, and 33% had PD. Light bars indicate
patients receiving combination therapy (N=19) and black bars
indicate patients receiving erlotinib alone. Data are shown as %
change in tumor volume.
[0084] FIGS. 12A to 12C are graphs showing data resulting from a
Phase 2 trial of MM-121+erlotinib in EGFR-wild-type non-small cell
lung cancer (NSCLC) patients. FIGS. 12A and 12B show data from all
patients treated with MM-121+erlotinib and erlotinib alone, showing
that in the unselected overall population there is not a
significant difference in PFS (FIG. 12A) and overall survival (FIG.
12B) between the two groups. FIG. 12C shows data sorted between
biomarker positive (BM+) patients (in this case, heregulin+) and
biomarker negative (BM-) patients (in this case, heregulin-). Shown
are BM+ patients treated with erlotinib alone, BM+ patients treated
with MM-121+erlotinib, BM- patients treated with erlotinib alone,
and BM- patients treated with MM-121+erlotinib. In contrast to the
PFS data for the overall population in FIG. 12A, FIG. 12C shows
that BM+ patients treated with MM-121+erlotinib had a longer PFS
than BM+ patients treated with erlotinib alone. The data set forth
in FIG. 12C demonstrate (inter alia) that of the patients treated
with erlotinib alone, BM- patients (light dashed line, in this
case, heregulin-) achieved much longer PFS than did BM+ patients
(light solid line, in this case, heregulin+), indicating that
heregulin is a predictive biomarker for this standard of care
therapy.
[0085] FIGS. 13A and 13B is a spreadsheet showing data for various
groupings of patients in the studies set forth above. The left-hand
column "study" indicates which of these three studies as follows:
08=Example 5, 101=Example 6, and 03=Example 8. Column "N" indicates
number of patients in the group: "N.BM+" indicates number of BM+
patients in the group; "N.BM-" indicates number of BM- patients in
the group; "BM+prev" indicates the percentage of BM+ patients in
the group; "HR" indicates hazard ratio; "HR 95% CI" indicates the
95 percent confidence interval for the hazard ratio; "P" indicates
the P value for the hazard ratio; "Median PFS BM-" indicates the
median progression free survival for BM- patients in the group; and
"Median PFS BM+" indicates the median progression free survival for
BM+ patients in the group.
[0086] FIGS. 14A to 14D are graphs showing results from a titration
of MM-121 treatment on spheroid cell cultures, both with and
without exogenous heregulin. Each cell line, NCI-N87 cells (FIG.
14A), SKBR3 cells (FIG. 14B), OVCAR8 cells (FIG. 14C), and HCC1937
cells (FIG. 14D), were mock transduced (gray circles, either GFP or
empty vector), transduced with wild-type PI3K (black squares), or
transduced with PI3K containing the H1047R activating mutation
(black triangles).
[0087] FIGS. 15A to 15D are graphs showing analysis of ErbB3
expression in HRG-stimulated OVCAR8 cells, as measured by
quantitative RT-PCR (FIG. 15A, RNA expressed as fold change of ddCT
normalized to GFP/EV) and western blot (FIG. 15B, ratio of ErbB3 to
actin normalized to N87-GFP). FIG. 15C shows re-expression of ErbB3
into control and PI3K-H1047R cells, and FIG. 15D shows
HRG-stimulated growth in H1047R mutant cells transduced with empty
ErbB3-reexpression vector (EV-NEG) and H1047R mutant cells
transduced with ErbB3 (E30X). Cells were treated with serum-free
medium alone (SFM), SFM+HRG, or SFM+HRG+MM-121.
DETAILED DESCRIPTION
[0088] Provided herein are methods for selecting and/or optimizing
therapy for patients having cancer (e.g., non-hematological
cancers) by determining whether the patient will benefit from
treatment with an ErbB3 inhibitor (e.g., an antibody, such as
MM-121), based on particular biomarker scores obtained from a
biological sample of the patient (i.e., ErbB2, ErbB3, ErbB4, HRG,
or any combination thereof). Also provided are methods of treating
patients having cancer based on particular biomarker scores
obtained from a biological sample of the patient (i.e., ErbB2,
ErbB3, ErbB4, HRG, or any combination thereof).
[0089] Definitions
[0090] "ErbB3" and "HER3" both refer to human ErbB3 protein, as
described in U.S. Pat. No. 5,480,968.
[0091] "ErbB3 inhibitor" indicates a therapeutic agent that
inhibits, downmodulates, suppresses or downregulates activity or
expression of ErbB3, e.g., an agent that does one or more of the
following: reduces cellular ErbB3 levels, reduces ligand binding to
ErbB3, and reduces ErbB3-mediated intracellular signal
transduction. The term is intended to include small molecule kinase
inhibitors, antibodies, interfering RNAs (shRNA, siRNA), soluble
receptors, and the like. An exemplary ErbB3 inhibitor is an
anti-ErbB3 antibody.
[0092] An "anti-ErbB3 antibody" is an antibody that
immunospecifically binds to the ectodomain of ErbB3 and an
"anti-ErbB2 antibody" is an antibody that immunospecifically binds
to the ectodomain of ErbB2. The antibody may be an isolated
antibody. Exemplary anti-ErbB3 antibodies inhibit ligand mediated
phosphorylation of ErbB3 by HRG, and some (such as MM-121) also
inhibit phosphorylation of ErbB3 mediated by one or more of the
EGF-like ligands EGF, TGF.alpha., betacellulin, heparin-binding
epidermal growth factor, biregulin, epigen, epiregulin, and
amphiregulin.
[0093] An "antibody," is a protein consisting of one or more
polypeptides comprising binding domains substantially encoded by
immunoglobulin genes or fragments of immunoglobulin genes, wherein
the protein immunospecifically binds to an antigen. One type of
naturally occurring immunoglobulin structural unit (e.g., an IgG)
comprises a tetramer that is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kD) and
one "heavy" chain (about 50-70 kD). "V.sub.L" and V.sub.H" refer to
the variable regions of these light and heavy chains respectively.
"Antibodies" include intact proteins as well as antigen-binding
fragments, which may be produced by digestion of intact proteins,
e.g., with various peptidases, or may be synthesized de novo either
chemically or using recombinant DNA expression technology. Such
fragments include, for example, F(ab).sub.2 dimers and Fab
monomers, and single chain antibodies. Single chain antibodies
exist, generally due to genetic engineering, as a single
polypeptide chain, e.g., single chain Fv antibodies (scFv) in which
a V.sub.H fragment and a V.sub.L fragment are joined together
(directly or through a peptide linker) to form a continuous
polypeptide that retains immunospecific binding activity.
[0094] "Immunospecific" or "immunospecifically" refer to binding
via domains substantially encoded by the variable region(s) of
immunoglobulin genes or fragments of immunoglobulin genes to one or
more epitopes of a protein of interest, but which do not
specifically bind to unrelated molecules in a sample containing a
mixed population of antigenic molecules. Typically, an antibody
binds immunospecifically to a cognate antigen with a K.sub.D with a
value of no greater than 100 nM, or preferably no greater than 50
nM, (a higher K.sub.D value indicates weaker binding) as measured
e.g., by a surface plasmon resonance assay or a cell binding
assay.
[0095] The term "platinum-based agent" refers to an organoplatinum
compound, including for example carboplatin, cisplatin, oxaliplatin
and nedaplatin.
[0096] Aromatase inhibitors are a class of drugs that inhibit the
production of estrogen by blocking the activity of aromatase, an
enzyme required for estrogen biosynthesis. As gynecological (e.g.,
breast and ovarian) cancers often require estrogen to grow,
aromatase inhibitors can inhibit growth of such tumors.
[0097] The terms "suppress", "suppression", "inhibit" and
"inhibition" as used herein, refer to any statistically significant
decrease in biological activity (e.g., tumor cell growth),
including full blocking of the activity. For example, "inhibition"
can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 100% in biological activity.
[0098] The term "patient" indicates a human subject receiving
either prophylactic or therapeutic treatment.
[0099] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative (prophylactic)
measures such as those described herein. The methods of "treatment"
employ administration to a patient of an ErbB3 inhibitor as
provided herein, for example, a patient having cancer, in order to
prevent, cure, delay, reduce the severity of, or ameliorate one or
more symptoms of the cancer, or in order to prolong the survival of
a patient beyond that expected in the absence of such
treatment.
[0100] The term "effective amount," as used herein, refers to that
amount of an agent, such as an anti-ErbB3 antibody, which is
sufficient to product a therapeutic benefit when administered to a
patient.
[0101] The terms "anti-cancer agent" and "antineoplastic agent"
refer to drugs used to treat malignancies, such as cancerous
growths.
[0102] The term "obtaining" as used in reference to biomarker
scores herein and in the claims, indicates the procurement of one
or more biomarker scores, whether directly or indirectly.
Biomarkers may be directly measured and scored by laboratory
personnel. The biomarker scores measured by the laboratory
personnel may be made available to at least one other party (e.g.,
a healthcare provider) as data (e.g., in written or electronic
format). In such embodiments, a second party "obtains" the scores
by consulting the data, e.g., by reading the data or hearing them
read.
[0103] The term "and/or", as used herein, means either or both.
[0104] "CI" indicates confidence interval.
[0105] "CV" indicates coefficient of variation.
[0106] "dH.sub.2O" indicates distilled water.
[0107] "FFPE" indicates formalin fixation and paraffin embedding
(or formalin fixed and paraffin embedded).
[0108] "Fl-IHC" indicates fluorescence-based quantitative
immunohistochemistry.
[0109] "HCT" refers to the HercepTest.RTM. assay, which is a
commercially available (DAKO) semi-quantitative immunohistochemical
assay for determination of HER2 protein (c-erbB-2 oncoprotein)
expression levels.
[0110] "HR" indicates hazard ratio--see, e.g., Spruance, et al.,
"Hazard Ratio in Clinical Trials". Antimicrob Agents Chemother
(2010) 48 (8):2787-2792.
[0111] "HRG" or "HRG1" indicates any and all isotypes of heregulin
(neuregulin-1, "NRG"), a set of naturally occurring ligands of
ErbB3.
[0112] "PCR" indicates polymerase chain reaction in any
experimental embodiment of the method first set forth in Mullis,
1987, U.S. Pat. No. 4,683,202).
[0113] "qIHC" indicates chromogenic quantitative
immunohistochemistry.
[0114] "qPCR indicates quantitative fluorogenic RT-PCR.
[0115] "RMSE" indicates root mean square error.
[0116] "RT-PCR" indicates reverse transcription followed by PCR of
the resulting reverse transcripts.
Various aspects and embodiments are described in further detail in
the following subsections.
[0117] Biomarkers
[0118] The methods described herein involve one or more particular
biomarkers, levels of which are measured in a biological sample
from the patient.
[0119] Scores for any single one of the biomarkers ErbB2, ErbB3,
ErbB4 and HRG can be used in the methods provided herein.
[0120] Additionally, scores for each of any combination of the
biomarkers described herein can be used. In one embodiment, the
scores of at least two biomarkers are used (e.g., HRG and ErbB2;
HRG and ErbB3; HRG and ErbB4; ErbB2 and ErbB3; ErbB2 and ErbB4; or
ErbB3 and ErbB4). In other embodiments, the scores of at least
three biomarkers are used.
[0121] As described above in the Summary, in embodiments in which a
plurality of scores are used, levels of ErbB2 may be determined by
reference to cancer type rather than by measuring ErbB2 in a
biological sample. In particular, in accordance with this aspect,
cancers other than breast cancer, bladder cancer, sarcoma,
endometrial cancer, esophageal cancer, gastric cancer,
gastro-esophageal junction cancer, ovarian cancer, lung cancer,
colorectal cancer, pancreatic cancer, testicular germ cell cancer,
gastric cancer, and multiple myeloma are scored as having fewer
than 126,000 ErbB2 receptors per cell or as ErbB2 1+. In one
embodiment, non-small cell lung cancers (NSCLCs) are also so
scored.
[0122] Mutational Status
[0123] The biological sample may comprise tumor cells that are
characterized by DNA sequencing, or other methods well known in the
art (such as hybridization assays) as comprising at least one
activating mutation in the catalytic subunit of human
phosphoinisitide-3-kinase (PI3KCA). Such PI3KCA activating
mutations include Exon 9 mutations and Exon 20 mutations. Exemplary
activating Exon 9 mutations include E545K, E542K, and Q546R. Other
exemplary mutations in Exon 9 include E545G, E545K/D549H Q546K, and
P539R. Exemplary activating Exon 20 mutations include H1047R and
G1049R. Other exemplary mutations in Exon 20 include H1047L, M1043V
and M1043I.
[0124] Biological Samples
[0125] The expression of one or more biomarkers may be determined
in a biological sample (biopsy) obtained from a subject. Such a
sample is typically further processed after it is obtained from the
subject. Biopsy samples suitable for detecting and quantitating the
biomarkers described herein may be fresh, frozen, or fixed.
Suitable samples are preferably sectioned. Alternatively, samples
may be solubilized and/or homogenized and subsequently
analyzed.
[0126] In one embodiment, a freshly obtained biopsy sample embedded
in a cryoprotectant such as OCT.RTM. or Cryomatrix.RTM. and frozen
using, for example, liquid nitrogen or difluorodichloromethane. The
frozen sample is serially sectioned in a cryostat. In another
embodiment, samples are fixed and embedded prior to sectioning. For
example, a tissue sample may be fixed in, for example, formalin,
gluteraldehyde, ethanol or methanol, serially dehydrated (e.g.,
using alcohol and or xylenes) and embedded in, for example,
paraffin.
[0127] In one embodiment, the sample is a microtome section of a
biopsy (e.g., FFPE prior to microtome sectioning). In another
embodiment, the biopsy was obtained within 30, 60, or 90 days prior
to treating the patient.
[0128] Detecting and Scoring Biomarkers [0129] Nucleic Acid
Assays
[0130] In various embodiments, expression of the biomarker is
detected at the nucleic acid level. For example, the biomarker
score for HRG can be assessed based on HRG RNA levels. In one
embodiment, RNA is detected using an RNA-ISH assay as discussed in
further detail below.
[0131] Another method for determining the level of RNA in a sample
involves the process of nucleic acid amplification from homogenized
tissue, e.g., by RT-PCR (reverse transcribing the RNA and then,
amplifying the resulting cDNA employing PCR or any other nucleic
acid amplification method, followed by the detection of the
amplified molecules.
[0132] In particular aspects, RNA expression is assessed by
quantitative fluorogenic RT-PCR (qPCR) e.g., by using the
TaqMan.TM. System. Such methods typically utilize pairs of
oligonucleotide primers that are specific for the nucleic acid of
interest. Further details of such assays are provided below in the
Examples. [0133] 1. Assay Specificity
[0134] In one approach, quantitative image analysis was used with
the RNA-ISH assay to count individual dots in cells, which
generally correspond to individual transcripts. A quantitative
comparison of HRG RNA levels in four control cell lines measured by
RNA-ISH and qPCR showed an R.sup.2 of 0.91 (FIG. 3), wherein
R.sup.2 is the coefficient of correlation for linear regression of
spots/cell area vs. Log 10 qPCR. [0135] 2. Limits of Detection
[0136] RNAscope.RTM. assays are designed to detect individual RNA
transcripts (each dot generally represents one transcript). Thus,
the lower limit of detection is 0 HRG RNA transcripts (0-1 dots per
cell), corresponding to a pathologist score of 0. The upper limit
of detection occurs when single dots in a cell become
indistinguishable from each other. This corresponds to a
pathologist score of 4+. [0137] 3. Reproducibility and Error
Measures
[0138] Assay reproducibility can be assessed using the reference
TMA of cell line plugs (reproducibility testing on human tumor
tissue is ongoing) and software-aided quantification of number of
spots per cell. Twenty-five reference TMAs are stained on different
days, performed either manually or on a VENTANA autostainer, by two
different operators. Both versions of the assay showed <20% CV
as detailed in Table 1.
TABLE-US-00001 TABLE 1 Observed Variance of 25 Independent HRG
RNA-ISH Assay across Experiments. Mean of RMSE Platform Readout N
spots/cell RMSE CV Manual HRG 381 5.4289 0.9523 18% VENTANA HRG 66
7.6307 1.0970 14% autostainer
[0139] Protein Assays
[0140] Expression of the biomarker also can be detected at the
protein level. Accordingly, the score for ErbB2, ErbB3, and or
ErbB4 can be assessed based on detected levels of protein. In a
particular embodiment, expression of protein levels is measured
using immunohistochemistry (IHC). Immunohistochemistry is a
technique for detecting proteins in cells of a tissue section by
using antibodies that specifically bind to the proteins. Exemplary
IHC assays, such as Fl-IHC and qIHC are described in further detail
below.
[0141] Exemplary IHC assays, such as Fl-IHC and qIHC are described
in further detail below in the Examples.
[0142] ErbB3 Inhibitors
[0143] Methods provided herein can be used to predict efficacy of
therapeutic treatment using any suitable ErbB3 inhibitor or
combination of inhibitors.
[0144] In one embodiment, the ErbB3 inhibitor is an anti-ErbB3
antibody, e.g., a monoclonal antibody. An exemplary anti-ErbB3
monoclonal antibody is SAR256212 (MM-121), described further in WO
2008/100624 and U.S. Pat. No. 7,846,440 (Ab #6), and having V.sub.H
and V.sub.L sequences as shown in SEQ ID NOs: 1 and 2,
respectively. Alternately, the anti-ErbB3 monoclonal antibody is an
antibody that competes with MM-121 for binding to ErbB3. In another
embodiment, the anti-ErbB3 antibody is an antibody comprising the
V.sub.H and V.sub.L CDR sequences of MM-121 in the same relative
order as they are present in MM-121, and which are shown in SEQ ID
NOs: 3-5 (V.sub.H CDR1, 2, 3) and 6-8 (V.sub.L CDR1, 2, 3),
respectively. MM-121 administration may be intravenous at exactly
or about 6 mg/kg or 12 mg/kg weekly, or 12 mg/kg or 24 mg/kg
biweekly. Additional dosing regimens are described below. Other
examples of anti-ErbB3 antibodies include Ab #3, Ab #14, Ab #17 and
Ab #19, also described further in WO 2008/100624 and U.S. Pat. No.
7,846,440, and having V.sub.H and V.sub.L sequences as shown in SEQ
ID NOs: 9 and 10, 17 and 18, 25 and 26, and 33 and 34,
respectively. In another embodiment, the anti-ErbB3 antibody is an
antibody comprising the V.sub.H and V.sub.L CDR sequences of Ab #3
(shown in SEQ ID NOs: 11-13 and 14-18, respectively) or antibody
comprising the V.sub.H and V.sub.L CDR sequences of Ab #14 (shown
in SEQ ID NOs: 19-21 and 22-24, respectively) or an antibody
comprising the V.sub.H and V.sub.L CDR sequences of Ab #17 (shown
in SEQ ID NOs: 27-29 and 30-32, respectively) or an antibody
comprising the V.sub.H and V.sub.L CDR sequences of Ab #19 (shown
in SEQ ID NOs: 35-37 and 38-40, respectively), each of said CDRs
being present in the same relative order as they are present in the
corresponding Ab # antibody.
[0145] Alternately, the anti-ErbB3 antibody is a monoclonal
antibody or antigen binding portion thereof which binds an epitope
of human ErbB3 comprising residues 92-104 of SEQ ID NO:41 and is
characterized by inhibition of proliferation of a cancer cell
expressing ErbB3. The cancer cell may be a MALME-3M cell, an AdrR
cell, or an ACHN cell and the proliferation may be reduced by at
least 10% relative to control. In an additional embodiment this
isolated monoclonal antibody or antigen binding portion thereof
binds an epitope comprising residues 92-104 and 129 of SEQ ID
NO:41.
[0146] Other examples of useful anti-ErbB3 antibodies include the
antibodies 1B4C3 and 2D1D12 (U3 Pharma AG), both of which are
described in US Patent Application Publication No. 20040197332 by
Ullrich et al., and monoclonal antibodies (including humanized
versions thereof), such as AMG-888 (U3 Pharma AG and Amgen), 8B8
(Genentech) as described in U.S. Pat. No. 5,968,511, AV-203 (Aveo
Oncology), MEHD7945A (Genentech/Roche), and MM-141 (Merrimack
Pharmaceuticals) as described in U.S. Pat. No. 8,476,409.
[0147] In yet another embodiment, the anti-ErbB3 antibody can
comprise a mixture, or cocktail, of two or more anti-ErbB3
antibodies, each of which binds to a different epitope on ErbB3. In
one embodiment, the mixture, or cocktail, comprises three
anti-ErbB3 antibodies, each of which binds to a different epitope
on ErbB3.
[0148] In another embodiment, the ErbB3 inhibitor comprises a
nucleic acid molecule, such as an RNA molecule, that inhibits the
expression or activity of ErbB3. RNA antagonists of ErbB3 have been
described in the art (see e.g., US Patent Application Publication
No. 20080318894). Moreover, interfering RNAs specific for ErbB3,
such as shRNAs or siRNAs that specifically inhibits the expression
and/or activity of ErbB3, have been described in the art.
[0149] In yet another embodiment, the ErbB3 inhibitor comprises a
soluble form of ErbB3 that inhibits signaling through the ErbB3
pathway. Such soluble ErbB3 molecules have been described in the
art (see e.g., U.S. Pat. No. 7,390,632, U.S. Patent Application
Publication No. 20080274504 and U.S. Patent Application Publication
No. 20080261270, each by Maihle et al., and U.S. Patent Application
Publication No. 20080057064 by Zhou).
[0150] The ErbB3 inhibitor can be administered to the patient by
any route suitable for the effective delivery of the inhibitor to
the patient. For example, many small molecule inhibitors are
suitable for oral administration. Antibodies and other biologic
agents typically are administered parenterally, e.g.,
intravenously, intraperitoneally, subcutaneously or
intramuscularly. Various routes of administration, dosages and
pharmaceutical formulations suitable for use in the methods
provided herein are described in further detail below.
[0151] Pharmaceutical Compositions: Prior to administration, ErbB3
inhibitors can be formulated with a pharmaceutical carrier (i.e.,
into a pharmaceutical composition). In one embodiment, the ErbB3
inhibitor in the composition is an anti-ErbB3 antibody, e.g.,
MM-121 (SAR256212) or an antibody comprising the V.sub.H and
V.sub.L CDRs of MM-121 positioned in the antibody in the same
relative order as they are present in MM-121 so as to provide
immunospecific binding of ErbB3. Additional non-limiting exemplary
anti-ErbB3 antibodies and other forms of ErbB3 inhibitors are
described in detail above.
[0152] MM-121 for intravenous infusion (e.g., over the course of
one hour) is supplied as a clear liquid solution in sterile,
single-use vials containing 10.1 ml of MM-121 at a concentration of
25 mg/ml in an aqueous solution of 20 mM histidine, 150 mM sodium
chloride, pH 6.5, which should be stored at 2-8.degree. C.
[0153] Pharmaceutical compositions comprising an ErbB3 inhibitor
can be administered alone or in combination therapy. For example,
the combination therapy can include a composition provided herein
comprising an ErbB3 inhibitor and at least one or more additional
therapeutic agents, such as one or more chemotherapeutic agents
known in the art, discussed in further detail in Subsection IV
below. Pharmaceutical compositions can also be administered in
conjunction with radiation therapy and/or surgery.
[0154] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation.
[0155] Exemplary dosage ranges for administration of an antibody
include: 10-1000 mg (antibody)/kg (body weight of the patient),
10-800 mg/kg, 10-600 mg/kg, 10-400 mg/kg, 10-200 mg/kg, 30-1000
mg/kg, 30-800 mg/kg, 30-600 mg/kg, 30-400 mg/kg, 30-200 mg/kg,
50-1000 mg/kg, 50-800 mg/kg, 50-600 mg/kg, 50-400 mg/kg, 50-200
mg/kg, 100-1000 mg/kg, 100-900 mg/kg, 100-800 mg/kg, 100-700 mg/kg,
100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100-300 mg/kg and
100-200 mg/kg. Exemplary dosage schedules include once every three
days, once every five days, once every seven days (i.e., once a
week), once every 10 days, once every 14 days (i.e., once every two
weeks), once every 21 days (i.e., once every three weeks), once
every 28 days (i.e., once every four weeks) and once a month.
[0156] Estrogen Inhibitors
[0157] The estrogen inhibitor may be an estrogen receptor blocker
such as tamoxifen, a selective estrogen receptor modulator such as
raloxifene or an aromatase inhibitor such as anastrozole,
letrozole, exemestane, vorozole, formestane, or fadrozole. Each of
the preceding estrogen inhibitors is in current clinical use and
may be administered in accordance with the manufacturer's
instructions.
[0158] Taxanes/Taxoids
[0159] Taxanes (used interchangeably herein with (and broadly
incorporating the meaning of) the term "taxoids") are diterpene
derivatives, including natural products obtained from plants of the
genus Taxus (yews), and include paclitaxel (Taxol.RTM.), docetaxel
(Taxotere.RTM.), cabazitaxel (Jevtana.RTM.), and Abraxane.RTM. (a
formulation of paclitaxel bound to albumin). Each of the preceding
taxanes is in current clinical use and may be administered in
accordance with the manufacturer's instructions. Other taxanes in
development include, EndoTAG.RTM.-1, a formulation of paclitaxel
encapsulated in positively charged lipid-based complexes being
developed by MediGene; and Tesetaxel.RTM., an orally bioavailable
semisynthetic taxane derivative being developed by Genta Inc.
[0160] Combination Therapy
[0161] In certain embodiments, the methods and uses provided herein
for treating a patient with cancer can comprise administration of
an ErbB3 inhibitor and at least one additional anti-cancer agent
that is not an ErbB3 inhibitor.
[0162] In one embodiment, the at least one additional anti-cancer
agent comprises at least one chemotherapeutic drug. Non-limiting
examples of such chemotherapeutic drugs include taxanes; estrogen
inhibitors; platinum-based chemotherapy drugs (e.g., cisplatin,
carboplatin); and tyrosine kinase inhibitors; e.g., imatinib
(Gleevec.RTM.), sunitinib (Sutent.RTM.), dasatinib and
(Sprycel.RTM.).
[0163] In another aspect, the at least one additional anti-cancer
agent is paclitaxel. In a particular embodiment, the method
comprises at least one cycle, wherein the cycle is a period of 4
weeks, wherein for each cycle the anti-ErbB3 antibody is
administered every other week at a dose of 20 mg/kg and paclitaxel
is administered once per week at a dose of 80 mg/m.sup.2.
[0164] Paclitaxel injection USP is a clear colorless to slightly
yellow viscous solution. It is supplied as a nonaqueous solution
intended for dilution with a suitable parenteral fluid prior to
intravenous infusion. Paclitaxel is available in 30 mg (5 mL), 100
mg (16.7 mL), and 300 mg (50 mL) multidose vials. Each mL of
sterile nonpyrogenic solution contains 6 mg Paclitaxel, 527 mg of
polyoxyl 35 castor oil, NF1 and 49.7% (v/v) dehydrated alcohol,
USP.
[0165] Paclitaxel has the following structural formula:
##STR00001##
[0166] Paclitaxel is a white to off-white crystalline powder with
the molecular formula C47H51NO14 and a molecular weight of 853.9.
It is highly lipophilic, insoluble in water, and melts at around
216.degree. C. to 217.degree. C.
[0167] In another embodiment, the at least one additional
anti-cancer agent is exemestane.
[0168] Exemestane is marketed by Pfizer as Aromasin.RTM. tablets
for oral administration. These tablets each contain 25 mg of
exemestane, an irreversible, steroidal aromatase inactivator
chemically described as 6-methylenandrosta-1,4-diene-3,17-dione.
Its molecular formula is C.sub.20H.sub.24O.sub.2 and its structural
formula is:
##STR00002##
[0169] Exemestane is a white to slightly yellow crystalline powder
with a molecular weight of 296.41. It is freely soluble in
N,N-dimethylformamide, soluble in methanol, and practically
insoluble in water. Each Aromasin.RTM. tablet contains the
following inactive ingredients: mannitol, crospovidone, polysorbate
80, hypromellose, colloidal silicon dioxide, microcrystalline
cellulose, sodium starch glycolate, magnesium stearate,
simethicone, polyethylene glycol 6000, sucrose, magnesium
carbonate, titanium dioxide, methylparaben, and polyvinyl
alcohol.
[0170] In another embodiment, the anti-ErbB3 antibody is
administered at an initial loading dose of 40 mg/kg and a weekly
dose of 20 mg/kg thereafter and one tablet (25 mg) of exemestane is
administered daily.
[0171] As used herein, combined administration (coadministration,
combination therapy) includes simultaneous administration of the
compounds in the same or different dosage form, or separate
administration of the compounds (e.g., sequential administration).
For example, the ErbB3 inhibitor can be administered in combination
with the exemestane or with paclitaxel, wherein both the ErbB3
inhibitor and the exemestane or the paclitaxel are formulated for
separate administration and are administered concurrently or
sequentially in either order. Such concurrent or sequential
administration preferably results in both the compounds (e.g., an
ErbB3 inhibitor and exemestane or an ErbB3 inhibitor and
paclitaxel) being simultaneously present in treated patients.
Patient Populations
[0172] Provided herein are effective methods for treating cancer in
a human patient and for selecting patients to be so treated. In one
embodiment, the human patient suffers from a cancer selected from
the group consisting of non-small cell lung cancer (NSCLC), renal
cell carcinoma (RCC), melanoma (e.g., cutaneous or intraocular
malignant melanoma), colorectal cancer, serous ovarian carcinoma,
liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer
of the head or neck, breast cancer, lung cancer, uterine cancer,
colon cancer, rectal cancer, cancer of the anal region, esophageal
cancer, gastric cancer, gastro-esophageal junction cancer,
testicular cancer, uterine cancer, carcinoma of the fallopian
tubes, carcinoma of the endometrium, carcinoma of the cervix,
carcinoma of the vagina, carcinoma of the vulva, cancer of the
esophagus, cancer of the small intestine, cancer of the endocrine
system, cancer of the thyroid gland, cancer of the parathyroid
gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer
of the urethra, cancer of the penis, solid tumors of childhood,
cancer of the bladder, cancer of the kidney or ureter, carcinoma of
the renal pelvis, neoplasm of the central nervous system (CNS),
spinal axis tumor, glioma, pituitary adenoma, Kaposi's sarcoma,
epidermoid cancer, squamous cell cancer, and mesothelioma. The
disclosed methods are also applicable to treatment of metastatic
cancers. In a particular embodiment, the cancer is ovarian cancer.
In another particular embodiment, the cancer is breast cancer. The
breast cancer may be either or both of ER+ and PR+ breast cancer
("ER+ and/or PR+"). The breast cancer may be HER2 negative. The
breast cancer may be either or both of 1) ER+ and/or PR+ and 2)
HER2 negative. Methods for testing ER and PR status are used as a
matter of clinical routine in the treatment of gynecological
tumors. Such methods may be carried out in accordance with the
well-established guidelines of Hammond, ME et al., "American
Society of Clinical Oncology/College of American Pathologists
Guideline Recommendations for Immunological Testing of Estrogen and
Progesterone Receptors in Breast Cancer" Arch Pathol Lab Med. 2010;
134:E1-E16. HER2 status may be determined using HCT, with a score
of 3+ being considered HER2 positive and a score of 2+ or 1+ or 0
being considered HER2 negative.
[0173] In one embodiment, a human patient for treatment using the
subject methods and compositions has evidence of recurrent or
persistent disease following primary chemotherapy.
[0174] In another embodiment, a human patient for treatment using
the subject methods and compositions has had at least one prior
platinum based chemotherapy regimen for management of primary or
recurrent disease.
[0175] In another embodiment, the patient has a cancer that is
platinum-resistant or refractory. In one example, the
platinum-resistant cancer is ovarian cancer.
[0176] In another embodiment, a human patient for treatment using
the subject methods and compositions has evidence of recurrent or
persistent disease following a) primary treatment, e.g., with an
anti-estrogen therapy or b) an adjuvant treatment with a
non-steroidal aromatase inhibitor and/or tamoxifen.
[0177] In another embodiment, the cancer undergoing treatment is
advanced. In one aspect, the term "advanced" cancer denotes a
cancer above Stage II. In another, "advanced" refers to a stage of
disease where chemotherapy is typically recommended, which is any
one of the following: 1. in the setting of recurrent disease: any
stage or grade; 2. stage IC or higher, any grade; 3. stage IA or
IB, grade 2 or 3; or 4. in the setting of incomplete surgery or
suspected residual disease after surgery (where further surgery can
not be performed): any stage or grade.
[0178] Outcomes
[0179] The efficacy of the treatment methods provided herein can be
assessed using any suitable means. In one embodiment, the treatment
produces at least one therapeutic effect selected from the group
consisting of reduction in growth rate of tumor, reduction in size
of tumor, reduction in number of metastatic lesions over time,
increase in duration of progression-free survival, and increase in
overall response rate.
[0180] With respect to target lesions, responses to therapy may
include:
[0181] Complete Response (CR): Disappearance of all target lesions.
Any pathological lymph nodes (whether target or non-target) must
have reduction in short axis to <10 mm;
[0182] Partial Response (PR): At least a 30% decrease in the sum of
the diameters of target lesions, taking as reference the baseline
sum diameters;
[0183] Progressive Disease (PD): At least a 20% increase in the sum
of the diameters of target lesions, taking as reference the
smallest sum on study (this includes the baseline sum if that is
the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm. (Note: the appearance of one or more new lesions is also
considered progression); and
[0184] Stable Disease (SD): Neither sufficient shrinkage to qualify
for PR, nor sufficient increase to qualify for PD, taking as
reference the smallest sum diameters while on study. (Note: a
change of 20% or less that does not increase the sum of the
diameters by 5 mm or more is coded as stable disease). To be
assigned a status of stable disease, measurements must have met the
stable disease criteria at least once after study entry at a
minimum interval of 6 weeks.
[0185] With respect to non-target lesions, responses to therapy may
include:
[0186] Complete Response (CR): Disappearance of all non-target
lesions and normalization of tumor marker level. All lymph nodes
must be non-pathological in size (<10 mm short axis). If tumor
markers are initially above the upper normal limit, they must
normalize for a patient to be considered in complete clinical
response;
[0187] Non-CR/Non-PD: Persistence of one or more non-target
lesion(s) and/or maintenance of tumor marker level above the normal
limits; and
[0188] Progressive Disease (PD): Appearance of one or more new
lesions and/or unequivocal progression of existing non-target
lesions. Unequivocal progression should not normally trump target
lesion status. It must be representative of overall disease status
change, not a single lesion increase.
[0189] In exemplary outcomes, patients treated according to the
methods disclosed herein may experience improvement in at least one
sign of a cancer, such as platinum resistant/refractory advanced
ovarian cancer.
[0190] In one embodiment, the patient so treated exhibits CR, PR,
or SD.
[0191] In another embodiment, the patient so treated experiences
tumor shrinkage and/or decrease in growth rate, i.e., suppression
of tumor growth. In yet another embodiment, one or more of the
following can occur: the number of cancer cells is reduced; tumor
size is reduced; cancer cell infiltration into peripheral organs is
inhibited, retarded, slowed, or stopped; tumor metastasis is slowed
or inhibited; tumor growth is inhibited; recurrence of tumor is
prevented or delayed; or one or more of the symptoms associated
with cancer is relieved to some extent.
[0192] In other embodiments, such improvement is measured by a
reduction in the quantity and/or size of measurable tumor lesions.
Measurable lesions are defined as those that can be accurately
measured in at least one dimension (longest diameter is to be
recorded) as >10 mm by either or both of CT scan (CT scan slice
thickness no greater than 5 mm) and caliper measurement via
clinical exam, or as >20 mm by chest X-ray. The size of
non-target lesions, e.g., pathological lymph nodes, can also be
measured for improvement. In one embodiment, lesions can be
measured on chest x-rays or CT or MRI outputs.
[0193] In other embodiments, cytology or histology can be used to
evaluate responsiveness to a therapy. The cytological confirmation
of the neoplastic origin of any effusion that appears or worsens
during treatment when the measurable tumor has met criteria for
response or stable disease can be considered to differentiate
between response or stable disease (an effusion may be a side
effect of the treatment) and progressive disease.
[0194] The following examples are merely illustrative and should
not be construed as limiting the scope of this disclosure in any
way as many variations and equivalents will become apparent to
those skilled in the art upon reading the present disclosure.
[0195] All patents, patent applications and publications cited
herein are incorporated herein by reference in their
entireties.
EXAMPLES
Example 1
Chromogenic RNA-In Situ Hybridization Assay (RNA-ISH)
[0196] HRG RNA was detected using a chromogenic RNA-In Situ
Hybridization Assay (RNA-ISH). A chromogenic RNA-ISH assay may be
used to stain an FFPE tissue section for an RNA of interest. For
each RNA-ISH assay, a scoring system was applied by a certified
pathologist. The system scores levels as the discrete variables 0,
1+, 2+, 3+, or 4+.
[0197] Heregulin (HRG) is an ErbB3 ligand that activates ErbB3,
thereby initiating intracellular signaling in tumor cells. This may
occur in an autocrine fashion, in which the HRG produced by a cell
activates the same cell, or it may occur in a paracrine fashion, in
which HRG produced by one cell (e.g., a stromal cell in a tumor)
activates neighboring cells (e.g., tumor cells). Accordingly, it is
be desirable to measure HRG expression in both tumor cells and
stromal cells in the same biopsy. This can be achieved by
visualizing HRG transcripts (e.g., in FFPE patient samples) using
RNA in situ hybridization (RNA-ISH) and scoring patient samples
based on the observed hybridization levels.
[0198] FIG. 1 shows an example of HRG RNA staining in an ovarian
cancer biopsy section. The staining pattern is strikingly
non-uniform, with a small subset of cells expressing high levels of
HRG transcripts (dark blotches) and the majority of cells not
expressing detectable levels of transcripts. [0199] 1. Overview of
the Assay
[0200] FFPE tumor samples are scored for HRG RNA levels using the
following variant of an Advanced Cell Diagnostics.RTM. ("ACD"
Hayward, Calif.) RNAscope.RTM. assay. In this assay, cells are
permeabilized and incubated with a set of oligonucleotide "Z"
probes (see, e.g., U.S. Pat. No. 7,709,198) specific for HRG. Using
"Z" probes, as well as using multiple sets of probes per
transcript, increases the specificity of the assay over standard
ISH methods. One HRG probe set that can be used in this assay is
ACD Part Number 311181. Another HRG probe set prepared by ACD (and
used in RNAscope.RTM. assays to generate the data presented below
and in the Figures) includes 62 probes (31 pairs), each 25 bases in
length, that target a 1919 base long region of the HRG transcript
comprising nucleotides 442-2977 of SEQ ID NO:42 and that together
detect 15 separate HRG isoforms (.alpha., .beta.1, .beta.1b,
.beta.1c, .beta.1d, .beta.2, .beta.2b, .beta.3, .beta.3b, .gamma.,
.gamma.2, .gamma.3, ndf43, ndf34b, and GGF2). Following Z probe
incubation, a pre-amplifier is added that can only hybridize to a
pair of adjacent Z probes bound to the target transcript. This
minimizes amplification of non-specific binding. Several sequential
amplification steps are then performed based on sequence-specific
hybridization to the pre-amplifier, followed by enzyme-mediated
chromogenic detection that enables semi-quantitative measurement of
HRG RNA levels in the tumor tissue.
[0201] Step 1: FFPE tissue sections are deparaffinized and
pretreated to block endogenous phosphatases and peroxidases and to
unmask RNA binding sites. Step 2: Target-specific double Z probes
are applied, which specifically hybridize to the target RNA at
adjacent sequences. Step 3: Targets are detected by sequential
applications of a preamplifier oligonucleotide, amplifier
oligonucleotides, a final HRP-conjugated oligonucleotide, and DAB.
Step 4: Slides are visualized using a light microscope and scored
by a pathologist.
[0202] To score the assay, a reference tissue microarray (TMA) of
four cell lines is stained alongside the tumor sample. These cell
lines express different levels of HRG, ranging from low to high. A
pathologist then assigns the patient sample a score based on a
visual comparison with the reference TMA. [0203] 2. Sample
Preparation and Staining
[0204] Patient sample preparation and pathologist review procedures
are similar to qIHC assays. Upon biopsy or surgical resection,
patient tumor samples are immediately placed in fixative (10%
neutral buffered formalin) typically for 20-24 hours at room
temperature. Samples are then transferred to 70% ethanol and
embedded in paraffin as per standard hospital procedures. Before
the assay is performed, 4-.mu.m sections of the sample are prepared
and mounted on positively charged 75.times.25 mm glass slides.
These are baked for improved tissue adhesion (10-30 min at
65.degree. C.), dipped in paraffin for tissue preservation, and
stored at room temperature under nitrogen. One of the sections is
used for routine H&E staining, which a pathologist reviews for
tumor content, quality, and clinical diagnosis. The pathologist
differentiates areas of tumor, stroma, and necrosis. Following this
review, an adjacent or nearby tissue section (within 20 .mu.m of
the H&E section) is used for the assay.
[0205] Pretreat solutions, target probes, and wash buffers for
RNAscope.RTM. assays are obtained from ACD. The assay can be run
manually, or using a VENTANA autostainer (Discovery XT). For the
manual assay, 40.degree. C. incubations are performed in a metal
slide tray inside a HybEZ oven (ACD). For the automated assay,
incubation temperatures are controlled by the autostainer. ACD
software is used to run the RNAscope.RTM. assays on the VENTANA
autostainer.
[0206] To begin the assay, samples are deparaffinized by baking at
65.degree. C. for 30 min, followed by sequential immersion in
xylenes (2.times.20 min) and 100% ethanol (2.times.3 min). After
air-drying, tissues are covered with Pretreat1 solution, which
blocks endogenous enzymes (phosphatases and peroxidases which would
produce background with chromogenic detection reagents), incubated
for 10 min at room temperature, then rinsed twice by immersion in
dH.sub.2O. Slides are then incubated in boiling Pretreat2 solution
for 15 min, which unmasks binding sites, and transferred
immediately to containers of dH.sub.2O.
[0207] After washing by immersion in dH.sub.2O (2.times.2 min),
tissue is covered with Pretreat3 solution and incubated in a HybEZ
oven at 40.degree. C. for 30 min. Pretreat3 solution contains a
protease, which strips the RNA transcripts of protein and exposes
them to the target probes. After washing the slides 2.times.2 min
in dH.sub.2O, the tissues are covered with the 15 isoform-detecting
HRG RNAscope.RTM. probes described above. Serial tissue sections
are incubated with positive control probes (protein phosphatase 1B
(PP1B) ACD Part Number 313901), negative control probes (bacterial
gene DapB--ACD Part Number 310043), or HRG probes for 2 h at
40.degree. C. Slides are washed (2.times.2 min) with
1.times.RNAscope.RTM. wash buffer before incubating with Amp1
reagent. Amp1 incubation conditions (30 min, 40.degree. C.) favor
binding only to pairs of adjacent probes bound to RNA transcripts.
Slides are washed by immersion in RNAscope.RTM. wash buffer before
incubating with subsequent amplification reagents.
[0208] For signal amplification, each of the sequentially applied
reagents binds to the preceding reagent and amplifies the signal
present at the previous step. Amplification steps may include Amp2
(15 min, 40.degree. C.), Amp3 (30 min, 40.degree. C.), Amp4 (15
min, 40.degree. C.), AmpS (30 min, room temperature), and Amp6 (15
min, room temperature). The final reagent, Amp6, can be conjugated
to horseradish peroxidase (HRP). To visualize the transcripts, the
slides are then incubated with the ACD staining reagent, which
contains diaminobenzidine (DAB), for 10 min at room temperature.
Chromogen development is stopped by rinsing with dH.sub.2O. Nuclei
are then counterstained with hematoxylin, which is blued with
dilute ammonium chloride. Stained slides are immersed in 80%
ethanol (2.times.5 min), 100% ethanol (2.times.5 min), and xylenes
(2.times.5 min) before coverslipping with Cytoseal non-aqueous
mounting medium (Thermo Scientific, 8312-4). [0209] 3. Generation
of Biomarker Values
[0210] The biomarker values to be generated are a composite of
pathologist scores. To score the assay, a TMA comprising plugs of
four different cell lines is included in each staining run. Cell
line plugs are prepared prior to generating a TMA. Cultured cells
grown to a sub-confluent density are harvested by trypsinization,
rinsed in PBS, and fixed for 16-24 hr at 4.degree. C. before
rinsing in PBS and resuspending in 70% ethanol. Cells are then
centrifuged for 1-2 minutes at approximately 12,000 rpm to produce
a dense cell pellet, which is then coated with low-melting point
agarose. The agarose pellets are stored in 70% ethanol at 4.degree.
C., and embedded in paraffin before constructing the TMA.
[0211] The arrays are constructed, e.g., using a Manual Tissue
Arrayer (MTA-1, Beecher Instruments), with which a 0.6 mm punch is
used to take a portion of the cell pellet and plug it into an empty
recipient paraffin block. The pathologist uses the images of the
TMA to provide a score ranging from 0 (undetectable) to 4 (high).
As shown in FIG. 2, RT112 (RT-112, CLS Cell Lines Service GmbH,
Eppelheim, Germany) is assigned a score of 1, OVCAR3 (ATCC.RTM.
HTB-161.TM.) a score of 2, TK10 (TK.10 cells, NCI, Bethesda, Md.) a
score of 3, and NCI-522 (ATCC.RTM. CRL-5810.TM.) a score of 4. The
pathologist provides two scores for the top two populations of
tumor cells, and one score for the top population of stromal cells
(when available), along with the percentage of cells in each
population. So, for example, a patient sample may have 20% tumor
with a score of 3, 40% tumor with a score of 2, and 60% stroma with
a score of 2. Scores are provided for the target probe (HRG), as
well as the positive control probe (PP1B) and the negative control
probe (DapB). [0212] 4. Preliminary Results
[0213] Identification of heregulin expression as a driver of a
difficult-to-treat cancer phenotype and development of a
prospective companion diagnostic for the heregulin-ErbB3 targeting
drug seribantumab. In three Phase 2 studies, the novel heregulin
assay was able to identify patients with heregulin-positive tumors
where the addition of seribantumab to standard-of-care therapies
may provide benefit. Heregulin was detected at significant levels
across multiple solid tumor types, with a prevalence of between
30-60% of patients, potentially defining a critical patient
phenotype that may be difficult to treat. Tumor biopsies were
measured by RNA-ISH. A fully validated RNA-ISH assay is currently
being used to identify heregulin-positive patients in a Phase 2
randomized trial of seribantumab in patients with NSCLC as further
discussed below. [0214] 5. Alternative RNA-ISH Assay
[0215] RNAscope.RTM. is a novel RNA-ISH method that achieves
single-molecule RNA visualization in individual cells through use
of a novel probe design strategy and a hybridization-based signal
amplification system to simultaneously amplify signals and suppress
background. Many of the steps in RNAscope.RTM. are similar to those
in immunohistochemistry (IHC). RNAscope.RTM. can be used with
formalin-fixed, paraffin-embedded (FFPE) tissue samples on glass
slides, and the stained slides can be visualized under a standard
bright-field microscope using RNAscope.RTM. with chromogenic
labels.
[0216] The RNAscope.RTM. ISH method is intended to enable analysis
of gene expression in situ in routine clinical specimen types with
high sensitivity and specificity while retaining spatial expression
patterns.
[0217] The RNAscope.RTM. method is similar to conventional
riboprobe RNA ISH methods, but with an improved signal-to-noise
ratio achieved by amplifying target-specific signals without
background noise from nonspecific hybridization. A novel target
probe design strategy, referred to as a "double-Z" or "ZZ" probe
design, is coupled with multiple layers of signal
amplification.
[0218] In step 1, cells or tissues are fixed and undergo heat
induced RNA retrieval to allow for target probe access. In step 2,
target-specific RNA probes consisting of pooled oligonucleotides
(Z) hybridize in pairs (ZZ) to multiple adjacent sequences on
target RNA. In step 3, a preamplifier molecule binds to each pair
of ZZ target probes, multiple signal amplifier molecules hybridize
to each preamplifier molecule and multiple label probes then
hybridize to each amplifier molecule. Each label probe is
conjugated to an enzyme. In step 4, signal is detected using a
standard bright-field microscope after incubation with chromogenic
enzyme substrate.
[0219] An RNAscope.RTM. target probe consists of a pool of
oligonucleotides ("oligos") designed to hybridize to a select
region of the target RNA molecule. Each of the oligos consists of
three parts: (1) an 18- to 25-nucleotide region that is
complementary to the target RNA; (2) a spacer sequence; and (3) a
14-nucleotide tail sequence (conceptualized as a Z). Signal
amplification is dependent on contiguous hybridization of an oligo
pair (ZZ) to a target region of 50 nucleotides. Each oligo in a ZZ
pair possesses a different tail sequence. The two tail sequences
together form a 28-base hybridization site for a "preamplifier"
(PreAMP) oligo. The PreAMP contains multiple binding sites for
amplifier (AMP) oligo hybridization. Each AMP oligo, in turn,
contains multiple sites for hybridization of label probe.
[0220] A typical RNAscope.RTM. target probe set will span
approximately 1 kb of the RNA molecule and comprise 20 oligo pairs.
This probe and amplifier design has a theoretical yield of up to
20,000 labels localized to each target RNA molecule. The probe is
highly specific to target mRNA molecule with no background signal
associated with nonspecific hybridization. We can estimate the
probability of oligo non-specific hybridization from the
probability of finding a non-exact match (i.e., off-target) in the
genome at a certain threshold. Based on the Basic Local Alignment
Search Tool (BLAST) model, a single Z (25 mer) has a probability of
5.8.times.10.sup.-11 for finding an off-target binding site with
.about.70% identity, the probability of finding two contiguous such
binding sites for a ZZ pair is 3.3.times.10.sup.-21. Given all
known RNA species in the human transcriptome, one may expect to
find 0.6 binding site for a single Z, but only 3.6.times.10.sup.-11
(essentially 0) binding site for a double Z pair. In addition, a
single 14-base tail sequence will not form a stable hybrid with a
PreAMP oligo under the assay conditions employed.
[0221] The label probe is conjugated to horseradish peroxidase
(HRP) for chromogenic reactions with 3,3-diaminobenzidine (DAB).
The HRP-labeled probes have the advantage of enabling viewing with
a standard bright-field microscope similar to IHC procedures. This
makes RNAscope.RTM. assay results easy to score and archive in a
clinical setting.
[0222] RNAscope.RTM. target probe design is accomplished by
selecting oligos comprising a target probe set that have sequences
with compatible melting temperature (Tm) and avoid sequences that
would result in cross-hybridization to off-target sequences under
the stringent assay conditions employed in the RNAscope.RTM.
assay.
[0223] Sufficient visible signal with HRP/DAB is generated with
only three (3) oligo pairs. Therefore, a target probe consisting of
10-20 oligo pairs enables a highly robust RNA molecule detection
assay even in the presence of RNA fragmentation, partial
degradation and protein binding impeding the binding of many of the
oligos comprising a target probe set.
[0224] The Leica HRG RNA-ISH assay is a chromogenic, RNA in situ
hybridization assay used on the Leica BOND-III for the
semi-quantitative determination of HRG mRNA levels in FFPE NSCLC
tissue specimens. The HRG RNA-ISH assay kit will contain minimally
the deparaffinization, pre-treatment, hybridization probes,
amplification, and detection components:
TABLE-US-00002 TABLE 2 Kit Components Component Reagent Detail
Description Hs-NRG1Probe RNAscope .RTM. 2.5 LS Hs- Target probe
Hs-PPIB Probe RNAscope 2.5 LS Positive Positive Control Control
Probe Hs-PPIB Probe dapB Probe RNAscope 2.5 LS Negative Negative
Control Control Probe dapB Probe BOND RNAscope .RTM. BOND RNAscope
.RTM. Detection System-- Detection System-- BROWN BROWN BOND
RNAscope BOND RNAscope Protease Protease (NRG1) Assay Control
Negative Slide Positive
[0225] A. Assay Controls
[0226] A single slide comprised of at least two human cancer cell
lines, one (1) HRG negative and one (1) HRG positive.
[0227] B. Reagents/Materials:
[0228] The RNA-ISH detection kit integral for performing the HRG
assay is comprised of the reagents described in Table 2. Additional
reagent components integral for performing the HRG RNA-ISH assay
are: [0229] BOND Universal Covertiles [0230] BOND Epitope Retrieval
2 Solution [0231] BOND Dewax Solution [0232] BOND Wash Solution
[0233] BOND Aspirating Probe Cleaning System [0234] BOND Mixing
Stations [0235] Standard solvents for use in IHC: graded ethanol,
xylene (or xylene substitutes), mounting medium and distilled or
de-ionized water
[0236] C. HRG RNA ISH Assay Interpretation of Results and Scoring
System [0237] 1. Microscope
[0238] Sample assessment and signal enumeration can be evaluated
via brightfield microscopy utilizing objectives ranging from low to
high magnification (2.5.times. to 40.times.). Lower magnifications
will be utilized to assess sample histomorphology (tumor vs.
non-tumor) while 20.times. magnification will be utilized for mRNA
signal evaluation across the entire tumor, and 40.times.
magnification for scoring confirmation. [0239] 2. Scoring
Guideline
[0240] The Leica BOND Heregulin (NRG1) RNA-ISH Assay can use a
qualitative (binary as Negative or Positive) approach to scoring
applied to dapB, Hs-PPIB, and Hs-NRG1 stained slides. Positive
staining is defined by one or more dots per cell in .gtoreq.10% of
tumor cells.
[0241] For exploratory purposes, scoring may be quantitative and
captured as 0, 1+ or 2+ correlating to negative, low positive and
high positive staining.
TABLE-US-00003 TABLE 3 HRG (NRG-1) Scoring System Exploratory
Exploratory Assessment Criteria Score Criteria Negative No staining
or .gtoreq. 1dot/cell 0 No staining or .gtoreq. 1 dot in in <10%
tumor cells <10% tumor cells Positive Staining in .gtoreq. 1
dot/cell 1+ 1-3 dots/cell in .gtoreq.10% tumor cells in .gtoreq.10%
tumor cells 2+ .gtoreq.4 dots/cell in .gtoreq.10% tumor cells A
sample should contain a minimum of 50 tumor cells to be eligible
for analysis
Example 2
Fluorescence-Based Quantitative IHC (Fl-IHC) Assay for ErbB2
[0242] Fl-IHC can be used to measure ErbB2 protein levels in tumor
cells in FFPE tissue. Fl-IHC is an imaging-based assay and provides
a measure of the number of protein molecules per tumor cell in each
sample. This assay, schematized in FIG. 4, is quantitative and
yields a continuous variable.
[0243] Either surgically resected tumor tissue or core needle
biopsies are collected from patients, fixed in formalin for 24 h,
and embedded in paraffin blocks using standard procedures. FFPE
blocks are sectioned (4 .mu.m thickness), mounted on glass slides,
and co-stained for DNA, cytokeratin, and the ErbB2 receptor. Final
detection of these markers is based on fluorescence. The slides are
imaged using an Aperio.RTM. ScanScope.RTM. FL set at 20.times.
magnification and analyzed using quantitative digital image
analysis. The automated image analysis algorithm applies a regular
grid across the tissue region where each square tile is
approximately the size of a single cell. Each tile is classified as
either cytokeratin positive (CK+) or negative (CK-) using a fixed
threshold for the CK staining (see details below). The fluorescence
measurements of the CK+ tiles are then converted to an absolute
scale (receptors per cell) using a standard curve generated from a
tissue microarray (TMA), stained and imaged at the same time as the
patient sample, that is composed of cell lines with known ErbB2
protein levels. [0244] 1. Sample Preparation and Staining
[0245] Following surgical resection or core needle biopsy, patient
tumor samples are placed immediately in fixative (10% Neutral
Buffered Formalin) for 20-24 hours at room temperature. Samples are
then transferred to 70% ethanol and subsequently embedded in
paraffin as per standard hospital procedures. Before the assay is
performed, 4-.mu.m sections of the sample are prepared and mounted
on positively charged 75.times.25 mm glass slides. These are baked
for improved tissue adhesion (10-30 min at 65.degree. C.), and
dipped in xylenes to preserve antigens (2-5 min at room
temperature). Sections to be used for the assay are dipped in
paraffin to preserve them until the assay is ready to be performed.
One section is used for routine H&E staining, which a
pathologist reviews for tumor content, quality, and clinical
diagnosis. The pathologist differentiates areas of tumor, stroma,
and necrosis. Following this review, an adjacent or nearby tissue
section (within 20 .mu.m of the H&E section) is used for the
assay.
[0246] Prior to staining, the sample is deparaffinized by baking
for 30 min at 65.degree. C., followed by sequential immersion in
xylenes (2.times.20 min), 100% ethanol (2.times.2-5 min), 80%
ethanol (2.times.2-5 min) and re-hydrated by immersion in water.
Heat induced antigen retrieval in Tris-EDTA buffer, pH 9 (Fisher
TA-250-PM4X) is performed in a PT Module (Thermo Scientific) set to
102.degree. C. for 25 min, with the no-boil setting enabled.
Subsequent steps are performed on a Dako Autostainer, with all
incubations at room temperature. Slides are pre-rinsed with
1.times.TBS-T (Fisher, TA-999-TT) before blocking for endogenous
peroxidases (10 min incubation in Peroxidazed 1 (BioCare Medical,
PX968M)). The autostainer rinses each slide with 13 mL TBS-T twice
before blowing air for several seconds to displace it immediately
before addition of the next reagent. Nonspecific protein binding is
blocked with Background Sniper (10 min, BioCare Medical, BS966M).
Slides are rinsed twice in TBS-T. Primary antibodies for ErbB2 and
pan-cytokeratin (CK) are applied to the slides together diluted in
DaVinci.TM. Green diluent (BioCare Medical, PD900M) for 1 h. The
primary antibody for ErbB2 is a rabbit-derived monoclonal antibody
from Fisher Scientific (catalog #RM-9103-S) applied at a dilution
of 1:300. The primary antibody for CK is a mouse-derived monoclonal
antibody anti Hu IgG1 Kappa from DAKO (catalog #M351501) applied at
a dilution of 1:50. Slides are rinsed twice in TBS-T. The detection
antibody cocktail consists of Alexa Fluor.RTM. 555-GAM IgG (H+L)
(Invitrogen.RTM., A-21422) diluted 1:200 into DAKO
Envision.sup.+.RTM. anti-rabbit HRP labeled polymer (K400311,
supplied ready to use). Following a 30-min incubation with
detection antibodies, slides are rinsed twice in TBS-T before
visualization of the ErbB2 signal by 10 min incubation with
Perkin-Elmer.RTM. CY5-Tyramide (SAT705A, used as per manufacturer's
instructions with reconstituted Tyramide diluted 1:50 into supplied
diluent). The slides are rinsed twice in TBS-T. Slides are removed
from the autostainer and coverslips applied over mounting medium.
DAPI nuclear stain is included in the Prolong Gold mounting medium
(Invitrogen.RTM., P36935). After allowing the mounting medium to
set (in the dark at room temperature overnight), slides are imaged
using an Aperio.RTM. fluorescence scanning microscope (Aperio.RTM.
ScanScope.RTM. Fla.). [0247] 2. Generation of Biomarker Values
[0248] The biomarker values to be generated are the
receptor-per-cell expressions of target receptors in the
cytokeratin-positive cells. The images of the fluorescently stained
biopsies are analyzed using Definiens-Developer image analysis
software (Definiens, Inc., Carlsbad, Calif.). The algorithm
measures the mean fluorescent intensity of regularly sampled square
tiles overlaid on the tissue region of the image. Each tile is
approximately the size of a single cell. Regular grid sampling of
the image removes the complexity and uncertainty of accurate
single-cell segmentation of the image while preserving the ability
to measure spatial intensity variations. The tiles are classified
as either cytokeratin positive (CK+) or negative (CK-) using a
fixed cytokeratin threshold computed as the intersect of two normal
distributions fit to combined normalized quantile distributions
(sampled every 5%) across all patient biopsies within the current
MM-121 clinical trials. The biomarker and cytokeratin fluorescence
for each tile is normalized for each biopsy using a standard curve
generated with a cell-line based tissue microarray (TMA). A
biomarker quantile analysis of all CK+ tiles are reported for each
biopsy and the most predictive quantile level is determined
empirically for each trial. [0249] 3. Reproducibility and Error
Measures
[0250] Assay reproducibility can be determined by staining and
analyzing about 40 independent sections of replicate TMAs
containing plugs from 12 cell lines in triplicate over the course
of >12 months. The RMSE and CV of the measured fluorescence
values for all repeated cell lines across all TMAs were measured
for the ErbB2 assay. In parallel, two similar assays, one for EGFR
and one for ErbB3, were also assessed. The reproducibility and
error measurements for all three assays are provided in Table
4.
TABLE-US-00004 TABLE 4 Reproducibility and Error Measurements of
Fluorescence Values of qIHC Assays for EGFR, ErbB2, and ErbB3 Using
TMAs of Cell Line Plugs. Receptor N Mean Fluorescence (log.sub.10)
RMSE CV EGFR 1232 4.0919 0.1058 2.6% HER2 1222 3.4500 0.1485 4.3%
HER3 1183 3.9745 0.0891 2.2%
[0251] 4. Dynamic Range and Limits of Detection
[0252] The limits of detection and dynamic range of the qIHC assay
can be defined by the lower and upper bounds of the microscope
camera sensor and by the lower and upper expression levels of
receptors in the cell lines used on the standard curve TMA. The
upper bound of the 16-bit camera used in the Aperio.RTM.
ScanScope.RTM. FL microscope is 65,535 intensity units (216-1). The
lower bounds were determined empirically by measuring the
background fluorescence of .about.41 slides containing biopsy
tissue from three independent staining batches. The average lower
bounds and dynamic ranges were computed for DNA (DAPI), cytokeratin
(Cy3) and biomarker (Cy5) channels (Table 5). As above, this
calculation was performed for three separate assays for each of
EGFR, ErbB2, and ErbB3.
TABLE-US-00005 TABLE 5 Dynamic Range of Measurements in the DNA,
Cytokeratin, and Biomarker channels and Target expressions in cell
lines of control curve TMA Dynamic range of Dynamic range of the
fluorescence target expressions values in the cell lines of Lower
Lower TMA (log.sub.10 Bound Bound receptors per cell) Ab Upper
Lower Lower Upper Upper Lower Channel Stain N Bound Bound 95% 95%
Bound Bound DNA EGFR 41 65535 619.80 544.75 705.18 (DAPI) DNA ErbB3
41 65535 1346.48 1183.59 1531.79 (DAPI) DNA HER2 42 65535 2149.81
1892.34 2442.31 (DAPI) Cytokeratin EGFR 41 65535 317.04 274.35
366.44 (Cy3) Cytokeratin ErbB3 41 65535 379.19 328.10 438.23 (Cy3)
Cytokeratin HER2 42 65535 396.93 344.03 457.93 (Cy3) Biomarker EGFR
41 65535 364.75 314.85 422.57 5.651 1.968 (Cy5) Biomarker ErbB3 41
65535 247.46 213.60 286.68 4.688 3.729 (Cy5) Biomarker HER2 42
65535 302.96 261.94 350.35 6.143 3.491 (Cy5)
[0253] The lower and upper expression levels of ErbB receptors
included on the TMAs define the dynamic range of the standard
curve. Any value inside the range can be interpolated whereas
values outside require extrapolation. The reference values were
measured using quantitative flow cytometry (Table 6).
TABLE-US-00006 TABLE 6 Lower and Upper Receptor Expression Levels
for Cell Lines Used on the Standard Curve TMA. Units are receptors
per cell with cell line name in parenthesis. Value EGFR HER2 ErbB3
Low 93 3,100 5,360 (CHOK-1) (CHOK-1) (IGROV-1) High 448,000
1,390,000 48,700 (ACHN) (BT474-M3) (MDA-MB-453)
Example 3
ErbB2, ErbB3 and ErbB4 Chromogenic Quantitative IHC (qIHC)
[0254] qIHC can be used to detect protein levels of ErbB2, ErbB3
and ErbB4. qIHC uses a standard brown-stain technology to indicate
protein levels in FFPE tissue sections. For each qIHC assay used in
this study, the TMA-based scoring system described above was
applied by a certified pathologist. As described, this system
yields scores based on staining intensity (0, 1, 2, 3, 4). In the
clinical assay results described herein, the same pathologist
scored all patient samples for a given assay. Table 7 lists the
materials used in these studies.
TABLE-US-00007 TABLE 7 Materials Chart: Incubation Reagent Vendor
Dilution Time/Step Name Catalog # Factor N/A DI Water in-house N/A
N/A N/A Tris Buffered Saline and FISHER 1:20 Tween 20 (20X)
SCIENTIFIC TA-999-TT N/A ErbB2: PT Module Buffer 4, FISHER 1:300
Tris EDTA, pH9 SCIENTIFIC TA-250-PM4X ErbB3: PT Module Buffer 4,
FISHER 1:100 Tris EDTA, pH9 SCIENTIFIC TA-250-PM4X ErbB4: PT Module
Buffer 1, FISHER Citrate, pH6 SCIENTIFIC TA-250-PM1X 10 min
Peroxidazed .RTM. 1 BIOCARE RTU MEDICAL PX968 H, M 10 min
Background Sniper BIOCARE RTU MEDICAL B5966 H, M 60min DA VINCI
GREEN BIOCARE RTU MEDICAL PD900 H, M ErbB2: Cytokeratin (Mono DAKO
M351501 ErbB2: Ms Anti-Hu IgG1 Kappa) 1:50 ErbB3: Her3/ErbB3
(D22C5) CELL SIGNALING 1:100 Rabbit mAb TECHNOLOGY 12708BC ErbB4:
HER-4/c-erbB-4 FISHER 1:200 (Rabbit Polyclonal Antibody) SCIENTIFIC
RB-9045-P1 30 min ErbB2: Alexa Fluor .RTM. 555 INVITROGEN 1:200 GAM
IgG (H + L) A-21422 ErbB2-4: ENVISION + Anti- DAKO K400311 RTU
Rabbit HRP Labeled Polymer 10 min ErbB2: Cyanide 5 Tyramide PERKIN
ELMER 1:50 Reagent SAT705A ErbB3/4: Liquid DAB + DAKO K3468
Substrate Chromogen System 6 min Automation Hematoxylin DAKO S3301
RTU N/A CYTOSEAL XYL FISHER RTU SCIENTIFIC 8312-4 N/A Rectangular
cover glass Various N/A (sized to tissue) N/A *1 drop DAB + per mL
of substrate solution.
[0255] Additional details for the materials are as follows: [0256]
ErbB2: Her-2/c-erB-2/neu, Rabbit monoclonal antibody (Fisher
Scientific, Cat. #RM-9103-S) and Cytokeratin (Mono Mouse Anti-Hu
IgG1 Kappa) (Dako, Cat. #M351501) [0257] ErbB3: HER-3/ErbB-3 Rabbit
monoclonal antibody (RB mAb) (Cell Signaling Technology, catalog
#12708BC) [0258] ErbB4: HER-4/c-erbB-4 Rabbit polyclonal antibody
(RB pAb) (FISHER SCIENTIFIC, catalog #RB-9045-P1) [0259] ENVISION+
System-HRP Labelled Polymer Anti-Rabbit (DAKO, catalog #K4003)
[0260] Liquid DAB+ Substrate Chromogen System (DAB Chromogen and
Substrate Buffer solutions in separate containers) (DAKO, catalog
#K3468) [0261] Automation Hematoxylin Histological Staining Reagent
(DAKO, catalog #S3301) [0262] Tris Buffered Saline and Tween 20
(20.times.; TBS-T) (FISHER SCIENTIFIC, catalog #TA-999-TT) [0263]
ErbB2/3: PT Module Buffer 4, Tris EDTA, pH9 (FISHER SCIENTIFIC,
catalog #TA-250-PM4X) [0264] ErbB4: PT Module Buffer 1, Citrate, pH
6 (FISHER SCIENTIFIC, catalog #TA-250-PM1X) [0265] PEROXIDAZED 1
(BIOCARE MEDICAL, catalog #PX968 H, M) [0266] BACKGROUND SNIPER
(BIOCARE MEDICAL, catalog #BS966 H, M) [0267] DA VINCI GREEN
Diluent (BIOCARE MEDICAL, catalog #PD900 H, M) [0268] FLEX 100
Alcohol Solution (FISHER SCIENTIFIC, catalog #8101) [0269] FLEX 80
Alcohol Solution (FISHER SCIENTIFIC, catalog #8301R) [0270] Xylene
(SIGMA, catalog #534056) [0271] ErbB3/4: CYTOSEAL XYL (FISHER
SCIENTIFIC, catalog #8312-4) [0272] ErbB2: Cyanine 5 Tyramide
(Perkin Elmer, Cat. #SAT705A) [0273] ErbB2: :PROLONG GOLD Antifade
Reagent with DAPI ((Invitrogen, Cat. #P36935) [0274] Glass
coverslips (VWR No. 1)
[0275] The assays were performed according to the following
methods.
[0276] 1. Deparaffinize/hydrate slides [0277] 1.1. If necessary,
use a razor blade to scrape paraffin wax off of the back of the
slides. Scrape the wax off of the front around the tissue region if
it is visible. If the tissue is not clearly visible under the wax,
do not scrape any wax from the front. [0278] 1.2. Incubate slides
for 30-50 min at 65.degree. C. in a metal slide rack (or
equivalent) in the oven to melt wax covering tissues. [0279] 1.3.
Transfer slides to a Tissue-Tek (or equivalent) slide rack. Immerse
slides in the following solutions with occasional gentle agitation:
[0280] xylene, twice for 20-30 min each [0281] 100% ethanol, twice
for 2-5 minutes [0282] 80% ethanol, twice for 2-5 minutes [0283]
Distilled water for 2-5 minutes [0284] 1.4. Apply programmed DAKO
slide labels to the front frosted end of the slides and place them
on the DAKO slide rack(s).
[0285] 2. Antigen retrieval (AR): [0286] 2.1. Perform AR in the PT
module using [0287] ErbB2/3: PT module buffer 4 (Tris EDTA at pH
9.+-.0.05) [0288] ErbB4: PT module buffer 1 (Citrate at pH
6.+-.0.05) [0289] using the following settings: [0290] Incubation
time: 25 minutes [0291] Incubation temperature: 102.degree. C.
[0292] No-boil function: Enabled [0293] 2.2. Once the program has
run and the solution cooled down to 65.degree. C. remove the slide
rack(s) from the PT module and place in the DAKO buffer wash
basin(s) containing 1.times.TBS-T for 3-5 minutes.
[0294] 3. Reagent Preparation: This should be done when slides are
in the PT module. [0295] 3.1. In DAKOLINK program, select all
slides to be stained from the "Pending" tab and click "Reagents"
button at the bottom of the screen. This will generate a list and
volumes needed per reagent (this assumes two 1504, drop zones for a
total of 3004, per slide). Print this list. [0296] 3.2. Select the
appropriate number and size of DAKO user fillable bottles. [0297]
3.3. Scan the barcode of each bottle and plug the volume required
of that particular reagent into the "usable quantity" box. This
will then factor in the dead volume of that particular size bottle
into your "Fill quantity" or total volume. [0298] 3.4. Calculate
the individual amounts of reagents needed for your assay. [0299]
Endogenous peroxidase block (Peroxidazed 1), protein block
(Background Sniper), secondary antibody (ENVISION+System-HRP
Labelled Polymer Anti-Rabbit) and automation hematoxylin are ready
to use reagents and may be filled right away. [0300] Primary
antibody will be diluted in blocking diluent at the desired
dilution factor. [0301] Liquid DAB+ Substrate Chromogen working
reagent: add 1 drop (or 20 .mu.L) of the DAB Chromogen per mL of
Substrate Buffer.
[0302] 4. Autostainer run preparation: Once all reagents are made
and AR process is complete, place the reagent bottles into the DAKO
AutostainerLink 48. [0303] 4.1. Fill the 10 L buffer carboy with
1.times.TBS-T (add more if required) [0304] 4.2. Fill the 10 L
water carboy with DI water [0305] 4.3. Remove all the rack(s) from
the wash basin(s) and place on the autostainer. [0306] 4.4. Click
on the "Instruments" tab in the DAKOLink software and click "Start"
button.
[0307] 5. Automated staining: The following is the summary of the
DAKO AutostainerLink48 chromogenic staining protocol. Note: The
autostainer calculates the appropriate rinse step after each
critical incubation step therefore the rinse incubation time is
arbitrary.
[0308] 6. Remove the mounting medium from freezer and allow it to
come to room temperature.
[0309] 7. Once the staining is complete, transfers Dako slide
racks(s) from the autostainer to the Dako wash basin(s) fill with
1.times.TBS-T.
[0310] 8. Mount each slides with 55-75 .mu.L of room temperature
ProLong Gold Antifade reagent with DAPI.
[0311] 9. Allow mounting medium to set on a level surface in a
dark, dry place.
TABLE-US-00008 TABLE 8 Summary of ErbB2 protocol Incubation Volume
Category Reagent (min) (.mu.L) Rinse Buffer 1 Endogenous BioCare
Medical 10 150 enzyme block Peroxidazed 1 Rinse Buffer 1 Rinse
Buffer 1 Protein Block BioCare Medical 10 150 Background Sniper
Rinse Buffer 1 Rinse Buffer 1 Primary Neomarkers Rabbit Anti- 60
150 antibody C-ErbB2 (SP3) Rinse Buffer 1 Rinse Buffer 1 Secondary
DAKO EnVision Anti- 30 150 Reagent Rabbit + A555GAM Rinse Buffer 1
Rinse Buffer 1 Substrate- Perkin Elmer Cyanine 5 10 150 chromogen
Tyramide Reagent Rinse Buffer 1 Rinse Buffer 1
TABLE-US-00009 TABLE 9 Summary of ErbB3/4 protocol: Volume (.mu.L)
Incubation (per drop Category Reagent (min) zone) Rinse Buffer 1
Endogenous BIOCARE MEDICAL 10 150 enzyme block PEROXIDAZED 1 Rinse
Buffer 1 Rinse Buffer 1 Protein Block BIOCARE MEDICAL 10 150
BACKGROUND SNIPER Rinse Buffer 1 Rinse Buffer 1 Primary ErbB3: RB
mAb to ErbB3 60 150 antibody ErbB4: RB pAb to ErbB4 (in DA VINCI
GREEN) Rinse Buffer 1 Rinse Buffer 1 Labeled DAKO ENVISION anti- 30
150 polymer rabbit HRP Rinse Buffer 1 Rinse Buffer 1 Rinse Buffer 1
Substrate- Substrate working solution 10 300 chromogen (mix) Rinse
DI water 1 Rinse DI water 1 Counterstain DAKO Hematoxylin 6 300
S3301 Rinse DI water 1 Rinse DI water 1 Rinse DI water 1
[0312] ErbB3/4: Once the staining is complete, perform the
following for dehydration step: [0313] Incubate slides in 80%
ethanol twice, each for 2 minutes. [0314] Incubate slides in 100%
ethanol twice, each for 2 minutes. [0315] Incubate slides in xylene
twice, each for 5 minutes. [0316] Mount each slide with 1-2 drops
of CYTOSEAL XYL and a coverslip.
Example 4
RT-PCR Assays
[0317] Quantitative RT-PCR (real-time polymerase chain reaction)
assays were developed to measure ErbB receptors and ligand
transcript levels in FFPE patient samples. Assays were carried out
by AltheaDX.RTM. using the TaqMan.RTM. Low Density Array (TLDA)
format. Each SOP referred to in this example is an AltheaDX.RTM.
SOP.
Overview of RT-PCR Assay: A section from a FFPE patient specimen is
macrodissected and RNA is extracted from a tumor-containing region.
Target and reference gene transcripts are reverse transcribed into
cDNA and pre-amplified by gene-specific PCR. The products of this
pre-amplification step are then transferred into TaqMan.RTM. Low
Density Array TLDA plate format (Wechser et al, 2004) and each
target is quantified by qPCR. To normalize for different RNA amount
across biopsies, three reference genes were also measured (GUSB,
B2M, HPRT1). TaqMan.RTM. Gene Expression Assays consist of a pair
of unlabeled PCR primers and a TaqMan.RTM. probe with a FAM.TM.
(56-FAM) dye label on the 5' end and a non-fluorescent quencher
(3BHQ_1) on the 3' end. RNA from samples of interest is reverse
transcribed into cDNA and pre-amplified by PCR. The product of this
amplification step then serves as the template for real-time
PCR.
[0318] Selection of Primers and Probes: The gene information for
both target and endogenous reference (control) genes are provided
in Table 10 below. Forward and reverse PCR primers are selected by
sequence alignment and BLAST sequence homology searches throughout
the entire human genome. The assay primers and probes are listed in
Table 11 below. The forward and reverse primers for HRG1 are
designed to hybridize within a single exon because the consensus
region of all HRG1 isoforms exists in a single exon. As a result,
the HRG1 assay can detect both mRNA and genomic DNA. All other
forward and reverse primers are designed in separate exons to
insure specific detection of mRNA and not genomic DNA. For
mRNA-specific HRG1 analysis, all samples are tested by an
independent run of the HRG1 assay without reverse transcription to
confirm absence of genomic DNA contamination.
[0319] The melting temperature (Tm) of the primers was designed to
be around 60.degree. C., and the (Tm) for the TaqMan.RTM.
hydrolysis probes is designed to be 5 to 7.degree. C. higher. The
size range of the target amplicon was designed to be between 80 and
100 bp (an appropriate size for FFPE samples). All synthetic
oligonucleotides were purchased from Integrated DNA Technologies,
Inc.
[0320] Total RNA extraction and qualification: Total RNA extraction
from samples is conducted per SOP 914023 "Extraction of RNA from
Tissue & Cells Using Qiagen RNeasy.RTM. Mini Kit," and total
RNA extraction from FFPE tissue is conducted per SOP 914017 "RNA
Extraction from Formalin Fixed Paraffin Embedded Tissue (FFPE)
using EPICENTRE MasterPure.RTM. RNA Kit." Isolated total RNA is
quantified per SOP 914052 "Determining Nucleic Acid Concentration
Using the NanoDrop 1000 Spectrophotometer." Assessment of sample
RNA quality is performed per SOP 914049 "Assessment of RNA Using
the Agilent.RTM. 2100 Bioanalyzer with RNA 6000 Nano & Pico
LabChip.RTM. Kits."
[0321] Genomic DNA contamination in RNA samples is determined by
singleplex Taqman.RTM. RT-PCR using the HRG1 primer/probe set
without reverse-transcriptase. If human genomic DNA is detected in
sample RNA with Ct<35, additional DNase I treatment is conducted
per SOP 914010, "DNase I Treatment of RNA," and RNA is then
re-purified per SOP 914009 "Nucleic Acid Extraction Using Phenolic
Reagents."
[0322] cDNA synthesis and multiplex pre-amplification: Extracted
and qualified RNAs are converted to cDNA and pre-amplified per SOP
914039 "One Step Reverse Transcriptase Polymerase Chain Reaction"
with a minor modification. Total 25 .mu.L reactions containing
various amount of RNA, 1.times.QIAGEN RT-PCR buffer, 2 .mu.M of all
forward and reverse primers mixture, 0.4 mM of dNTP, 1.times.
enzyme mix, and 0.4 units/.mu.L of RNase inhibitor in 96-well
plates are placed on a ThermoCycler (ABI 9700) pre-heated at
50.degree. C. After a 30-minute incubation, reactions are heated to
95.degree. C. for 15 minutes to inactivate the reverse
transcriptase and activate the Hot-start Taq polymerase. The
synthesized cDNAs are amplified by 14 cycles of 94.degree. C. for
30 sec, 55.degree. C. for 30 sec, and 72.degree. C. for 30 sec. The
reactions are kept at 4.degree. C. until they are needed for the
next process.
[0323] TaqMan.RTM. real-time PCR assay on TLDA card: TLDA
(TaqMan.RTM. Low Density Array) cards are custom manufactured by
Applied Biosystems (ABI). Each lane consists of 12 quadruplicate
assays including two repeats of HRG1B1 and HER3. The GAPDH assay is
a TLDA manufacturing control that is excluded from further
analysis.
[0324] The 25 .mu.l of pre-amplified reactions are mixed with 25
.mu.L of dH2O and 50 .mu.L of 2.times. Universal qPCR master mix
(ABI). A total of 100 .mu.L of mixture is loaded into each TLDA
lane reservoir. The TLDA card is then placed into a swing bucket
rotor, and centrifuged twice for 1 minute at 1200 RPM. After
sealing the card, the reservoirs are removed with scissors. The
trimmed TLDA card is placed into the real-time PCR instrument
(ViiA7.RTM., ABI), and run at: 50.degree. C. for 2 minutes,
94.degree. C. for 10 minutes, followed by 40 cycles of 94.degree.
C. for 30 sec and 57.degree. C. for 60 sec. Data collection and
analysis is performed using ViiA7.RTM. software and DataAssist.RTM.
v2.0 from ABI.
TABLE-US-00010 TABLE 10 List of Target and Reference Genes for
RT-PCR Assays Gene Genotype Category Gene name symbols covered
Target Epidermal growth HER3, HER3 factor receptor 3 ErbB3
(NM_001982) Target Heregulin 1 HRG1 HRG1-.alpha. HRG1-.beta.1
HRG1-.beta.2 HRG1-.beta.3 HRG1-.gamma. Target Heregulin 1-.beta.1
HRG1.beta.1 HRG1-.beta.1 Target Epidermal growth HER1, factor
receptor 1 ErbB1, EGFR Target Epidermal growth HER2 factor receptor
2 Target .beta.-cellulin BTC Reference Glucoronidase-.beta. GUSB
GUSb (control) (NM_000181) Reference .beta.-2-microglobulin B2M,
B2M (control) .beta.2M (NM_004048) Reference Hypothantin-guanine
HPRT1 HPRT1 (control) phosphoribosyltranferase 1 (NM_000194)
[0325] Total RNA extraction and qualification: Total RNA extraction
from samples was conducted as described in AltheaDx SOP 914023.
Extraction of RNA from Tissue & Cells Using Qiagen.RTM.
RNeasy.RTM. Mini Kit, and total RNA extraction from FFPE tissue was
conducted as described in SOP 914017 RNA Extraction from Formalin
Fixed Paraffin Embedded Tissue (FFPE) using EPICENTRE
MasterPure.TM. RNA Kit. Isolated total RNA was quantified using a
NanoDrop.RTM. (ND-1000) spectrophotometer as described in SOP
914052 Determining Nucleic Acid Concentration Using the NanoDrop
1000 Spectrophotometer. Assessment of sample RNA quality was
performed using Agilent 2100 Bioanalyzer with RNA 6000 Nano and
Pico LabChip kit according to SOP 914049 (Assessment of RNA Using
the Agilent 2100 Bioanalyzer with RNA 6000 Nano & Pico LabChip
Kits).
[0326] Genomic DNA contamination in RNA samples was determined by
single-plex TaqMan real-time PCR using the HRG1 primer/probe
(below) set without reverse-transcriptase. If human genomic DNA was
detected in sample RNA with Ct<35, additional DNase I treatment
would be conducted according to SOP 914010, DNase I Treatment of
RNA, then RNA would be re-purified with phenolic reagents according
to SOP 914009 (Nucleic Acid Extraction Using Phenolic Reagents).
Probes for RT-PCR are set forth below in Table 11, in which the
third probe of each sequential group of 3 probes (indicated by a
name ending in "P" or "P" followed by one or two digits) has a 5'
terminal 56-FAM fluor and a 3' terminal 3BHQ_1 fluor attached
thereto.
TABLE-US-00011 TABLE 11 List of primers and probes for RT-PCR
assay. SEQ # of ID nucle- Tm Oligo Name Sequence NO: otides
.degree. C. HER1-F3 CTATGTGCAGAGGA 27 61.7 ATTATGATCTTTC HER1-R11
GCTAAGGCATAGGA 24 61.2 ATTTTCGTAG HER1-P10 TGCAGGTTTTCCAA 26 67.4
AGGAATTCGCTC HER2-F11 GGAAACCTGGAACT 21 61.7 CACCTAC HER2-R13
CCTGCCTCACTTGG 20 63.0 TTGTGA HER2-P10 ACCAATGCCAGCCT 22 68.4
GTCCTTCC HER3-F5 GCAACTCTCAGGCA 19 62.4 GTGTG HER3-R5
TGGTATTGGTTCTC 21 61.9 AGCATCG HER3-AP3 CGGTCACACTCAGG 23 67.6
CCATTCAGA HRG1-F1 CTTGTAAAATGTGC 23 62.8 GGAGAAGGA HRG1-R1
ATCTCGAGGGGTTT 23 64.1 GAAAGGTCT HRG1-P TGTGAATGGAGGGG 26 69.3
AGTGCTTCATGG HRG1B1 F4 GTGCAAGTGCCCAA 24 64.4 ATGAGTTTAC HRG1B1 R5
CTCCATAAATTCAA 25 62.1 TCCCAAGATGC HRG1B1 ASP TGGCCATTACGTAG 26
69.8 TTTTGGCAGCGA BTC-F3b TGGGAATTCCACCA 20 61.3 GAAGTC BTC-R1b
GCCTTTCCGCTTTG 19 60.9 ATTGT BTC-P2 ACTGTGCAGCTACC 26 69.7
ACCACACCAATC GUSB-F4A GGAATTTTGCCGAT 24 62.7 TTCATGACTG GUSB-R5
GTCTCTGCCGAGTG 20 61.3 AAGATC GUSB-P CACCGACGAGAGTG 20 67.9 CTGGGG
B2M-F1 TGACTTTGTCACAG 23 63.7 CCCAAGATA B2M-R1 AATCCAAATGCGGC 20
61.2 ATCTTC B2M-P1 TGATGCTGCTTACA 27 68.7 TGTCTCGATCCCA HPRT-F1
CCTTGGTCAGGCAG 22 61.9 TATAATCC HPRT-R1 TCTGGCTTATATCC 23 61.9
AACACTTCG HPRT-SP1 AAGCTTGCTGGTGA 23 67.0 AAAGGACCC
Example 5
Clinical Trial--Ovarian Cancer
[0327] A clinical trial was performed that was designed to allow,
inter alia, determination of associations between biomarker
profiles and clinical responses. Biomarker data were measured in
pre-treatment biopsies and (when available) in archived tumor
samples. Results pertaining to predictive biomarkers measured in
pre-treatment biopsies are described herein.
[0328] The following study was performed to assess biomarkers to be
used to predict response to ErbB3 inhibitor (MM-121) therapy.
[0329] Assays
[0330] Four types of assay (each set forth above) were used to
assess the six primary biomarkers assessed in this study (EGFR,
ErbB2, ErbB3, ErbB4, HRG and BTC):
[0331] 1. Fluorescence-based quantitative immunohistochemistry
(Fl-IHC)
[0332] 2. Chromogenic quantitative immunohistochemistry (qIHC).
[0333] 3. Chromogenic RNA-in situ hybridization (RNA-ISH).
[0334] 4. Real-time quantitative polymerase chain reaction
(RT-PCR).
[0335] Scoring Systems for Chromogenic Assays
[0336] For the two chromogenic assays (qIHC and RNA-ISH), two
different overall scores were used for biomarker analyses: [0337]
Composite score=highest score x % tumor cells exhibiting highest
score+second highest score x % tumor cells exhibiting second
highest score. [0338] Top-10 score=highest score in at least 10% of
tumor cells.
[0339] A summary of the assays used in this study is provided in
Table 12.
TABLE-US-00012 TABLE 12 Primary Biomarker Assays FI-IHC qIHC
RNA-ISH RT-PCR Biomarker (protein) (protein) (RNA) (RNA) EGFR C C
ErbB2 C C ErbB3 C D D C ErbB4 D HRG C D C HRG-1.beta.1 C (HRG
isotype) BTC D C C = Continuous variable | D = Discrete variable
(pathologist-scored as 0, 1+, 2+, 3+ or 4+)
[0340] Biomarker Values
[0341] Biomarker analyses were performed on the 220 subjects in the
safety population of this trial. The number of subjects with
biomarker data for each type of assay is summarized in FIG. 5A. The
number of subjects for whom serum biomarker data are available is
provided for reference. The primary cause underlying missing data
was insufficient tumor material in the biopsies.
[0342] Of the six biomarkers, EGFR was the least prevalent. It was
at or below the limit of detection, either by RT-PCR or IHC, in
>60% of the samples.
[0343] Univariate Analyses
[0344] In total, six primary biomarkers were measured using 15
assays. For the 5 chromogenic assays (qIHC and RNA-ISH), two
different overall scores were used: composite score and top-10
score. Accordingly, 20 different variables were assessed at this
stage. A Cox proportional hazard model was used to rank the
biomarkers. This method determines if PFS varies with biomarker
values differently in the treatment arm relative to the control
arm. A P-value cutoff of 0.4 was used to determine which biomarkers
would be prioritized for subsequent bivariate analyses. The results
of these biomarker ranking experiments are provided in FIG. 5B.
[0345] Overall, four biomarkers were prioritized for further
analysis based on the data summarized in FIG. 5B: 1) ErbB2 qIHC, 2)
HRG RNA-ISH (top-10), 3) ErbB3 qIHC (top-10), and 4) ErbB4 qIHC
(top-10). For all of the pathologist-scored assays, the top-10
score correlated better with HR than the composite score. In
addition, the imaging-based methods (IHC and ISH) provided better
correlations than RT-PCR, which relies on homogenized tissue. One
explanation for this observation is that imaging-based methods can
account for variability in the tumor cell content of a specimen,
whereas RT-PCR cannot.
[0346] Relationship Between ErbB2 Levels and HR
[0347] The relationship between ErbB2 levels and local hazard ratio
(HR) is shown in FIG. 6A. This plot shows local HR (the HR within a
defined window of ErbB2 levels), rather than cumulative HR (the HR
for all patients either above or below a given ErbB2 level). As can
be seen in FIG. 6A, local HR decreases (favors the treatment arm)
as ErbB2 levels decrease. The level of ErbB2, as a single
biomarker, at which the HR crosses 1 (no treatment effect) is
approximately 5.1 on a log.sub.10 scale, which corresponds to about
126,000 receptors per cell. This is consistent with pre-clinical
predictions from computational modeling and pre-clinical data
showing that MM-121 loses potency when ErbB2 levels rise above
about 100,000 to about 200,000 receptors per cell. Of the patients
for which ErbB2 data are available (n=174), 53% fall below a
threshold of 5.1. Coincidentally, this threshold corresponds to the
approximate boundary defining the difference between a score of 1+
and a score of 2+ of the HercepTest.RTM. assay ("HCT").
Accordingly, HCT can be used as an alternative HER2 assay in this
and other aspects of the disclosed methods, with HCT score of less
than 2+ indicating a favorable HER2 level for treatment with an
ErbB3 inhibitor such as MM-121.
[0348] The relationship between ErbB2 levels and HR was not
observed at the interim analysis of this trial. At the time of the
interim analysis, very few patients (n=37) had ErbB2 levels above
5.1. In the final dataset, more patients (n=81) were observed with
a level above this value, providing increased resolution in the
local HR scans.
[0349] Relationships Between Biomarker Measurements and HRs for
HRG, ErbB3, and ErbB4
[0350] The relationships between biomarker measurements and local
HRs for HRG, ErbB3, and ErbB4 (all pathologist-scored assays) are
shown in FIG. 6B. The solid dots indicate local HRs, whereas the
speckled dots show the percentage of patients with the given
biomarker value. The different sizes of the various dots provide a
heuristic for prevalence at each biomarker value that is redundant
to the prevalence scale. Black lines represent the 95% CI of the HR
estimates.
[0351] With regard to HRG, subjects with undetectable levels of HRG
(score of 0) had a HR that favored control, whereas those with
detectable levels of HRG (score of 1-2 or >2) had HRs that
favored treatment. This is consistent with the concept underlying
MM-121, which was designed to block HRG-driven ErbB3 signaling. The
HR is similar for a score of 1-2 and a score of >2. The
detection limit of the assay thus provides a natural cut-point for
HRG of either HRG present or absent. Of the patients for which HRG
data are available (n=157), 62% have detectable levels of HRG
(score of 1 or higher).
[0352] With regard to ErbB4, subjects with undetectable levels of
ErbB4 (score of 0) had a HR that favored treatment, whereas those
with detectable levels of ErbB4 (score>0) had a HR that favored
control. This is consistent with the pre-clinical prediction that
high ErbB4 levels provide an alternative way for HRG to signal
independently of ErbB3 and hence render cells less sensitive to
MM-121. Of the patients for which ErbB4 data are available (n=128),
49% have undetectable levels of ErbB4 (score of 0).
[0353] Finally, with regard to ErbB3, the data show that low or
undetectable levels of ErbB3 (score <2) favor the control,
whereas medium to high levels of ErbB3 favor treatment. The result
is complex, however, as medium levels of ErbB3 (score of 2) favor
treatment to a greater extent than high levels of ErbB3 (score of 3
or 4). A similar result was observed using data from the
fluorescence-based qIHC assay for ErbB3. Although the decrease in
HR from low to medium levels of ErbB3 was observed at interim, the
increase in HR from medium to high levels of ErbB3 was not
observed. The observed increase in HR with high levels of ErbB3 in
the final dataset occurs at levels above about 20,000 receptors per
cell. At the time of the interim analysis, very few patients (n=34)
had ErbB3 levels above this inflection point. In the final dataset,
more patients (n=101) were observed with levels above this value,
providing increased resolution in the local HR scans.
[0354] Conclusion of Univariate Analyses
[0355] Overall, low levels of ErbB2 (53% of patients), detectable
levels of HRG (62%), medium to high levels of ErbB3 (80%), and
undetectable levels of ErbB4 (49%) were all independently found to
favor treatment over control. The results show that intermediate
levels of ErbB3 favor treatment to a greater extent than high ErbB3
levels.
[0356] Bivariate Analyses (Models with Two Biomarkers)
[0357] Pairwise interactions between biomarkers were evaluated as
follows. In total, there are six two-biomarker models that can be
constructed using four biomarkers: ErbB2&HRG, ErbB2&ErbB3,
ErbB2&ErbB4, HRG&ErbB3, HRG&ErbB4, and ErbB3&ErbB4.
To understand pairwise relationships between biomarkers, cumulative
HR was plotted as a function of one biomarker and then repeated at
different values of the other biomarker. The results of these
calculations are shown in FIG. 7A.
[0358] Focusing on the ErbB2 and HRG model as an example (top-left
plot), the cumulative HR scans were determined by selecting a
subpopulation of patients based on their HRG status and then
plotting cumulative HR across ErbB2 levels. The x-axes represent a
scan of ErbB2 levels, showing the overall percentage of patients
with ErbB2 levels below each value in the scan. The dashed lines
represent patients with any score for HRG (i.e., all the patients
with BM measurements). The right side of the plot shows all of
these patients (HR.apprxeq.1, prevalence=100%). Moving from right
to left, the ErbB2 threshold above which patients are excluded
progressively decreases. Thus, the midway point of this plot
(Prevalence=50%) represents all the patients with ErbB2 levels
below the median. The cumulative HR decreases as ErbB2 high
patients are successively excluded. The thickest solid line on this
plot shows the same procedure performed only on the patients with a
HRG score of 1 or higher (i.e., detectable levels of HRG). The
right end of this plot starts at a prevalence of 62% because 38% of
patients had undetectable levels of HRG. Moving from right to left
along the red line, the ErbB2 threshold above which patients are
excluded progressively decreases, as before. The thinnest solid
line represents the same procedure, performed using subjects with a
HRG score.gtoreq.2 (41% of subjects). The starting point for this
scan (right end of the thinnest solid line) is therefore at 41%
prevalence. This plot shows that patients with detectable levels of
HRG and low levels of ErbB2 derive meaningful clinical benefit from
MM-121 (HR<0.5). The thinnest solid line (HRG score.gtoreq.2) is
shifted to the left relative to the thickest solid line because
fewer patients have a HRG score.gtoreq.2 than a HRG score.gtoreq.1.
The thinnest solid line does not drop appreciably below the
thickest solid line, showing that a detectable level of HRG is
roughly equivalent to a high level of HRG in this context (i.e.,
here higher HRG scores do not predict greater benefit than lower,
but detectable HRG levels).
[0359] The other five plots were prepared in a similar fashion to
the ErbB2 and HRG plot. For the ErbB2 and ErbB3 and ErbB2 and ErbB4
plots, cumulative HR was scanned on ErbB2 levels (as above). For
the ErbB3 and HRG and ErbB3 and ErbB4 plots, cumulative HR was
scanned on ErbB3 values. For the ErbB4 and HRG plot, cumulative HR
was scanned on ErbB4 values.
[0360] Overall, the strongest pairwise interaction (resulting in
the most favorable balance between HR and prevalence) was observed
for the interaction between ErbB2 and HRG. To explore this
interaction further, local HR was plotted as a function of ErbB2
levels for all subjects and for HRG-positive subjects
(score.gtoreq.1) (FIG. 7G). The dots and dashes are observed HRs,
while the thin and heavy lines are smoothed renderings of these
data accounting for noise in the ErbB2 measurements. Notably, the
thick line is shifted down relative to the thin line at low levels
of ErbB2, indicating that HRG status augments ErbB2 status in the
identification of patients that derive benefit from MM-121. Based
on this plot, a HRG score of .gtoreq.1 and an ErbB2 threshold of
log.sub.10 5.1 (corresponding to about 126,000 receptors per cell)
were chosen for subsequent analyses.
[0361] Based on these two thresholds, a biomarker profile positive
(BM+) subpopulation is defined for subsequent analyses as: (ErbB2
levels.ltoreq.log.sub.10 5.1) AND (HRG score.gtoreq.1) ("ErbB2 low"
AND "HRG positive"). Accordingly, a biomarker profile negative
(BM-) subpopulation is defined as (ErbB2 levels >log.sub.10 5.1)
OR (HRG score<1) This definition results in a BM+ prevalence of
34% in the clinical trial population.
[0362] Survival Characteristics of BM+ and BM- Subpopulations
[0363] Progression-free survival: The Kaplan-Meier PFS plot for the
overall safety population after 60 weeks is provided in FIG. 8A. In
this and plots 8B and 8C, the treatment arm (paclitaxel+MM-121) is
represented by a solid black line and the control arm (paclitaxel)
is represented by a dashed line. In FIG. 8G (progression free
survival at 10+ months) and FIG. 8H (overall survival at 10+
months), the treatment arm is represented by a blue line and the
control arm is represented by a black line.
[0364] Kaplan-Meier PFS plots for the BM+ and BM- profile
subpopulations are provided in FIGS. 8B and 8C for data collected
at 60 weeks. The BM+ profile subpopulation had a 0.37 [95% CI
0.2-0.8] HR and the BM- subpopulation had a 1.54 [95% CI 1.0-2.4]
HR. In comparing the control arms in these two populations, the BM+
profile subpopulation performs worse than the BM- profile
subpopulation (i.e., BM+ profile is indicative of poor prognosis
when treated only with paclitaxel). A Kaplan-Meier PFS plot for the
BM+ and BM- profile subpopulations is provided in FIG. 8I.
[0365] No imbalances were observed between treatment and control
arms in the BM+ profile population for a) number of lines of prior
therapy, b) time to first metastatic event, histology, or c) age. A
slight imbalance was observed in dichotomized age (>60 vs.
.ltoreq.60) and stage of disease (stage IV vs. stage I, II,
III).
[0366] Best Response Rates
[0367] Best response rates for treatment and control arms are shown
in FIG. 8F.
[0368] Although the numbers are low, the PR and SD rates are higher
in the BM+ subpopulation than in the BM- subpopulation on the
treatment arm. This contrasts with the control arm, where the PR
rate is higher in the BM- subpopulation than in the BM+
subpopulation.
[0369] Consequence of ErbB3 and ErbB4 Status in the BM+
Subpopulation
[0370] To evaluate the potential significance of not including
ErbB3 and ErbB4 measurements in the definition of a biomarker
profile positive subpopulation (ErbB2 low and HRG positive), HRs
were estimated for subgroups in the BM+ and BM- subpopulations as
determined by ErbB3 or ErbB4 status. The results are provided in
Table 13.
TABLE-US-00013 TABLE 13 HR estimates in subgroups of BM+ and BM-
patients as defined by ErbB3 and ErbB4 status. BM+ or - Prevalence
(HER2 low Subgroup Subgroup in BM sub- N N HR HR & HRG+) BM BM
score population (MM-121) (ctrl) (trt:ctrl) (95% CI) BM- ErbB4 0
0.49 28 13 1.05 0.13-8.8 BM- ErbB4 .gtoreq.1 0.51 27 15 2.20
0.27-18 BM+ ErbB4 0 0.48 14 6 0.13 0.02-1.1 BM+ ErbB4 .gtoreq.1
0.52 16 6 0.65 0.08-5.5 BM- ErbB3 <2 0.17 11 4 NA NA-NA BM-
ErbB3 .gtoreq.2 0.83 46 28 0.86 0.11-7.0 BM+ ErbB3 <2 0.21 10 0
NA NA-NA BM+ ErbB3 .gtoreq.2 0.79 24 13 0.33 0.04-2.7 (trt =
treatment, ctrl = control)
[0371] The HR for the ErbB4 positive subgroup (score.gtoreq.1)
within the BM+ subpopulation is 0.65 [0.08-5.5] in favor of the
treatment arm. This subgroup represents 52% of the BM+ patients.
Thus, there is no evidence that this subgroup is adversely affected
by treatment. Conversely, for the subgroup of ErbB4 negative
patients (score=0) in the BM- subpopulation, the estimated HR is
1.05 (0.13-8.8). There is therefore no evidence that these patients
would receive benefit from MM-121, even though they are excluded
from treatment by the definition of BM+ (ErbB2 low & HRG
positive). This subgroup constitutes 49% of the BM-
subpopulation.
[0372] For the ErbB3 low/negative subgroup (score<2) in the BM+
subpopulation (21%), the HR is not calculable as there are no
patients within this subgroup enrolled in the control arm. For the
ErbB3 med/high subgroup (score.gtoreq.2) in the BM- subpopulation,
the estimated HR is 0.86 (0.11-7.0). Thus, there is no evidence
that these patients would receive substantial benefit from MM-121,
even though they are excluded from treatment by the definition of
BM+ (ErbB2 low & HRG positive).
[0373] Summary of Results
[0374] Overall, four biomarkers were identified that favored
treatment relative to control. These include low ErbB2 (less than
2+ as measured by qIHC, =about 126,000 ErbB2 receptors per cell,
=HCT less than 2+) representing 53% of patients, HRG positive (1+
or higher as measured by RNA-ISH) representing 62% of patients,
ErbB4 negative (less than 1+ as measured by IHC) representing 49%
of patients, and ErbB3 medium to high (2+ or higher as measured by
IHC) representing 80% of patients.
[0375] The interpretation of the ErbB3 results was found to be more
complex than for the other biomarkers, and it was determined that
ErbB3 negative/low (20%) favors control and ErbB3 medium/high (80%)
favors treatment, but ErbB3 medium favors treatment more so than
ErbB3 high.
[0376] No major imbalances in clinical covariates are observed in
the BM+ subpopulation and there is no evidence suggesting that low
ErbB3 status or high ErbB4 status places patients in the BM+
subpopulation at risk.
[0377] A two-biomarker model (ErbB2 low and HRG positive)
identifies a subpopulation of patients that benefit from MM-121 (HR
of 0.37 [95% CI of 0.2-0.8]; prevalence of 34%). The subpopulation
of patients, defined by log.sub.10(ErbB2)<5.1 and HRG
detectable, resulted in HR of 0.37 (95% CI of 0.2-0.8) with a
prevalence of 34%.
[0378] The HR in the biomarker negative population (66%) was 1.54
(95% CI of 1.0-2.4)
[0379] The results of the study suggest that ligand-driven ErbB3
signaling mediates tumor cell survival and so renders tumors less
responsive to chemotherapy (weekly paclitaxel). MM-121 is designed
to block ligand-driven ErbB3 signaling and therefore may provide
benefit to patients in which ErbB3 signaling is providing a
mechanism of resistance to chemotherapy.
[0380] A biomarker profile of low ErbB2 (less than or equal to
about 126,000 receptors per cell by chromogenic qIHC as disclosed
herein, or below 2+ by HCT) and HRG positive (+1 or higher by
chromogenic RNA-ISH) has been identified as an exemplary biomarker
signature indicative of an increased likelihood of responsiveness
to ErbB3 inhibitor therapy.
[0381] In summary, this study determined that HRG mRNA levels
predicted response to MM-121, which was further enhanced in
patients with low ErbB2 levels. BM+ patients were defined as having
detectable HRG mRNA and low ErbB2 protein in pre-treatment
biopsies, and while BM+ patients responded poorly to paclitaxel
alone, these same patients benefited from the combination therapy
of MM-121 and paclitaxel. Results from this study further implicate
heregulin-driven ErbB3 signaling as a mechanism of resistance to
standard-of-care therapies such as paclitaxel in advanced,
platinum-resistant ovarian cancer.
Blockade of this pathway by MM-121 enhances sensitivity to
paclitaxel in this molecularly-defined patient population. These
data, together with findings from other MM-121 Phase 2 studies,
establish the role for heregulin-dependent ErbB3 signaling as a
critical survival pathway mediating resistance to
anti-proliferative therapies across indications.
Example 6
Clinical Trial--Breast Cancer: A Randomized Trial of
Exemestane+/-Seribantumab (MM-121) in Postmenopausal Women with
Locally Advanced or Metastatic ER/PR+ HER2- Breast Cancer: Final
Analysis and Extended Subgroup Analysis
[0382] A randomized, double-blind phase 2 trial of
exemestane+/-MM-121 in postmenopausal women with locally advanced
or metastatic estrogen receptor positive (ER+) and/or progesterone
receptor positive (PR+), HER2 negative breast cancer was performed.
118/145 patients were randomized to receive treatment. Patients
were randomized in a 1:1 fashion to receive MM-121 plus exemestane
(M, n=59) or placebo plus exemestane (P, n=59). Of the 118 patients
randomized, 118/118 (100%) were successfully randomized and
included in the intent to treat (ITT) population. The safety
population, 115/118 (97.5%) patients (56 (M), 59 (P)) is a subset
of the ITT population that received at least one dose (including a
partial dose) of study medication (MM-121/placebo or exemestane).
This population is for safety analyses, as well as the primary
population for all efficacy parameters.
[0383] An objective of the trial was to assess biomarker profiles
as predictors of clinical responses to MM-121 and/or exemestane.
Biomarker analyses were performed using the safety population
(n=115). Analyses were performed on the five pre-specified primary
biomarkers, all mechanistically linked to ErbB3 signaling, as
measured in FFPE archived tissue blocks. Biomarkers were assessed
based on local HR scans. Thresholds for defining biomarker profile
positive populations were chosen based on these scans and on
results obtained in the above-described ovarian cancer trial. A
single two-variable model was assessed using HRG and ErbB2, based
on the findings from the above-described ovarian cancer trial. HRs
in the biomarker profile positive and negative populations were
calculated using a Cox proportional hazard model that included
biomarker groups by treatment and stratification factors as
additive factors. Only patients with non-missing biomarkers were
used in each calculation.
[0384] For these analyses, ErbB2 levels were determined using a
fluorescence-based quantitative immunohistochemistry assay (as
described above in Example 2) and HRG RNA was determined using
RT-PCR (as described above in Example 4). Both assays were
performed using FFPE archived tissue blocks. The RNA-ISH assay that
was used in the ovarian cancer trial described above in Example 5
failed to provide usable data in this context, presumably because
the assay is not robust to the degraded RNA found in old, archived
tissue.
[0385] Effects of Single Biomarkers: Local HR Scans
[0386] Biomarker effects were assessed by preparing local HR scans
for each biomarker independently. Scans for HRG RT-PCR and ErbB2
qIHC are shown in FIG. 9. Both HRG and ErbB2 presented in the same
direction as observed in the ovarian cancer trial described above
and are consistent with pre-clinical predictions. HRG mRNA was
measured in archived tissue using RT-PCR rather than RNA-ISH (as
used in the ovarian cancer trial). The positive control for the
RNA-ISH assay failed in 17 of the 48 evaluable samples. Of the
remaining 31 samples, the RNA-ISH positive control scores were
generally lower than those observed in the ovarian pre-treatment
biopsies.
[0387] Effects of Biomarkers: BM+ and BM- Subpopulations
[0388] Biomarker profile positive (BM+) and biomarker profile
negative (BM-) subpopulations were defined by dichotomizing
biomarker values for HRG and ErbB2. For HRG, a cut point of -5
(RT-PCR score) was chosen based on the local HR scan (value at
which HR=1). For ErbB2, the cut point used in the analysis of the
ovarian cancer trial was used: log.sub.10(ErbB2)=5.1 corresponding
to about 126,000 receptors per cell. HRs and prevalence of
biomarker-defined subpopulations are provided in Table 14, below.
Two separate analyses of ErbB2 levels are provided: (1) ErbB2
levels based on the qIHC assay; and (2) for patients where qIHC
data are missing, but HCT results were available, ErbB2 levels were
adapted from the reported HCT results, with a score of HCT 2+ being
deemed equal to log.sub.10(ErbB2)=5.1.
TABLE-US-00014 TABLE 14 HRs and prevalence of biomarker-defined
subpopulations. BM population BM positive BM negative Group N HR
95% CI N % HR 95% CI N % HR 95% CI no selection 115 0.75 0.48- 1.15
HRG > -5 57 0.68 0.38- 21 37% 0.35 0.13- 36 63% 0.99 0.47- 1.23
0.94 2.08 ErbB2 < 5.1 (qlHC) 72 1.16 0.67- 53 74% 0.84 0.44- 19
26% 3.00 1.01- 2.01 1.61 8.93 ErbB2 < 5.1 103 0.82 0.52- 84 82%
0.62 0.37- 19 18% 3.00 1.01- (HCT) 1.3 1.04 8.93 ErbB2 < 5.1
(qlHC) 44 0.86 0.44- 14 32% 0.32 0.09- 30 68% 1.30 0.59- & HRG
> -5 1.68 1.12 2.86 ErbB2 < 5.1 (HCT) 55 0.66 0.36- 17 31%
0.32 0.1-1 38 69% 0.89 0.44- & HRG > -5 1.2 1.79
[0389] Description of BM Population
[0390] The BM population (biomarker-assessed population) is a
subset of the safety population and includes all patients with
measured HRG levels and either measured ErbB2 qIHC levels or
available HCT results as described above. A total of 55/115
patients (48%) were part of the BM population. The HR of the BM
population was 0.66 [95% CI 0.36-1.20]. Of the 55 patients with
ErbB2 and HRG data, 17 (31%) were BM+ and 38 (69%) were BM-.
Relative to control arm, patients in the treatment arm of the BM+
group have a higher proportion of non-bone-only lesions, a higher
proportion of last treatment in the adjuvant setting, a lower
proportion of anti-estrogen as the most recent therapy, and a
higher proportion of patients with ECOG=0. Using a Cox proportional
hazard model that includes additive terms for treatment, biomarker
groups, and these clinical covariates as a sensitivity analysis,
the estimated HR in the BM+ group is 0.38 [95% CI 0.12-1.24].
[0391] Efficacy Analysis of the BM+ Subpopulation
[0392] All efficacy analyses for the BM subgroups were performed
using a Cox proportional hazard model that included both of the
pre-specified strata as additive effects in combination with the
treatment effect. The ErbB2 low & HRG high BM+ subpopulation
was defined as patients with log.sub.10(ErbB2).ltoreq.5.1 and HRG
RT-PCR>-5. Kaplan-Meier PFS plots for the BM+ and BM-
subpopulations are provided in FIG. 10 for data collected at about
60 weeks (10A-10B) and for data collected at about 18 months
(10C-10E). For PFS at about 60 weeks the BM+ subpopulation HR was
0.32 [95% CI 0.10-1.00] (FIG. 10A) and the BM- subpopulation HR was
0.89 [95% CI 0.44-1.79] (FIG. 10B). PFS for the overall population
in the study was calculated for data collected at end of study and
shows that patients in the treatment arm had overall better
outcomes than patients in the control arm (FIG. 10C). A similar
result is seen for overall survival (FIG. 10D). Data for BM+
patients in the control arm (thin solid line), BM+ patients in the
treatment arm (thick solid line), BM- patients in the control arm
(thin dashed line), and BM- patients in the treatment arm (thick
dashed line) are shown in FIG. 10E, and illustrate that BM+
patients do considerably worse on the standard of care therapy
(exemestane alone) than do BM- patients. FIG. 10F depicts overall
survival in patients who progressed in the adjuvant setting prior
to entering study. FIG. 10G depicts overall survival in patients
who progressed in the metastatic setting prior to entering study.
FIG. 10H depicts progression free survival in patients who
progressed in the adjuvant setting prior to entering study. FIG.
10I depicts progression free survival in patients who progressed in
the metastatic setting prior to entering study.
[0393] Objective Response Rates
[0394] In BM+ patients the objective response rate was 11% (1/9) on
the treatment arm (M+E) and 0% (0/8) on the control arm (E). In BM-
patients the objective response rate was 4.8% (1/21) on the
treatment arm (M+E) and 5.9% (1/17) on the control arm (E).
[0395] Additional Data: final analysis and extended subgroup
analysis of a Phase 2 study of seribantumab in combination with
exemestane in metastatic HER2-negative, ER/PR+ breast cancer. The
study was a randomized, double-blinded, placebo-controlled study
that evaluated whether the combination of seribantumab and
exemestane was more effective in prolonging progression free
survival (PFS) than exemestane in ER/PR+ metastatic breast cancer
(n=118) who had previously failed anti-estrogen therapy. Patients
who were heregulin-positive had a 74% decrease in risk of
progression (n=34 patients, HR 0.26; 95% CI [0.11-0.63], p=0.003)
than patients who did not receive seribantumab. Notably, in the
full patient population, overall survival data trended in favor of
the seribantumab arm with a 59% decrease in risk of death (HR 0.41;
95% CI [0.19-0.90]) versus patients on the standard-of-care arm. In
multiple subgroup analyses, including analyses by age and
metastatic disease state, the seribantumab arm showed longer
overall survival than the control arm.
[0396] Summary of Adverse Events
[0397] There was no difference in any grade or grade 3 or higher
incidence of TEAEs. There was no difference in serious TEAEs. All
fatal AEs were disease progression-related. Overall well tolerated
combination of seribantumab and exemestane.
TABLE-US-00015 TABLE 15 Seribantumab + Placebo + Exemestane
Exemestane N = 56 N = 59 Subjects with at least one AE 48 ( 85.7)
51 ( 86.4) Subjects with at least one 48 ( 85.7) 50 ( 84.7) TEAE
Subjects with CTCAE grade 14 ( 25.0) 15 ( 25.4) 3 or higher TEAE
Subjects with TEAE related 40 ( 71.4) 32 ( 54.2) to study drug (a)
Subjects with serious TEAE 7 ( 12.5) 11 ( 18.6) Subjects with TEAE
leading 2 ( 3.6) 1 ( 1.7) to death Subjects with TEAE leading 2 (
3.6) 0 to dose discontinuation
CONCLUSIONS
[0398] A BM+ subpopulation was identified using the same
combination of two primary biomarkers as identified in the ovarian
cancer trial described in the preceding Example (ErbB2 low &
HRG positive/high), albeit with different cutoffs for HRG as
necessitated by the differing outputs of the RNA-ISH and RT-PCR HRG
transcript assays used in the two trials. In this trial, HRG
carried the most predictive information, with ErbB2 levels serving
as a potential modifier. HRG high (RT-PCR score.gtoreq.-5) and
ErbB2 low (log.sub.10 (ErbB2).ltoreq.5.1) favored treatment (N=17;
HR=0.32 [0.10-1.00]; prevalence=31%), whereas HRG low and/or ErbB2
high favored control (N=38; HR=0.89 [0.44-1.79]). The median PFS in
the BM+ population was 33 [8-NA] weeks (7.59 months) (M+E) and 12
weeks [8-NA] (2.76 months) (E). The prevalence of 31% likely
represents a lower boundary for this population, as the assays were
performed on archived tissue rather than biopsies obtained
immediately prior to treatment (treatments between archived tissue
collection and current start of treatment may increase the number
of patients with high HRG). The median PFS in the BM+ population
was 33 weeks (7.59 months) (MM-121+exemestane) and 12 weeks (2.76
months) (exemestane control). The HR in the BM- population was 0.89
[95% CI 0.44-1.79].
Example 7
HRG-.beta.1 Intracellular Domain ("Stump") Fluorescence Staining
Assay
[0399] Introduction: HRG is initially synthesized as a
transmembrane protein. The extracellular domain of this protein is
proteolytically cleaved to yield HRG, leaving behind a
transmembrane domain still imbedded in the cell membrane, and
intracellular domain within the cell cytoplasm. Measuring this
remaining "stump" of the HRG precursor protein was done to
determine if levels of the HRG stump could serve as a predictive
biomarker in accordance with the disclosure herein. Results of this
assay indicated that this was not the case, and that tumor levels
of HRG stump measured by the following procedure are not predictive
of responsiveness to ErbB3 inhibition.
TABLE-US-00016 TABLE 16 Materials Chart: Incubation Reagent
(numbered Dilution Time/Step per materials list below) Factor N/A
DI Water N/A N/A 5 1:20 25 min 6 1:100 10 min 7 RTU 10 min 8 RTU 60
min 9 RTU 1 1:1500 2 1:50 30 min 3 1:200 4 RTU 10 min 10 1:50 N/A
11 1:5000 N/A 12 N/A N/A 16 N/A
[0400] Materials List: [0401] 1. Anti-NRG-.beta.1 monoclonal
antibody clone 60-10 (start conc.=1 mg/mL) [0402] 2.
Anti-human-cytokeratin monoclonal antibody clone AE1/AE3 (Dako,
catalog #M351501) [0403] 3. Alexa Fluor.RTM. 555 Goat Anti-Mouse
IgG (H+L) (Invitrogen, catalog #A-21422) [0404] 4. EnVision+
System-HRP Labelled Polymer Anti-Rabbit (Dako, catalog #K4003)
[0405] 5. Tris Buffered Saline and Tween 20 (20.times.; TBS-T)
(Fisher Scientific, catalog #TA-999-TT) [0406] 6. PT Module Buffer
4, Tris-EDTA, pH 9 (Fisher Scientific, catalog #TA-250-PM4X) [0407]
7. Peroxidazed 1 (BioCare Medical, catalog #PX968 H, M) [0408] 8.
Background Sniper (BioCare Medical, catalog #BS966 H, M) [0409] 9.
Da Vinci Green Diluent (BioCare Medical, catalog #PD900 H, M)
[0410] 10. Cyanine 5 Tyramide (Perkin Elmer, catalog #SAT705A)
[0411] 11. Hoechst 33342 (Invitrogen, catalog #H3570) [0412] 12.
ProLong Gold Antifade reagent (Invitrogen, catalog #P36934) [0413]
13. Flex 100 Alcohol Solution (Fisher Scientific, catalog #8101)
[0414] 14. Flex 80 Alcohol Solution (Fisher Scientific, catalog
#8301R) [0415] 15. Xylene (VWR, catalog #534056) [0416] 16. Glass
coverslips (VWR No. 1)
[0417] Methods:
[0418] 1. Deparaffinize/Hydrate Slides [0419] 1.1. If necessary,
use a razor blade to scrape paraffin wax off of the back of the
slides. Scrape the wax off of the front around the tissue region if
it is visible. If the tissue is not clearly visible under the wax,
do not scrape any wax from the front. [0420] 1.2. Incubate slides
for 30-50 min at 65.degree. C. in a metal slide rack (or
equivalent) in the oven to melt wax covering tissues. [0421] 1.3.
Transfer slides to a Tissue-Tek.RTM. (or equivalent) slide rack.
Immerse slides in the following solutions with occasional gentle
agitation: [0422] xylene, twice for 20-30 min each [0423] 100%
ethanol, twice for 2-5 minutes [0424] 80% ethanol, twice for 2-5
minutes [0425] Distilled water for 2-5 minutes [0426] 1.4. Apply
programmed Dako slide labels to the front frosted end of the slides
and place them on the Dako slide rack(s).
[0427] 2. Antigen retrieval (AR): [0428] 2.1. Perform AR in the PT
module using PT module buffer 4 (Tris EDTA at pH 9.+-.0.05) using
the following settings: [0429] Incubation time: 25 minutes [0430]
Incubation temperature: 102.degree. C. [0431] No-boil function:
Enabled [0432] 2.2. Once the program has run and the solution
cooled down to 65.degree. C. remove the slide rack(s) from the PT
module and place in the Dako buffer wash basin(s) containing
1.times.TBS-T for 3-5 minutes.
[0433] 3. Reagent Preparation: This should be done when slides are
in the PT module. [0434] 3.1. In DakoLink.RTM. program, select all
slides to be stained from the "Pending" tab and click "Reagents"
button at the bottom of the screen. This will generate a list and
volumes needed per reagent (this assumes two 150 .mu.L drop zones
for a total of 300 .mu.L per slide). Print this list. [0435] 3.2.
Select the appropriate number and size of Dako user fillable
bottles. [0436] 3.3. Scan the barcode of each bottle and plug the
volume required of that particular reagent into the "usable
quantity" box. This will then factor in the dead volume of that
particular size bottle into your "Fill quantity" or total volume.
[0437] 3.4. Calculate the individual amounts of reagents needed for
your assay. [0438] Endogenous peroxidase block (Peroxidazed.RTM. 1)
and protein block (Background Sniper) are ready to use reagents and
may be filled right away. [0439] Primary antibody will be a
cocktail consisting of the target antibody, a compatible tumor mask
and blocking diluent. [0440] Secondary antibody will be a cocktail
of a molecular probe and the Dako EnVision+ secondary that
corresponds to the target being detected.
[0441] 4. Autostainer run preparation: Once all reagents are made
and AR process is complete, place the reagent bottles into the Dako
AutostainerLink 48. [0442] 4.1. Fill the 10 L buffer carboy with
1.times.TBS-T (add more if required) [0443] 4.2. Fill the 10 L
water carboy with DI water [0444] 4.3. Remove all the rack(s) from
the wash basin(s) and place on the autostainer. [0445] 4.4. Click
on the "Instruments" tab in the DakoLink.RTM. software and click
"Start" button.
[0446] 5. Automated staining: The following is the summary of the
Dako AutostainerLink48 NRG IF staining protocol. Note: The
autostainer selects each rinse reagent and also calculates each
rinse time and volume, therefore rinse reagent is indicated as an
alternative and the rinse incubation times and volumes are not
indicated.
TABLE-US-00017 TABLE 17 Protocol Reagent (numbered Incubation
Volume Category per materials list above) (min) (.mu.L) Rinse 1 or
5 -- -- Endogenous enzyme block 7 10 150 Rinse 1 or 5 -- -- Rinse 1
or 5 -- -- Protein Block 8 10 150 Rinse 1 or 5 -- -- Rinse 1 or 5
-- -- Primary antibody 1 60 150 Rinse 1 or 5 -- -- Rinse 1 or 5 --
-- Rinse 1 or 5 -- -- Secondary Reagent 4 30 150 Rinse 1 or 5 -- --
Rinse 1 or 5 -- -- Rinse 1 or 5 -- -- Substrate-chromogen 10 10 150
Rinse 1 or 5 -- -- Rinse 1 or 5 -- -- Rinse 1 or 5 -- --
Counterstain 11 5 150 Rinse 1 or 5 -- --
[0447] 6. Remove the ProLong Gold antifade reagent from freezer and
allow to come to room temperature.
[0448] 7. Once the staining is complete, transfers Dako slide
racks(s) from the autostainer to the Dako wash basin(s) fill with
1.times.TBS-T.
[0449] 8. Mount each slide with 55-75 .mu.L of room temperature
ProLong Gold.RTM. antifade reagent.
[0450] 9. Allow mounting medium to cure in a dark, dry and well
ventilated place on a level surface.
Example 8
Clinical Trial--Non-Small Cell Lung Cancer
[0451] A global, multi-center, open-label study was performed of
MM-121 and erlotinib in patients having non-small cell lung cancer
(NSCLC). A group participating in the study was comprised of 132
patients with wild-type epidermal growth factor receptor (EGFR) who
progressed on .gtoreq.1 platinum-based standard of care therapy and
were EGFR tyrosine kinase inhibitor (TKI)-naive. Patients were
randomized 2:1 to receive daily erlotinib alone at 150 mg, or 20
mg/kg MM-121 every other week plus daily erlotinib at 100 mg (see,
e.g., International Publication No. WO/2012/154587). Data were
collected at 10+ months.
[0452] An objective of the trial was to assess biomarker profiles
as predictors of clinical responses to the combination therapy
compared to erlotinib alone. As ErbB3 signaling was expected to be
active in only a subset of patients, pre-treatment biopsies were
collected from all patients to evaluate a pre-specified set of
biomarkers mechanistically linked to ErbB3 signaling: heregulin
(HRG), betacellulin, EGFR, ErbB2, and ErbB3. (Other objectives of
the trial were to compare the progression-free survival (PFS)
between the combination therapy (MM-121+erlotinib) and erlotinib
alone, as well as overall survival (OS) and safety data.)
[0453] Based on the findings in ovarian cancer (Example 5) and
breast cancer (Example 6) that HRG mRNA is predictive of beneficial
effects of MM-121, biomarker analyses focused on HRG mRNA.
Therefore, in this Example, Biomarker positive (BM+) is used
interchangeably with Heregulin positive (HRG+). BM+ patients were
defined as having detectable HRG mRNA by RNA-ISH (RNA in situ
hybridization) as described above in Example 1.
Summary of Results:
[0454] As in the studies with ovarian cancer and breast cancer, it
was found that HRG mRNA levels correlated with a beneficial effect
of MM-121 treatment. HRG-positive patients (53.7%) were defined as
having detectable levels of HRG mRNA by RNA-ISH in pretreatment
biopsies. While HRG-positive patients responded poorly to erlotinib
alone, most HRG-positive patients benefited from the addition of
MM-121 to the erlotinib treatment. FIG. 11 is a graphical
representation of the outcomes for individual BM+ patients on the
study, with patients receiving erlotinib alone represented by black
bars and patients receiving MM-121+erlotinib represented by light
bars. Of the 19 patients that received MM-121+erlotinib, only 6 had
progressive disease (as shown by the horizontal line at 20% on the
y-axis), in contrast to 5 out of the 10 patients that received
erlotinib alone. Only 1 patient receiving erlotinib alone had a
partial response, in contrast to 6 patients receiving combination
therapy.
[0455] PFS for the overall population in the study was calculated
for data collected at about 10 months and shows that patients in
the treatment arm had overall better outcomes than patients in the
control arm (FIG. 12A). A similar result is seen for overall
survival (FIG. 12B). Data for BM+ patients in the control arm (thin
solid line), BM+ patients in the treatment arm (thick solid line),
BM- patients in the control arm (thin dashed line), and BM-
patients in the treatment arm (thick dashed line) are shown in FIG.
12C, and illustrate that BM+ patients do considerably worse on the
standard of care therapy (erlotinib alone) than do BM- patients.
Results from this study further implicate heregulin-driven ErbB3
signaling as a mechanism of resistance to standard of care
therapies, such as erlotinib, in EGFR wild-type NSCLC. As disclosed
herein, blockade of this pathway by MM-121 confers sensitivity to
erlotinib in this molecularly-defined patient population. As the
impact of erlotinib treatment in EGFR wild-type NSCLC remains
modest, however, future studies should not rely on erlotinib as
backbone therapy. These data, together with findings from other
MM-121 Phase 2 studies, establish the role for heregulin-dependent
ErbB3 signaling as a critical survival pathway mediating resistance
to anti-proliferative therapies across indications.
Example 9
In Vitro Analysis of PI3KCA Mutant Cells
[0456] N87, SKBR3, OVCAR8 cells and HCC1937 cancer cells were
obtained from ATCC or NCI and maintained in culture per supplier's
suggestions. For cell viability experiments, log phase growing
cells were plated onto 96 well micro-honeycomb patterned, low
adhesion culture plates (Scivax USA, Inc) or ultra-low attachment,
round bottom plates (Corning). After 48 hours, heregulin-EGF domain
(R&D Systems), and/or MM-121 (Merrimack Pharmaceuticals) was
introduced to the cells. Cell viability assays were performed 4
days after treatment using the Cell Titer Glo.RTM. kit (Promega).
For the measurement of growth rates, Cell Titer Glo.RTM. was
performed on spheroids after 2, 5, and 7 days in culture, and
normalized to control cells on the same plate.
[0457] NCI-N87 cells were transduced with either full-length
PIK3CA-H1047R mutant or wild type (GeneCopoeia, Inc.) expressing
lentiviruses that were also engineered to express PAC-PA-turboGFP
(OriGene Technologies, Inc.). OVCAR8, HCC1937, and SKBR3 cells were
transduced with PIK3CA-H1047R mutant or wild type retroviruses
generated from a PIK3CA-IRES-tag2GFP vector (GeneWiz).
PIK3CA-H1047R- or wild type expressing polyclonal cell lines were
established after selecting for puromycin and FACS sorting for
GFP-expressing cells. Control cell lines were engineered to express
either GFP (NCI-N87) or empty vector (OVCAR8, HCC1937, and SKBR3)
in the same manner as the PI3K expressing cells. Cells were
maintained in puromycin-containing medium.
[0458] Results are shown in FIG. 14. Both wild-type and
PI3K-activating mutant cells that were incubated with HRG had, in a
four day growth assay, variable growth responses that were
statistically indistinguishable from each other. Co-incubation of
the cells with MM-121 blocked the HRG-stimulated growth, regardless
of PI3K mutation. These results indicate that activating mutations
in PI3K do not preclude HRG-stimulated growth, and do not prevent
MM-121 activity.
Example 10
ErbB3 Expression is Reduced in HRG Non-Responsive PI3K-111047R
Cells
[0459] N87, SKBR3, OVCAR8 cells and HCC1937 cancer cells were grown
as described in Example 9. To measure mRNA levels by quantitative
reverse-transcriptase PCR (RT-PCR), RNA was extracted from
spheroids using the RNAEasy.RTM. kit (Qiagen). ErbB3, actin, and
GAPDH RNA levels were measured using the Taqman.RTM. assay (Applied
Biosystems) on a ViiA 7 Real Time PCR System. For signaling
experiments, log phase growing cells were plated onto 24 well
micro-honeycomb patterned culture plates (Scivax USA, Inc) for 6
days, incubated with heregulin, then lysed in RIPA buffer
(Sigma-Aldrich) supplemented with protease and phosphatase
inhibitors (Roche) and 1% SDS (Sigma-Aldrich). All antibodies used
in western blots were obtained from Cell Signaling Technologies,
with the exception of the total ErbB3 antibody (AbCam). Protein
levels were measured via quantitative western blot and levels were
normalized to actin.
[0460] The heregulin receptor ErbB3 is negatively regulated by the
FOXO feedback loop, in both cells that express endogenous PI3K and
the engineered cells described in Example 9. In the PI3K-H1047R
mutant expressing cells, there was a dramatic increase in
phospho-FoxO in each of the cell lines, but an even more dramatic
increase in those cell lines that didn't respond well to heregulin.
The levels of phospho-FoxO correlated with levels of ErbB3 mRNA, as
measured by real-time quantitative PCR in PI3K-H1047R NCI-N87,
OVCAR8, and HCC1937 cells. The cells expressing mutant PI3K had
reduced levels of ErbB3 transcript as compared to control cells
(FIG. 15A). In addition, there was a consistent reduction in ErbB3
protein levels in OVCAR8 and HCC1937 PI3K-H1047R cells, in
comparison to control ErbB3 levels (FIG. 15B). There was no
reduction in ErbB3 protein levels in SKBR3 and NCI-N87 cells.
[0461] To determine whether ErbB3 levels were the limiting factor
in signaling in PI3K mutant cells, rather than a downstream
blockage of pAKT production, ErbB3 was re-expressed in PI3K-H1047R
cells (FIG. 15C). Lysates from engineered OVCAR8 cells (control
(EV) and PI3K-H1047R, expressing empty vector (NEG) or ErbB3 (E3)).
p110, pAKT, and pERK levels were unchanged with added ErbB3. As
shown in FIG. 15D, control cells (empty vector, EV) were stimulated
by HRG but PI3K-H1047R cells were stimulated to a much lesser
extent. Re-expression of ErbB3 partially rescued the HRG-stimulated
growth, demonstrating that the level of ErbB3 is a limiting factor
in signaling. Importantly, as shown in the figure, MM-121 abrogates
the heregulin stimulated growth in these mutant cells.
[0462] Taken together, these results show that activating mutations
in PI3K do not preclude potential benefit from ErbB3-directed
therapy, but that patients with HRG-positive, PI3K-mutant cancer
will benefit from testing of ErbB3 levels to ensure a minimum
threshold of +2 is met so that patients will benefit from MM-121
and other ErbB3-directed therapies.
TABLE-US-00018 SUMMARY OF SEQUENCE LISTING MM-121 V.sub.H amino
acid sequence (SEQ ID NO: 1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSSISSSG
GWTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATIFDYWG QGTLVTVSS
MM-121 V.sub.L amino acid sequence (SEQ ID NO: 2)
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQ
RPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGGTKVTV L MM-121
V.sub.H CDR1 (SEQ ID NO: 3) HYVMA MM-121 V.sub.H CDR2 (SEQ ID NO:
4) SISSSGGWTLYADSVKG MM-121 V.sub.H CDR3 (SEQ ID NO: 5) GLKMATIFDY
MM-121 V.sub.L CDR1 (SEQ ID NO: 6) TGTSSDVGSYNVVS MM-121 V.sub.L
CDR2 (SEQ ID NO: 7) EVSQRPS MM-121 V.sub.L CDR3 (SEQ ID NO: 8)
CSYAGSSIFVI Ab # 3 V.sub.H amino acid sequence (SEQ ID NO: 9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNMRWVRQAPGKGLEWVSVIYPSG
GATRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYYYYGMDVWGQ GTLVTVSS Ab
# 3 V.sub.L amino acid sequence (SEQ ID NO: 10)
QSVLTQPPSASGTPGQRVTISCSGSDSNIGRNYIYWYQQFPGTAPKLLIYRNNQR
PSGVPDRISGSKSGTSASLAISGLRSEDEAEYHCGTWDDSLSGPVFGGGTKLTVL Ab #3
V.sub.H CDR1 (SEQ ID NO: 11) AYNMR Ab # 3 V.sub.H CDR2 (SEQ ID NO:
12) VIYPSGGATRYADSVKG Ab # 3 V.sub.H CDR3 (SEQ ID NO: 13) GYYYYGMDV
Ab # 3 V.sub.L CDR1 (SEQ ID NO: 14) SGSDSNIGRNYIY Ab # 3 V.sub.L
CDR2 (SEQ ID NO: 15) RNNQRPS Ab # 3 V.sub.L CDR3 (SEQ ID NO: 16)
GTWDDSLSGPV Ab # 14 V.sub.H amino acid sequence (SEQ ID NO: 17)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSYISPSG
GHTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLETGLLVDAFD
IWGQGTMVTVSS Ab # 14 V.sub.L amino acid sequence (SEQ ID NO: 18)
QYELTQPPSVSVYPGQTASITCSGDQLGSKFVSWYQQRPGQSPVLVMYKDKRRPS
EIPERFSGSNSGNTATLTISGTQAIDEADYYCQAWDSSTYVFGTGTKVTVL Ab # 14 V.sub.H
CDR1 (SEQ ID NO: 19) AYGMG Ab # 14 V.sub.H CDR2 (SEQ ID NO: 20)
YISPSGGHTKYADSVKG Ab # 14 V.sub.H CDR3 (SEQ ID NO: 21)
VLETGLLVDAFDI Ab # 14 V.sub.L CDR1 (SEQ ID NO: 22) SGDQLGSKFVS Ab #
14 V.sub.L CDR2 (SEQ ID NO: 23) YKDKRRPS Ab # 14 V.sub.L CDR3 (SEQ
ID NO: 24) QAWDSSTYV Ab # 17 V.sub.H amino acid sequence (SEQ ID
NO: 25) EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMGWVRQAPGKGLEWVSYISPSG
GITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLNYYYGLDVWGQ GTTVTVSS Ab
# 17 V.sub.L amino acid sequence (SEQ ID NO: 26)
QDIQMTQSPSSLSASVGDRITITCQASQDIGDSLNWYQQKPGKAPRLLIYDASNL
ETGVPPRFSGSGSGTDFTFTFRSLQPEDIATYFCQQSANAPFTFGPGTKVDIK Ab # 17
V.sub.H CDR1 (SEQ ID NO: 27) WYGMG Ab # 17 V.sub.H CDR2 (SEQ ID NO:
28) YISPSGGITVYADSVKG Ab # 17 V.sub.H CDR3 (SEQ ID NO: 29)
LNYYYGLDV Ab # 17 V.sub.L CDR1 (SEQ ID NO: 30) QASQDIGDSLN Ab # 17
V.sub.L CDR2 (SEQ ID NO: 31) DASNLET Ab # 17 V.sub.L CDR3 (SEQ ID
NO: 32) QQSANAPFT Ab # 19 V.sub.H amino acid sequence (SEQ ID NO:
33) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMWWVRQAPGKGLEWVSYIGSSG
GPTYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGRGTPYYFDSWG QGTLVTVSS
Ab # 19 V.sub.L amino acid sequence (SEQ ID NO: 34)
QYELTQPASVSGSPGQSITISCTGTSSDIGRWNIVSWYQQHPGKAPKLMIYDVSN
RPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFGGGTKLTVL Ab # 19
V.sub.H CDR1 (SEQ ID NO: 35) RYGMW Ab # 19 V.sub.H CDR2 (SEQ ID NO:
36) YIGSSGGPTYYVDSVKG Ab # 19 V.sub.H CDR3 (SEQ ID NO: 37)
GRGTPYYFDS Ab # 19 V.sub.L CDR1 (SEQ ID NO: 38 TGTSSDIGRWNIVS Ab #
19 V.sub.L CDR2 (SEQ ID NO: 39) DVSNRPS Ab # 19 V.sub.L CDR3 (SEQ
ID NO: 40) SSYTSSSTWV ErbB3 (SEQ ID NO: 41)
SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADL
SFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSH
ALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCP
PCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQ
DTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNF
VVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNID
GFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPP
HMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISAN
RQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGP
GQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSG
SDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGP
ELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRR
YLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGES
IKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQY
LPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKS
PSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGV
TVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENI
RPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPEL
DLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGE
SCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRS
RSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSRE
GTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGS
DLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPA
SEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANA QRT HRG
cDNA (GenBank accession number NM-013956)
(SEQ ID NO: 42) 1 gaggccaggg gagggtgcga aggaggcgcc tgcctccaac
ctgcgggcgg gaggtgggtg 61 gctgcggggc aattgaaaaa gagccggcga
ggagttcccc gaaacttgtt ggaactccgg 121 gctcgcgcgg aggccaggag
ctgagcggcg gcggctgccg gacgatggga gcgtgagcag 181 gacggtgata
acctctcccc gatcgggttg cgagggcgcc gggcagaggc caggacgcga 241
gccgccagcg gtgggaccca tcgacgactt cccggggcga caggagcagc cccgagagcc
301 agggcgagcg cccgttccag gtggccggac cgcccgccgc gtccgcgccg
cgctccctgc 361 aggcaacggg agacgccccc gcgcagcgcg agcgcctcag
cgcggccgct cgctctcccc 421 ctcgagggac aaacttttcc caaacccgat
ccgagccctt ggaccaaact cgcctgcgcc 481 gagagccgtc cgcgtagagc
gctccgtctc cggcgagatg tccgagcgca aagaaggcag 541 aggcaaaggg
aagggcaaga agaaggagcg aggctccggc aagaagccgg agtccgcggc 601
gggcagccag agcccagcct tgcctccccg attgaaagag atgaaaagcc aggaatcggc
661 tgcaggttcc aaactagtcc ttcggtgtga aaccagttct gaatactcct
ctctcagatt 721 caagtggttc aagaatggga atgaattgaa tcgaaaaaac
aaaccacaaa atatcaagat 781 acaaaaaaag ccagggaagt cagaacttcg
cattaacaaa gcatcactgg ctgattctgg 841 agagtatatg tgcaaagtga
tcagcaaatt aggaaatgac agtgcctctg ccaatatcac 901 catcgtggaa
tcaaacgaga tcatcactgg tatgccagcc tcaactgaag gagcatatgt 961
gtcttcagag tctcccatta gaatatcagt atccacagaa ggagcaaata cttcttcatc
1021 tacatctaca tccaccactg ggacaagcca tcttgtaaaa tgtgcggaga
aggagaaaac 1081 tttctgtgtg aatggagggg agtgcttcat ggtgaaagac
ctttcaaacc cctcgagata 1141 cttgtgcaag tgcccaaatg agtttactgg
tgatcgctgc caaaactacg taatggccag 1201 cttctacaag catcttggga
ttgaatttat ggaggcggag gagctgtacc agaagagagt 1261 gctgaccata
accggcatct gcatcgccct ccttgtggtc ggcatcatgt gtgtggtggc 1321
ctactgcaaa accaagaaac agcggaaaaa gctgcatgac cgtcttcggc agagccttcg
1381 gtctgaacga aacaatatga tgaacattgc caatgggcct caccatccta
acccaccccc 1441 cgagaatgtc cagctggtga atcaatacgt atctaaaaac
gtcatctcca gtgagcatat 1501 tgttgagaga gaagcagaga catccttttc
caccagtcac tatacttcca cagcccatca 1561 ctccactact gtcacccaga
ctcctagcca cagctggagc aacggacaca ctgaaagcat 1621 cctttccgaa
agccactctg taatcgtgat gtcatccgta gaaaacagta ggcacagcag 1681
cccaactggg ggcccaagag gacgtcttaa tggcacagga ggccctcgtg aatgtaacag
1741 cttcctcagg catgccagag aaacccctga ttcctaccga gactctcctc
atagtgaaag 1801 gtatgtgtca gccatgacca ccccggctcg tatgtcacct
gtagatttcc acacgccaag 1861 ctcccccaaa tcgccccctt cggaaatgtc
tccacccgtg tccagcatga cggtgtccat 1921 gccttccatg gcggtcagcc
ccttcatgga agaagagaga cctctacttc tcgtgacacc 1981 accaaggctg
cgggagaaga agtttgacca tcaccctcag cagttcagct ccttccacca 2041
caaccccgcg catgacagta acagcctccc tgctagcccc ttgaggatag tggaggatga
2101 ggagtatgaa acgacccaag agtacgagcc agcccaagag cctgttaaga
aactcgccaa 2161 tagccggcgg gccaaaagaa ccaagcccaa tggccacatt
gctaacagat tggaagtgga 2221 cagcaacaca agctcccaga gcagtaactc
agagagtgaa acagaagatg aaagagtagg 2281 tgaagatacg cctttcctgg
gcatacagaa ccccctggca gccagtcttg aggcaacacc 2341 tgccttccgc
ctggctgaca gcaggactaa cccagcaggc cgcttctcga cacaggaaga 2401
aatccaggcc aggctgtcta gtgtaattgc taaccaagac cctattgctg tataaaacct
2461 aaataaacac atagattcac ctgtaaaact ttattttata taataaagta
ttccacctta 2521 aattaaacaa tttattttat tttagcagtt ctgcaaatag
aaaacaggaa aaaaactttt 2581 ataaattaaa tatatgtatg taaaaatgtg
ttatgtgcca tatgtagcaa ttttttacag 2641 tatttcaaaa cgagaaagat
atcaatggtg cctttatgtt atgttatgtc gagagcaagt 2701 tttgtacagt
tacagtgatt gcttttccac agtatttctg caaaacctct catagattca 2761
gtttttgctg gcttcttgtg cattgcatta tgatgttgac tggatgtatg atttgcaaga
2821 cttgcaactg tccctctgtt tgcttgtagt agcacccgat cagtatgtct
tgtaatggca 2881 catccatcca gatatgcctc tcttgtgtat gaagttttct
ttgctttcag aatatgaaat 2941 gagttgtgtc tactctgcca gccaaaggtt
tgcctcattg ggctctgaga taatagtaga 3001 tccaacagca tgctactatt
aaatacagca agaaactgca ttaagtaatg ttaaatatta 3061 ggaagaaagt
aatactgtga tttaaaaaaa act
Sequence CWU 1
1
691119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Val Met Ala Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Ser Ser Ser Gly Gly Trp Thr Leu Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Gly Leu Lys Met Ala Thr Ile Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
2111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 2Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Val Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Ile Ile
Tyr Glu Val Ser Gln Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala
Gly Ser 85 90 95 Ser Ile Phe Val Ile Phe Gly Gly Gly Thr Lys Val
Thr Val Leu 100 105 110 35PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 3His Tyr Val Met Ala 1 5 417PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 4Ser Ile Ser Ser Ser Gly Gly Trp Thr Leu Tyr Ala Asp Ser
Val Lys 1 5 10 15 Gly 510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Gly Leu Lys Met Ala Thr Ile Phe Asp Tyr 1 5 10
614PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 6Thr Gly Thr Ser Ser Asp Val Gly Ser
Tyr Asn Val Val Ser 1 5 10 77PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Glu Val Ser Gln Arg Pro Ser 1 5 811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 8Cys Ser Tyr Ala Gly Ser Ser Ile Phe Val Ile 1 5 10
9118PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 9Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Arg Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val
Ile Tyr Pro Ser Gly Gly Ala Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
10110PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 10Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys
Ser Gly Ser Asp Ser Asn Ile Gly Arg Asn 20 25 30 Tyr Ile Tyr Trp
Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Ile Ser 50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65
70 75 80 Ser Glu Asp Glu Ala Glu Tyr His Cys Gly Thr Trp Asp Asp
Ser Leu 85 90 95 Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Ala Tyr Asn Met Arg 1 5 1217PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Val Ile Tyr Pro Ser Gly Gly Ala Thr Arg Tyr Ala Asp Ser
Val Lys 1 5 10 15 Gly 139PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 13Gly Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5
1413PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 14Ser Gly Ser Asp Ser Asn Ile Gly Arg
Asn Tyr Ile Tyr 1 5 10 157PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 15Arg Asn Asn Gln Arg Pro Ser 1 5 1611PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Gly Thr Trp Asp Asp Ser Leu Ser Gly Pro Val 1 5 10
17122PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 17Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Gly Met Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Val Leu Glu Thr Gly Leu Leu Val Asp Ala
Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 18106PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 18Gln Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Tyr Pro Gly Gln 1 5 10 15 Thr Ala Ser
Ile Thr Cys Ser Gly Asp Gln Leu Gly Ser Lys Phe Val 20 25 30 Ser
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Met Tyr 35 40
45 Lys Asp Lys Arg Arg Pro Ser Glu Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln
Ala Ile 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser
Ser Thr Tyr Val 85 90 95 Phe Gly Thr Gly Thr Lys Val Thr Val Leu
100 105 195PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 19Ala Tyr Gly Met Gly 1 5
2017PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Tyr Ile Ser Pro Ser Gly Gly His Thr
Lys Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Val Leu Glu Thr Gly Leu Leu Val Asp Ala Phe Asp Ile 1 5
10 2211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 22Ser Gly Asp Gln Leu Gly Ser Lys Phe
Val Ser 1 5 10 238PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 23Tyr Lys Asp Lys Arg Arg
Pro Ser 1 5 249PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 24Gln Ala Trp Asp Ser Ser
Thr Tyr Val 1 5 25118PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 25Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Gly
Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Tyr Ile Ser Pro Ser Gly Gly Ile Thr Val Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Asn Tyr Tyr Tyr Gly Leu Asp
Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
26108PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 26Gln Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Ile Thr Ile
Thr Cys Gln Ala Ser Gln Asp Ile Gly Asp 20 25 30 Ser Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45 Ile Tyr
Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Pro Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Phe Arg Ser Leu Gln 65
70 75 80 Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Ser Ala Asn
Ala Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 275PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Trp Tyr Gly Met Gly 1 5
2817PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 28Tyr Ile Ser Pro Ser Gly Gly Ile Thr
Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 299PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 29Leu Asn Tyr Tyr Tyr Gly Leu Asp Val 1 5
3011PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 30Gln Ala Ser Gln Asp Ile Gly Asp Ser
Leu Asn 1 5 10 317PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 31Asp Ala Ser Asn Leu Glu
Thr 1 5 329PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 32Gln Gln Ser Ala Asn Ala
Pro Phe Thr 1 5 33119PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 33Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly
Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Tyr Ile Gly Ser Ser Gly Gly Pro Thr Tyr Tyr Val Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Arg Gly Thr Pro Tyr Tyr Phe
Asp Ser Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
34110PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 34Gln Tyr Glu Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Ile Gly Arg Trp 20 25 30 Asn Ile Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr
Ser Ser 85 90 95 Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 355PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 35Arg Tyr Gly Met Trp 1 5 3617PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 36Tyr Ile Gly Ser Ser Gly Gly Pro Thr Tyr Tyr Val Asp Ser
Val Lys 1 5 10 15 Gly 3710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 37Gly Arg Gly Thr Pro Tyr Tyr Phe Asp Ser 1 5 10
3814PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 38Thr Gly Thr Ser Ser Asp Ile Gly Arg
Trp Asn Ile Val Ser 1 5 10 397PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 39Asp Val Ser Asn Arg Pro Ser 1 5 4010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 40Ser Ser Tyr Thr Ser Ser Ser Thr Trp Val 1 5 10
411323PRTHomo sapiens 41Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro
Gly Thr Leu Asn Gly 1 5 10 15 Leu Ser Val Thr Gly Asp Ala Glu Asn
Gln Tyr Gln Thr Leu Tyr Lys 20 25 30 Leu Tyr Glu Arg Cys Glu Val
Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45 Thr Gly His Asn Ala
Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60 Thr Gly Tyr
Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu 65 70 75 80 Pro
Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90
95 Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu
100 105 110 Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly
Gly Val 115 120 125 Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp
Thr Ile Asp Trp 130 135 140 Arg Asp Ile Val Arg Asp Arg Asp Ala Glu
Ile Val Val Lys Asp Asn 145 150 155 160 Gly Arg Ser Cys Pro Pro Cys
His Glu Val Cys Lys Gly Arg Cys Trp 165 170 175 Gly Pro Gly Ser Glu
Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190 Pro Gln Cys
Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205 His
Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215
220 Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys
225 230 235 240 Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu
Glu Pro Asn 245 250 255 Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys
Val Ala Ser Cys Pro 260 265 270 His Asn Phe Val Val Asp Gln Thr Ser
Cys Val Arg Ala Cys Pro Pro 275 280 285
Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys 290
295 300 Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser
Arg 305 310 315 320 Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe
Val Asn Cys Thr 325 330 335 Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile
Thr Gly Leu Asn Gly Asp 340 345 350 Pro Trp His Lys Ile Pro Ala Leu
Asp Pro Glu Lys Leu Asn Val Phe 355 360 365 Arg Thr Val Arg Glu Ile
Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro 370 375 380 Pro His Met His
Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly 385 390 395 400 Gly
Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn 405 410
415 Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala
420 425 430 Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His
His Ser 435 440 445 Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu
Glu Arg Leu Asp 450 455 460 Ile Lys His Asn Arg Pro Arg Arg Asp Cys
Val Ala Glu Gly Lys Val 465 470 475 480 Cys Asp Pro Leu Cys Ser Ser
Gly Gly Cys Trp Gly Pro Gly Pro Gly 485 490 495 Gln Cys Leu Ser Cys
Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr 500 505 510 His Cys Asn
Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520 525 Glu
Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala 530 535
540 Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe
545 550 555 560 Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly
Val Leu Gly 565 570 575 Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val
Gln Asn Glu Cys Arg 580 585 590 Pro Cys His Glu Asn Cys Thr Gln Gly
Cys Lys Gly Pro Glu Leu Gln 595 600 605 Asp Cys Leu Gly Gln Thr Leu
Val Leu Ile Gly Lys Thr His Leu Thr 610 615 620 Met Ala Leu Thr Val
Ile Ala Gly Leu Val Val Ile Phe Met Met Leu 625 630 635 640 Gly Gly
Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg 645 650 655
Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp 660
665 670 Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu
Thr 675 680 685 Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe
Gly Thr Val 690 695 700 His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser
Ile Lys Ile Pro Val 705 710 715 720 Cys Ile Lys Val Ile Glu Asp Lys
Ser Gly Arg Gln Ser Phe Gln Ala 725 730 735 Val Thr Asp His Met Leu
Ala Ile Gly Ser Leu Asp His Ala His Ile 740 745 750 Val Arg Leu Leu
Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr 755 760 765 Gln Tyr
Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg 770 775 780
Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala 785
790 795 800 Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg
Asn Leu 805 810 815 Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln
Val Gln Val Ala 820 825 830 Asp Phe Gly Val Ala Asp Leu Leu Pro Pro
Asp Asp Lys Gln Leu Leu 835 840 845 Tyr Ser Glu Ala Lys Thr Pro Ile
Lys Trp Met Ala Leu Glu Ser Ile 850 855 860 His Phe Gly Lys Tyr Thr
His Gln Ser Asp Val Trp Ser Tyr Gly Val 865 870 875 880 Thr Val Trp
Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu 885 890 895 Arg
Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala 900 905
910 Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys
915 920 925 Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu
Ala Asn 930 935 940 Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr
Leu Val Ile Lys 945 950 955 960 Arg Glu Ser Gly Pro Gly Ile Ala Pro
Gly Pro Glu Pro His Gly Leu 965 970 975 Thr Asn Lys Lys Leu Glu Glu
Val Glu Leu Glu Pro Glu Leu Asp Leu 980 985 990 Asp Leu Asp Leu Glu
Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr Leu 995 1000 1005 Gly Ser
Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro Arg 1010 1015 1020
Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro Met 1025
1030 1035 Asn Gln Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val
Ser 1040 1045 1050 Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu
His Pro Met 1055 1060 1065 Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser
Glu Gly His Val Thr 1070 1075 1080 Gly Ser Glu Ala Glu Leu Gln Glu
Lys Val Ser Met Cys Arg Ser 1085 1090 1095 Arg Ser Arg Ser Arg Ser
Pro Arg Pro Arg Gly Asp Ser Ala Tyr 1100 1105 1110 His Ser Gln Arg
His Ser Leu Leu Thr Pro Val Thr Pro Leu Ser 1115 1120 1125 Pro Pro
Gly Leu Glu Glu Glu Asp Val Asn Gly Tyr Val Met Pro 1130 1135 1140
Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg Glu Gly Thr Leu 1145
1150 1155 Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr Glu Glu Glu
Asp 1160 1165 1170 Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg
Arg His Ser 1175 1180 1185 Pro Pro His Pro Pro Arg Pro Ser Ser Leu
Glu Glu Leu Gly Tyr 1190 1195 1200 Glu Tyr Met Asp Val Gly Ser Asp
Leu Ser Ala Ser Leu Gly Ser 1205 1210 1215 Thr Gln Ser Cys Pro Leu
His Pro Val Pro Ile Met Pro Thr Ala 1220 1225 1230 Gly Thr Thr Pro
Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln Arg 1235 1240 1245 Asp Gly
Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly Ala Cys 1250 1255 1260
Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly 1265
1270 1275 Pro Gly His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys
Thr 1280 1285 1290 Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp
Asn Pro Asp 1295 1300 1305 Tyr Trp His Ser Arg Leu Phe Pro Lys Ala
Asn Ala Gln Arg Thr 1310 1315 1320 423093DNAHomo sapiens
42gaggccaggg gagggtgcga aggaggcgcc tgcctccaac ctgcgggcgg gaggtgggtg
60gctgcggggc aattgaaaaa gagccggcga ggagttcccc gaaacttgtt ggaactccgg
120gctcgcgcgg aggccaggag ctgagcggcg gcggctgccg gacgatggga
gcgtgagcag 180gacggtgata acctctcccc gatcgggttg cgagggcgcc
gggcagaggc caggacgcga 240gccgccagcg gtgggaccca tcgacgactt
cccggggcga caggagcagc cccgagagcc 300agggcgagcg cccgttccag
gtggccggac cgcccgccgc gtccgcgccg cgctccctgc 360aggcaacggg
agacgccccc gcgcagcgcg agcgcctcag cgcggccgct cgctctcccc
420ctcgagggac aaacttttcc caaacccgat ccgagccctt ggaccaaact
cgcctgcgcc 480gagagccgtc cgcgtagagc gctccgtctc cggcgagatg
tccgagcgca aagaaggcag 540aggcaaaggg aagggcaaga agaaggagcg
aggctccggc aagaagccgg agtccgcggc 600gggcagccag agcccagcct
tgcctccccg attgaaagag atgaaaagcc aggaatcggc 660tgcaggttcc
aaactagtcc ttcggtgtga aaccagttct gaatactcct ctctcagatt
720caagtggttc aagaatggga atgaattgaa tcgaaaaaac aaaccacaaa
atatcaagat 780acaaaaaaag ccagggaagt cagaacttcg cattaacaaa
gcatcactgg ctgattctgg 840agagtatatg tgcaaagtga tcagcaaatt
aggaaatgac agtgcctctg ccaatatcac 900catcgtggaa tcaaacgaga
tcatcactgg tatgccagcc tcaactgaag gagcatatgt 960gtcttcagag
tctcccatta gaatatcagt atccacagaa ggagcaaata cttcttcatc
1020tacatctaca tccaccactg ggacaagcca tcttgtaaaa tgtgcggaga
aggagaaaac 1080tttctgtgtg aatggagggg agtgcttcat ggtgaaagac
ctttcaaacc cctcgagata 1140cttgtgcaag tgcccaaatg agtttactgg
tgatcgctgc caaaactacg taatggccag 1200cttctacaag catcttggga
ttgaatttat ggaggcggag gagctgtacc agaagagagt 1260gctgaccata
accggcatct gcatcgccct ccttgtggtc ggcatcatgt gtgtggtggc
1320ctactgcaaa accaagaaac agcggaaaaa gctgcatgac cgtcttcggc
agagccttcg 1380gtctgaacga aacaatatga tgaacattgc caatgggcct
caccatccta acccaccccc 1440cgagaatgtc cagctggtga atcaatacgt
atctaaaaac gtcatctcca gtgagcatat 1500tgttgagaga gaagcagaga
catccttttc caccagtcac tatacttcca cagcccatca 1560ctccactact
gtcacccaga ctcctagcca cagctggagc aacggacaca ctgaaagcat
1620cctttccgaa agccactctg taatcgtgat gtcatccgta gaaaacagta
ggcacagcag 1680cccaactggg ggcccaagag gacgtcttaa tggcacagga
ggccctcgtg aatgtaacag 1740cttcctcagg catgccagag aaacccctga
ttcctaccga gactctcctc atagtgaaag 1800gtatgtgtca gccatgacca
ccccggctcg tatgtcacct gtagatttcc acacgccaag 1860ctcccccaaa
tcgccccctt cggaaatgtc tccacccgtg tccagcatga cggtgtccat
1920gccttccatg gcggtcagcc ccttcatgga agaagagaga cctctacttc
tcgtgacacc 1980accaaggctg cgggagaaga agtttgacca tcaccctcag
cagttcagct ccttccacca 2040caaccccgcg catgacagta acagcctccc
tgctagcccc ttgaggatag tggaggatga 2100ggagtatgaa acgacccaag
agtacgagcc agcccaagag cctgttaaga aactcgccaa 2160tagccggcgg
gccaaaagaa ccaagcccaa tggccacatt gctaacagat tggaagtgga
2220cagcaacaca agctcccaga gcagtaactc agagagtgaa acagaagatg
aaagagtagg 2280tgaagatacg cctttcctgg gcatacagaa ccccctggca
gccagtcttg aggcaacacc 2340tgccttccgc ctggctgaca gcaggactaa
cccagcaggc cgcttctcga cacaggaaga 2400aatccaggcc aggctgtcta
gtgtaattgc taaccaagac cctattgctg tataaaacct 2460aaataaacac
atagattcac ctgtaaaact ttattttata taataaagta ttccacctta
2520aattaaacaa tttattttat tttagcagtt ctgcaaatag aaaacaggaa
aaaaactttt 2580ataaattaaa tatatgtatg taaaaatgtg ttatgtgcca
tatgtagcaa ttttttacag 2640tatttcaaaa cgagaaagat atcaatggtg
cctttatgtt atgttatgtc gagagcaagt 2700tttgtacagt tacagtgatt
gcttttccac agtatttctg caaaacctct catagattca 2760gtttttgctg
gcttcttgtg cattgcatta tgatgttgac tggatgtatg atttgcaaga
2820cttgcaactg tccctctgtt tgcttgtagt agcacccgat cagtatgtct
tgtaatggca 2880catccatcca gatatgcctc tcttgtgtat gaagttttct
ttgctttcag aatatgaaat 2940gagttgtgtc tactctgcca gccaaaggtt
tgcctcattg ggctctgaga taatagtaga 3000tccaacagca tgctactatt
aaatacagca agaaactgca ttaagtaatg ttaaatatta 3060ggaagaaagt
aatactgtga tttaaaaaaa act 30934327DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 43ctatgtgcag aggaattatg atctttc 274424DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 44gctaaggcat aggaattttc gtag 244526DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 45tgcaggtttt ccaaaggaat tcgctc 264621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 46ggaaacctgg aactcaccta c 214720DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 47cctgcctcac ttggttgtga 204822DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 48accaatgcca gcctgtcctt cc 224919DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 49gcaactctca ggcagtgtg 195021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 50tggtattggt tctcagcatc g 215123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 51cggtcacact caggccattc aga 235223DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 52cttgtaaaat gtgcggagaa gga 235323DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 53atctcgaggg gtttgaaagg tct 235426DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 54tgtgaatgga ggggagtgct tcatgg 265524DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 55gtgcaagtgc ccaaatgagt ttac 245625DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 56ctccataaat tcaatcccaa gatgc 255726DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 57tggccattac gtagttttgg cagcga 265820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 58tgggaattcc accagaagtc 205919DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 59gcctttccgc tttgattgt 196026DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 60actgtgcagc taccaccaca ccaatc 266124DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 61ggaattttgc cgatttcatg actg 246220DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 62gtctctgccg agtgaagatc 206320DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 63caccgacgag agtgctgggg 206423DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 64tgactttgtc acagcccaag ata 236520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 65aatccaaatg cggcatcttc 206627DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 66tgatgctgct tacatgtctc gatccca 276722DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 67ccttggtcag gcagtataat cc 226823DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 68tctggcttat atccaacact tcg 236923DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
probe" 69aagcttgctg gtgaaaagga ccc 23
* * * * *