U.S. patent application number 15/160523 was filed with the patent office on 2016-11-10 for combination therapies comprising anti-erbb3 agents.
The applicant listed for this patent is Merrimack Pharmaceuticals, Inc.. Invention is credited to Alexandra HUHALOV, Charlotte MCDONAGH, Bo ZHANG.
Application Number | 20160326262 15/160523 |
Document ID | / |
Family ID | 50184467 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160326262 |
Kind Code |
A1 |
ZHANG; Bo ; et al. |
November 10, 2016 |
COMBINATION THERAPIES COMPRISING ANTI-ERBB3 AGENTS
Abstract
Disclosed are methods and compositions for inhibiting the growth
of a tumor (e.g., a malignant tumor) in a subject. In particular,
combination therapies for treating a tumor in a subject by
co-administering an agent selected from i) an effective amount of
an anti-estrogen agent; ii) an effective amount of a receptor
tyrosine kinase inhibitor; iii) an effective amount of a MEK/PI3
kinase/AKT inhibitor; iv) an effective amount of MM-151; v) an
effective amount of an mTOR inhibitor; and/or vi) an effective
amount of trastuzumab or TMD1, and/or combinations thereof; and an
effective amount of a bispecific anti-ErbB2/anti-ErbB3 antibody.
Also disclosed is a bispecific anti-ErbB2/anti-ErbB3 antibody for
use in the therapy of a tumor in combination with an agent selected
from i) an effective amount of an anti-estrogen agent; ii) an
effective amount of a receptor tyrosine kinase inhibitor; iii) an
effective amount of a MEK/PI3 kinase/AKT inhibitor; iv) an
effective amount of MM-151; v) an effective amount of an mTOR
inhibitor; and/or vi) an effective amount of trastuzumab or TMD1,
and/or combinations thereof.
Inventors: |
ZHANG; Bo; (Lynnfield,
MA) ; MCDONAGH; Charlotte; (Winchester, MA) ;
HUHALOV; Alexandra; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merrimack Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
50184467 |
Appl. No.: |
15/160523 |
Filed: |
May 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14015776 |
Aug 30, 2013 |
9345766 |
|
|
15160523 |
|
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|
|
61695242 |
Aug 30, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4196 20130101;
A61K 31/519 20130101; A61K 31/567 20130101; C07K 16/32 20130101;
A61K 2039/505 20130101; A61K 39/3955 20130101; C07K 16/2863
20130101; C07K 2317/24 20130101; C07K 2317/31 20130101; A61K 45/06
20130101; A61K 31/138 20130101; A61K 31/565 20130101; A61K 39/39558
20130101; A61K 31/436 20130101; C07K 16/40 20130101; A61K 2039/507
20130101; A61K 31/7068 20130101; A61K 31/337 20130101; A61K 31/517
20130101; A61K 2300/00 20130101; A61K 39/39558 20130101; C07K
16/468 20130101; A61K 33/24 20130101; C07K 2317/76 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; A61K 31/517 20060101 A61K031/517; A61K 31/519 20060101
A61K031/519; A61K 31/337 20060101 A61K031/337; A61K 31/565 20060101
A61K031/565; A61K 31/4196 20060101 A61K031/4196; A61K 33/24
20060101 A61K033/24; A61K 31/7068 20060101 A61K031/7068; A61K
39/395 20060101 A61K039/395; A61K 31/138 20060101 A61K031/138 |
Claims
1.-41. (canceled)
42. An aqueous solution comprising a bispecific
anti-ErbB2/anti-ErbB3 antibody at a first concentration and an
agent selected from one or more of i) an effective amount of an
anti-estrogen agent; ii) an effective amount of a receptor tyrosine
kinase inhibitor; iii) an effective amount of a MEK/P13 kinase/AKT
inhibitor; iv) MM-151; v) an effective amount of an mTOR inhibitor;
and/or vi) an effective amount of trastuzumab or ado-trastuzumab
emtansine, at a second concentration, wherein each concentration is
an effective concentration and when the aqueous solution is blood
plasma in a subject, the subject does not experience a toxicity
that is sufficiently harmful to require a change in a therapy being
administered to the subject, which toxicity is mediated by a
drug-drug interaction in the subject between the bispecific
anti-ErbB2/anti-ErbB3 antibody and the agent.
43.-82. (canceled)
83. The aqueous solution of claim 42, wherein the anti-estrogen
agent is fulvestrant or tamoxifen.
84. The aqueous solution of claim 42, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody comprises the amino acid sequence
set forth in SEQ ID NO:1.
85. The aqueous solution of claim 42, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody is chosen from the group consisting
of A5-HSA-ML3.9, ML3.9-HSA-A5, A5-HSA-B1 D2, B1 D2-HSA-A5,
B12-HSA-B1D2, B1 D2-HSA-B12, A5-HSA-F5B6H2, F5B6H2-HSA-A5,
H3-HSA-F5B6H2, F5B6H2-HSA-H3, F4-HSA-F5B6H2, F5B6H2-HSA-F4, B1
D2-HSA-H3, and H3-HSA-B1 D2.
86. The aqueous solution of claim 42, wherein the receptor tyrosine
kinase inhibitor is selected from the group consisting of
erlotinib, afatinib, dasatinib, gefitinib, imatinib, pazopinib,
lapatinib, sunitinib, nilotinib, and sorafenib.
87. The aqueous solution of claim 86, wherein the receptor tyrosine
kinase inhibitor is lapatinib.
88. The aqueous solution of claim 42, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody is MM-111.
89. The aqueous solution of claim 42, wherein the subject is a
human.
90. The aqueous solution of claim 42, wherein the aqueous solution
creates a substantially additive or superadditive effect.
91. The aqueous solution of claim 42, wherein the anti-estrogen
agent is letrozole, exemestane, anastrozole, aminoglutethimide,
testolactone, vorozole, formestane, or fadrozole.
92. The aqueous solution of claim 42, wherein the MEK/P13
kinase/AKT inhibitor is selected from one or more of selumetinib
(AZD6244), buparlisib (BKM-120), pictilisib (GDC-0941), trametinib
(GSK1120212), MK-2206, PD0325901, and triciribine, and combinations
thereof.
93. The aqueous solution of claim 42, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody inhibits heregulin activation of
ErbB2 and ErbB3.
94. The aqueous solution of claim 92, wherein the aqueous solution
comprises the bispecific anti-ErbB2/anti-ErbB3 antibody and
trametinib.
95. The aqueous solution of claim 94, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody is at a first concentration and the
trametinib is at a second concentration and when a first tissue
culture medium is prepared comprising the bispecific
anti-ErbB2/anti-ErbB3 antibody at the first concentration and the
trametinib at the second concentration and is contacted with cancer
cells of a cell line in a cell culture, cell growth or cell
proliferation or production of pErbB3 or production of pAKT in the
cells is inhibited as compared to when cells of the cell line in a
cell culture are contacted with a second tissue culture medium that
is essentially the same as the first medium except that it does not
comprise the bispecific anti-ErbB2/anti-ErbB3 antibody.
96. The aqueous solution of claim 94, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody comprises the amino acid sequence
set forth in SEQ ID NO: 1.
97. The aqueous solution of claim 94, wherein the bispecific
anti-ErbB2/anti-ErbB3 antibody is MM-111.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/695,242, filed Aug. 30, 2012, the contents of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The various aspects of the invention disclosed herein relate
to methods and compositions for the treatment of cancers.
BACKGROUND OF THE INVENTION
[0003] Approximately 75% of breast cancers are estrogen receptor
(ER) positive. Other cancers are also ER positive (ER+). Estrogen
receptors mediate intracellular signaling that can increase the
frequency of cell division and drive tumor growth. Although
anti-endocrine therapies such as tamoxifen, fulvestrant, and
letrozole have demonstrated significant efficacy in treating ER+
breast cancer patients, intrinsic or acquired resistance to such
therapies has limited their success.
[0004] The prevalence of amplification of the human epidermal
growth factor receptor 2 (HER2, or ErbB2) in breast cancer and
other cancers has resulted in the research and development of drugs
that have ErbB2 as a therapeutic target. Although both the
anti-ErbB2 monoclonal antibody trastuzumab or TMD1 and the
ErbB1/ErbB2 dual receptor tyrosine kinase inhibitor lapatinib have
met with success in the clinic, many patients fail to benefit from
these drugs. Additionally, the majority of patients with tumors
that initially respond will eventually recrudesce after extended
treatment using these therapies.
[0005] The ErbB2/ErbB3 heterodimer is the most potent ErbB receptor
pairing with respect to strength of interaction, impact on receptor
tyrosine phosphorylation, and effects on downstream signaling
through mitogen activated protein kinase and phosphoinositide-3
kinase pathways. Heregulin is the primary ligand for ErbB3, and
activates signaling by ErbB2/ErbB3 heterodimers. Current
ErbB2-targeted therapies do not effectively inhibit heregulin
activated signaling. MM-111 is a bispecific anti-ErbB2/anti-ErbB3
antibody that abrogates heregulin binding to ErbB2/ErbB3 and
inhibits heregulin activation of ErbB2/ErbB3 without significantly
affecting ErbB2 biological activity. In preclinical models of
HER-2+ gastric, breast, ovarian and lung cancers, MM-111 inhibits
ErbB3 phosphorylation, cell cycle progression, and tumor
growth.
[0006] Thus, a need exists for therapies and therapeutic strategies
providing improved inhibition of ErbB3 activation (e.g.,
ligand-induced activation) as well as for therapies and therapeutic
strategies providing improved inhibition of estrogen receptor
signaling activity or of ErB 1 and ErbB2 receptor signaling
activity.
[0007] In the treatment of cancers, the co-administration of
pluralities of anti-cancer drugs (combination therapy) often
provides better treatment outcomes than monotherapy. Such outcomes
can be subadditive, additive, or superadditive. That is to say that
the combined effects of two anti-cancer drugs, each of which
provides a quantifiable degree of benefit, can be less than, equal
to, or greater than the sum of the benefits of each drug. For
example, two drug, each of which when used alone to treat a lethal
cancer provides an average one year extension of progression free
survival, could together provide a <24 month extension (e.g., an
18 month extension), about a 24 month extension, or a >24 month
extension (e.g., a 30 month extension) of progression free
survival. Typically, combination therapies for cancer treatment
provide significantly subadditive outcomes. Outcomes that are near
additive, additive, or superadditive are most desirable, but only
occur rarely. In addition, many drugs are known to alter the
bioavailability, or otherwise affect the safety profile of other
drugs when both drugs are co-administered. As new drugs are first
used in combination therapies, unforeseen, hazardous drug-drug
interactions may be observed that result in drug-drug
interaction-mediated toxicity in the patient.
[0008] Thus approaches for safely administering combination
therapies comprising administration of ErbB2/ErbB3
heterodimer-targeted agents for cancer treatment, and especially
combinations that yield near-additive, additive, or superadditive
outcomes are needed.
SUMMARY OF THE INVENTION
[0009] Provided herein are methods and compositions effective for
the inhibition of ErbB3 activation and also effective for the
inhibition of estrogen receptor activation. Also provided are
methods and compositions effective for the inhibition of ErbB3
activation and also effective for the inhibition of ErB1 and/or
ErbB2 activation. These methods and compositions are useful for the
treatment of tumors, e.g., malignant tumors, as well as for the
treatment of other cancers.
[0010] In a first embodiment, a method of treating a subject with a
malignant tumor is provided, where the tumor is an ErbB2 expressing
or ErbB2 over-expressing tumor (e.g., HER.sup.++ or HER.sup.+++
tumors) and the tumor may be a melanoma, clear cell sarcoma, head
and neck, endometrial, prostate, breast, ovarian, gastric, colon,
colorectal, lung, bladder, pancreatic, salivary gland, liver, skin,
brain or renal tumor. The method comprises co-administering to the
subject an effective amount an agent selected from i) an effective
amount of an anti-estrogen agent; ii) an effective amount of a
receptor tyrosine kinase inhibitor; iii) an effective amount of a
MEK/PI3 kinase/AKT inhibitor (e.g., those inhibitors described in
the appendix and including, e.g., AZD6244, BKM-120, GDC-0941,
GSK1120212, MK-2206, PD0325901, and Triciribine, and combinations
thereof); iv) MM-151; v) an effective amount of an mTOR inhibitor
(e.g., one or more of the mTOR inhibitors described in the
appendix); and/or vi) an effective amount of trastuzumab or TMD1,
and combinations thereof, in combination with an effective amount
of an anti-ErbB3 agent, e.g., a bispecific anti-ErbB2/anti-ErbB3
antibody (e.g., the antibody comprising the amino acid sequence set
forth in SEQ ID NO:1) and optionally an effective amount of
trastuzumab or TMD1.
[0011] In one aspect, the combination of the bispecific
anti-ErbB2/anti-ErbB3 antibody and either the effective amount of
an anti-estrogen agent or the effective amount of the receptor
tyrosine kinase inhibitor, and optionally the effective amount of
trastuzumab or TMD1, is characterized as follows: when a first
tissue culture medium is prepared comprising the bispecific
anti-ErbB2/anti-ErbB3 antibody (e.g., the antibody comprising the
amino acid sequence set forth in SEQ ID NO:1) at a first
concentration and either the anti-estrogen agent at a second
concentration or the receptor tyrosine kinase inhibitor (e.g.,
lapatinib) at a third concentration (wherein each concentration is
the same or different as each other concentration), and the medium
is contacted with cancer cells of a cell line in a cell culture,
cell growth or cell proliferation or production of pErbB3 or
production of pAKT in the cells is inhibited, or the percentage of
cells in the culture that are apoptotic is increased. In certain
aspects, cell growth or cell proliferation or production of pErbB3
or production of pAKT in the cells is inhibited, or the percentage
of cells in the culture that are apoptotic is increased to a
greater degree than cell growth, or cell proliferation or
production of pErbB3 or production of pAKT in the cells is
inhibited, or percentage of cells in the culture that are apoptotic
is increased, to a lesser degree when cancer cells of the cell line
in a cell culture are contacted with each of a second medium that
is essentially the same as the first medium except that it does not
comprise a bispecific anti-ErbB2/anti-ErbB3 antibody, and a third
medium that is essentially the same as the first medium except that
it does not comprise any anti-estrogen agent and it does not
comprise any receptor tyrosine kinase inhibitor.
[0012] In another aspect, all effective amounts are either mouse
effective amounts or human effective amounts. In another aspect,
all effective amounts are mouse effective amounts and the
combination of the bispecific anti-ErbB2/anti-ErbB3 antibody
(optionally the antibody comprising the amino acid sequence set
forth in SEQ ID NO:1) and either the effective amount of an
anti-estrogen agent or the effective amount of the receptor
tyrosine kinase inhibitor, is characterized as follows: when
co-administered to BT474-M3 xenograft tumor bearing mice with a
tumor of a measured volume, the combination is more effective at
inhibiting tumor volume increase after 32 days of co-administration
than is the mouse effective amount of the bispecific
anti-ErbB2/anti-ErbB3 antibody administration without the
co-administration of either the effective amount of an
anti-estrogen agent or the effective amount of the receptor
tyrosine kinase inhibitor. In another aspect, a mouse effective
amount of trastuzumab or TMD1 is co-administered with the
bispecific anti-ErbB2/anti-ErbB3 antibody.
[0013] In a second embodiment, a bispecific anti-ErbB2/anti-ErbB3
antibody (optionally the antibody comprising SEQ ID NO:1) is
provided for use in combination therapy of a cancer (optionally a
melanoma, clear cell sarcoma, head and neck, endometrial, prostate,
breast, ovarian, gastric, colon, colorectal, lung, bladder,
pancreatic, salivary gland, liver, skin, brain or renal tumor),
where the combination therapy comprises concomitant use of an
effective amount an agent selected from i) an effective amount of
an anti-estrogen agent; ii) an effective amount of a receptor
tyrosine kinase inhibitor; iii) an effective amount of a MEK/PI3
kinase/AKT inhibitor (e.g., those inhibitors described in the
appendix and including, e.g., AZD6244, BKM-120, GDC-0941,
GSK1120212, MK-2206, PD0325901, and Triciribine, and combinations
thereof); iv) an effective amount of MM-151; v) an effective amount
of an mTOR inhibitor (e.g., one or more of the mTOR inhibitors
described in the appendix); and/or vi) an effective amount of
trastuzumab or TMD1, and combinations thereof.
[0014] In a third embodiment, an aqueous solution is provided
comprising a bispecific anti-ErbB2/anti-ErbB3 antibody (optionally
the antibody comprising the amino acid sequence set forth in SEQ ID
NO:1) at a first concentration and an agent selected from i) an
effective amount of an anti-estrogen agent; ii) an effective amount
of a receptor tyrosine kinase inhibitor; iii) an effective amount
of a MEK/PI3 kinase/AKT inhibitor (e.g., those inhibitors described
in the appendix and including, e.g., AZD6244, BKM-120, GDC-0941,
GSK1120212, MK-2206, PD0325901, and Triciribine, and combinations
thereof); iv) an effective amount of MM-151; v) an effective amount
of an mTOR inhibitor (e.g., one or more of the mTOR inhibitors
described in the appendix); and/or vi) an effective amount of
trastuzumab or TMD1, and combinations thereof, at a second
concentration. In certain aspects, when a first tissue culture
medium is prepared comprising the bispecific anti-ErbB2/anti-ErbB3
antibody at the first concentration and the agent at the second
concentration, and the medium is contacted with cancer cells of a
cell line in a cell culture, cell growth or cell proliferation or
production of pErbB3 or production of pAKT in the cells is
inhibited, or percentage of cells in the culture that are apoptotic
is increased. In certain aspects, cell growth or cell proliferation
or production of pErbB3 or production of pAKT in the cells is
inhibited, or the percentage of cells in the culture that are
apoptotic is increased, to a lesser degree when cells of the cell
line in a cell culture are contacted with a second tissue culture
medium that is essentially the same as the first medium of except
that it does not comprise the agent(s). In another aspect, cell
growth or cell proliferation or production of pErbB3 or production
of pAKT in the cells is inhibited, or the percentage of cells in
the culture that are apoptotic is increased, to a lesser degree
when cells of the cell line in a cell culture are contacted with a
third tissue culture medium that is essentially the same as the
first medium of except that it does not comprise any bispecific
anti-ErbB2/anti-ErbB3 antibody.
[0015] In another aspect, the aqueous solution is blood plasma in a
subject, and the subject does not experience a toxicity that is
sufficiently harmful to require a change in a therapy being
administered to the subject, which toxicity is mediated by a
drug-drug interaction in the subject between the bispecific
anti-ErbB2/anti-ErbB3 antibody and the anti-estrogen agent or the
receptor tyrosine kinase inhibitor.
[0016] In another aspect, the aqueous solution further comprises
trastuzumab or TMD1 at a third concentration, and the medium also
comprises trastuzumab or TMD1 at the third concentration.
[0017] In another aspect, the method, combination therapy, or
aqueous solution does not comprise an aromatase inhibitor or an
estrogen receptor antagonist. In one embodiment the method,
combination therapy, or aqueous solution comprises
nab-paclitaxel.
[0018] In each embodiment and aspect thereof above, the
anti-estrogen agent may be an estrogen receptor antagonist (e.g.,
fulvestrant or tamoxifen) or an aromatase inhibitor (e.g., wherein
the aromatase inhibitor is letrozole, exemestane, anastrozole,
aminoglutethimide, testolactone, vorozole, formestane, or
fadrozole. Preferably the aromatase inhibitor is letrozole. Also in
each embodiment and aspect thereof above, the receptor tyrosine
kinase inhibitor is erlotinib, afatinib, dasatinib, gefitinib,
imatinib, pazopinib, lapatinib, sunitinib, nilotinib or sorafenib.
Preferably the receptor tyrosine kinase inhibitor is lapatinib.
Also in each embodiment and aspect thereof above, the bispecific
anti ErbB2/anti-ErbB3 antibody is the A5-HSA-ML3.9, ML3.9-HSA-A5,
A5-HSA-B1D2, B1D2-HSA-A5, B12-HSA-B1D2, B1D2-HSA-B12,
A5-HSA-F5B6H2, F5B6H2-HSA-A5, H3-HSA-F5B6H2, F5B6H2-HSA-H3,
F4-HSA-F5B6H2, F5B6H2-HSA-F4, B1D2-HSA-H3, H3-HSA-B1D2, or the
antibody comprising the amino acid sequence set forth in SEQ ID
NO:1. Each embodiment and aspect thereof above may also further
comprise use of capecitabine and/or cisplatin.
[0019] In each embodiment and aspect thereof above, one or more of
a)-i) that follow may optionally apply: a) the cell line is
BT474-M3; b) the culture is a spheroid culture, c) paclitaxel or
another taxane or another chemotherapeutic drug is co-administered,
optionally in accordance with the manufacturer's directions, d) the
agent i)-vi) is administered in accordance with the manufacturer's
directions, e) the trastuzumab or TMD1 is administered in
accordance with the manufacturer's directions, f) the
co-administration of the bispecific anti-ErbB2/anti-ErbB3 antibody
with the agent g)-vi) produces an about additive or a superadditive
effect, h) the bispecific anti-ErbB2/anti-ErbB3 antibody is the
antibody comprising SEQ ID NO:1 and is administered in accordance
with any of the regimens (e.g., modes, dosages, dosing intervals,
loading and maintenance doses and dosing schemes) described in
Examples 12 and 13, below, i) the lapatinib is administered in
accordance with any of the regimens (e.g., modes, dosages, dosing
intervals, loading and maintenance doses and dosing schemes)
described in Example 16, below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a graph showing that the combination of MM-111 and
tamoxifen inhibits tumor growth in vivo better than either MM-111
or tamoxifen does alone. The x-axis shows time post tumor implant
in days and the y-axis shows tumor volume in mm.sup.3 Mice were
treated with inhibitors beginning on day 7 post BT474-M3 cell
implant.
[0021] FIGS. 2A-2G are seven graphs showing that MM-111 combines
positively with anti-estrogen drugs in inhibiting
estrogen-stimulated spheroid growth in vitro. FIG. 2A shows the
effect of MM-111, tamoxifen (4-hydroxytamoxifen or 4OHT), or MM-111
and tamoxifen on in vitro spheroid growth. FIG. 2B shows the effect
of trastuzumab, tamoxifen, or trastuzumab and tamoxifen. FIG. 2C
shows the effect of MM-111, fulvestrant (FVT), or MM-111 and
fulvestrant. FIG. 2D shows the effect of trastuzumab, fulvestrant,
or trastuzumab and fulvestrant. FIG. 2E shows the effect of MM-111,
trastuzumab, or MM-111 and trastuzumab. FIG. 2F shows the effect of
MM-111, trastuzumab, and tamoxifen combined compared to that of any
of the double combinations. FIG. 2G shows the effect of MM-111,
trastuzumab, and fulvestrant combined compared to that of any of
the double combinations. The x-axes are a log scale of each drug
concentration for each experimental condition in nM and the y axis
is spheroid size as % of control spheroid size.
[0022] FIGS. 3A-3G are seven graphs showing that MM-111 combines
positively with anti-estrogen drugs in inhibiting heregulin
(HRG)-stimulated spheroid growth in vitro. FIG. 3A shows the effect
of MM-111, tamoxifen (4-hydroxytamoxifen or 4OHT), or MM-111 and
tamoxifen. FIG. 3B shows the effect of trastuzumab, tamoxifen, or
trastuzumab and tamoxifen. FIG. 3C shows the effect of MM-111,
fulvestrant (FVT), or MM-111 and fulvestrant. FIG. 3D shows the
effect of trastuzumab, fulvestrant, or trastuzumab and fulvestrant.
FIG. 3E shows the effect of MM-111, trastuzumab, or MM-111 and
trastuzumab. FIG. 3F shows the effect of MM-111, trastuzumab, and
tamoxifen combined compared to that of any of the double
combinations. FIG. 3G shows the effect of MM-111, trastuzumab, and
fulvestrant combined compared to that of any of the double
combinations. The x-axes are a log scale of each drug concentration
for each experimental condition in nM and the y axis is spheroid
size as % of control spheroid size.
[0023] FIGS. 4A-4G are seven graphs showing that MM-111 combines
positively with anti-estrogen drugs in inhibiting dual ligand
(estrogen and heregulin)-stimulated spheroid growth in vitro. FIG.
4A shows the effect of MM-111, tamoxifen, or MM-111 and tamoxifen.
FIG. 4B shows the effect of trastuzumab, tamoxifen, or trastuzumab
and tamoxifen. FIG. 4C shows the effect of MM-111, fulvestrant
(FVT), or MM-111 and fulvestrant. FIG. 4D shows the effect of
trastuzumab, fulvestrant, or trastuzumab and fulvestrant. FIG. 4E
shows the effect of MM-111, trastuzumab, or MM-111 and trastuzumab.
FIG. 4F shows the effect of MM-111, trastuzumab, and tamoxifen
combined compared to that of any of the double combinations. FIG.
4G shows the effect of MM-111, trastuzumab, and fulvestrant
combined compared to that of any of the double combinations. The
x-axes are a log scale of each drug concentration for each
experimental condition in nM and the y axis is spheroid size as %
of control spheroid size.
[0024] FIG. 5A is a graph summarizing the effect of MM-111,
trastuzumab, and tamoxifen combined on BT474M3 spheroids as
compared to that of any of the double combinations. The y-axis is %
inhibition of spheroid size normalized to stimulated control.
[0025] FIG. 5B is a graph summarizing the effect of MM-111,
trastuzumab, and fulvestrant combined compared to that of any of
the double combinations at inhibiting single ligand (estrogen or
heregulin) or dual-ligand (estrogen and heregulin)-stimulated
spheroid growth in vitro. The y-axis is % inhibition of spheroid
size normalized to stimulated control.
[0026] FIG. 6 is a graph showing that the combination of MM-111 and
lapatinib inhibits tumor growth in vivo. The x-axis shows the time
post tumor implant in days and the y-axis shows tumor volume in
mm.sup.3. Mice were treated with inhibitors on day 7 post tumor
implant.
[0027] FIGS. 7A and 7B are graphs showing the ability of lapatinib
to inhibit ErbB3 and AKT activation in heregulin-stimulated cells.
FIG. 7A is a graph comparing computer-generated dose-response
curves to experimental results in heregulin-stimulated BT474-M3
cells. FIG. 7B is a graph showing lapatinib inhibition (IC50) of
ErbB3 and AKT activation in heregulin-stimulated and unstimulated
cells following a 1-hour incubation with inhibitor.
[0028] FIGS. 8A-8H are a series of graphs showing MM-111 or
lapatinib inhibition of ErbB3 (FIGS. 8A-8D) or AKT (FIGS. 8E-8H)
activation in heregulin-stimulated cells incubated with inhibitor
for 15 minutes, 1 hour, 4 hours, and 24 hours.
[0029] FIGS. 8I and 8J are graphs showing a comparison of 1050 for
MM-111 and lapatinib at 1 hour and 24 hours for both BT474M3 cells
and ZR75-30 cells.
[0030] FIG. 9 is a graph showing the effect of MM-111 and lapatinib
combination treatment on AKT activation in heregulin-stimulated
BT474-M3 cells.
[0031] FIG. 10 is a graph showing the effect of lapatinib on cell
viability as a measure of proliferation of unstimulated and
heregulin-stimulated BT474-M3 cells.
[0032] FIG. 11 is a graph showing the effect of MM-111, lapatinib,
or the combination on BT474-M3 cell apoptosis. The number of dead
cells, cells in late apoptosis, early apoptosis, and live cells was
quantitated.
[0033] FIGS. 12A-12C are graphs showing that MM-111 combines
positively with anti-estrogen drugs and lapatinib in inhibiting
dual ligand (estrogen (E2) and heregulin (HRG))-stimulated spheroid
growth in vitro. FIG. 12A shows the effect of lapatinib alone or
the combination of lapatinib and fulvestrant (FVT). FIG. 12B shows
the effect of lapatinib alone or the combination of lapatinib and
MM-111. FIG. 12C shows the effect of lapatinib alone, the
combination of MM-111 and fulvestrant, or the triple combination of
MM-111, FVT, and lapatinib. Lapatinib is given in 3.3, 10, or 30 nM
doses. The x-axes are a log scale of each of MM-111 and/or FVT
concentration in nM and the y axis is spheroid size as % of control
(FBS alone) spheroid size.
[0034] FIGS. 13A-13D are graphs showing that MM-111 combines
positively with the aromatase inhibitor letrozole and the tyrosine
kinase inhibitor lapatinib in heregulin (HRG) and androstenedione
(A4)-stimulated BT474-M3-Aro cells that stably express human
aromatase, which converts androstenedione to estrogen. FIG. 13A
shows the effect of letrozole, MM-111, or the combination of
letrozole and MM-111. FIG. 13B shows the effect of lapatinib,
MM-111 or the combination of lapatinib and MM-111. FIG. 13C shows
the effect of lapatinib, letrozole, or the combination of lapatinib
and letrozole. FIG. 13D shows the effect of the dual combinations
of MM-111 and letrozole, MM-111 and lapatinib, lapatinib and
letrozole, and the triple combination of MM-111, lapatinib and
letrozole. The x-axes are a log scale of MM-111 concentration in
nM. The drug concentrations are a ratio of 10:20:1 MM-111 to
letrozole to lapatinib. The y axis is spheroid size as % of control
spheroid size.
DETAILED DESCRIPTION
[0035] As herein provided, bispecific anti-ErbB2/anti-ErbB3
antibodies (e.g., MM-111) are co-administered with one or more
additional therapeutic agents (e.g. an aromatase inhibitor or
tyrosine kinase inhibitor), to provide effective treatment to human
patients having a cancer.
[0036] The term "anti-ErbB3 agent" refers to any therapeutic agent
that binds to ErbB3 or binds to an ErbB3-specific ligand or blocks
the expression of ErbB3, and thereby inhibits the activity of
cellular signaling mediated by ErbB3. Non-limiting examples of
types of anti-ErbB3 agents include antibodies, bispecific
antibodies, ligand analogs, soluble forms of ErbB3 or the ErbB3
ectodomain, ErbB3 specific RNAi molecules, and similar biologic
agents.
[0037] The term "antibody" describes a polypeptide comprising at
least one antibody-derived antigen binding site (e.g.,
V.sub.H/V.sub.L region or Fv, or complementarity determining
region--CDR) that specifically binds to a specific antigen, e.g.,
ErbB3. "Antibodies" include whole antibodies and any antigen
binding fragment, e.g., Fab or Fv, or a single chain fragment
(e.g., scFv), as well as bispecific antibodies and similar
engineered variants, human antibodies, humanized antibodies,
chimeric antibodies Fabs, Fab'2s, ScFvs, SMIPs, Affibodies.RTM.,
nanobodies, or a domain antibodies, and may be of any of the
following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,
IgAsec, IgD, and IgE. The antibody may be a naturally occurring
antibody or may be an antibody that has been altered (e.g., by
mutation, deletion, substitution, conjugation to a non-antibody
moiety). For example, an antibody may include one or more variant
amino acids (compared to a naturally occurring antibody) which
change a property (e.g., a functional property) of the antibody.
For example, numerous such alterations are known in the art which
affect, e.g., half-life, effector function, and/or immune responses
to the antibody in a patient. The term "antibody" thus includes
whole antibodies and any antigen binding fragment (i.e.,
"antigen-binding portion," e.g., Fabs) or single chains thereof
(e.g., scFvs) as well as bispecific antibodies and similar
engineered variants, provided that they retain the binding
specificity of an antibody.
[0038] 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. Such binding to ErbB3 or ErB2 exhibits a Kd with a value
of no greater than 50 nM as measured by a surface plasmon resonance
assay or a cell binding assay. Exemplary anti-ErbB3 antibodies
inhibit EGF-like ligand mediated phosphorylation of ErbB3, e.g.,
anti-ErbB2 antibodies that inhibit the binding of heregulin to
ErbB2/ErbB3 heterodimers. EGF-like ligands include EGF, TGF.alpha.,
betacellulin, heparin-binding epidermal growth factor, biregulin,
epigen, epiregulin, and amphiregulin, which typically bind to ErbB
1 and induce heterodimerization of ErbB 1 with ErbB3.
[0039] The term "bispecific antibody" as used herein refers to a
protein comprising two antigen-binding sites, a first binding site
exhibiting immunospecific binding to a first antigen or epitope and
a second binding site exhibiting immunospecific binding to a second
antigen or epitope distinct from the first. An
"anti-ErbB2/anti-ErbB3 bispecific antibody" is an antibody that
comprises two binding sites, one that immunospecifically binds to
the ectodomain of ErbB3 and another that immunospecifically binds
to the ectodomain of ErbB2. Preferably, a bispecific ErbB3, ErbB2
antibody is the antibody comprising SEQ ID NO:1.
[0040] An "anti-estrogen agent" as used herein refers to an agent
that prevents or reduces production of estrogen or prevents or
reduces signaling mediated by estrogen receptors. Anti-estrogen
agents include but are not limited to estrogen receptor antagonists
and aromatase inhibitors. Estrogen receptor antagonists include but
are not limited to raloxifene, fulvestrant, tamoxifen, afimoxifene
(4-hydoroxytamoxifen), arzoxifene, toremifene, and lasofoxone.
Preferably, the estrogen receptor antagonist is tamoxifen or
fulvestrant. Aromatase inhibitors work by blocking the synthesis of
estrogen in an animal (e.g., a mouse or a human) This lowers
estrogen levels in the animal and thereby inhibits the growth of
estrogen-driven cancers. Examples of aromatase inhibitors include
but are not limited to exemestane, anastrozole, letrozole,
aminoglutethimide, testolactone, vorozole, formestane, and
fadrozole. Preferably, the aromatase inhibitor is exemestane or
letrozole.
[0041] By "cancer" is meant any condition characterized by
abnormal, unregulated, malignant cell growth.
[0042] By "malignant tumor" is meant any cancer that takes the form
of a tumor.
[0043] The term "effective amount" refers to an amount of a drug
effective to achieve a desired effect, e.g., to ameliorate disease
in a subject. Where the disease is a cancer, the effective amount
of the drug may inhibit (e.g., slow to some extent, inhibit or
stop) one or more of the following characteristics: cancer cell
growth, cancer cell proliferation, cancer cell motility, cancer
cell infiltration into peripheral organs, tumor metastasis, and
tumor growth. Where the disease is a cancer, the effective amount
of the drug may alternately do one or more of the following when
administered to a subject: slow or stop tumor growth, reduce tumor
size (e.g., volume or mass); relieve to some extent one or more of
the symptoms associated with the cancer, extend progression free
survival, result in an objective response (including a partial
response or a complete response), and increase overall survival
time. To the extent the drug may prevent growth and/or kill
existing cancer cells, it is cytostatic and/or cytotoxic.
[0044] A "mouse effective amount" refers to an amount of a drug
effective to achieve a desired effect when the subject is a
mouse.
[0045] A "human effective amount" refers to an amount of a drug
effective to achieve a desired effect when the subject is a human
patient.
[0046] The terms "combination therapy," "concomitant use,"
"co-administration," co-administering," "co-administered," and the
like, refer to the administration of at least two therapeutic
agents to a subject either simultaneously or within a time period
during which the effects of the earlier-administered therapeutic
agent are still operative in the subject when a later-administered
therapeutic agent is administered.
[0047] A "receptor tyrosine kinase inhibitor" as used herein refers
to a member of a class of drugs that specifically inhibit receptor
tyrosine kinases and thus reduce or eliminate the activation of
various signal transduction pathways. Receptor tyrosine kinase
inhibitors useful for the treatment of cancer as disclosed herein
include but are not limited to the small molecule inhibitors
erlotinib, afatinib, dasatinib, gefitinib, imatinib, pazopinib,
lapatinib, sunitinib, nilotinib and sorafenib. Receptor tyrosine
kinase inhibitors also include antibody-based therapeutics such as
cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab).
Preferably, the receptor tyrosine kinase inhibitor is
lapatinib.
[0048] "Dosage" or "dosing regimen" refers to parameters for
administering a drug in defined quantities per unit time (e.g., per
hour, per day, per week, per month, etc.) to a patient. Such
parameters include, e.g., the size of each dose. Such parameters
also include the configuration of each dose, which may be
administered as one or more units, e.g., taken at a single
administration, e.g., orally (e.g., as one, two, three or more
pills, capsules, etc.) or injected (e.g., as a bolus). Dosage sizes
may also relate to doses that are administered continuously (e.g.,
as an intravenous infusion over a period of minutes or hours). Such
parameters further include frequency of administration of separate
doses, which frequency may change over time. A "dosing cycle" or
"dosing interval" is the period of time that comprises one cycle of
treatment (e.g., 21 days or 28 days) for a dosing regimen.
[0049] "Dose" refers to an amount of a drug given in a single
administration.
[0050] Preferred cancer cells of cell lines are cells of ErbB2
expressing cell lines such as ErbB2 overexpressing cell lines,
e.g., BT474-M3 (ATCC.RTM. #CRL-HTB-20.TM., derived from breast
ductal carcinoma cells), BT474-M3-Aro (BT474-M3 cells that stably
express human aromatase), ZR75-30 (ATCC.RTM. #CRL1504.TM., derived
from breast ductal carcinoma cells), SKOV-3 (ATCC.RTM. #HTB-77.TM.,
derived from metastatic ovarian adenocarcinoma cells), MCF7
(ATCC.RTM. #HTB-22.TM.) clone 18, MDA-MB-453 (ATCC.RTM.
#HTB-131.TM., derived from breast carcinoma cells), SK-BR-3
(ATCC.RTM. #HTB-30.TM., derived from breast adenocarcinoma cells),
and NCI-N87 (ATCC.RTM. #CRL-5822.TM., derived from gastric
carcinoma cells).
[0051] Cancers may include, for example, solid tumors such as:
sarcomas (e.g., clear cell sarcoma), carcinomas (e.g., renal cell
carcinoma), and lymphomas; tumors of the breast, colon, rectum,
lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,
bilecyst, bile duct, small intestine, urinary system (including the
kidney, bladder, and epithelium of the urinary tract), female
genital system (including the uterine neck, uterus, ovary,
chorioma, and gestational trophoblast), male genital system
(including the prostate, seminal vesicle, and testicles), endocrine
glands (including the thyroid gland, adrenal gland, and pituitary
body), skin (including angioma, melanoma, sarcoma originating from
bone or soft tissue, and Kaposi's sarcoma), brain and meninges
(including astrocytoma, neuroastrocytoma, spongioblastoma,
retinoblastoma, neuroma, neuroblastoma, neurinoma and
neuroblastoma), nerves, and eyes.
[0052] A cancer may be an estrogen receptor positive (ER+) cancer.
Such cancers exemplify candidates for therapy regimens that include
anti-estrogen agents. Such cancers may include but are not limited
to certain breast, ovarian, uterine, endometrial, lung, bone,
brain, bladder, liver and urogenital cancers.
[0053] A cancer may be an ErbB2 gene-amplified cancer and/or an
ErbB2-expressing or overexpressing cancer. ErbB2, also known as
HER2 or Neu, is a cell surface transmembrane receptor protein that
generates intracellular signals (e.g., upon ligand activation) via
its intracellular tyrosine kinase activity. In excess, such signals
can promote oncogenesis e.g., by triggering cell division. The
ErbB2 gene is amplified and/or overexpressed in many types of human
malignancies, including but not limited to breast, ovarian,
endometrial, pancreatic, colorectal, prostate, salivary gland,
kidney, and lung. ErbB2 overexpressing cancers are designated a
HER2.sup.+++ or HER2.sup.++ depending on the level of ErbB2
overexpression, with HER2.sup.+++ indicating the highest levels of
HER2 expression. HER2.sup.+++ and HER2.sup.++ status are typically
determined by an immunoassay such as immunohistochemistry, e.g.,
Herceptest.RTM.. ErbB2 gene amplification is may be determined by,
e.g., FISH (fluorescence in situ hybridization), with
HER2-amplified cancer cells being those that have more than two
HER2 gene copies being HER2-amplified, and cells and/or tumors
comprising HER2-amplified cancer cells being referred to as "FISH
positive."
[0054] A number of bispecific anti-ErbB2, antiErbB3 antibodies that
are scFv HSA conjugates are described in co-pending US patent
publication No. 2011-0059076, and PCT publication Nos.
WO2009/126920 and WO 2010/059315, each of which is incorporated
herein by reference in its entirety and each of which discloses
MM-111 (also referred to as B2B3-1) and other bispecific
anti-ErbB2/antiErbB3 antibodies that are scFv HSA conjugates and
that are suitable for use in the methods and compositions provided
herein, including the components of A5-HSA-ML3.9, ML3.9-HSA-A5,
A5-HSA-B1D2, B1D2-HSA-A5, B12-HSA-B1D2, B1D2-HSA-B12,
A5-HSA-F5B6H2, F5B6H2-HSA-A5, H3-HSA-F5B6H2, F5B6H2-HSA-H3,
F4-HSA-F5B6H2, F5B6H2-HSA-F4, B1D2-HSA-H3, and H3-HSA-B1D2. Other
suitable bispecific anti-ErbB2/antiErbB3 antibodies are disclosed
and claimed in U.S. Pat. Nos. 7,332,580 and 7,332,585, which are
incorporated herein by reference. MM-111 is currently undergoing
clinical trials, including an open-label Phase 1/2 and
pharmacologic study of MM-111 in patients with advanced, refractory
HER2 positive cancers, an open-label Phase 1/2 trial of MM-111 in
combination with trastuzumab (Herceptin.RTM.) in patients with
advanced HER2 positive breast cancer, and an open label, Phase 1/2
and pharmacologic study of MM-111 with three different combination
treatments: MM-111 in combination with cisplatin, capecitabine, and
trastuzumab, MM-111 in combination with lapatinib and trastuzumab,
and MM-111 in combination with paclitaxel and trastuzumab.
[0055] A bispecific anti-ErbB2/anti-ErbB3 antibody (e.g., MM-111)
can be co-administered with other therapeutic agents, (e.g, an
anti-estrogen receptor agent or a receptor tyrosine kinase
inhibitor) prior to (e.g., neoadjuvant therapy), concurrent with,
or following (e.g., adjuvant therapy) radiotherapy of, or surgical
intervention to remove, a malignant tumor.
[0056] Additional therapeutic agents suitable for combination with
anti-ErbB2/anti-ErbB3 antibodies may further include: 1) antibody
EGFR inhibitors (e.g. MM-151, Sym004, cetuximab, panitumumab,
zalutumumab, nimotuzumab, and matuzumab), additional small molecule
tyrosine kinase inhibitors such as PKI-166, PD-158780, EKB-569,
Tyrphostin AG 1478, and pan-HER kinase inhibitors (e.g. CI-1033 (PD
183805), AC480, HM781-36B, AZD8931 and PF299804); 2) microtubule
stabilizing agents (e.g. laulimalide, epothilone A, epothilone B,
discodermolide, eleutherobin, sarcodictyin A, sarcodictyin B,
paclitaxel, nab-paclitaxel or docetaxel); antimetabolites such as
5-fluorouracil (5-FU) and capecitabine; and platinum-based
therapeutics such as oxaliplatin, carboplatin and cisplatin.
Additional examples of therapeutic agents suitable for combination
with anti-ErbB2/anti-ErbB3 antibodies may be found in Table 5 and
the Appendix below.
[0057] MM-111 is suitable for both large scale production and
systemic therapy. MM-111 binds to
[0058] ErbB2/ErbB3 heterodimers and forms a trimeric complex with
ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling. The
antitumor activity of MM-111 requires the presence of both ErbB2
and ErbB3, but is particularly dependent on ErbB2 expression. The
affinity of its ErbB2 antigen-binding site is about 30 times higher
than the affinity of its ErbB3 antigen-binding site, but the ErbB2
antigen-binding site does not by itself inhibit ErbB2 activity when
bound to ErbB2. The strong binding of MM-111 to ErbB2 places the
ErbB3 antigen-binding site in close proximity to bound ErbB2/ErbB3
heterodimer, resulting in an avidity effect that potentiates the
binding of the ErbB3 antigen-binding site to the heterodimer ErbB3,
whereby a biological effect is produced. MM-111 is administered to
human subjects (patients) at an interval measured in days, as a
single loading dose of at least 20 mg/kg of MM-111 followed by at
least seven day intervals (e.g., every 2 weeks) by at least one
administration of a single maintenance dose of MM-111, where the
maintenance dose is generally smaller than the loading dose, e.g.,
at least 5 mg/kg less than the loading dose.
EXAMPLES
[0059] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of non-critical parameters that could
be changed or modified to yield essentially the same or similar
results.
MM-111 in Combination with Anti-Estrogen Therapeutics
Methods:
Spheroid In Vitro Tumor Model Assay
[0060] BT474-M3 wild type cells (2000 cells/well) are plated in
Ultra Low Cluster 96-well plate (Costar). After overnight
incubation, indicated treatments are introduced to the plate. Cells
are continued to culture for six days. Spheroids are then examined
by Nikon microscope and analyzed by MetaMorph Image Analysis
Software (Molecular Devices). The spheroid size from cells cultured
in medium containing 10% FBS is set as control.
Xenograft Model
[0061] BT474-M3 cells (2.times.10.sup.7 cells per mice) are
inoculated subcutaneously into Nu/Nu immunodeficient mice, which
are implanted with an estrogen pellet (0.72 mg; 60-day release) one
day before the experiment. Tumors are measured after seven days and
mice are randomized into four groups: those treated with placebo,
MM-111 (60 mg/kg, Q7D), 4-hydroxytamoxifen (5 mg; 60-day release
pellet), and combination of MM-111 and 4-hydroxytamoxifen,
respectively. Tumors are measured every three days and the
experiment is ended at day 32.
Example 1
MM-111 and Tamoxifen Combination Therapy Inhibits Tumor Growth In
Vivo
[0062] In order to compare the effect of MM-111 and tamoxifen
combination therapy on tumor growth in vivo, estrogen stimulated
mice were prepared in the xenograft model using the methods
described above or minor variations thereof. Mice were inoculated
with tumor forming BT474-M3 cells and on day 7 given a placebo
(vehicle control), MM-111, tamoxifen, or a combination of MM-111
and tamoxifen and tumor growth was measured over time. As shown in
FIG. 1, this in vivo BT474-M3 xenograft model showed resistance to
tamoxifen treatment but when mice were given a combination of
MM-111 and tamoxifen the combination treatment inhibited tumor
growth to a significantly greater extent. Statistical significance
(p<0.05) was observed for the combination group from day 28
onward when compared to vehicle control, from day 21 onward when
compared to MM-111 and from day 25 onward when compared to
tamoxifen.
Example 2
MM-111 Combines Positively with Anti-Estrogen Drugs in Inhibiting
Estrogen-Stimulated Spheroid Growth
[0063] Multicellular spheroids are used to simulate the growth and
microenvironmental conditions of tumors in vitro. To further
investigate the ability of MM-111 to inhibit cell growth when in
combination with anti-estrogen drugs, spheroids of BT474-M3 cells
were prepared using the methods described above or minor variations
thereof and treated with an ErbB2-binding therapeutic and/or an
anti-estrogen therapeutic. Spheroids of estrogen-stimulated cells
were treated with a dose range of MM-111, tamoxifen, or the
combination of MM-111 and tamoxifen (FIG. 2A); trastuzumab,
tamoxifen or the combination of trastuzumab and tamoxifen (FIG.
2B); MM-111, fulvestrant, or the combination of MM-111 and
fulvestrant (FIG. 2C); trastuzumab, fulvestrant, or the combination
of trastuzumab and fulvestrant (FIG. 2D); or MM-111, trastuzumab,
or the combination of MM-111 and trastuzumab (FIG. 2E). When used
as single agent alone, MM-111, trastuzumab, fulvestrant and
tamoxifen showed inhibitory effects on spheroid growth in the
estrogen-stimulated BT474-M3 spheroid assay. The combination of
tamoxifen or fulvestrant with MM-111 (FIGS. 2A and 2C,
respectively) or trastuzumab (FIGS. 2B and 2D, respectively)
increased the degree of growth inhibition, as did the combination
of MM-111 and trastuzumab (FIG. 2E). The inhibitory effects were
increased still further when estrogen-stimulated spheroids were
treated with the triple combination of MM-111, trastuzumab, and
tamoxifen (FIG. 2F) or MM-111, trastuzumab, and fulvestrant (FIG.
2G) as compared to the double combinations of drugs.
Example 3
MM-111 Combines Positively with Anti-Estrogen Drugs in Inhibiting
Heregulin-Stimulated Spheroid Growth
[0064] To further investigate the ability of MM-111 to inhibit cell
growth when in combination with anti-estrogen drugs, spheroids of
heregulin (HRG)-stimulated BT474-M3 cells were prepared using the
methods described above or minor variations thereof and treated
with a dose range of MM-111, tamoxifen, or the combination of
MM-111 and tamoxifen (FIG. 3A); trastuzumab, tamoxifen or the
combination of trastuzumab and tamoxifen (FIG. 3B); MM-111,
fulvestrant, or the combination of MM-111 and fulvestrant (FIG.
3C); trastuzumab, fulvestrant, or the combination of trastuzumab
and fulvestrant (FIG. 3D); or MM-111, trastuzumab, or the
combination of MM-111 and trastuzumab (FIG. 3E). MM-111 inhibited
heregulin-induced spheroid growth but tamoxifen (FIG. 3A),
trastuzumab (FIG. 3B), and fulvestrant (FIG. 3C) did not inhibit
heregulin stimulated spheroid growth. No significant combinational
effect was observed when MM-111 was used with tamoxifen (FIG. 3A)
or fulvestrant (FIG. 3C). The combination of trastuzumab and either
tamoxifen (FIG. 3B) or fulvestrant (FIG. 3D) failed to show
inhibitory activity significantly greater than either drug alone.
As shown in FIG. 3E, MM-111 but not trastuzumab showed inhibitory
activity in heregulin-stimulated spheroid growth. Improved
inhibitory effects were observed when both drugs were combined. In
comparison to the double combination of either MM-111or trastuzumab
with tamoxifen or fulvestrant, the triple combination of MM-111,
trastuzumab and either tamoxifen (FIG. 3F) or fulvestrant (FIG. 3G)
showed similar inhibitory effects as those of MM-111 and
trastuzumab in combination (FIG. 3E) on heregulin-stimulated
spheroid growth.
Example 4
MM-111 Combines Positively with Anti-Estrogen Drugs in Inhibiting
Dual Ligand (Estrogen and Heregulin)-Stimulated Spheroid Growth
[0065] Dual ligand (estrogen and heregulin) stimulated spheroids
were treated with a dose range of tamoxifen, MM-111 or the
combination of MM-111 and tamoxifen (FIG. 4A) or trastuzumab,
tamoxifen or the combination of trastuzumab and tamoxifen (FIG.
4B). While MM-111 and trastuzumab each inhibited spheroid growth
(FIG. 4A) the combination of MM-111 and tamoxifen showed greater
inhibitory effects than either drug alone. In contrast, trastuzumab
alone had no significant inhibitory effects and the combination of
trastuzumab and tamoxifen showed similar effects to tamoxifen
alone.
[0066] Dual ligand stimulated spheroids were then treated with a
dose range of fulvestrant, MM-111 or the combination of MM-111 and
fulvestrant (FIG. 4C) or fulvestrant, trastuzumab, or a combination
of fulvestrant or trastuzumab (FIG. 4D). Again, while MM-111 and
fulvestrant each separately inhibited spheroid growth the
combination of MM-111 and fulvestrant showed greater inhibitory
effects than either drug alone (FIG. 4C). Trastuzumab alone had no
significant inhibitory effects and the combination of trastuzumab
and fulvestrant showed similar effects to tamoxifen alone (FIG.
4D).
[0067] Dual ligand stimulated spheroids were then treated with
MM-111, trastuzumab, or a combination of MM-111 and trastuzumab.
MM-111 showed greater inhibitory effects than trastuzumab in dual
ligand-stimulated spheroid growth. Enhanced inhibitory effects were
observed when both drugs were combined (FIG. 4E).
[0068] In comparison to the double combination of MM-111or
trastuzumab with tamoxifen or fulvestrant, the triple combination
of MM-111, trastuzumab and either tamoxifen (FIG. 4F) or
fulvestrant (FIG. 4G) showed similar inhibitory effects to those of
MM-111 and trastuzumab in combination (FIG. 4E) on estrogen- and
heregulin-(dual ligand) stimulated spheroid growth.
[0069] The data in the preceding Examples demonstrate that
combination therapies comprising MM-111 and an anti-estrogen
therapeutic are more effective than each of these therapies alone.
The percent of spheroid growth inhibition induced by each treatment
under estrogen or heregulin stimulation is summarized in FIGS. 5A
and 5B and Table 1. MM-111 was required for inhibition of spheroids
stimulated with heregulin. For each stimulated condition tested,
the triple combination resulted in the greatest inhibition of
spheroid growth, providing a percent inhibition ranging from about
70% to about 90%.
TABLE-US-00001 TABLE 1 Percent inhibitor induced maximal spheroid
growth inhibition MM-111 + MM-111 + Trastuzumab + Triple
Trastuzumab anti-estrogen anti-estrogen combination Tamoxifen
combination E2 54% 49% 55% 73% HRG 65% 43% 0% 71% E2 + HRG 46% 43%
36% 79% Fulvestrant combination E2 54% 49% 55% 77% HRG 64% 34% 4%
71% E2 + HRG 46% 57% 47% 88%
[0070] The percent of spheroid growth inhibition (normalized to
untreated, stimulated control) was determined for 1 .mu.M doses of
inhibitor treatment.
[0071] The combination of MM-111 and tamoxifen resulted in potent
inhibition of tumor growth in vivo. Taken together, these data
demonstrate that the combination of MM-111 and anti-estrogen
therapies results in potent anti-tumor effects in vitro and in
vivo.
MM-111 in Combination with Lapatinib
Methods
Computational Modeling
[0072] A computational model of HRG-induced phospho-ErbB3
signaling, as well as a model of lapatinib, was used as previously
described (Schoeberl, et al 2009).
Cell Signaling Assay
[0073] Serum-starved cells are pre-incubated with serial dilutions
of MM-111, lapatinib or combinations at doses and treatment times
indicated, followed by stimulation with 5 nM heregulin 1-.beta.
(R&D Systems, Minneapolis, Minn.) for 10 minutes. Cell lysates
are probed for phospho-ErbB3 (pErbB3), and phospho-AKT (pAKT) by
ELISA as described previously (Schoeberl et al, 2009). Inhibitor
IC.sub.50 values are calculated by fitting dose-response data to a
4-parameter sigmoidal curve (GraphPad Prism.RTM., GraphPad
Software, Inc., La Jolla, Calif.).
Cell Proliferation Assay
[0074] Cells (8,000/well) are seeded into 96-well plates and
incubated overnight. Inhibitor is added at doses indicated and
cells are treated for 24 hours. For experiments with ligand
stimulation, cells are serum-starved overnight prior to addition of
inhibitor and 2 nM heregulin 1-.beta. (R&D Systems,
Minneapolis, Minn.) is added 1 hour post-inhibitor treatment in
media containing 5% FBS. Numbers of viable cells are measured as an
indicator of cell proliferation using the CellTiter-Glo.RTM.
Luminescent Cell Viability Assay (Promega, Madison, Wis.).
Apoptosis Assay
[0075] BT474-M3 cells (2000 cells/well) are plated in Ultra Low
Cluster 96-well plate (Costar.RTM., Corning, N.Y.). After overnight
incubation, spheroids are treated with inhibitor at concentrations
indicated for 72 hours. Spheroids are then trypsinized and combined
with floating cells. Cells are washed twice with cold PBS and
suspended in binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 2.5
mM CaCl.sub.2). Cells are then stained with FITC-conjugated Annexin
V and PI. Apoptotic cells are quantified on a FACSCalibur.TM. FACS
machine.
Xenograft Efficacy Studies
[0076] Tumor xenografts are established by subcutaneous injection
of BT474-M3 cells into the flank of 5-6 weeks old female athymic
nude mice (nu/nu; Charles River Labs, Wilmington, Mass.). Mice
receive a subcutaneous 60 day, slow-release estrogen implant in the
opposite flank (0.72 mg pellet; Innovation Research of America,
Sarasota, Fla.) 24 hours prior to the injection of cells. Once
tumors reach a mean volume of 150-500 mm.sup.3, mice are randomized
into groups of 8 or 10 and dosed by intraperitoneal injection once
every three days with vehicle, MM-111 or lapatinib. For lapatinib
combination studies, MM-111 is given once every seven days and
lapatinib daily by gavage at doses indicated.
Aromatase-Overexpressing BT474-M3 Cells and Proliferation Assay
[0077] BT474-M3 cells were transfected with PS100010 vector
containing human aromatase (gene accession No: NM_000103.2). Cells
with stable expression of aromatase (BT474-M3-Aro) were obtained
after selection with 400 .mu.g/ml geneticin. For cell proliferation
assay, BT474-M3-Aro cells (5000 cells/well) were plated in phenol
red-free RPMI-1640 medium containing 5% charcoal-stripped FBS into
96-well plate. After overnight incubation, indicated treatments
were introduced in the presence of androstenedione (A-4; 200 nM)
and heregulin (HRG; 2 nM). After three days of treatment, cell
viability was determined by WST-1 (Roche; Cat. #11 644 807 001)
according to manufacturer's instruction. Cell viability in the
presence of 5% charcoal-stripped FBS was set as control (100%).
Example 5
The Combination of MM-111 and Lapatinib Inhibits Tumor Growth In
Vivo
[0078] The combination of MM-111 with lapatinib was investigated in
vivo in the BT474-M3 breast cancer xenograft model using the
methods described above or minor variations thereof. MM-111 and
lapatinib were each dosed at an optimal efficacious dose weekly and
daily, respectively. The combination of MM-111 and lapatinib
provided more potency compared to either drug alone, reaching
statistical significance for MM-111 (p=3.9.times.10-4) and
lapatinib (p=5.1.times.10-3) on day 13 (FIG. 6). The percent change
in tumor volume from day 40 to day 7 (inoculation) was calculated
for each group. The combination of MM-111 and lapatinib resulted in
a percent change in tumor volume of -69% (about 70%), reflecting
tumor regressions, compared to -11% (about 10%) for lapatinib and
14% (about 15%) for MM-111.
Example 6
Simulations Predict Lapatinib has Suboptimal Activity in Inhibiting
Heregulin-Driven pErbB3 and pAKT
[0079] A dose range of lapatinib inhibition of pErbB3 activation
was predicted using the computational modeling described above. A
dose range of lapatinib was applied to BT474-M3 cells followed by
stimulation with 5 nM heregulin for 10 min. The amount of pErbB3
was measured by ELISA using the methods described above or minor
variations thereof. Model-generated dose-response curves overlay
the experimental data (FIG. 7A). A comparison of the inhibitory
activity of lapatinib in heregulin-stimulated or unstimulated
(basal) cells was performed to demonstrate that heregulin signaling
perturbs the activity of lapatinib. Untreated and
heregulin-stimulated cells were probed for pErbB3 and pAKT and the
IC50 was calculated (FIG. 7B). These data show that lapatinib alone
is not an effective inhibitor of heregulin-activated signaling.
Example 7
MM-111 is a More Potent Inhibitor of HRG-Driven ErbB3 and AKT
Phosphorylation than Lapatinib
[0080] In order to compare the ability of MM-111 and lapatinib to
inhibit heregulin-induced ErbB3 activation, BT474-M3, or an
additional ErbB2 overexpressing breast tumor cell line, ZR75-30
(ATCC.RTM. #CRL-1504.TM.), cells were incubated with serial
dilutions of either inhibitor for 15 minutes, 1 hour, 4 hours, and
24 hours followed by stimulation with 5 nM heregulin for 10 min.
Amounts of pAKT and pErbB3 were measured by ELISA essentially as
described. MM-111 potently reduced pErbB3 levels (inhibited ErbB3
phosphorylation) in BT474-M3 (IC.sub.50=3 nM) cells (FIGS. 8A-8D)
and ZR75-30 cells (IC.sub.50=5 nM) (FIGS. 8I and 8J). Good
reduction by MM-111 of pAKT levels (inhibition of AKT
phosphorylation) in BT474-M3 (IC.sub.50=10) (FIGS. 8E-8H) and in
ZR75-30 cells (IC.sub.50=4 nM) (FIGS. 8I and 8J) was also observed.
The ability of MM-111 to inhibit heregulin-induced ErbB3 activation
(phosphorylation) was superior to lapatinib by greater than an
order of magnitude and the relative IC.sub.50 for each inhibitor
(FIGS. 8I and 8J) was consistent following up to 24 hours
incubation with inhibitors, indicating treatment times had little
effect on the potency of the inhibitors.
Example 8
The Combination of MM-111 and Lapatinib Potently Inhibits pAKT
[0081] The effect of MM-111 combined with lapatinib on pAKT
inhibition (reduction of pAKT levels) was assessed in
heregulin-stimulated BT474-M3 cells. Cells were incubated for 2
hours with a dose range of MM-111, lapatinib or their combination
and pAKT was measured by ELISA. In the presence of heregulin, the
combination of MM-111 and lapatinib was extremely effective,
inhibiting pAKT well below basal levels at therapeutically relevant
concentrations (FIG. 9). Treatment with either MM-111 (1 .mu.M) or
lapatinib (1 .mu.M) alone resulted in similar levels of pAKT
inhibition (see FIGS. 8E-8H) while the combination resulted in
about 20% more inhibition of pAKT.
Example 9
The Ability of Lapatinib to Inhibit Cell Proliferation is Perturbed
Under Heregulin-Stimulated Conditions
[0082] The effect of lapatinib on cell proliferation was measured
in unstimulated and heregulin-stimulated BT474-M3 cells. Cells
grown in serum or in serum plus 2 nM heregulin were treated with
lapatinib across a dose range for 24 hours. Lapatinib treatment
resulted in about a 50% inhibition of unstimulated cells but its
effect was reduced to about 23% inhibition in heregulin-stimulated
BT474-M3 cells (FIG. 10).
Example 10
Treatment with the Combination of MM-111 and Lapatinib Results in
Increased Apoptosis
[0083] The effect of the MM-111 combination with lapatinib on
apoptosis was assessed in a BT474-M3 spheroid model. Spheroids were
prepared using the methods described above or minor variations
thereof and treated with MM-111 (100 nM), lapatinib (33 nM), or a
combination of 100 nM MM-111 and 33 nM lapatinib. Cells were then
stained with Annexin V and propidium iodide (PI) and quantitated
using FACS (FIG. 11, Table 2). Cell populations staining positive
with Annexin V and PI were quantified as late apoptotic, cell
populations staining positive with Annexin V but not PI were
quantified as early apoptotic, cell populations staining positive
for PI but not Annexin V were quantified as dead cells and
populations of cells not stained with either Annexin V or PI were
considered alive and not apoptotic (Table 2). Spheroids that were
treated with both MM-111 and lapatinib had a higher number of total
apoptotic cells (about 46%) compared to those treated with only
lapatinib (about 31%) or only MM-111 (about 20%; FIG. 10).
TABLE-US-00002 TABLE 2 Percent cell population after treatment with
MM-111, lapatinib or the combination Live cells Early apoptosis
Late apoptosis Dead cells Control 75.2 17.3 7.2 0.42 MM-111 78.9
12.9 7.5 0.74 Lapatinib 67.9 16.8 14.5 0.73 Combination 52.1 30.0
16.2 1.74
Example 11
MM-111 Combines Positively with Anti-Estrogen Drugs and Lapatinib
in Inhibiting Dual Ligand (Estrogen and Heregulin)-Stimulated
Spheroid Growth
[0084] To further investigate the ability of MM-111 to inhibit cell
growth when in combination with both anti-estrogen drugs and
tyrosine kinase inhibitors, spheroids of estrogen and
heregulin-stimulated BT474-M3 cells were prepared using the methods
described above or minor variations thereof and treated with 3.3
nM, 10 nM, or 30 nM lapatinib, either alone or in combination with
a dose range of fulvestrant (FVT) (FIG. 12A); 3.3 nM, 10 nM, or 30
nM lapatinib, either alone or in combination with a dose range of
MM-111 (FIG. 12B); or 3.3 nM, 10 nM, or 30 nM lapatinib, either
alone or in combination with a dose range of both MM-111 and
fulvestrant (FIG. 12C). In the presence of dual ligand stimulation
the combination of lapatinib and FVT did not greatly increase
inhibition of spheroid growth over lapatinib alone (FIG. 12A). In
contrast, the addition of MM-111 greatly increased the sensitivity
of the spheroids to lapatinib treatment (FIG. 12B), and the triple
combination of lapatinib, FVT and MM-111 showed an even greater
increase of spheroid growth inhibition over lapatinib alone.
Example 12
MM-111 Combines Positively with Anti-Estrogen Drugs in Inhibiting
Spheroid Growth in BT474-M3 Cells Overexpressing Human
Androstenedione
[0085] Androstenedione is a steroid hormone that is converted to
estrogen by aromatase. To further investigate the ability of MM-111
to inhibit spheroid growth, aromatase-expressing cells were treated
in the presence of androstenedione (A4) and heregulin (HRG) with
MM-111, letrozole, or the combination of MM-111 or letrozole (FIG.
13A); MM-111, lapatinib, or the combination of MM-111 and lapatinib
(FIG. 13B); lapatinib, letrozole, or the combination of lapatinib
and letrozole (FIG. 13C); and each of the dual combination plus the
triple combination of MM-111, lapatinib, and letrozole (FIG. 13D).
In cells treated with A4 and HRG, the letrozole treatment did not
result in significant inhibition of spheroid cell growth as
compared to control (untreated) cells, whereas cells treated with
MM-111 alone or the combination of MM-111 and letrozole inhibited
cell proliferation to a similar extent (FIG. 13A). Lapatinib
treatment of the cells did not result in growth inhibition except
at high concentrations, whereas treatment with MM-111 alone or in
combination resulted in similar levels of cell growth inhibition
except in higher concentrations where the combination showed
increased inhibition of cell growth over either of the single
treatments (FIG. 13B). Treatment with lapatinib alone, letrozole
alone, or the combination of lapatinib and letrozole did not result
in significant cell growth inhibition except at high concentration
(FIG. 13C). Similarly, as shown in FIG. 13D, the double combination
of lapatinib and letrozole resulted in cell growth inhibition only
at high drug concentration. In contrast the dual combinations of
MM-111 and letrozole or MM-111 and lapatinib both showed an
increase in cell growth inhibition as compared to control, and the
triple combination of MM-111, lapatinib, and letrozole inhibited
cell growth to an even greater degree.
Example 13
Amino Acid Sequence of MM-111(SEQ ID NO:1)
TABLE-US-00003 [0086]
QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVAN
INRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDR
GVGYFDLWGRGTLVTVSSASTGGGGSGGGGSGGGGSQSALTQPASVSGSP
GQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRF
SGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGAA
SDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEF
AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN
ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYF
YAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK
CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGD
LLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP
ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRL
AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLG
EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCA
EDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP
KEFQAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMD
DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGAE
VKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKY
SPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCA
KWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQ
KVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGS
KSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG
Dosing and Administration of MM-111 in Combination One or More
Additional Therapeutics
Example 14
Mode of Administration of MM-111
[0087] MM-111 is prepared as a formulation containing 25 mg/ml
MM-111 in a sterile aqueous solution comprising 20 mM L-histidine
hydrochloride, 150 mM sodium chloride, pH 6.5, which is stored at
2-8.degree. C.
[0088] MM-111 must be brought to room temperature prior to
administration. Containers (e.g., vials) of MM-111 must not be
shaken. The appropriate quantity of MM-111 is removed from the
container, diluted in 250 mL of 0.9% normal saline and administered
as an infusion using a low protein binding in-line filter (e.g., a
0.22 micrometer filter).
[0089] MM-111 is initially administered over about 90 minutes
(first administration). In the absence of an infusion reaction,
subsequent doses are administered over about 60 minutes.
[0090] A patient's body weight at the start of a dosing cycle is
used to calculate the dose used throughout the cycle. Should a
patient's body weight change by more than 10%, a new total dose is
calculated to reflect this change.
Example 15
Dosage and Administration of MM-111
[0091] Preferred plasma concentrations of MM-111 achieved during
treatment are at least 106 mg/L. It has now been discovered that
certain combinations of dose frequency and dosage will achieve and
maintain this plasma concentration during the course of treatment
in at least half, and preferably in more than 60%, 70% or 80% of
treated patients.
[0092] In certain embodiments a higher initial dose (loading
dose--LD) is given, followed as defined intervals by at least one
maintenance dose (MD). Intervals of dosing in days are typically
indicated as Q.times.D, wherein x represents an integer, so that a
Q.times.D of 7 indicates dosing every 7 days. Table 3A, Table 3B,
and Table 3C below show doses and dosing intervals of the
invention. In Table 3A, Table 3B, and Table 3C the indicated
loading doses are optional--initial doses are preferably made at
the indicated loading dose (LD), but may (e.g., as directed or at
the physician's discretion) be made at the maintenance dose (MD).
Table 3A provides a set of exemplary dosing intervals, loading
doses and maintenance doses. Table 3B provides a variation of Table
3A allowing for dosage variability (indicated as "about") of up to
+/-3 mg/mL. Table 3C appears below and provides a more extensive
set of exemplary dosing intervals, loading doses and maintenance
doses. In each cell of Table 3A, Table 3B, and Table 3C, the top
figure is the integer x in the interval Q.times.D (e.g., 18 as the
top figure in a cell indicates a dosing interval of Q18D or every
18 days), the middle figure represents the (optional) loading dose
(LD) in mg/kg, and the bottom figure represents the maintenance
dose (MD) in mg/kg. Thus the top cell in Table 3A indicates a
dosing interval (Q.times.D) of once every seven days, a loading
dose (optional) of 25 mg per kg of patient body weight, and a
maintenance dose of 20 mg per kg of patient body weight; while the
cell furthest to the right on the top row of Table 3C indicates a
dosing interval (Q.times.D) of once every seven days, a loading
dose (optional) of 30 mg per kg of patient body weight, and a
maintenance dose of 15 mg per kg of patient body weight.
TABLE-US-00004 TABLE 3A 7 25 20 7 40 30 14 60 45 14 90 75 21 120
105
TABLE-US-00005 TABLE 3B 7 about 25 about 20 7 about 40 about 30 14
about 60 about 44 14 about 90 about 75 21 about 120 about 105
TABLE-US-00006 TABLE 3C 7 7 7 7 7 7 7 7 7 7 7 7 7 10 15 20 25 30 15
20 25 30 35 20 25 30 5 5 5 5 5 10 10 10 10 10 15 15 15 7 7 7 7 7 7
7 7 7 7 7 7 7 35 40 25 30 35 40 45 30 35 40 45 50 55 15 15 20 20 20
20 20 25 25 25 25 25 25 7 7 14 14 14 14 14 14 14 14 14 14 14 60 65
35 40 45 50 55 60 65 70 75 40 45 25 25 30 30 30 30 30 30 30 30 30
35 35 14 14 14 14 14 14 14 14 14 14 14 14 14 50 55 60 65 70 75 45
50 55 60 65 70 75 35 35 35 35 35 35 40 40 40 40 40 40 40 14 14 14
14 14 14 14 14 14 14 14 14 14 50 55 60 65 70 75 55 60 65 70 75 60
65 45 45 45 45 45 45 50 50 50 50 50 55 55 14 14 14 14 14 14 14 14
21 21 21 21 21 70 75 65 70 75 70 75 75 60 65 70 65 70 55 55 60 60
60 65 65 70 55 55 55 60 60 21 21 21 21 21 21 75 70 75 80 85 90 60
65 70 75 80 85
Example 16
Dosage and Administration of MM-111 with Lapatinib and
Trastuzumab
[0093] Treatment for patients with trastuzumab-refractory
HER2-overexpressing breast cancer is a critical unmet need in the
field of breast oncology, and novel approaches to address this need
are required. Although selective tyrosine kinase inhibitors (TKIs)
have been highly effective for the treatment of certain tyrosine
kinase oncogene-driven cancers, their clinical anti-tumor efficacy
in the treatment of HER2-driven breast cancer has been
disappointing despite adequate biodistribution and apparent target
inhibition. Two completed phase II trials using the most potent
HER2 TKI, lapatinib, have reported response rates of only 4%-8% in
patients with trastuzumab-refractory HER2-overexpressing breast
cancer. It is now known that the effective treatment of HER2+
breast cancer is more complex and resilient than previously
thought. Recent evidence has highlighted the role of HER3 and a
robust signal buffering capacity inherent in the HER2-HER3 tumor
driver that protects it against a two log inhibition of HER2
catalytic activity, placing it beyond the therapeutic index of even
the most potent tyrosine kinase inhibitors (TKIs).
[0094] Typically, lapatinib is administered at a dosage of 1000 to
1500 mg in 250 mg tablets taken once daily. Lapatinib is often used
in combination with another cancer medication, capecitabine, which
is taken for 14 day periods with one week in between.
[0095] In order to test whether the full inactivation of the
HER2-HER3 driver can be achieved with much higher TKI dosing at an
intermittent dosing schedule is more efficacious than continuous
dosing, a modified dosing schedule is used wherein an increased
dose of lapatinib is administered on days 1-5 of a 14 day cycle,
said increased dose being a higher dose than the standard dose of
1000 to 1500 mg/day. In some embodiments, the higher lapatinib dose
is between 2000 and 9000 mg/d. For example, higher lapatinib dose
might be 2000, 2250, 3375, 3000, 3250, 3500, 3750, 4000, 4250,
4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000,
7250, 7500, 7750, 8000, 8250, 8500, 8750, or 9000 mg/day, and so
on.
[0096] In certain embodiments a loading dose is given on day 1 of
the 14-day cycle that is a higher dose than that given on
subsequent days, the maintenance dose. For example, a loading dose
given on day 1 of the 14 day cycle might be 7000 mg/day, followed
by a maintenance dose of 3000 mg/day. Non-limiting examples of
loading dose and maintenance dose combinations are listed in Table
4 below.
[0097] MM-111 is administered as described in Example 15. In some
embodiments the treatment further comprises trastuzumab.
Trastuzumab is typically given with an initial loading dose
followed by a maintenance dose. For example, trastuzumab may be
dosed at a loading dose of 8 mg/kg followed by a maintenance dose
of 6 mg/kg every three weeks.
TABLE-US-00007 TABLE 4 Exemplary lapatinib dosing schedule: loading
dose (top number) and maintenance dose (bottom number) in mg/d 2000
2000 2000 2500 2500 2500 3000 3000 3000 3000 3000 3500 3500 1000
1500 2000 1000 1500 2000 1000 1500 2000 2500 3000 1000 1500 3500
3500 3500 4000 4000 4000 4000 4000 4000 4500 4500 4500 4500 2000
2500 3000 1000 1500 2000 2500 3000 3500 1000 1500 2000 2500 4500
4500 4500 5000 5000 5000 5000 5000 5000 5000 5000 5500 5500 3000
3500 4000 1000 1500 2000 2500 3000 3500 4000 4500 1000 1500 5500
5500 5500 5500 5500 5500 5500 6000 6000 6000 6000 6000 6000 2000
2500 3000 3500 4000 4500 5000 1000 1500 2000 2500 3000 3500 6000
6000 6000 6000 7500 7500 7500 7500 7500 7500 7500 7500 7500 4000
4500 5000 5500 1000 1500 2000 2500 3000 3500 4000 4500 5000 7500
7500 7500 7500 8000 8000 8000 8000 8000 8000 8000 8000 8000 5500
6000 6500 7000 1000 1500 2000 2500 3000 3500 4000 4500 5000 8000
8000 8000 8000 8000 9000 9000 9000 9000 9000 9000 9000 9000 5500
6000 6500 7000 7500 1000 1500 2000 2500 3000 3500 4000 4500 9000
9000 9000 9000 9000 9000 9000 9000 5000 5500 6000 6500 7000 7500
8000 8500
Example 17
Dosage and Administration of MM-111with Cisplatin, Capecitabine,
and Trastuzumab
[0098] Administration of MM-111 with cisplatin, capecitabine, and
trastuzumab is done, for example, by the following method or minor
variations thereof.
[0099] Patients are administered therapy on a 21-day treatment
cycle. Cisplatin is administered on day 1 of each 21-day cycle by
intravenous (i.v.) infusion over two hours, at a dose of 80
mg/m.sup.2. Capecitabine is administered orally, twice daily, at a
dose of 1000 mg/m.sup.2. Up to 21-day cycles of cisplatin and
capecitabine are administered. Trastuzumab is administered i.v. at
week 1 at an 8 mg/kg loading dose over 90 minutes, followed by a
maintenance dose of 6 mg/kg every 21 days over 30-90 minutes.
MM-111 is administered as described in the above Examples. For
example, MM-111 is administered i.v. over 90 minutes for the first
dose and then weekly over 60 minutes thereafter.
Example 18
Dosage and Administration of MM-111 with Lapatinib and
Trastuzumb
[0100] Administration of MM-111 with lapatinib and trastuzumab is
done, for example, by the following method or minor variations
thereof. Trastuzumab is administered i.v. at a 4 mg/kg loading dose
on week 1 over 90 minutes, followed by a 2 mg/kg weekly maintenance
dose thereafter. Lapatinib is given by mouth either at 1000 mg
daily doses or at the one of the dose regimens described in Example
13. MM-111 is administered as described in the above Examples. For
example, MM-111 is administered i.v. over 90 minutes for the first
dose and then weekly over 60 minutes thereafter.
Example 19
Dosage and Administration of MM-111 with Paclitaxel and
Trastuzumab
[0101] Administration of MM-111 with paclitaxel and trastuzumab is
done, for example, by the following method or minor variations
thereof. Patients are administered therapy on a 28-day treatment
cycle. Paclitaxel dosing begins on day 1 of cycle 1. Paclitaxel is
administered at 80 mg/m.sup.2 weekly, as an i.v. infusion over 60
minutes. Trastuzumab is administered at a 4 mg/kg loading dose on
week 1, i.v. over 90 minutes, followed by a 2 mg/kg weekly
maintenance dose thereafter. MM-111 is administered as described in
the above Examples. For example, MM-111 is administered i.v. over
90 minutes for the first dose and then weekly over 60 minutes
thereafter.
Example 20
Co-Administration of MM-111 and a MEK/PI3k/AKT Inhibitor
[0102] MM-111, at dosages described herein (see, e.g., Example 15),
can be administered in combination with one or more MAP/ERK kinase
(MEK)/phosphatidylinositol 3-kinase (PI3k)/AKT inhibitors to a
patient in need thereof for the treatment of a cancer. MM-111 can
be administered in the same dosage form as the MEK/PI3k/AKT
inhibitor(s) or these agents can be administered in separate dosage
forms.
[0103] In preferred embodiments, the MEK/PI3k/AKT inhibitor(s) is
selected from AZD6244, BKM-120, GDC-0941, GSK1120212, MK-2206,
PD0325901, and Triciribine, and combinations thereof.
[0104] In another embodiment, MM-111 and a MEK/PI3k/AKT inhibitor
is administered to a patient for the treatment of a malignant
tumor, e.g., an ErbB2-expressing or ErbB2 over-expressing tumor
(e.g., HER.sup.++ or HER.sup.+++ tumors). The tumor may be a
melanoma, clear cell sarcoma, head and neck, endometrial, prostate,
breast, ovarian, gastric, colon, colorectal, lung, bladder,
pancreatic, salivary gland, liver, skin, brain or renal tumor.
Example 21
Coadministration of MM-111 and Other Therapeutic Agents
[0105] MM-111 (at dosages described herein; see, e.g., Example 15)
can be administered in combination with one or more additional
agents to a patient in need thereof for the treatment of a cancer.
In particular, MM-111 can be administered in combination with
MM-151 (oligoclonal anti-EGFR mixture), TDM-1 (Trastuzumab
emtansine; an antibody-drug conjugate of the antibody trastuzumab
linked to maytansine derivative (DM1)), and an mTOR inhibitor
(e.g., one or more of the mTOR inhibitors listed in the attached
appendix), and combinations thereof.
[0106] MM-151 is an oligoclonal therapeutic that is a mixture of
three fully human monoclonal antibodies designed to bind to
non-overlapping epitopes of the epidermal growth factor receptor,
or EGFR (also known as ErbB1). An oligoclonal therapeutic is a
mixture of two or more distinct monoclonal antibodies. MM-151 is
disclosed, e.g., in copending PCT Application No. PCT/US12/45235,
incorporated herein by reference.
[0107] MM-111 can be administered in the same dosage form as
MM-151, TDM-1, and/or the mTOR inhibitor(s), or the agents can be
administered in separate dosage forms.
[0108] In an embodiment, MM-111 and one or more of MM-151, TDM-1,
and/or the mTOR inhibitor(s) is administered to a patient for the
treatment of a malignant tumor, e.g., an ErbB2-expressing or ErbB2
over-expressing tumor (e.g., HER.sup.++ or HER.sup.+++ tumors). The
tumor may be a melanoma, clear cell sarcoma, head and neck,
endometrial, prostate, breast, ovarian, gastric, colon, colorectal,
lung, bladder, pancreatic, salivary gland, liver, skin, brain or
renal tumor.
[0109] In another embodiment, MM-111 and MM-151 are co-administered
to treat a solid tumor (e.g., an advanced refractory solid tumor)
in a patient in need thereof.
Endnotes
[0110] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth.
[0111] All patents patent applications and publications mentioned
herein are incorporated by reference to the same extent as if each
independent patent or patent application was specifically and
individually indicated to be incorporated by reference in its
entirety. In particular, WO 2012/116317 is incorporated herein by
reference in its entirety.
APPENDIX
Anticancer Agents
[0112] The Table and Appendix below describe effective an
anti-estrogen agents, receptor tyrosine kinase inhibitors; MEK/PI3
kinase/AKT inhibitors, and mTOR inhibitors that can be used in the
methods and compositions of the invention.
[0113] The bispecific anti-ErbB2/anti-ErbB3 antibody
co-administered in combination with an agent selected from i) an
effective amount of an anti-estrogen agent; ii) an effective amount
of a receptor tyrosine kinase inhibitor; iii) a MEK/PI3 kinase/AKT
inhibitor; iv) MM-151; v) an mTOR inhibitor; and/or vi) trastuzumab
or TMD1, and combinations thereof, can be further co-administered
with at least a third antineoplastic agent selected from any of
those disclosed in the Table and Appendix below.
TABLE-US-00008 TABLE 5 Exemplary antineoplastic agents for
treatment of breast cancer in combination with a bispecific
anti-ErbB2/anti-ErbB3 antibody. Exemplary Agent Therapeutic Class
(Generic/Tradename) Exemplary Dose Mitotic Inhibitors paclitaxel
(TAXOL .RTM.; 175 mg/m.sup.2 ABRAXANE .RTM.) docetaxel 60-100
mg/m.sup.2 (TAXOTERE .RTM.) Topoisomerase Inhibitors camptothecin
topotecan hydrochloride (HYCAMTIN .RTM.) etoposide (EPOSIN .RTM.)
Alkylating Agents cyclophosphamide 600 mg/m.sup.2 (CYTOXAN .RTM.)
Platinum-Based Agents Cisplatin 20-100 mg/m.sup.2 carboplatin 300
mg/m.sup.2 (PARAPLATIN .RTM.) nedaplatin (AQUPLA .RTM.) oxaliplatin
65-85 mg/m.sup.2 (ELOXATIN .RTM.) satraplatin (SPERA .RTM.)
triplatin tetranitrate Selective Estrogen tamoxifen 20-40 mg/day
Modulators (SERM) (NOLVADEX .RTM.) raloxifene (EVISTA .RTM.) 60
mg/day toremifene (FARESTON .RTM.) Antimetabolites methotrexate 40
mg/m.sup.2 Fluorouracil (5-FU) 500 mg/m.sup.2 Raltitrexed Antitumor
Antibiotics Doxorubicin 40-75 mg/m.sup.2 (ADRIAMYCIN .RTM.)
epirubicin 60-120 mg/m.sup.2 (ELLENCE .RTM.) Aromatase Inhibitors
aminoglutethimide 250-2000 mg/day (CYTADREN .RTM.) anastrozole 1
mg/day (ARIMIDEX .RTM.) letrozole (FEMARA .RTM.) 2.5 mg/day
Vorozole exemestane 25-50 mg/day (AROMASIN .RTM.) Testolactone
fadrozole (AFEMA .RTM.) Anti-VEGF Agents bevacizumab 10 mg/kg
(AVASTIN .RTM.) Anti-ErbB2 (HER2/neu) trastuzumab 2-8 mg/kg Agents
(HERCEPTIN .RTM.) Pertuzumab (OMNITARG .RTM.) Anti-ErbB3 (HER3)
U3-1287 (AMG 888) Agents
TABLE-US-00009 APPENDIX ANTICANCER AGENTS Other anticancer agents
for combination with a bispecific anti-ErbB2/anti-ErbB3 antibody
Brand Name(s) Manufacturer/Proprietor Anti-IGF1R Antibodies AMG 479
(fully humanized mAb) Amgen IMCA12 (fully humanized mAb) ImClone
NSC-742460 Dyax 19D12 (fully humanized mAb) CP751-871 (fully
humanized mAb) Pfizer H7C10 (humanized mAb) alphaIR3 (mouse)
scFV/FC (mouse/human chimera) EM/164 (mouse) MK-0646, F50035 Pierre
Fabre Medicament, Merck Small Molecules Targeting IGF1R NVP-AEW541
Novartis BMS-536,924 (1H-benzoimidazol-2-yl)- Bristol-Myers Squibb
1H-pyridin-2-one) BMS-554,417 Bristol-Myers Squibb Cycloligan
TAE226 PQ401 Anti-EGFR Antibodies INCB7839 Incyte Bevacizumab
Avastin .RTM. Genentech Cetuximab Erbitux .RTM. IMCLONE mAb 806
Matuzumab (EMD72000) Nimotuzumab (TheraCIM) Panitumumab Vectibix
.RTM. Amgen MM-151 Merrimack Sym004 Symphogen Zalutumumab Humax
Anti-ErbB3 Therapeutics U3-1287/AMG888 U3 Pharma/Amgen MM-121
Merrimack Pharmaceuticals Anti-ErbB2 Therapeutics trastuzumab
Herceptin .RTM. Genentech HKI-272 - neratinib Wyeth KOS-953 -
tanespimycin Kosan Biosciences Her/ErbB Dimerization Inhibitors
2C4, R1273 - Pertuzumab Pertuzumab, Omnitarg .RTM. Genentech, Roche
Small Molecules Targeting EGFR CI-1033 (PD 183805) Pfizer, Inc.
EKB-569 Gefitinib IRESSA .TM. AstraZeneca Lapatinib (GW572016)
GlaxoSmithKline Lapatinib Ditosylate Tykerb .RTM. SmithKline
Beecham Erlotinib HCl (OSI-774) Tarceva .RTM. OSI Pharms PD158780
PKI-166 Novartis Tyrphostin AG 1478 (4-(3-Chloroanillino)-
6,7-dimethoxyquinazoline) Afatinib (BIBW 2992) Boehringer Ingelheim
Small Molecules Targeting MEK CI-1040 (PD184352) AZD6244
(Selumetinib) RDEA119 (BAY 869766) GSK1120212 Glaxo Smith Kline
PD-0325901 GDC-0973 Genentech Anti-cMet Antibody Therapies AVEO
(AV299) AVEO AMG102 Amgen 5D5 (OA-5D5) Genentech Small Molecules
Targeting cMet PHA665752 ARQ-650RP ArQule ARQ 197 ArQule Alkylating
Agents BCNU.fwdarw. 1,3-bis t2-chloroethyl)- nitrosourea
Bendamustine Busulfan Myleran GlaxoSmithKline Carboplatin
Paraplatin Bristol-Myers Squibb Carboquone Carmustine CCNU.fwdarw.
1,-(2-chloroethyl)-3-cyclohexyl- 1-nitrosourea (methyl CCNU)
Chlorambucil Leukeran .RTM. Smithkline Beecham Chlormethine
Cisplatin (Cisplatinum, CDDP) Platinol Bristol-Myers
Cyclophosphamide Cytoxan Bristol-Myers Squibb Neosar Teva
Parenteral Dacarbazine (DTIC) Fotemustine Hexamethylmelamine
(Altretamine, HMM) Hexalen .RTM. MGI Pharma, Inc. Ifosfamide
Mitoxana .RTM. ASTA Medica Lomustine Mannosulfan Melphalan Alkeran
.RTM. GlaxoSmithKline Nedaplatin Nimustine Oxaliplatin Eloxatin
.RTM. Sanofi-Aventis US Prednimustine, Procarbazine HCL Matulane
Sigma-Tau Pharmaceuticals, Inc. Ribonucleotide Reductase Inhibitor
(RNR) Ranimustine Satraplatin Semustine Streptozocin Temozolomide
Treosulfan Triaziquone Triethylene Melamine ThioTEPA Bedford,
Abraxis, Teva Triplatin tetranitrate Trofosfamide Uramustine
Antimetabolites 5-azacytidine Flourouracil (5-FU)/Capecitabine
6-mercaptopurine (Mercaptopurine, 6-MP) 6-Thioguanine (6-TG)
Purinethol .RTM. Teva Cytosine Arabinoside (Cytarabine, Thioguanine
.RTM. GlaxoSmithKline Ara-C) Azathioprine Azasan .RTM. AAIPHARMA
LLC Capecitabine XELODA .RTM. HLR (Roche) Cladribine (2-CdA, 2-
Leustatin .RTM. Ortho Biotech chlorodeoxyadenosine)
5-Trifluoromethyl-2'-deoxyuridine Fludarabine phosphate Fludara
.RTM. Bayer Health Care Floxuridine (5-fluoro-2) FUDR .RTM.
Hospira, Inc. Methotrexate sodium Trexall Barr Pemetrexed Alimta
.RTM. Lilly Pentostatin Nipent .RTM. Hospira, Inc. Raltitrexed
Tomudex .RTM. AstraZeneca Tegafur Aromatose Inhibitor Ketoconazole
Glucocorticoids Dexamethasone Decadron .RTM. Dexasone, Wyeth, Inc.
Diodex, Hexadrol, Maxidex Prednisolone Prednisone Deltasone,
Orasone, Liquid Pred, Sterapred .RTM. Immunotherapeutics Alpha
interferon Angiogenesis Inhibitor Avastin .RTM. Genentech
IL-12.fwdarw. Interleukin 12 IL-2.fwdarw. Interleukin 2
(Aldesleukin) Proleukin .RTM. Chiron Receptor Tyrosine Kinase
Inhibitors AMG 386 Amgen Axitinib ((AG-013736) Pfizer, Inc
Bosutinib (SKI-606) Wyeth Brivanib alalinate (BMS-582664) BMS
Cediranib (AZD2171) Recentin AstraVeneca Dasatinib (BMS-354825)
Sprycel .RTM. Bristol-Myers Squibb Imatinib mesylate Gleevec
Novartis Lestaurtinib (CEP-701) Cephalon Motesanib diphosphate
(AMG-706) Amgen/Takeda Nilotinib hydrochloride monohydrate Tasigna
.RTM. Novartis Pazopanib HCL (GW786034) Armala GSK Semaxanib
(SU5416) Pharmacia, Sorafenib tosylate Nexavar .RTM. Bayer
Sunitinib malate Sutent .RTM. Pfizer, Inc. Vandetanib (AZD647)
Zactima AstraZeneca Vatalanib; PTK-787 Novartis; Bayer Schering
Pharma XL184, NSC718781 Exelixis, GSK Microtubule-Targeting Agents
Colchicine Docetaxel Taxotere .RTM. Sanofi-Aventis US Ixabepilone
IXEMPRA .TM. Bristol-Myers Squibb Larotaxel Sanofi-aventis
Ortataxel Spectrum Pharmaceuticals Nanoparticle paclitaxel
(ABI-007) Abraxane .RTM. Abraxis BioScience, Inc. Paclitaxel Taxol
.RTM. Bristol-Myers Squibb Tesetaxel Genta Vinblastine sulfate
Velban .RTM. Lilly Vincristine Oncovin .RTM. Lilly Vindesine
sulphate Eldisine .RTM. Lilly Vinflunine Pierre Fabre Vinorelbine
tartrate Navelbine .RTM. Pierre Fabre mTOR Inhibitors Deforolimus
(AP23573, MK 8669) ARIAD Pharmaceuticals, Inc Everolimus (RAD001,
RAD001C) Certican .RTM., Afinitor Novartis Sirolimus (Rapamycin)
Rapamune .RTM. Wyeth Pharama Temsirolimus (CCI-779) Torisel .RTM.
Wyeth Pharama Protein Synthesis Inhibitor L-asparaginase Elspar
.RTM. Merck & Co. Somatostatin Analogue Octreotide acetate
Sandostatin .RTM. Novartis Topoisomerase Inhibitors Actinomycin D
Camptothecin (CPT) Belotecan Daunorubicin citrate Daunoxome .RTM.
Gilead Doxorubicin hydrochloride Doxil .RTM. Alza Vepesid .RTM.
Bristol-Myers Squibb Etoposide Etopophos Hospira, Bedford, Teva
Parenteral, Etc. Irinotecan HCL (CPT-11) Camptosar .RTM. Pharmacia
& Upjohn Mitoxantrone HCL Novantrone EMD Serono Rubitecan
Teniposide (VM-26) Vumon .RTM. Bristol-Myers Squibb Topotecan HCL
Hycamtin .RTM. GlaxoSmithKline Chemotherapeutic Agents Adriamycin,
5-Fluorouracil, Cytoxin, Bleomycin, Mitomycin C, Daunomycin,
Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins
Clofarabine, Mercaptopurine, Pentostatin, Thioguanine, Cytarabine,
Decitabine, Floxuridine, Gemcitabine (Gemzar), Enocitabine,
Sapacitabine Hormonal Therapies Abarelix Plenaxis .TM. Amgen
Abiraterone acetate CB7630 BTG plc Afimoxifene TamoGel Ascend
Therapeutics, Inc. Anastrazole Arimidex .RTM. AstraZeneca Aromatase
inhibitor Atamestane plus toremifene Intarcia Therapeutics, Inc.
Arzoxifene Eli Lilly & Co. Asentar; DN-101 Novartis; Oregon
Health & Science Univ. Bicalutamide Casodex .RTM. AstraZeneca
Buserelin Suprefact .RTM. Sanofi Aventis Cetrorelix Cetrotide .RTM.
EMD Serono Exemestane Aromasin .RTM. Pfizer Exemestane Xtane Natco
Pharma, Ltd. Fadrozole (CGS 16949A) Flutamide Eulexin .RTM.
Schering Flutamide Prostacur Laboratorios Almirall, S.A.
Fulvestrant Faslodex .RTM. AstraZeneca Goserelin acetate Zoladex
.RTM. AstraZeneca Letrozole Femara .RTM. Novartis Letrozole
(CGS20267) Femara Chugai Pharmaceutical Co., Ltd. Letrozole
Estrochek Jagsonpal Pharmaceuticals, Ltd. Letrozole Letrozole
Indchemie Health Specialities Leuprolide acetate Eligard .RTM.
Sanofi Aventis Leuprolide acetate Leopril VHB Life Sciences, Inc.
Leuprolide acetate Lupron .RTM./Lupron Depot TAP Pharma Leuprolide
acetate Viador Bayer AG Megestrol acetate Megace .RTM.
Bristol-Myers Squibb Magestrol acetate Estradiol Valerate Jagsonpal
Pharmaceuticals, Ltd. (Delestrogen) Medroxyprogesterone acetate
Veraplex Combiphar MT206 Medisyn Technologies, Inc. Nafarelin
Nandrolone decanoate Zestabolin Mankind Pharma, Ltd. Nilutamide
Nilandron .RTM. Aventis Pharmaceuticals Raloxifene HCL Evista .RTM.
Lilly Tamoxifen Taxifen Yung Shin Pharmaceutical Tamoxifen Tomifen
Alkem Laboratories, Ltd. Tamoxifen citrate Nolvadex AstraZeneca
Tamoxifen citrate Soltamox EUSA Pharma, Inc. Tamoxifen citrate
Tamoxifen citrate Sopharma JSCo. SOPHARMA Toremifene citrate
Fareston .RTM. GTX, Inc. Triptorelin pamoate Trelstar .RTM. Watson
Labs Triptorelin pamoate Trelstar Depot Paladin Labs, Inc. Protein
Kinase B (PKB) Inhibitors Akt Inhibitor ASTEX Astex Therapeutics
Akt Inhibitors NERVIANO Nerviano Medical Sciences AKT Kinase
Inhibitor TELIK Telik, Inc. AKT Inhibitor Triciribine AKT DECIPHERA
Deciphera Pharmaceuticals, LLC Perifosine (KRX0401, D-21266) Keryx
Biopharmaceuticals, Inc., AEterna Zentaris, Inc. Perifosine with
Docetaxel Keryx Biopharmaceuticals, Inc., AEterna Zentaris, Inc.
Perifosine with Gemcitabine AEterna Zentaris, Inc. Perifosine with
Paclitaxel Keryx Biopharmaceuticals, Inc, AEterna Zentaris, Inc.
Protein Kinase-B inhibitor DEVELOGEN DeveloGen AG PX316
Oncothyreon, Inc. RX0183 Rexahn Pharmaceuticals, Inc. RX0201 Rexahn
Pharmaceuticals, Inc. VQD002 VioQuest Pharmaceuticals, Inc. XL418
Exelixis, Inc. ZEN027 AEterna Zentaris, Inc. Phosphatidylinositol
3-Kinase (PI3K) Inhibitors BEZ235 Novartis AG BGT226 Novartis AG
CAL101 Calistoga Pharmaceuticals, Inc. CHR4432 Chroma Therapeutics,
Ltd. Erk/PI3K Inhibitors ETERNA AEterna Zentaris, Inc. GDC0941
Genentech Inc./Piramed Limited/Roche Holdings, Ltd. Enzastaurin HCL
(LY317615) Enzastaurin Eli Lilly LY294002/Wortmannin Eli Lilly PI3K
Inhibitors SEMAFORE Semafore Pharmaceuticals PX866 Oncothyreon,
Inc. SF1126 Semafore Pharmaceuticals VMD-8000 VM Discovery, Inc.
XL147 Exelixis, Inc. XL147 with XL647 Exelixis, Inc. XL765
Exelixis, Inc. PI-103 Roche/Piramed BKM120 Cyclin-dependent kinase
inhibitors CYC200, r-roscovitine Seliciclib Cyclacel Pharma
NSC-649890, L86-8275, HMR-1275 Alvocidib NCI TLr9, CD289 IMOxine
Merck KGaA HYB2055 Idera IMO-2055 Isis Pharma 1018 ISS Dynavax
Technologies/UCSF PF-3512676 Pfizer Enzyme Inhibitor Lonafarnib
(SCH66336) Sarasar SuperGen, U Arizona Anti-TRAIL AMG-655 Aeterna
Zentaris, Keryx Biopharma Apo2L/TRAIL, AMG951 Genentech, Amgen
Apomab (fully humanized mAb Genentech Other Imprime PGG Biothera
CHR-2797 AminopeptidaseM1 Chroma Therapeutics E7820, NSC 719239
Integrin-alpha2 Eisai INCB007839 ADAM 17, TACE Incyte CNF2024,
BIIB021 Hsp90 Biogen IdeC MP470, HPK-56 Kit/Met/Ret Shering-Plough
SNDX-275/MS-275 HDAC Syndax Zarnestra, Tipifarnib, R115777 Ras
Janssen Pharma Volociximab; Eos 200-4, M200 alpha581 integrin
Biogen Idec; Eli Lilly/UCSF/PDL BioPharma Apricoxib (TP2001) COX-2
Inhibitor Daiichi Sankyo; Tragara Pharma
Sequence CWU 1
1
111095PRTArtificial SequenceSynthetic Construct 1Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Asn Ile Asn Arg Asp Gly Ser Ala Ser Tyr Tyr Val Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn
Ser 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 Asp Arg Gly Val Gly Tyr Phe
Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala
Ser Thr Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly
Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 130 135 140 Ser Val Ser
Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 145 150 155 160
Thr Ser Ser Asp Val Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 165
170 175 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asp
Arg 180 185 190 Pro Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser
Gly Asn Thr 195 200 205 Ala Ser Leu Ile Ile Ser Gly Leu Gln Ala Asp
Asp Glu Ala Asp Tyr 210 215 220 Tyr Cys Ser Ser Tyr Gly Ser Ser Ser
Thr His Val Ile Phe Gly Gly 225 230 235 240 Gly Thr Lys Val Thr Val
Leu Gly Ala Ala Ser Asp Ala His Lys Ser 245 250 255 Glu Val Ala His
Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 260 265 270 Leu Val
Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu 275 280 285
Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 290
295 300 Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr
Leu 305 310 315 320 Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg
Glu Thr Tyr Gly 325 330 335 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro Glu Arg Asn Glu Cys 340 345 350 Phe Leu Gln His Lys Asp Asp Asn
Pro Asn Leu Pro Arg Leu Val Arg 355 360 365 Pro Glu Val Asp Val Met
Cys Thr Ala Phe His Asp Asn Glu Glu Thr 370 375 380 Phe Leu Lys Lys
Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 385 390 395 400 Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 405 410
415 Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys
420 425 430 Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys
Gln Arg 435 440 445 Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg
Ala Phe Lys Ala 450 455 460 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe
Pro Lys Ala Glu Phe Ala 465 470 475 480 Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys Val His Thr Glu Cys 485 490 495 Cys His Gly Asp Leu
Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala 500 505 510 Lys Tyr Ile
Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 515 520 525 Cys
Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 530 535
540 Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe
545 550 555 560 Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala
Lys Asp Val 565 570 575 Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
Arg His Pro Asp Tyr 580 585 590 Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr Tyr Glu Thr Thr Leu 595 600 605 Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu Cys Tyr Ala Lys Val 610 615 620 Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 625 630 635 640 Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 645 650 655
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 660
665 670 Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys
Cys 675 680 685 Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu
Asp Tyr Leu 690 695 700 Ser Val Val Leu Asn Gln Leu Cys Val Leu His
Glu Lys Thr Pro Val 705 710 715 720 Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser Leu Val Asn Arg Arg 725 730 735 Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 740 745 750 Phe Gln Ala Glu
Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 755 760 765 Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 770 775 780
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp 785
790 795 800 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp
Lys Glu 805 810 815 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala
Ala Ser Gln Ala 820 825 830 Ala Leu Gly Leu Ala Ala Ala Leu Gln Val
Gln Leu Val Gln Ser Gly 835 840 845 Ala Glu Val Lys Lys Pro Gly Glu
Ser Leu Lys Ile Ser Cys Lys Gly 850 855 860 Ser Gly Tyr Ser Phe Thr
Ser Tyr Trp Ile Ala Trp Val Arg Gln Met 865 870 875 880 Pro Gly Lys
Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser 885 890 895 Asp
Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val 900 905
910 Asp Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro
915 920 925 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly
Tyr Cys 930 935 940 Thr Asp Arg Thr Cys Ala Lys Trp Pro Glu Trp Leu
Gly Val Trp Gly 945 950 955 960 Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Ser Gly 965 970 975 Gly Gly Ser Gly Gly Gly Gly
Ser Gln Ser Val Leu Thr Gln Pro Pro 980 985 990 Ser Val Ser Ala Ala
Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly 995 1000 1005 Ser Ser
Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln 1010 1015 1020
Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn 1025
1030 1035 Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser
Gly 1040 1045 1050 Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg Ser
Glu Asp Glu 1055 1060 1065 Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Tyr
Thr Leu Ser Gly Trp 1070 1075 1080 Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 1085 1090 1095
* * * * *