U.S. patent application number 14/424344 was filed with the patent office on 2015-07-23 for combination therapies comprising anti-erbb3 agents.
This patent application is currently assigned to Merrimack Pharmaceuticals, Inc.. The applicant listed for this patent is Merrimack Pharmaceuticals, Inc.. Invention is credited to Alexandra Huhalov, Charlotte McDonagh, Bo Zhang.
Application Number | 20150202287 14/424344 |
Document ID | / |
Family ID | 50184467 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202287 |
Kind Code |
A1 |
Zhang; Bo ; et al. |
July 23, 2015 |
COMBINATION THERAPIES COMPRISING ANTI-ERBB3 AGENTS
Abstract
Disclosed are methods and compositions for inhibiting the growth
of a tumor (e.g., a malignant 5 tumor) in a subject. In particular,
combination therapies for treating a tumor in a subject by
coadministering 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 T-DM 1, and/or combinations thereof; and an
effective amount of a 10 bispecific anti-ErbB2/anti-ErbB3
antibody.
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 |
|
|
Assignee: |
Merrimack Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
50184467 |
Appl. No.: |
14/424344 |
Filed: |
August 30, 2013 |
PCT Filed: |
August 30, 2013 |
PCT NO: |
PCT/US2013/057714 |
371 Date: |
February 26, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61695242 |
Aug 30, 2012 |
|
|
|
Current U.S.
Class: |
424/136.1 ;
435/375 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/138 20130101; A61K 2039/505 20130101; C07K 16/468 20130101;
A61K 31/517 20130101; A61K 2039/507 20130101; A61K 31/565 20130101;
A61K 39/39558 20130101; C07K 2317/76 20130101; A61K 31/436
20130101; A61K 39/39558 20130101; A61K 39/3955 20130101; A61K
31/4196 20130101; C07K 16/2863 20130101; A61K 33/24 20130101; C07K
16/40 20130101; C07K 2317/31 20130101; A61K 2300/00 20130101; A61K
31/519 20130101; C07K 2317/24 20130101; A61K 31/567 20130101; A61K
31/7068 20130101; C07K 16/32 20130101; A61K 31/337 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/567 20060101 A61K031/567; A61K 31/138 20060101
A61K031/138; A61K 31/4196 20060101 A61K031/4196; A61K 31/517
20060101 A61K031/517; A61K 31/436 20060101 A61K031/436; A61K 33/24
20060101 A61K033/24; A61K 31/519 20060101 A61K031/519; C07K 16/28
20060101 C07K016/28; A61K 31/337 20060101 A61K031/337; A61K 45/06
20060101 A61K045/06; C07K 16/40 20060101 C07K016/40; A61K 31/7068
20060101 A61K031/7068 |
Claims
1. A method of treating a subject having a malignant tumor, the
method comprising administering to the subject an effective amount
a combination therapy comprising and effective amount of each of
one or more agents selected from i) an anti-estrogen agent, ii) a
receptor tyrosine kinase inhibitor, iii) a MEK inhibitor, iv) a PI3
kinase inhibitor, v) an AKT inhibitor, vi) an mTOR inhibitor, vii)
trastuzumab, viii) ado-trastuzumab emtansine, ix) capecitabine, x)
cisplatin, and xi) nab-paclitaxel; and an effective amount of an
agent that is a bispecific anti-ErbB2/anti-ErbB3 antibody that
comprises the amino acid sequence set forth in SEQ ID NO:1.
2. The method of claim 1, wherein the anti-estrogen agent is an
estrogen receptor antagonist or an aromatase inhibitor.
3. The method of claim 2, wherein the estrogen receptor antagonist
is fulvestrant or tamoxifen.
4. The method of claim 2, wherein the aromatase inhibitor is
letrozole, exemestane, anastrozole, aminoglutethimide,
testolactone, vorozole, formestane, or fadrozole.
5. The method of claim 4, wherein the aromatase inhibitor is
letrozole.
6. The method of claim 1, wherein the administration to the subject
of the combination therapy does not create a drug-drug
interaction-mediated toxicity in the subject.
7. The method of claim 1, wherein the administration to the subject
of the combination therapy creates a substantially additive or
superadditive effect as compared to the separate administration of
each of the combination therapy agents alone.
8. The method of any one of claims 1 to 7, wherein the receptor
tyrosine kinase inhibitor is erlotinib, afatinib, dasatinib,
gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, or
sorafenib.
9. The method of claim 8, wherein the receptor tyrosine kinase
inhibitor is lapatinib.
10. The method of any one of claims 1 to 9, further comprising
co-administration to the patient of an effective amount of either
or both of capecitabine and cisplatin.
11. Use of a bispecific anti-ErbB2/anti-ErbB3 antibody for
combination therapy together with one or more agents selected from
i) an anti-estrogen agent, ii) a receptor tyrosine kinase
inhibitor, iii) a MEK inhibitor, iv) a PI3 kinase inhibitor, v) an
AKT inhibitor, vi) an mTOR inhibitor, vii) trastuzumab, viii)
ado-trastuzumab emtansine, ix) capecitabine, and x) cisplatini) an
anti-estrogen agent, ii) a receptor tyrosine kinase inhibitor, iii)
a MEK inhibitor, iv) a PI3 kinase inhibitor, v) an AKT inhibitor,
vi) an mTOR inhibitor, vii) trastuzumab, viii) ado-trastuzumab
emtansine, ix) capecitabine, and x) cisplatin; for treatment of a
malignant tumor.
12. The use of claim 11, wherein the anti-estrogen agent is an
estrogen receptor antagonist or an aromatase inhibitor.
13. The use of claim 12, wherein the estrogen receptor antagonist
is fulvestrant or tamoxifen.
14. The use of claim 12, wherein the aromatase inhibitor chosen
from the group consisting of letrozole, exemestane, anastrozole,
aminoglutethimide, testolactone, vorozole, formestane, and
fadrozole.
15. The use of claim 14, wherein the aromatase inhibitor is
letrozole.
16. The use of any one of claims 11 to 15, wherein the receptor
tyrosine kinase inhibitor is chosen from the group consisting of
erlotinib, afatinib, dasatinib, gefitinib, imatinib, pazopinib,
lapatinib, sunitinib, nilotinib and sorafenib.
17. The use of claim 16, wherein the receptor tyrosine kinase
inhibitor is lapatinib.
18. An aqueous solution comprising a bispecific
anti-ErbB2/anti-ErbB3 antibody and one or more of i) an
anti-estrogen agent, ii) a receptor tyrosine kinase inhibitor, iii)
a MEK inhibitor, iv) a PI3 kinase inhibitor, v) an AKT inhibitor,
vi) an mTOR inhibitor, vii) trastuzumab, viii) ado-trastuzumab
emtansine, ix) capecitabine, x) cisplatin, and xi)
nab-paclitaxel.
19. A method of inhibiting the growth of a malignant tumor
comprising tumor cells, said method comprising contacting the tumor
cells with the aqueous solution of claim 18.
20. The method of claim 1, wherein said MEK inhibitor is
selumetinib, trametinib, UO126, or PD0325901, said PI3K inhibitor
is BKM-120 or GDC-0941, and said AKT inhibitor is triciribine or
MK-2206.
21. The use of claim 11, wherein said MEK inhibitor is selumetinib,
trametinib, UO126, or PD0325901, said PI3K inhibitor is BKM-120 or
GDC-0941, and said AKT inhibitor is triciribine or MK-2206.
22. The aqueous solution of claim 18, wherein said MEK inhibitor is
selumetinib, trametinib, UO126, or PD0325901, said PI3K inhibitor
is BKM-120 or GDC-0941, and said AKT inhibitor is triciribine or
MK-2206.
23. The method of claim 1, comprising co-administering the MEK
inhibitor and the bispecific anti-ErbB2/anti-ErbB3 antibody.
24. The method of claim 1, comprising co-administering the PI3K
inhibitor and the bispecific anti-ErbB2/anti-ErbB3 antibody.
25. The method of claim 1, comprising co-administering the mTOR
inhibitor and the bispecific anti-ErbB2/anti-ErbB3 antibody.
26. The method of claim 1, comprising co-administering trastuzumab
or ado-trastuzumab emtansine and the bispecific
anti-ErbB2/anti-ErbB3 antibody.
27. The method of claim 1, wherein said mTOR inhibitor is BEZ235,
AZD8055, everolimus, temsirolimus, sirolimus, or ridaforolimus.
28. The use of claim 11, wherein said mTOR inhibitor is BEZ235,
AZD8055, everolimus, temsirolimus, sirolimus, or ridaforolimus.
29. The aqueous solution of claim 18, wherein said mTOR inhibitor
is BEZ235, AZD8055, everolimus, temsirolimus, sirolimus, or
ridaforolimus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 61/695,242, filed Aug. 30, 2012, which is hereby
incorporated by reference in its entirety.
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, the anti-ErbB2
monoclonal antibody drug conjugate T-DM1 (ado-trastuzumab
emtansine) 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] ErbB2 can function as a homodimer or as a heterodimer with
related epidermal growth factor-type receptors such as ErbB3. 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 intracellular
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.
Such signaling is believed to drive proliferation of cancer cells.
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 ErB1 and ErbB2 receptor signaling
activity, as well as inhibition of the downstream intracellular
signaling pathways such as the mitogen activated protein kinase and
phosphoinositide-3 kinase pathways.
[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., HER2.sup.+++ or HER2.sup.++
tumors) and the tumor may be a melanoma, clear cell sarcoma, head
and neck, endometrial, prostate, breast, ovarian, esophageal,
gastric, gastro-esophageal, 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 anti-estrogen agent, ii) a
receptor tyrosine kinase inhibitor, iii) a MEK inhibitor, (e.g.,
selumetinib, trametinib, PD0325901, UO126), iv) a PI3 kinase
inhibitor (e.g., BEZ235, BKM120, GDC0941), v) an AKT inhibitor
(e.g., MK-2206, triciribine), vi) an mTOR inhibitor (e.g., BEZ235,
AZD8055, everolimus, temsirolimus, sirolimus, ridaforolimus), vii)
trastuzumab, viii) T-DM1, ix) capecitabine, x) cisplatin, and xi)
MM-151; 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., an antibody comprising the
amino acid sequence set forth in SEQ ID NO:1). Additional agents
for use in combination with the anti-ErbB3 agent, e.g., a
bispecific anti-ErbB2/anti-ErbB3 antibody, are described in the
Appendix.
[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 T-DM1, 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 T-DM1 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,
esophageal, gastro-esophageal junction, 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 anti-estrogen agent, ii) a receptor tyrosine
kinase inhibitor, iii) a MEK inhibitor, iv) a PI3 kinase inhibitor,
v) an AKT inhibitor, vi) an mTOR inhibitor, vii) trastuzumab, viii)
T-DM1, ix) capecitabine, x) cisplatin, and xi) MM-151; and
combinations thereof.
[0014] In a third embodiment, an aqueous solution is provided
comprising a bispecific anti-ErbB2/anti-ErbB3 antibody (optionally
an antibody comprising the amino acid sequence set forth in SEQ ID
NO:1) at a first concentration and an agent selected from i) an
anti-estrogen agent, ii) a receptor tyrosine kinase inhibitor, iii)
an effective amount of a MEK inhibitor, iv) a PI3 kinase inhibitor,
v) an AKT inhibitor, vi) an mTOR inhibitor, vii) trastuzumab, viii)
T-DM1, ix) capecitabine, x) cisplatin, and xi) MM-151; 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 T-DM1 at a 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)-xi) is administered in accordance with the manufacturer's
directions, e) the trastuzumab or T-DM1 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] FIG. 2 is 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] FIG. 3 is 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] FIG. 4 is 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. 5 is a graph summarizing the effect of MM-111,
trastuzumab, and tamoxifen combined compared to that of any of the
double combinations or 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.
[0025] 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.
[0026] FIG. 7 evaluates the ability of lapatinib to inhibit ErbB3
and AKT activation in heregulin-stimulated cells. 7a is a graph
comparing computer-generated dose-response curves to experimental
results in heregulin-stimulated BT474-M3 cells. 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.
[0027] FIG. 8 is a series of graphs showing MM-111 or lapatinib
inhibition of ErbB3 (8a) or AKT (8b) activation in
heregulin-stimulated cells incubated with inhibitor for 15 minutes,
1 hour, 4 hours, and 24 hours. FIG. 8c shows a comparison of IC50
for MM-111 and lapatinib at 1 hour and 24 hours for both BT474M3
cells and ZR75-30 cells.
[0028] FIG. 9 is a graph showing the effect of MM-111 and lapatinib
combination treatment on AKT activation in heregulin-stimulated
BT474-M3 cells.
[0029] 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.
[0030] 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.
[0031] FIG. 12 is three 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.
[0032] FIG. 13 is four 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.
[0033] FIG. 14 is two graphs showing that MM-111 combines
positively with the PI3K/mTOR inhibitor BEZ235 and trastuzumab in
unstimulated (FIG. 14a) and heregulin-stimulated (FIG. 14b) NCI-N87
cells in vitro. Cells were plated and either untreated (Control,
open circle), treated with BEZ235 alone (open diamond), or treated
with BEZ235 (5 nM FIG. 14a, 20 nM FIG. 14b) and either trastuzumab
(closed circle), MM-111 (closed square), or the combination of
MM-111 and trastuzumab (closed diamond). Closed circles
(trastuzumab) may appear as squares or rectangles on trastuzumab
curves when they comprise error bars. The x-axes are a log scale of
the concentrations of each of MM-111 and/or trastuzumab.
[0034] FIG. 15 is two graphs showing that MM-111 combines
positively with the mTOR inhibitor everolimus and trastuzumab in
unstimulated (FIG. 15a) and heregulin-stimulated (FIG. 15b) NCI-N87
cells in vitro. Cells were plated and either untreated (Control,
open circle), treated with everolimus alone (open diamond), or
treated with everolimus (1 nM FIG. 15a, 5 nM FIG. 15b) and either
trastuzumab (closed circle), MM-111 (closed square), or the
combination of MM-111 and trastuzumab (closed diamond). Closed
circles may appear as squares or rectangles on trastuzumab curves
when they comprise error bars. The x-axes are a log scale of the
concentrations of each of MM-111 and/or trastuzumab.
[0035] FIG. 16 is two graphs showing that MM-111 combines
positively with the MEK inhibitor GSK1120212 (trametinib) in a
mouse xenograft study. Phospho-ERK was measured as a function of
ERK activity via a Luminex.RTM. immunosandwich assay at 4 hours
after the initial treatment (FIG. 16A) and 24 hours after the
initial treatment (FIG. 16B) as a function of ERK activity via a
Luminex immunosandwich assay. The x-axes show type of treatment and
the y-axes show mean fluorescent intensity.
[0036] FIG. 17 is two graphs showing that MM-111 combines
positively with T-DM1 (ado-trastuzumab emtansine) in both the
absence (FIG. 17A) and presence (FIG. 17B) of endogenous heregulin.
The x-axes show a log scale of T-DM1 concentration and the y-axes
show viability as % of control.
[0037] FIG. 18 is a series of 3D graphs showing that MM-111
combines positively with TDM-1 (ado-trastuzumab emtansine) in the
presence of exogenous heregulin. NCI-N87 (FIGS. 18A and 18B) or
BT-474-M3 (FIGS. 18C and 18D) cells were incubated with a dose
range of both T-DM1 and heregulin, either without MM-111 (FIGS. 18A
and 18C) or with MM-111 (FIGS. 18B and 18D). The x-axes are the
concentration of T-DM1, the y-axes are cell viability as % of
untreated cells, and the z-axes are heregulin concentration. The
data presented in these graphs is also set forth below in Tables
1-4.
DETAILED DESCRIPTION
[0038] 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. Such co-administrations beneficially have
an additive or superadditive effect on suppressing tumor cell
growth, which effect on suppressing tumor cell growth is measured,
e.g., in a mouse xenograft model e.g., using BT-474 or NCI-N87
cells.
[0039] 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.
[0040] 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.
[0041] 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 ErbB1
and induce heterodimerization of ErbB1 with ErbB3.
[0042] 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. An exemplary bispecific ErbB3, ErbB2
antibody is an antibody comprising SEQ ID NO:1.
[0043] 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.
[0044] By "cancer" is meant any condition characterized by
abnormal, unregulated, malignant cell growth.
[0045] By "malignant tumor" is meant any cancer that takes the form
of a tumor.
[0046] 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.
[0047] A "mouse effective amount" refers to an amount of a drug
effective to achieve a desired effect when the subject is a
mouse.
[0048] A "human effective amount" refers to an amount of a drug
effective to achieve a desired effect when the subject is a human
patient.
[0049] 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.
[0050] 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.
[0051] "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.
[0052] "Dose" refers to an amount of a drug given in a single
administration.
[0053] In combination, the components of the combination of the
anti-ErbB2/ErbB3 antibody have an additive or superadditive effect
on suppressing tumor cell growth, as compared to monotherapy with
the anti-ErbB2/ErbB3 antibody or treatment with the other agents in
the absence of antibody therapy. By "additive" is meant a result
that is greater in extent (e.g., in the degree of reduction of
tumor mitotic index or of tumor growth or in the degree of tumor
shrinkage or the frequency and/or duration of symptom-free or
symptom-reduced periods) than the best separate result achieved by
monotherapy with each individual component, while "superadditive"
is used to indicate a result that exceeds in extent the sum of such
separate results. In one embodiment, the additive effect is
measured as slowing or stopping of tumor growth or tumor cell
proliferation. The additive effect can also be measured as, e.g.,
reduction in size of a tumor, reduction of tumor mitotic index,
reduction in number of metastatic lesions over time, increase in
overall response rate, or increase in median or overall
survival.
[0054] One non-limiting example of a measure by which effectiveness
of a therapeutic treatment can be quantified is by calculating the
log 10 cell kill, which is determined according to the following
equation:
log 10 cell kill=TC(days)/3.32.times.Td
[0055] in which T C represents the delay in growth of the cells,
which is the average time, in days, for the tumors of the treated
group (T) and the tumors of the control group (C) to have reached a
predetermined value (1 g, or 10 mL, for example), and Td represents
the time, in days necessary for the volume of the tumor to double
in the control animals. When applying this measure, a product is
considered to be active if log 10 cell kill is greater than or
equal to 0.7 and a product is considered to be very active if log
10 cell kill is greater than 2.8. Using this measure, a
combination, used at its own maximum tolerated dose, in which each
of the constituents is present at a dose generally less than or
equal to its maximum tolerated dose, exhibits therapeutic synergy
when the log 10 cell kill is greater than the value of the log 10
cell kill of the best constituent when it is administered alone. In
an exemplary case, the log 10 cell kill of the combination exceeds
the value of the log 10 cell kill of the best constituent of the
combination by at least 0.1 log cell kill, at least 0.5 log cell
kill, or at least 1.0 log cell kill.
[0056] 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).
[0057] 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, gastric esophageal
junction, 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.
[0058] 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.
[0059] 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 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."
[0060] 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, 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. 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, an open label, Phase 1/2 and
pharmacologic study of MM-111 with five 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, MM-111
in combination with lapatinib, paclitaxel and trastuzumab, and
MM-111 in combination with docetaxel and trastuzumab, and an open
label, Phase 2 study of MM-111 and paclitaxel with or without
trastuzumab in patients with HER-2 expressing carcinomas of the
distal esophagus, gastroesophageal junction and stomach.
[0061] 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.
[0062] 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
(pelitinib), tyrphostin AG-1478, and pan-HER kinase inhibitors
(e.g. CI-1033 (canertinib, PD 183805), AC480/BMS-599626, HM781-36B,
AZD8931 (sapitinib) and PF299804 (dacomitinib)); 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; 3) platinum-based
therapeutics such as oxaliplatin, carboplatin and cisplatin; 4)
mTOR inhibitors such as BEZ235 (a dual PI3K/mTOR inhibitor),
AZD8055, everolimus, temsirolimus, siroplimus/rapamycin, and
ridaforolimus; and 5) additional anti-ErbB2 therapeutic agents,
such as trastuzumab, pertuzumab, and T-DM1 (ado-trastuzumab
emtansine). Additional examples of therapeutic agents suitable for
combination with anti-ErbB2/anti-ErbB3 antibodies may be found in
Table 5 and the Appendix below.
[0063] MM-111 is suitable for both large scale production and
systemic therapy. MM-111 binds to 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
[0064] 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
[0065] 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
[0066] 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
[0067] 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
[0068] 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
[0069] 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-111 or
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
[0070] 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.
[0071] 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).
[0072] 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).
[0073] In comparison to the double combination of MM-111 or
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.
[0074] 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 FIG. 5 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%
The percent of spheroid growth inhibition (normalized to untreated,
stimulated control) was determined for 104 doses of inhibitor
treatment.
[0075] 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
[0076] 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
[0077] 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 143 (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
[0078] 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--(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
[0079] 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
[0080] 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
[0081] BT474-M3 cells were transfected with PS100010 vector
containing human aromatase (gene accession No: NM.sub.--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
[0082] 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 (FIG. 6b). 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
[0083] 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
[0084] 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. #
[0085] CRL-1504.RTM.), 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 (FIG. 8a) and
ZR75-30 cells (IC.sub.50=5 nM) (FIG. 8c). Good reduction by MM-111
of pAKT levels (inhibition of AKT phosphorylation) in BT474-M3
(IC.sub.50=10) (FIG. 8b) and in ZR75-30 cells (IC.sub.50=4 nM)
(FIG. 8d) 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 (FIG. 8c) 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
[0086] 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 FIG. 8b) 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
[0087] 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
[0088] 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
[0089] 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
[0090] 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 [0091]
QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDS
VKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTGGGGS
GGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVS
DRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGAASDAHK
SEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHT
LFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE
ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAK
QRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRA
DLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK
DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNL
IKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAED
YLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFTFHADICTL
SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS
QAALGLAAALQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYP
GDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEWL
GVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSW
YQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVF
GGGTKLTVLG
Dosing and Administration of MM-111 in Combination with One or More
Additional Therapeutics
Example 14
Mode of Administration of MM-111
[0092] 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.
[0093] 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).
[0094] 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.
[0095] 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
[0096] 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.
[0097] 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 QxD, wherein x represents an integer, so that a QxD 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 QxD (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
(QxD) 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 (QxD) 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 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-00005 TABLE 3A 7 25 20 7 40 30 14 60 45 14 90 75 21 120
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
[0098] 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).
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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-111 with Cisplatin, Capecitabine,
and Trastuzumab
[0103] Administration of MM-111 with cisplatin, capecitabine, and
trastuzumab is done, for example, by the following method or minor
variations thereof.
[0104] 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
[0105] 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
[0106] 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
Coadministration of MM-111 and Other Therapeutic Agents
[0107] 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 (ado-trastuzumab
emtansine, an antibody-drug conjugate of the antibody trastuzumab
linked to maytansine derivative (DM1)), an mTOR inhibitor (e.g.,
AZD8055, sirolimus, everolimus, temsirolimus, ridaforolimus, or the
dual PI3K/mTOR inhibitor BEZ235), and combinations thereof.
[0108] 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.
[0109] 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.
[0110] 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 or HER 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.
[0111] 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.
[0112] The effect of MM-111 combined with trastuzumab and the dual
PI3K/mTOR inhibitor BEZ235 on cell proliferation was assessed by
the methods described above or with minor variations thereof in
unstimulated and heregulin-stimulated NCI-N87 cells in vitro. Cells
were incubated for 2 hours with a dose range of MM-111,
trastuzumab, or their combination, in the presence of BEZ235 as
shown in FIG. 14. Cells were plated and either untreated (Control,
open circle), treated with BEZ235 alone (open diamond), or treated
with BEZ235 (5 nM FIG. 14a, 20 nM FIG. 14b) and either trastuzumab
(closed circle), MM-111 (closed square), or the combination of
MM-111 and trastuzumab (closed diamond). Treatment with the triple
combination of MM-111, trastuzumab, and BEZ235 resulted in a
significant reduction in cell number compared to control cells in
both the presence and absence of heregulin stimulation.
[0113] The effect of MM-111 combined with the mTOR inhibitor
everolimus and trastuzumab was assessed in unstimulated (FIG. 15a)
and heregulin-stimulated (FIG. 15b) BT474M3 cells in vitro. Cells
were plated and either untreated (Control, open circle), treated
with everolimus alone (open diamond), or treated with everolimus (1
nM FIG. 15a, 5 nM FIG. 15b) and either trastuzumab (closed circle),
MM-111 (closed square), or the combination of MM-111 and
trastuzumab (closed diamond). Treatment with the triple combination
of MM-111, trastuzumab, and everolimus resulted in a significant
reduction in cell number compared to control cells in both the
presence and absence of heregulin stimulation.
Example 21
Combination Treatment with MM-111 and MEK/PI3k/AKT Pathway
Inhibitors
[0114] 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 pathway 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
pathway inhibitor(s) or these agents can be administered in
separate dosage forms.
[0115] In one embodiment, MM-111 and a MEK/PI3k/AKT pathway
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.
[0116] To determine whether the combination of MM-111 with a MEK
inhibitor results in decreased Erk1/2 activity in a tumor model,
MM-111 and the MEK inhibitor trametinib, and were tested in a mouse
xenograft model as described below:
Cell Culture and Xenograft Study
[0117] BT474-M3 cells are transduced with full length HRG1-.beta.1
(Origene)-constructs (encoding PAC-PA-turboGFP for selection) by
lentiviral infection. A HRG overexpressing polyclonal cell line
(BT474-M3-GFP-HRG) is established after puromycin selection and
sorting for high GFP expressing cell population. A control cell
line (BT474-M3-GFP) is engineered to express EGFP in the same
manner. Cells are maintained in culture in RPMI supplemented with
10% FBS, penicillin, streptomycin and puromycin. MM-111 is produced
in-house at Merrimack Pharmaceuticals and MK2206, trametinib, and
UO126 (UO126-EtOH) are purchased from Selleckchem. 7-9 week old
female NCRNU-mice (Taconic) are implanted subcutaneously with 0.72
mg 60-day release 17 .beta.-estradiol pellets (Innovative Research
of America).
MM-111 Combination Treatment with Trametinib
[0118] 48 hours after implantation of the .beta.-estradiol pellets,
15.times.106 BT474-M3-GFP-HRG cells were implanted in the second
axillary mammary fat pad. When tumors reached an average volume of
450-500 mm3 (day 0), mice were randomized into groups to receive
control (phosphate buffered saline, intraperitoneally), MM-111 (48
mg/kg, intraperitoneally using phosphate buffered saline as
vehicle), trametinib (3 mg/kg, by oral gavage in 0.5%
methylcellulose/0.1% Tween.RTM.-80) or a combination of MM-111 and
trametinib. A set (3 per group) of mice are euthanized 4 h after
the initial treatment. Tumors were excised and snap-frozen in
liquid nitrogen for subsequent protein analysis. Another set of
animals continued on their assigned treatment regimens for another
7 days receiving phosphate buffered saline control
(intraperitoneally) on days 3 and 6, MM-111 (48 mg/kg,
intraperitoneally) on days 3 and 6, trametinib (3 mg/kg, by oral
gavage) on days 1, 2, 5 and 6. 24 hours after the last treatment,
tumors were excised and snap-frozen in liquid nitrogen for
subsequent protein analysis. Frozen tumor samples were pulverized
and protein lysates were prepared in Tissue Extraction Reagent
(Invitrogen) supplemented with Protease Inhibitor Cocktail Set III
(Calbiochem, EMD) and Halt.TM. Phosphatase Inhibitor Cocktail
(Thermo Scientific). Lysates were diluted in assay buffer (1%
Bovine Serum Albumin in Tris buffered saline, pH 7.4, containing
0.1% Tween-20) to 1 mg/ml concentration and analyzed for phopsho-
and total Erk1/2 using Luminex.RTM. immunosandwich assay on a
FLEXMAP 3D instrument (Luminex). Antibodies from Cell Signaling
Technologies were used to detect total Erk1/2 (3374) and
pT202/Y204-sites on Erk1/2 (4370BF). 4 h after the start of
treatment, addition of MM-111 to trametinib showed a trend to
decreasing Erk1/2 activity (decreasing pT202/Y204-Erk1/2, p=0.086,
Student's t-test) compared to treatment withtrametinib alone (FIG.
16A). 24 h after the last treatment, addition of MM-111 to
trametinib significantly decreased Erk1/2 activity (decreasing
pT202/Y204-Erk1/2, p<0.001, Student's t-test) compared to
treatment with trametinib alone (FIG. 16B).
Example 22
Combination Treatment with MM-111 and ErbB2-Targeted Therapeutic
Agents
[0119] MM-111, at dosages described herein (see, e.g., Example 15),
can be administered in combination with one or more additional
ErbB2-targeted therapeutic agents, such as trastuzumab, T-DM1,
pertuzumab, etc. In order to assess the effect of MM-111 in
combination with an additional ErbB2-targeted therapeutic, cells
with (FIG. 17B) and without (FIG. 17A) heregulin (HRG) were treated
with a dose escalation of T-DM1 and then treated with MM-111 as
follows.
Materials
[0120] NCI-N87 cells are transduced with full length HRG1-.beta.1
(Origene)-constructs (encoding PAC-PA-turboGFP for selection) by
lentiviral infection. A HRG overexpressing polyclonal cell line
(NCI-N87-GFP-HRG) is established after puromycin selection and
sorting for high GFP expressing cell population. A control cell
line (NCI-N87-GFP) is engineered to express EGFP in the same
manner. Cells are maintained in culture in RPMI supplemented with
10% fetal bovine serum, penicillin, streptomycin and puromycin (1
.mu.g/ml).
[0121] MM-111 is manufactured at Merrimack Pharmaceuticals.
Recombinant human NRG-1-.beta.1 EGF domain is from R&D Systems.
T-DM1 (KADCYLA, Genentech) is obtained from pharmacy.
CellTiter-Glo.RTM. Luminescent Cell Viability Assay is from
Promega.
[0122] 700 NCI-N87 or BT-474-M3 cells were seeded on 384-well flat
clear bottom polystyrene microplates in 20 .mu.l of culture media
(RPMI supplemented with 10% fetal bovine serum and penicillin and
streptomycin). Cells were allowed to adhere 16-20 hours at
+37.degree. C., 5% CO.sub.2. Cells were stimulated for 4 hours by
adding 5 .mu.l of culture media containing recombinant human
NRG-1-.beta.1 EGF domain (HRG1) bringing the final HRG1 stimulation
concentration to 0, 0.313, 0.625, 1.25, 2.5, 5 or 10 nM. After 4
hours, T-DM1 was added in 5 .mu.l of culture media with or without
MM-111 bringing the T-DM1 concentration to 0, 0.001524, 0.004572,
0.013717, 0.041152, 0.123457, 0.37037, 1.11111, 3.333333 or 10
.mu.g/ml and the MM-111 concentration to 0 or 1 .mu.M. All
conditions were assayed in biological quadruplicates. Cells were
incubated for 72 h at +37.degree. C., 5% CO.sub.2. Cell viability
was determined by CellTiter-Glo.RTM. Luminescent Cell Viability
Assay by adding 20 .mu.l of reconstituted CellTiter-Glo reagent per
well, incubating plated for 10 minutes at RT in an orbital shaker
and reading the luminescence signal using Biotek.TM. Synergy H1
Hybrid Reader.
[0123] 700 NCI-N87-GFP or NCI-N87-GFP-HRG1 cells were seeded on
384-well flat clear bottom polystyrene microplates in 20 .mu.l of
culture media (RPMI supplemented with 10% fetal bovine serum and
penicillin and streptomycin). Cells were allowed to adhere 16-20
hours at +37.degree. C., 5% CO2. T-DM1 was added in 5 .mu.l of
culture media with or without MM-111 bringing the T-DM1
concentration to 0, 0.001524, 0.004572, 0.013717, 0.041152,
0.123457, 0.37037, 1.11111, 3.333333 or 10 .mu.g/ml and the MM-111
concentration to 0 or 1 .mu.M. All conditions were assayed in
biological quadruplicates. Cells were incubated for 72 h at
+37.degree. C., 5% CO.sub.2. Cell viability was determined by
CellTiter-Glo.RTM. Luminescent Cell Viability Assay by adding 20 ul
of reconstituted CellTiter-Glo reagent per well, incubating plated
for 10 minutes at RT in an orbital shaker and reading the
luminescence signal using Biotek Synergy H1 Hybrid Reader.
[0124] As shown in FIG. 17, In the absence of endogenous heregulin,
treatment of cells with T-DM1 reduces cell viability up to
approximately 54.6% with 0.1 .mu.g/mL T-DM1, at which point the
remaining cells become resistant. The addition of MM-111 restores
sensitivity of the cells to T-DM1. This effect is significantly
increased in the presence of endogenous heregulin where treatment
of cells with T-DM1 reduces cell viability up to approximately
47.3% with 0.04111152 .mu.g/mL T-DM1, at which point the remaining
cells become resistant. The addition of MM-111 restores sensitivity
of the cells to T-DM1 further reducing cell viability by
approximately 30% to approximately 80% (FIG. 17B).
[0125] To test the combination therapy with endogenous heregulin
(HRG), NCI-N87 cells and BT-474-M3 cells were prepared as described
above and treated with a dose range of both T-DM1 and heregulin in
the presence and absence of MM-111 (3D graphs, FIG. 18). Cell
viability was tested and numbers are given as % of control and
summarized in Tables 1-4 below. As can be seen in FIG. 18, while
exogenous HRG decreases the activity of T-DM1, co-treatment of the
cells with a combination of MM-111 and T-DM1 greatly reduced cell
viability in the presence of endogenous heregulin in both NCI-N87
(FIG. 18B) and BT-474-M3 cells (FIG. 18D).
TABLE-US-00008 TABLE 1 NCI-N87 cells without MM-111 T- HRG DM1 (nM)
0 0.001524 0.004572 0.013717 0.041152 0.123457 0.37037 1.111111
3.333333 10 (.mu.g/ml) 0 100.0001 93.36976 75.62197 51.2155
50.04061 42.64956 46.5761 42.8517 40.80667 43.84619 0.313 105.2683
100.3208 92.59442 79.62461 74.71907 64.7733 50.76463 55.8238
57.24998 57.69454 0.625 110.9354 106.4739 106.0159 84.03995
61.45342 71.29415 58.90545 65.075 62.77755 67.86139 1.25 105.9291
102.2426 94.15791 81.15374 64.90047 66.01914 69.29675 69.40256
58.90233 68.76759 2.5 95.5206 90.80036 84.09815 83.76757 69.34041
74.67369 67.23904 72.16217 66.05516 65.68811 5 99.9019 94.98233
89.09868 80.41984 71.01775 73.54437 68.99217 75.72861 64.94796
63.21516 10 114.0075 88.14249 101.4929 80.41022 69.82144 64.32714
61.13489 62.15488 59.45985 68.79469
TABLE-US-00009 TABLE 2 NCI-N87 cells with MM-111 T- HRG DM1 (nM) 0
0.001524 0.004572 0.013717 0.041152 0.123457 0.37037 1.111111
3.333333 10 (.mu.g/ml) 0 99.99994 95.67672 78.87085 64.23961
46.34934 41.8017 32.86071 35.79531 35.56626 32.20002 0.313 101.4158
98.3686 87.83576 74.46012 52.5808 44.82781 41.52759 37.6864
34.50943 36.02112 0.625 105.024 108.5211 86.78032 66.85997 55.32827
45.12097 40.0349 38.27817 38.6461 36.36282 1.25 110.2781 107.8277
99.94741 73.38117 51.00783 43.11336 43.031 40.2014 37.52794 35.5527
2.5 118.0867 117.2117 99.28302 90.13422 63.70836 52.75964 43.73032
41.26843 36.41708 38.52877 5 126.5145 128.6171 118.6616 95.04545
70.74315 51.52235 43.87818 43.0434 42.54104 40.95163 10 146.1417
140.3695 122.2993 95.17032 70.81566 52.81674 47.2081 43.46258
42.60197 42.12973
TABLE-US-00010 TABLE 3 BT-474-M3 cells without MM-111 T- HRG DM1
(nM) 0 0.001524 0.004572 0.013717 0.041152 0.123457 0.37037
1.111111 3.333333 10 (.mu.g/ml) 0 100 113.2862 98.62592 94.0068
59.46435 46.28945 46.03028 40.94169 38.39982 37.44111 0.313
119.8347 116.4401 115.5001 120.231 97.75329 98.67097 94.19504
95.98138 95.80208 84.02226 0.625 123.4093 117.8323 116.7982
112.1619 108.0231 104.9562 107.4638 97.33409 94.15624 94.01721 1.25
116.9189 108.1483 107.0419 108.9035 104.0392 109.1902 91.67416
94.82696 100.5096 88.67155 2.5 114.6797 113.1709 104.357 114.8767
108.3647 96.44198 100.0466 99.7191 99.2052 89.80503 5 117.0592
123.3681 119.6607 111.0487 111.2081 98.30082 102.3885 99.45587
90.43802 99.39723 10 116.0498 130.2768 115.6098 111.159 104.5468
101.7859 99.51807 101.389 98.00674 87.40396
TABLE-US-00011 TABLE 4 BT-474-M3 cells with MM-111 T- HRG DM1 (nM)
0 0.001524 0.004572 0.013717 0.041152 0.123457 0.37037 1.111111
3.333333 10 (.mu.g/ml) 0 100.0001 105.1766 103.9555 103.6039
77.39243 66.65368 57.99249 52.81845 61.04489 49.52486 0.313
116.4349 118.4495 115.8389 106.7296 91.78999 68.56924 61.57601
59.03206 58.89703 52.39394 0.625 123.4726 120.7381 123.5142
115.1022 91.45484 68.63193 61.65257 53.11713 54.6567 47.24296 1.25
132.3855 131.1941 131.9924 125.6464 100.4235 78.41731 64.38723
54.49712 56.83025 54.51656 2.5 131.572 136.6084 146.7699 134.055
109.0098 80.11692 73.45718 64.84097 55.26672 53.33503 5 146.0121
152.0284 151.8639 138.9854 116.4752 86.2649 70.99611 74.74774
61.73628 55.30379 10 157.2453 154.9672 149.674 145.9666 121.6431
91.79307 81.23282 69.08484 71.97691 61.17057
Endnotes
[0126] 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.
[0127] 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
[0128] 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.
[0129] 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 T-DM1, 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-00012 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.; ABRAXANE .RTM.) 175 mg/m.sup.2 docetaxel (TAXOTERE
.RTM.) 60-100 mg/m.sup.2 Topoisomerase Inhibitors camptothecin
topotecan hydrochloride (HYCAMTIN etoposide (EPOSIN .RTM.)
Alkylating Agents cyclophosphamide (CYTOXAN .RTM.) 600 mg/m.sup.2
Platinum-Based Agents Cisplatin 20-100 mg/m.sup.2 carboplatin
(PARAPLATIN .RTM.) 300 mg/m.sup.2 nedaplatin (AQUPLA .RTM.)
oxaliplatin (ELOXATIN .RTM.) 65-85 mg/m.sup.2 satraplatin (SPERA
.RTM.) triplatin tetranitrate Selective Estrogen Modulators (SERM)
tamoxifen (NOLVADEX .RTM.) 20-40 mg/day 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 (ADRIAMYCIN .RTM.) 40-75
mg/m.sup.2 epirubicin (ELLENCE .RTM.) 60-120 mg/m.sup.2 Aromatase
Inhibitors aminoglutethimide (CYTADREN .RTM.) 250-2000 mg/day
anastrozole (ARIMIDEX .RTM.) 1 mg/day letrozole (FEMARA .RTM.) 2.5
mg/day Vorozole exemestane (AROMASIN .RTM.) 25-50 mg/day
Testolactone fadrozole (AFEMA .RTM.) Anti-VEGF Agents bevacizumab
(AVASTIN .RTM.) 10 mg/kg Anti-ErbB2 (HER2/neu) Agents trastuzumab
(HERCEPTIN .RTM.) 2-8 mg/kg Pertuzumab (OMNITARG .RTM.) Anti-ErbB3
(HER3) Agents U3-1287 (AMG 888)
APPENDIX
Anticancer Agents
TABLE-US-00013 [0130] 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. EMCLONE 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 T-DM1--ado-trastuzumab
emtansine Kadcyla .RTM. Genentech Her/ErbB Dimerization Inhibitors
2C4, R1273--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 (trametinib) Glaxo
Smith Kline PD-0325901 GDC-0973 Genentech UO126-EtOH Cell Signaling
Technology Anti-cMet Antibody Therapies AVEO (AV299) AVEO AMG102
Amgen 5D5 (OA-5D5) Genentech Small Molecules Targeting cMet
PHA665752 ARQ-650RP ArQule ARQ 197 ArQule Alkyaling 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 Cytoxan
Bristol-Myers Squibb Cyclophosphamide 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 Ridaforolimus)
Everolimus (RAD001, RAD001C) Certican .RTM., Afinitor Novartis
Sirolimus (Rapamycin) Rapamune .RTM. Wyeth Pharma Temsirolimus
(CCI-779) BEZ235 Torisel .RTM. Wyeth Pharma AZD8055 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., AEtenta Zentaris, Inc. Perifosine with
Docetaxel Keryx Biopharmaceuticals, Inc., AEtenta Zentaris, Inc.
Perifosine with Gemcitabine AEtenta Zentaris, Inc. Perifosine with
Paclitaxel Keryx Biopharmaceuticals, Inc, AEtenta 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 AEtenta 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 Cvelin-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) ICOX-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
* * * * *