U.S. patent application number 17/129096 was filed with the patent office on 2021-07-15 for combination therapy with an anti-her2 antibody-drug conjugate and a bcl-2 inhibitor.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Gail Lewis Phillips, Deepak Sampath, Ingrid Wertz.
Application Number | 20210213130 17/129096 |
Document ID | / |
Family ID | 1000005490021 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210213130 |
Kind Code |
A1 |
Phillips; Gail Lewis ; et
al. |
July 15, 2021 |
COMBINATION THERAPY WITH AN ANTI-HER2 ANTIBODY-DRUG CONJUGATE AND A
BCL-2 INHIBITOR
Abstract
The present invention is directed to a combination therapy
involving an anti-HER2 antibody-drug conjugate and a selective
Bcl-2 inhibitor for the treatment of a patient suffering from
cancer, particularly, a HER2-expressing cancer.
Inventors: |
Phillips; Gail Lewis; (South
San Francisco, CA) ; Sampath; Deepak; (South San
Francisco, CA) ; Wertz; Ingrid; (South San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
1000005490021 |
Appl. No.: |
17/129096 |
Filed: |
December 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15849018 |
Dec 20, 2017 |
10898570 |
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17129096 |
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PCT/US2016/041184 |
Jul 6, 2016 |
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15849018 |
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62189610 |
Jul 7, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 2039/505 20130101; A61K 2300/00 20130101; A61P 35/00 20180101;
A61K 47/6851 20170801; A61K 47/6803 20170801; C07K 2317/24
20130101; A61K 31/635 20130101; C07K 2317/73 20130101; C07K 16/32
20130101; G01N 2800/52 20130101; G01N 33/57415 20130101; G01N
2333/4703 20130101; A61K 47/6809 20170801; A61K 39/39558 20130101;
G01N 33/57446 20130101; A61K 31/496 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/32 20060101 C07K016/32; G01N 33/574 20060101
G01N033/574; A61K 47/68 20060101 A61K047/68; A61K 31/635 20060101
A61K031/635; A61K 45/06 20060101 A61K045/06; A61P 35/00 20060101
A61P035/00; A61K 31/496 20060101 A61K031/496 |
Claims
1-50. (canceled)
51. A method for the diagnosis of a subject with a HER2-positive
tumor as being resistant or susceptible to treatment with an
anti-HER2 antibody-drug conjugate, comprising (i) obtaining a tumor
sample from said subject, (ii) measuring the expression level of
the Bcl-2 gene or its product in said tumor sample relative to a
control sample, and (iii) diagnosing said tumor as being resistant
to treatment with an anti-HER2 antibody-drug conjugate when the
measured expression level of Bcl-2 in said tumor sample is at least
2 fold greater than the expression level in said control sample, or
diagnosing said tumor as being susceptible to treatment with an
anti-HER2 antibody-drug conjugate when the measured expression
level of Bcl-2 in said tumor sample is less than 2 fold greater
than the expression level in said control sample.
52. The method of claim 51, wherein said subject is a human
patient.
53. The method of claim 51, wherein said control sample is a tumor
sample of the same cell type that is not resistant to treatment
with said anti-HER2 antibody-drug conjugate.
54. The method of claim 51, wherein the tumor is breast cancer or
gastric cancer.
55. The method of claim 51, wherein the tumor sample is a
formalin-fixed, paraffin-embedded tumor sample.
56. The method of claim 51, further comprising the step of
measuring the expression level of the HER2 gene or its product in
said tumor sample.
57. The method of claim 51, further comprising the step of treating
said subject with an anti-HER2 antibody-drug conjugate and a
selective Bcl-2 inhibitor when the measured expression level of
Bcl-2 in said tumor sample is at least 2 fold greater than the
expression level in said control sample.
58. The method of claim 51, further comprising the step of treating
said patient with an anti-HER2 antibody-drug conjugate when the
measured expression level of Bcl-2 in said tumor sample is less
than 2 fold greater than the expression level in said control
sample.
59. The method of claim 51, wherein the anti-HER2 antibody-drug
conjugate is trastuzumab-MCC-DM1.
60. The method of claim 51, wherein the selective Bcl-2 inhibitor
is
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof.
Description
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 28, 2016, is named GNE-0416-WO.txt and is 6,298 bytes in
size.
TECHNICAL FIELD
[0002] The present invention is directed to a combination therapy
involving an anti-HER2 antibody-drug conjugate and a Bcl-2
inhibitor for the treatment of cancer. In a particular embodiment,
the invention concerns methods of using trastuzumab-MCC-DM1
(trastuzumab emtansine; KADCYLA.RTM.) and a selective Bcl-2
inhibitor for the treatment of HER2-positive cancer, such as
HER2-positive breast cancer or gastric cancer.
BACKGROUND
[0003] Anti-HER2 Antibody-Drug Conjugates
[0004] The HER2 (ErbB2) receptor tyrosine kinase is a member of the
epidermal growth factor receptor (EGFR) family of transmembrane
receptors. Overexpression of HER2 is observed in approximately 20%
of human breast cancers (hereinafter referred to as HER2-positive
breast cancer) and is implicated in the aggressive growth and poor
clinical outcomes associated with these tumors (Slamon et al (1987)
Science 235:177-182). HER2 protein overexpression can be determined
using an immunohistochemistry based assessment of fixed tumor
blocks (Press M F, et al (1993) Cancer Res 53:4960-70).
[0005] Trastuzumab (CAS 180288-69-1, HERCEPTIN.RTM., huMAb4D5-8,
rhuMAb HER2, Genentech) is a recombinant DNA-derived, IgG1 kappa,
monoclonal antibody that is a humanized version of a murine
anti-HER2 antibody (4D5) that selectively binds with high affinity
in a cell-based assay (Kd=5 nM) to the extracellular domain of HER2
(U.S. Pat. Nos. 5,677,171; 5,821,337; 6,054,297; 6,165,464;
6,339,142; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 7,074,404;
Coussens et al (1985) Science 230:1132-9; Slamon et al (1989)
Science 244:707-12; Slamon et al (2001) New Engl. J. Med.
344:783-792). Trastuzumab has been shown, in both in vitro assays
and in animals, to inhibit the proliferation of human tumor cells
that overexpress HER2 (Hudziak t al (1989) Mol Cell Biol 9:1165-72;
Lewis et al (1993) Cancer Immunol Immunother; 37:255-63; Baselga et
al (1998) Cancer Res. 58:2825-2831). Trastuzumab is a mediator of
antibody-dependent cellular cytotoxicity, ADCC (Lewis et al (1993)
Cancer Immunol Immunother 37(4):255-263; Hotaling et al (1996)
[abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471;
Pegram M D, et al (1997) [abstract]. Proc Am Assoc Cancer Res;
38:602; Sliwkowski et al (1999) Seminars in Oncology 26(4), Suppl
12:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews:
Molecular Cell Biology, Macmillan Magazines, Ltd., Vol.
2:127-137).
[0006] HERCEPTIN.RTM. was approved in 1998 for the treatment of
patients with HER2-overexpressing metastatic breast cancers
(Baselga et al, (1996) J. Clin. Oncol. 14:737-744) that have
received extensive prior anti-cancer therapy, and has since been
used in over 300,000 patients (Slamon D J, et al. N Engl J Med
2001; 344:783-92; Vogel C L, et al. J Clin Oncol 2002; 20:719-26;
Marty M, et al. J Clin Oncol 2005; 23:4265-74; Romond E H, et al. T
N Engl J Med 2005; 353:1673-84; Piccart-Gebhart M J, et al. N Engl
J Med 2005; 353:1659-72; Slamon D, et al. [abstract]. Breast Cancer
Res Treat 2006, 100 (Suppl 1): 52). In 2006, the FDA approved
HERCEPTIN.RTM. (trastuzumab, Genentech Inc.) as part of a treatment
regimen containing doxorubicin, cyclophosphamide and paclitaxel for
the adjuvant treatment of patients with HER2-positive,
node-positive breast cancer.
[0007] An alternative approach to antibody-targeted therapy is to
utilize antibodies for delivery of cytotoxic drugs specifically to
antigen-expressing cancer cells. Antibody-drug conjugates, or ADCs,
are monoclonal antibodies to which highly potent cytotoxic agents
have been conjugated. ADCs represent a novel approach to conferring
tumor selectivity on systemically administered anti-tumor
therapeutics. Utilizing surface antigens that are tumor-specific
and/or overexpressed, ADCs are designed to focus the delivery of
highly potent cytotoxic agents to tumor cells. The potential of
this approach is to create a more favorable therapeutic window for
such agents than could be achieved by their administration as free
drugs.
[0008] Maytansinoids, derivatives of the anti-mitotic drug
maytansine, bind to microtubules in a manner similar to vinca
alkaloid drugs (Issell B F et al (1978) Cancer Treat. Rev.
5:199-207; Cabanillas F et al. (1979) Cancer Treat Rep, 63:507-9.
DM1 is a thiol-containing maytansinoid derived from the naturally
occurring ester ansamitocin P3 (Remillard S, Rebhun L I, Howie G A,
et al. (1975) Science 189(4207):1002-1005.3; Cassady J M, Chan K K,
Floss H G. (2004) Chem Pharm Bull 52(1):1-26.4). The related plant
ester, maytansine, has been studied as a chemotherapeutic agent in
approximately 800 patients, administered at a dose of 2.0 mg/m2
every 3 weeks either as a single dose or for 3 consecutive days
(Issell B F, Crooke S T. (1978) Maytansine. Cancer Treat Rev
5:199-207). Despite preclinical activity, the activity of
maytansine in the clinic was modest at doses that could be safely
delivered. The dose-limiting toxicity (DLT) was gastrointestinal,
consisting of nausea, vomiting, and diarrhea (often followed by
constipation). These toxicities were dose dependent but not
schedule dependent. Peripheral neuropathy (predominantly sensory)
was reported and was most apparent in patients with preexisting
neuropathy. Subclinical transient elevations of hepatic
transaminase, alkaline phosphatase, and total bilirubin were
reported. Constitutional toxicities, including weakness, lethargy,
dysphoria, and insomnia, were common. Less common toxicities
included infusion-site phlebitis and mild myelosuppression. Further
development of the drug was abandoned in the 1980s because of the
narrow therapeutic window.
[0009] Trastuzumab-MCC-DM1 (T-DM1, trastuzumab emtansine,
ado-trastuzumab emtansine, KADCYLA.RTM.), a novel antibody-drug
conjugate (ADC) for the treatment of HER2-positive breast cancer,
is composed of the cytotoxic agent DM1 (a thiol-containing
maytansinoid anti-microtubule agent) conjugated to trastuzumab at
lysine side chains via an MCC linker, with an average drug load
(drug to antibody ratio) of 3.5. After binding to HER2 expressed on
tumor cells, T-DM1 undergoes receptor-mediated internalization,
resulting in intracellular release of cytotoxic DM1-containing
catabolites and subsequent cell death.
[0010] In a Phase I study of T-DM1 (TDM3569g), the maximum
tolerated dose (MTD) of T-DM1 administered by IV infusion every 3
weeks (q3w) was 3.6 mg/kg. A DLT (Dose-Limiting Toxicity) consisted
of transient thrombocytopenia in patients treated at 4.8 mg/kg.
Treatment with 3.6 mg/kg q3w was well tolerated and associated with
significant clinical activity. (Krop (2010) J. Clin. Oncol.
28(16):2698-2704). That same study also showed that weekly dosing
with 2.4 mg/kg was also well tolerated and had anti-tumor activity.
(Beeram (2012) Cancer 118(23):5733-5740.)
[0011] A Phase II study (TDM4374g) demonstrated that T-DM1,
administered at 3.6 mg/kg q3w, had single-agent anti-tumor activity
in a heavily pre-treated patient population having HER2-positive
metastatic breast cancer. (Krop (2012) 30(26):3234-3241.) A Phase
III study (TDM4370g) demonstrated that T-DM1, administered at 3.6
mg/kg q3w, significantly prolonged progression-free survival and
overall survival with less toxicity compared to treatment with
lapatinib plus capecitabine in patients with HER2-positive advanced
breast cancer previously treated with trastuzumab and a taxane.
(Verma (2012) New England Journal of Medicine 367:1783-1791.)
[0012] The U.S. Food and Drug Administration approved
ado-trastuzumab emtansine, marketed under the tradename
KADCYLA.RTM., on Feb. 22, 2013 for the treatment of patients with
HER2-positive, metastatic breast cancer who previously received
treatment with trastuzumab and a taxane.
[0013] Bcl-2 Inhibitors
[0014] The Bcl-2 family of proteins regulates programmed cell death
triggered by developmental cues and in response to multiple stress
signals (Cory. S., and Adams, J. M., Nature Reviews Cancer 2 (2002)
647-656; Adams, Genes und Development 17 (2003) 2481-2495; Danial,
N. N., and Korsmeyer, S. J., Cell 116 (2004) 205-219). Whereas cell
survival is promoted by Bcl-2 itself and several close relatives
(Bcl-xL, Bcl-W, Mcl-1 and A1), which bear three or four conserved
Bcl-2 homology (BH) regions, apoptosis is driven by two other
sub-families. The initial signal for cell death is conveyed by the
diverse group of BH3-only proteins, including Bad, Bid, Bim, Puma
and Noxa, which have in common only the small BH3 interaction
domain (Huang and Strasser, Cell 103 (2000) 839-842). However, Bax
or Bak, multi-domain proteins containing BH1-BH3, are required for
commitment to cell death (Cheng, et al., Molecular Cell 8 (2001)
705-711; Wei, M. C., et al., Science 292 (2001) 727-730; Zong, W.
X., et al., Genes and Development 15 148 (2001) 1-1486). When
activated, they can permeabilize the outer membrane of mitochondria
and release pro-apoptogenic factors (e.g. cytochrome C) needed to
activate the caspases that dismantle the cell (Wang, K., Genes and
Development 15 (2001) 2922-2933; (Adams, 2003 supra); Green, D. R.,
and Kroemer, G., Science 305 (2004) 626-629).
[0015] Interactions between members of these three factions of the
Bcl-2 family dictate whether a cell lives or dies. When BH3-only
proteins have been activated, for example, in response to DNA
damage, they can bind via their BH3 domain to a groove on their
pro-survival relatives (Sattler, et al., Science 275 (1997)
983-986). How the BH3-only and Bcl-2-like proteins control the
activation of Bax and Bak, however, remains poorly understood
(Adams, 2003 supra). Most attention has focused on Bax. This
soluble monomeric protein (Hsu, Y. T., et al., Journal of
Biological Chemistry 272 (1997) 13289-13834; Wolter, K. G., et al.,
Journal of Cell Biology 139 (1997) 1281-92) normally has its
membrane targeting domain inserted into its groove, probably
accounting for its cytosolic localization (Nechushtan, A., et al.,
EMBO Journal 18 (1999) 2330-2341; Suzuki, et al., Cell 103 (2000)
645-654; Schinzel, A., et al., J Cell Biol 164 (2004) 1021-1032).
Several unrelated peptides/proteins have been proposed to modulate
Bax activity, reviewed in Lucken-Ardjomande, S., and Martinou, J.
C., J Cell Sci 118 (2005) 473-483, but their physiological
relevance remains to be established. Alternatively, Bax may be
activated via direct engagement by certain BH3-only proteins
(Lucken-Ardjomande, S., and Martinou, J. C, 2005 supra), the best
documented being a truncated form of Bid, tBid (Wei, M. C., et al.,
Genes und Development 14 (2000) 2060-2071; Kuwana, T., et al., Cell
111 (2002) 331-342; Roucou, X., et al., Biochemical Journal 368
(2002) 915-921; Cartron, P. F., et al., Mol Cell 16 (2004)
807-818). As discussed elsewhere (Adams 2003 supra), the oldest
model, in which Bcl-2 directly engages Bax (Oltvai, Z. N., et al.,
Cell 74 (1993) 609-619), has become problematic because Bcl-2 is
membrane bound while Bax is cytosolic, and their interaction seems
highly dependent on the detergents used for cell lysis (Hsu, Y. T.,
and Youle, 1997 supra). Nevertheless, it is well established that
the BH3 region of Bax can mediate association with Bcl-2 (Zha, H.,
and Reed, J., Journal of Biological Chemistry 272 (1997) 31482-88;
Wang, K., et al., Molecular und Cellular Biology 18 (1998)
6083-6089) and that Bcl-2 prevents the oligomerization of Bax, even
though no hctcrodimcrs can be detected (Mikhailov, V., et al.,
Journal of Biological Chemistry 276 (2001) 18361-18374). Thus,
whether the pro-survival proteins restrain Bax activation directly
or indirectly remains uncertain.
[0016] Although Bax and Bak seem in most circumstances to be
functionally equivalent (Lindsten, T., et al., Molecular Cell 6
(2000) 1389-1399; Wei, M. C., et al., 2001 supra), substantial
differences in their regulation would be expected from their
distinct localization in healthy cells. Unlike Bax, which is
largely cytosolic, Bak resides in complexes on the outer membrane
of mitochondria and on the endoplasmic reticulum of healthy cells
(Wei, M. C., et al., 2000 supra; Zong, W. X., et al., Journal of
Cell Biology 162 (2003) 59-69). Nevertheless, on receipt of
cytotoxic signals, both Bax and Bak change conformation, and Bax
translocates to the organellar membranes, where both Bax and Bak
then form homo-oligomers that can associate, leading to membrane
permeabilization (Hsu, Y. T., et al., PNAS 94 (1997) 3668-3672;
Wolter, K. G., et al., 1997 supra; Antonsson, B., et al., Journal
of Biological Chemistry 276 (2001) 11615-11623; Nechushtan, A., et
al., Journal of Cell Biology 153 (2001) 1265-1276; Wei, M. C., et
al., 2001 supra; Mikhailov, V., et al., Journal of Biological
Chemistry 278 (2003) 5367-5376).
[0017] There exist various Bcl-2 inhibitors, which all have the
same property of inhibiting prosurvival members of the Bcl-2 family
of proteins and are therefore promising candidates for the
treatment of cancer. Such Bcl-2 inhibitors are e.g. Oblimersen,
SPC-2996, RTA-402, Gossypol, AT-101, Obatoclax mesylate, A-371191,
A-385358, A-438744, ABT-737, ABT-263 (navitoclax), AT-101, BL-11,
BL-193, GX-15-003, 2-Methoxyantimycin A.sub.3, HA-14-1, KF-67544,
Purpurogallin, TP-TW-37, YC-137 and Z-24, and are described e.g. in
Zhai, D., et al., Cell Death and Differentiation 13 (2006)
1419-1421.
[0018] The link between other the Bcl-2 family proteins and cancer
is also well established and amply documented (Strasser, A. 2011
EMBO J. 30, 3667-3683), and inhibitors of other Bcl family members
are also known. Bcl-X.sub.L-selective inhibitors A-1155463 and
A-1331852 are described, for example, in Leverson et al., Science
Translational Medicine Vol 7, Issue 279 279ra40. Selective
benzothiazole hydrazone inhibitors of Bcl-X.sub.L are disclosed in
Sleebs et al., J. Med. Chem. 2013, 56, 5514-5540. For the
description of other Bcl-X.sub.L inhibitors see, e.g. Koehler et
al., ACS Med. Chem. Lett. 2014, 5, 662-667; and Tao et al, ACS Med.
Chem. Lett. 2014, 5, 1088-10. MCl-1 inhibitors and their uses as
cancer therapeutics are described, for example, in Leverson et al.,
Cell Death and Disease (2015) 6, e1590; Bruncko et al., J. Med.
Chem. 2015, 58, 2180-2194; Petros et al., Boorganic & Medicinal
Chemistry Letters 24 (2014) 1484-1488; Abulwerdi et al., Mol Cancer
Ther 2014; 13:565-5; Abuilwerdi et al., J. Med. Chem. 2014, 57,
4111-4133; Burke et al., J. Med. Chem. 2015, 58, 3794-3805; Friberg
et al., J. Med. Chem. 2013, 56, 15-30; and belmar et al.,
Pharmacology & Theraputics 145 (2015) 76-84. Mcl-1/Bcl-xL dual
inhibitors are disclosed by Tanaka et al., J. Med. Chem. 2013, 56,
9635-9645.
SUMMARY OF THE INVENTION
[0019] In one aspect, the invention concerns a method for the
treatment of a cancer in a human in need thereof comprising
administering to such human an effective amount of an anti-HER2
antibody-drug conjugate and an inhibitor of a Bcl family
protein.
[0020] In another aspect, the invention concerns a method for the
treatment of a cancer in a human in need thereof comprising
administering to such human an effective amount of an anti-HER2
antibody-drug conjugate and a selective Bcl-2 inhibitor.
[0021] In one embodiment, the cancer is HER2 positive cancer.
[0022] In another embodiment, the cancer is HER2 positive breast
cancer or gastric cancer.
[0023] In yet another embodiment, the HER2-positive breast cancer
or gastric cancer has a HER2 immunohistochemistry (IHC) score of 2+
or 3+ and/or an in situ hybridization (ISH) amplification ratio
(her2:CEP17 in situ hybridization (ISH) amplification ratio) of
2.0.
[0024] In a further embodiment, the HER2-positive cancer, such as
breast cancer or gastric cancer, is resistant to treatment with an
anti-HER2 antibody-drug conjugate administered as a single
agent.
[0025] In a still further embodiment, the HER2-positive cancer,
such as breast cancer or gastric cancer, is sensitive to treatment
with an anti-HER2 antibody-drug conjugate administered as a single
agent, and the combination of the anti-HER2 antibody-drug conjugate
and the selective Bcl-2 inhibitor can be administered to a patient
naive to treatment with the anti-HER2 antibody-drug conjugate.
[0026] In a different embodiment, the anti-HER2 antibody-drug
conjugate and the selective Bcl-2 inhibitor show synergistic
activity, including, but not limited to, synergistic activity in a
HER2-positive cancer, such as breast cancer or gastric cancer, that
is resistant to treatment with an anti-HER2 antibody-drug conjugate
administered as a single agent.
[0027] In all embodiments, the anti-HER2 antibody-drug conjugate
can, for example, be trastuzumab-MCC-DM1.
[0028] In all embodiments, the selective Bcl-2 inhibitor can, for
example, be
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dime-
thylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-py-
ran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof.
[0029] In another aspect, the invention concerns a method for the
treatment of HER2 positive cancer in a human in need thereof
comprising administering to the human an effective amount of
trastuzumab-MCC-DM1 and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof.
[0030] In one embodiment, the cancer is HER2 positive breast cancer
or gastric cancer.
[0031] In another embodiment, the HER2-positive breast cancer or
gastric cancer has a HER2 immunohistochemistry (IHC) score of 2+ or
3+ and/or an In situ hybridization (ISH) amplification ratio
(her2:CEP17 in situ hybridization (ISH) amplification ratio) of
22.0.
[0032] In yet another embodiment, the HER2 positive cancer, such as
breast cancer or gastric cancer, is resistant to treatment with
trastuzumab-MCC-DM1 administered as a single agent.
[0033] In a further embodiment, the HER2-positive cancer, such as
breast cancer or gastric cancer, is sensitive to treatment with
trastuzumab-MCC-DM1 administered as a single agent, and the
combination of the trastuzumab-MCC-DM1 and
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof can be administered to a patient naive to
treatment with trastuzumab-MCC-DM1.
[0034] In a still further embodiment, the HER2-positive cancer,
such as breast cancer or gastric cancer, is sensitive to treatment
with an anti-HER2 antibody-drug conjugate administered as a single
agent, and the combination of the anti-HER2 antibody-drug conjugate
and the selective Bcl-2 inhibitor can be administered to a patient
naive to treatment with the anti-HER2 antibody-drug conjugate. In a
further embodiment, the trastuzumab-MCC-DM1 and the
2-1H-pyrrolo[2,3-b]pyridin-5-yloxy)4-(4-((2-(4-chlorophenyl)-4,4-dimethyl-
cyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran--
4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptably salt thereof show synergistic activity, including, but
not limited to, synergistic activity in a HER2-positive cancer,
such as breast cancer or gastric cancer.
[0035] In a still further embodiment, the trastuzumab-MCC-DM1 and
the
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)4-(4-((2-(4-chlorophenyl)-4,4-dimethyl-
cyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran--
4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof are co-administered.
[0036] In another embodiment, the trastuzumab-MCC-DM1 and the
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof are administered simultaneously.
[0037] In yet another embodiment, the trastuzumab-MCC-DMI and the
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof are administered consecutively.
[0038] In another aspect, the invention concerns the use of a
combination of an anti-HER2 antibody-drug conjugate and an
inhibitor of a Bcl family protein in the preparation of a
medicament for the treatment of cancer.
[0039] In one embodiment, the Bcl family protein is a Bcl-2 like
protein, such as Mcl-1, Bcl-xl, Bcl-w (BCL2L2), or Bcl-xs,
preferably Mcl-1 or Bcl-xl.
[0040] In another aspect, the invention concerns the use of a
combination of an anti-HER2 antibody-drug conjugate and a selective
Bcl-2 inhibitor in the preparation of a medicament for the
treatment of cancer.
[0041] In one embodiment, the invention concerns the use of a
combination of trastuzumab-MCC-DM1 and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof in the preparation of a medicament for the
treatment of cancer.
[0042] In all embodiments, the cancer may be HER2 positive
cancer.
[0043] In all embodiments, the cancer may, for example, be breast
cancer or gastric cancer.
[0044] In all embodiments, the HER2 positive cancer, such as breast
cancer or gastric cancer, may be resistant to treatment with
trastuzumab-MCC-DM1 administered as a single agent.
[0045] In all embodiments, the HER2-positive cancer, such as breast
cancer or gastric cancer, may be sensitive to treatment with
trastuzumab-MCC-DM1 administered as a single agent, and the
combination of the trastuzumab-MCC-DM1 and
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof may be used to treat a patient naive to
treatment with trastuzumab-MCC-DM1.
[0046] In another aspect, the invention concerns the use of a
combination of an anti-HER2 antibody-drug conjugate and an
inhibitor of a Bcl family protein in the preparation of a
medicament for the treatment of cancer.
[0047] In one embodiment, the Bcl family protein is a Bcl-2 like
protein, such as Mcl-1, Bcl-xl, Bcl-w (BCL2L2), or Bcl-xs,
preferably Mcl-1 or Bcl-xl.
[0048] In a further aspect, the invention concerns a combination of
an anti-HER2 antibody-drug conjugate and a selective Bcl-2
inhibitor for use in the treatment of cancer.
[0049] In one embodiment, the combination of trastuzumab-MCC-DM1
and
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof is for use in the treatment of cancer.
[0050] In all combinations, the cancer may, for example, be HER2
positive cancer, such as HER2 positive breast cancer or gastric
cancer.
[0051] In a particular embodiment, the cancer, such as breast
cancer or gastric cancer, is resistant to treatment with the
anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1, when
administered as a single agent.
[0052] In another embodiment, the HER2-positive cancer, such as
breast cancer or gastric cancer, may be sensitive to treatment with
the anti-HER2 antibody-drug conjugate, e.g. trastuzumab-MCC-DM1,
when administered as a single agent, and the combination may be
used to treat a patient naive to treatment the anti-HER2
antibody-drug conjugate, e.g. trastuzumab-MCC-DM1.
[0053] In another aspect, the invention concerns a method for the
diagnosis of a HER2-positive tumor resistant to treatment with an
anti-HER2 antibody-drug conjugate, comprising determining in a
tumor sample obtained from a patient with HER2-positive cancer the
expression level of the Bcl-2 gene or its product relative to the
expression level in a control sample, and diagnosing said cancer as
resistant to treatment with said anti-HER2 antibody-drug conjugate
when the expression level in said tumor sample is at least 2 fold,
or at least 3 fold or at least 4 fold, or at least 5 fold greater
than the expression level in said control sample.
[0054] In a further aspect, the invention concerns a method for the
diagnosis of a HER2-positive tumor susceptible to treatment with an
anti-HER2 antibody-drug conjugate, comprising determining in a
tumor sample obtained from a patient with HER2-positive cancer the
expression level of the Bcl-2 gene or its product relative to the
expression level in a control sample, and diagnosing said cancer as
susceptible to treatment with said anti-HER2 antibody-drug
conjugate when the expression level in said tumor sample is less
than 2 fold, or at least 3 fold, or at least 4 fold, or at least 5
fold greater than the expression level in said control sample.
[0055] In a still further aspect, the invention concerns a method
for the diagnosis of a subject with a HER2-positive tumor as being
resistant or susceptible to treatment with an anti-HER2
antibody-drug conjugate, comprising (i) obtaining a tumor sample
from said subject, (ii) measuring the expression level of the Bcl-2
gene or its product in said tumor sample relative to a control
sample, and (iii) diagnosing said tumor as being resistant to
treatment with an anti-HER2 antibody-drug conjugate when the
measured expression level of Bcl-2 in said tumor sample is at least
2 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold
greater than the expression level in said control sample, or
diagnosing said tumor as being susceptible to treatment with an
anti-HER2 antibody-drug conjugate when the measured expression
level of Bcl-2 in said tumor sample is less than 2 fold, or less
than 3 fold, or less than 4 fold, or less than 5 fold greater than
the expression level in said control sample.
[0056] In one embodiment, the subject is a human patient.
[0057] In another embodiment, the control sample is a tumor sample
of the same cell type that is not resistant to treatment with said
anti-HER2 antibody-drug conjugate.
[0058] In yet another embodiment, the tumor is breast cancer or
gastric cancer.
[0059] In a further embodiment, the tumor sample is a
formalin-fixed, paraffin-embedded tumor sample.
[0060] In all embodiments, the diagnostic method may further
comprise a step of measuring the expression level of the HER2 gene
or its product in the tumor sample.
[0061] In all embodiments, the diagnostic method may further
comprise a step of treating the subject with an anti-HER2
antibody-drug conjugate and a selective Bcl-2 inhibitor when the
measured expression level of Bcl-2 in the tumor sample is at least
2 fold, or at least 3 fold, or at least 4 fold, or at least 4 fold,
or at least 5 fold greater than the expression level in said
control sample.
[0062] In all embodiments, the diagnostic method may further
comprise a step of treating said patient with an anti-HER2
antibody-drug conjugate when the measured expression level of Bcl-2
in the tumor sample is less than 2-times greater than the
expression level in said control sample.
[0063] In one embodiment, the anti-HER2 antibody-drug conjugate is
trastuzumab-MCC-DM1.
[0064] In another embodiment, the selective Bcl-2 inhibitor
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof.
[0065] In another aspect, the invention concerns a kit for the in
vitro diagnosis or prognosis of a HER2 positive tumor resistant to
treatment with an anti-HER2 antibody-drug conjugate in a biological
sample obtained from a patient, which comprises a specific binding
partner for the Bcl-2 gene or its expression product.
[0066] In one embodiment, the binding partner is an anti-Bcl-2
antibody.
[0067] In another embodiment, the binding partner is a nucleic acid
hybridizing to the Bcl-2 gene.
[0068] In a further aspect, the invention relates to a kit
comprising an anti-HER2 antibody drug conjugate and a selective
Bcl-2 inhibitor for the combination treatment of a patient with a
HER2 expressing cancer.
[0069] In an embodiment of the present invention, the kit further
comprises a pharmaceutically acceptable carrier. The kit may
further include a sterile diluent, which is preferably stored in a
separate additional container. The kit may further Include a
package insert comprising printed instructions directing the use of
the combined treatment as a method for a HER2 expressing cancer,
such as HER2 expressing breast or gastric cancer.
[0070] Just as in other aspects, the HER2 expressing cancer may,
for example, be breast cancer or gastric cancer, and in various
embodiments the anti-HER2 antibody drug conjugate may be
trastuzumab-MCC-DM1 and/or the selective Bcl-2 inhibitor may be
2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethy-
lcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-
-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof.
[0071] In a particular embodiment, the HER2 expressing cancer to be
treated, such as breast cancer or gastric cancer, is resistant to
treatment with the anti-HER2 antibody-drug conjugate or the
trastuzumab-MCC-DM1, when administered as a single agent.
[0072] In another embodiment, the HER2 expressing cancer, such as
breast cancer or gastric cancer, may be sensitive to treatment with
the anti-HER2 antibody-drug conjugate, e.g. trastuzumab-MCC-DM1,
when administered as a single agent, and the combination may be
used to treat a patient naive to treatment the anti-HER2
antibody-drug conjugate, e.g. trastuzumab-MCC-DM1.
BRIEF DESCRIPTION OF THE FIGURES
[0073] FIGS. 1A and 1B show expression of Bcl-2 family pro-survival
molecules in T-DM1 resistant KPL-4 and BT-474M1 human breast cancer
cells (HER2 positive) relative to parental cells. FIG. 1A shows
mRNA expression assessed by TaqMan qRT-PCR analysis; FIG. 1B shows
protein expression by Western blot analysis.
[0074] FIG. 2 presents results of a cell viability assay showing
that the T-DM1+GDC-0199 combination has a synergistic effect in
KPL-4 T-DM1-resistant human breast cancer cells, while no synergism
is observed in KPL-4 parental cells.
[0075] FIG. 3 presents results of a caspase activation assay
measuring activation of caspases 3 and 7. The results show that at
24 hours of drug treatment, T-DM1-resistant KPL-4 human breast
cancer cells are re-sensitized to T-DM1 in the presence of
GDC-0199, whereas at 24 hours, there is no effect of
T-DM1+/-GDC-0199 in KPL-4 parental cells.
[0076] FIGS. 4A and 4B presents the results of a caspase activation
assay measuring activation of caspases 3 and 7 in T-DM1 resistant
KPL-4 human breast cancer cells relative to parental cells after 48
hours of drug treatment. The results show dose-dependent increases
in caspases 3 and 7 activation with the addition of GDC-0199 to
T-DM1, with minimal effect of T-DM1 alone. In KPL-4 parental cells,
T-DM1 induces robust activation of caspases 3 and 7 with minimal
increase upon addition of GDC-0199.
[0077] FIG. 5A presents the results of a caspase activation assay
measuring activation of caspases 3 and 7 in Clone #17 T-DM1
resistant KPL-4 human breast cancer cell line treated with 1 ug/mL
T-DM1 alone or in combination with the indicated doses of GDC-0199
for 48 hours. The results show dose-dependent increases in caspases
3 and 7 activation with the addition of GDC-0199 to T-DM1, with
minimal effect of T-DM1 alone.
[0078] FIG. 5B shows the effect of T-DM1 (5 mg/kg administered
once), GDC-0199 (100 mg/kg qd.times.21) or the combination on tumor
growth (as measured by tumor volume) of Clone #17 T-DM1 resistant
KPL-4 human breast cancer xenografts in SCID beige mice.
Combination drug treatment resulted in tumor stasis, with no
activity of single agent treatment.
[0079] FIGS. 6A and 6B show the results of a caspase activation
assay measuring activation of caspases 3 and 7 in Clone #8 T-DM1
resistant KPL-4 human breast cell line at 0.1 pg/mL and 1pg/mL
T-DM1 concentrations, respectively, alone or in combination with
the indicated concentrations of GDC-199.
[0080] FIG. 6C shows the effect of T-DM1 (5 mg/kg q3w.times.2),
GDC-0199 (100 mg/kg qd.times.21) or the combination on tumor growth
(as measured by tumor volume) of Clone #8 T-DM1 resistant KPL-4
human breast cancer xenografts in SCID beige mice.
[0081] FIG. 7A shows the expression of Bcl-2 in formalin-fixed
paraffin-embedded T-DM1 resistant KPL-4 xenograft tumor samples
(Clones #8 and #17) determined by immunohistochemistry (IHC), using
DAB detection method.
[0082] FIG. 7B shows the expression of HER2 (ErbB2) in
formalin-fixed paraffin-embedded T-DM1 resistant KPL-4 xenograft
tumor samples (Clones #8 and #17) determined by
immunohistochemistry (IHC), using DAB detection method.
[0083] FIG. 8 shows protein expression as measured by Western blot
analysis of Bcl-2 and HER2 in Clone #17 KPL-4 T-DM1-resistant
xenograft tumors, treated with GDC-0199, T-DM1 or
T-DM-1+GDC-0199.
[0084] FIG. 9A presents results of a caspase 3/7 activation
luminescent in vitro apoptosis assay, testing the effect of five
separate concentrations of GDC-0199 (.mu.M) in combination with 9
different concentrations of T-DM1 after 24 hours of treatment in
HER2+MDA-MB-361 breast cancer cells (T-DM1 naive cells). The
results demonstrate enhanced apoptosis greater than T-DM1 alone
with all combinations tested.
[0085] FIG. 9B presents the results of a kinetic caspase 3/7
activation fluorescent in vitro apoptosis assay, testing the effect
of three different concentrations of GDC-0199 (0.63 .mu.M, 1.25
.mu.M, 2.5 LM), alone and in combination with T-DM1 (0.1 .mu.g/mL),
in HER2+MDA-MB-361 breast cancer cells. The results demonstrate
enhanced caspase activation greater than T-DM1 alone with all
combinations tested.
[0086] FIG. 10A presents the results of a caspase 3/7activation
luminescent in vitro apoptosis assay, testing the effect of five
separate concentrations of GDC-0199 (.mu.M) in combination with 9
different concentrations for 24 hours of treatment in HER2+HCC1569
breast cancer cells (T-DM1 naive cells). The results demonstrate no
induction of apoptosis by T-DM1 alone but enhanced apoptosis with
all combinations tested.
[0087] FIG. 10B presents the results of a kinetic caspase 3/7
fluorescent in vitro apoptosis assay, testing the effect of three
different concentrations of GDC-0199 (0.63 .mu.M, 1.25 .mu.M, 2.5
.mu.M), alone and in combination with T-DM1 (0.1 .mu.g/mL), in
HER2+HCC1569 breast cancer cells. The results demonstrate
dose-dependent enhanced caspase activation with combination
treatment.
[0088] FIG. 11 shows the effect of T-DM1 (1 mg/kg, 3 mg/kg, 7
mg/kg, iv q3w.times.1) and GDC-0199 (100 mg/kg, po, qd.times.21),
alone or in combination, on tumor growth in HER2+MDA-MB-361 breast
cancer xenograft model. Significant tumor growth delay was observed
with GDC-0199 combined with 7 mg/kg T-DM1.
[0089] FIG. 12 shows Western blot analysis of the effects of T-DM1,
with or without GDC-0199, on Bcl-2 family members (total and
phospho-Bcl-2 and -Bcl-xL, total Mcl-1 and Bim) in HER2+breast
cancer cell lines BT-474, EFM192A, KPL-4, HCC1569, HCC1954,
MDA-361, UACC-812, ZR-75-30.
[0090] FIG. 13 shows the amino acid sequence of trastuzumab light
chain (SEQ ID NO: 1).
[0091] FIG. 14 shows the amino acid sequence of trastuzumab heavy
chain (SEQ ID NO: 2).
DETAILED DESCRIPTION OF THE INVENTION
[0092] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents which may be
included within the scope of the present invention as defined by
the claims. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials
described.
[0093] All references cited throughout the disclosure are expressly
incorporated by reference herein in their entirety. In the event
that one or more of the incorporated literature, patents, and
similar materials differs from or contradicts this application,
including but not limited to defined terms, term usage, described
techniques, or the like, this application controls.
Definitions
[0094] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0095] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0096] The anti-HER2 antibody used in the antibody-drug conjugates
herein is a monoclonal antibody.
[0097] The term "chimeric antibody" refers to a monoclonal antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are especially preferred. Such
murine/human chimeric antibodies are the product of expressed
immunoglobulin genes comprising DNA segments encoding murine
immunoglobulin variable regions and DNA segments encoding human
immunoglobulin constant regions. Other forms of "chimeric
antibodies" encompassed by the present invention are those in which
the class or subclass has been modified or changed from that of the
original antibody. Such "chimeric" antibodies are also referred to
as "class-switched antibodies." Methods for producing chimeric
antibodies involve conventional recombinant DNA and gene
transfection techniques now well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0098] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric and bifunctional
antibodies.
[0099] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Disk, M. A., and van de
Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374) and can be
produced by a variety of techniques, including phage display. Based
on such technology, human antibodies against a great variety of
targets can be produced. Examples of human antibodies are for
example described in Kellermann, S. A., et al., Curr Opin
Biotechnol. 13 (2002) 593-597. For the use of phage display
technology to produce and select human antibodies see, e.g., Winter
et al., Ann Review Immunol, 1994, 12:433-455; and for the
production of fully human antibodies from transgenic mouse and
phage display platforms see, e.g., Lonberg, Current Opinion
Immunol, 2008, 20(4):450-459.
[0100] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as, for
example, antibodies isolated from a host cell such as a NS0 or CHO
cell or from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions derived from
human germline immunoglobulin sequences in a rearranged form.
[0101] As used herein, "specifically binding" or "binds
specifically to" refers a binding that is sufficiently selective to
a target as to distinguish it from a binding to unwanted or
nonspecific targets. In one embodiment, an antibody that binds
specifically to a target will have a binding affinity for that
target (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM
(e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M to 10.sup.-13 M,
e.g., from 10.sup.-9 M to 10.sup.-13M). In yet another embodiment,
the KD is 10.sup.-10 mol/l or lower (e.g. 10.sup.-12 mol/l). The
binding affinity is determined with a standard binding assay, such
as Scatchard plot analysis on cells expressing the target
antigen.
[0102] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded. In one
embodiment, it is double-stranded DNA.
[0103] The "constant domains" are not involved directly in binding
the antibody to an antigen but are involved in the effector
functions (ADCC, complement binding, and CDC).
[0104] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Frccman and Co., page 91
(2007).)
[0105] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include:
[0106] (a) hypervariable loops occurring at amino acid residues
26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and
96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
[0107] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56
(L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991));
[0108] (c) antigen contacts occurring at amino acid residues 27c-36
(L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101
(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
[0109] (d) combinations of (a), (b), and/or (c), including HVR
amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),
26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102
(H3).
[0110] The term "anti-HER2 antibody" according to the invention is
an antibody that binds specifically to HER2 antigen.
[0111] As defined herein, the terms "trastuzumab", "HERCEPTIN.RTM."
and "huMAb4D5-8" are used interchangeably. Such antibody preferably
comprises the light and heavy chain amino acid sequences shown in
FIG. 13 (SEQ ID NO: 1) and FIG. 14 (SEQ ID NO. 2),
respectively.
[0112] The "epitope 4D5" or "4D5 epitope" or "4D5" is the region in
the extracellular domain of HER2 to which the antibody 4D5 (ATCC
CRL 10463) and trastuzumab bind. This epitope is close to the
transmembrane domain of HER2, and within Domain IV of HER2. To
screen for antibodies which bind to the 4D5 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to assess whether the antibody binds to the 4D5
epitope of HER2 (e.g. any one or more residues in the region from
about residue 529 to about residue 625, inclusive, of HER2).
[0113] The "epitope 2C4" or "2C4 epitope" is the region in the
extracellular domain of HER2 to which the antibody 2C4 binds. In
order to screen for antibodies which bind to the 2C4 epitope, a
routine cross-blocking assay such as that described in Antibodies,
A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to assess whether the antibody binds to the 2C4
epitope of HER2. Epitope 2C4 comprises residues from domain II in
the extracellular domain of HER2. The 2C4 antibody and Pertuzumab
bind to the extracellular domain of HER2 at the junction of domains
I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).
[0114] As defined herein, the terms "T-DM1," "trastuzumab-MCC-DM1,"
"ado-trastuzumab emtansine," "trastuzumab emtansine," and "KADCYLA"
are used interchangeably, and refer to trastuzumab linked through
the linker moiety MCC to the maytansinoid drug moiety DM1,
including all mixtures of variously loaded and attached
antibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8 drug
moieties are covalently attached to the antibody trastuzumab (U.S.
Pat. No. 7,097,840; US 2005/0276812; US 2005/0166993).
[0115] The term "Bcl-2" as used herein refers to the Bcl-2 protein
(Swiss Prot ID No. P10415), a member of the Bcl-2 family of
proteins (Cory, S., and Adams, J. M., Nature Reviews Cancer 2
(2002) 647-656; Adams, Genes und Development 17 (2003) 2481-2495;
Danial, N. N., and Korsmeyer, S. J., Cell 116 (2004) 205-219;
Petros, A. M., Biochim Biophys Acta 1644 (2004) 83-94).
[0116] The term "selective Bcl-2 inhibitor" as used herein refers
to polypeptides and small molecules inhibiting prosurvival members
of the Bcl-2 family of proteins. Preferably, the selective Bcl-2
inhibitor is
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide. (a.k.a. ABT-199 or
GDC-0199), or a pharmaceutically acceptable salt thereof, a Bcl-2
inhibitor of formula I, which is described in International
Publication No. WO2010/0138588 and in US publication No.
US2010/0305122, which are incorporated by reference herein.
##STR00001##
[0117] Herein, an "anti-tumor agent" refers to a drug used to treat
cancer. Non-limiting examples of anti-tumor agents herein include
chemotherapy agents, HER dimerization inhibitors, HER antibodies,
antibodies directed against tumor associated antigens,
anti-hormonal compounds, cytokines, EGFR-targeted drugs,
anti-angiogenic agents, tyrosine kinase inhibitors, growth
inhibitory agents and antibodies, cytotoxic agents, antibodies that
induce apoptosis, COX inhibitors, farnesyl transferase inhibitors,
antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf
or ras inhibitors, liposomal doxorubicin, topotecan, taxane, dual
tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab,
trastuzumab, erlotinib, and bevacizumab.
[0118] A "chemotherapy" is use of a chemotherapeutic agent useful
in the treatment of cancer.
[0119] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer, regardless of mechanism of action. Classes
of chemotherapeutic agents include, but are not limited to:
alkylating agents, antimetabolites, spindle poison plant alkaloids,
cytotoxic/antitumor antibiotics, topoisomerase inhibitors,
antibodies, photosensitizers, and kinase inhibitors. Examples of
chemotherapeutic agents include: erlotinib (TARCEVA.RTM.,
Genentech/OSI Pharm.), docetaxel (TAXOTERE.RTM., Sanofi-Aventis),
5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine
(GEMZAR.RTM., Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),
cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1),
carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.), temozolomide
(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]
nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR.RTM.,
TEMODAL.RTM., Schering Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,
NOLVADEX.RTM., ISTUBAL.RTM., VALODEX.RTM.), and doxorubicin
(ADRIAMYCIN.RTM.), Akti-1/2, HPPD, and rapamycin.
[0120] More examples of chemotherapeutic agents include:
oxaliplatin (ELOXATIN.RTM., Sanofi), bortezomib (VELCADE.RTM.,
Millennium Pharm.), sutent (SUNITINIB.RTM., SU11248, Pfizer),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), XL-518 (MEK inhibitor, Exelixis, WO 2007/044515),
ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca),
SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K
inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK
222584 (Novartis), fulvestrant (FASLODEX.RTM., AstraZeneca),
leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE.RTM.,
Wyeth), lapatinib (TYKERB.RTM., GSK572016, Glaxo Smith Kline),
lonafarnib (SARASAR.TM., SCH 66336, Schering Plough), sorafenib
(NEXAVAR.RTM., BAY43-9006, Bayer Labs), gefitinib (IRESSA.RTM.,
AstraZeneca), irinotecan (CAMPTOSAR.RTM., CPT-11, Pfizer),
tipifamib (ZARNESTRA.TM., Johnson & Johnson), ABRAXANE.TM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
vandetanib (rINN, ZD6474, ZACTIMA.RTM., AstraZeneca),
chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus
(TORISEL.RTM., Wyeth), pazopanib (GaxoSmithKline), canfosfamide
(TELCYTA, Telik), thiotepa and cyclosphosphamide (CYTOXAN.RTM.,
NEOSAR.RTM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analog topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogs); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogs, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlomaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma1I,
calicheamicin omegaI1 (Angew Chem. Intl. Ed. Engl. (1994)
33:183-186); dynemicin, dynemicin A; bisphosphonates, such as
clodronate; an esperamicin; as well as neocarzinostatin chromophore
and related chromoprotein enediyne antibiotic chromophores),
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate, hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene,
Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(T-2 toxin, verracurin A, roridin A and anguidine); urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide;
thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine
(NAVELBINE.RTM.); novantrone; teniposide; edatrexate; daunomycin;
aminopterin; capecitabine (XELODA.RTM., Roche); ibandronate;
CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine
(DMFO); retinoids such as retinoic acid; and pharmaceutically
acceptably salts, acids and derivatives of any of the above.
[0121] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include breast cancer, squamous cell cancer (e.g., epithelial
squamous cell cancer), lung cancer including small-cell lung
cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the
lung and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer. In a preferred embodiment, the cancer is breast
cancer. In another preferred embodiment, the cancer is gastric
cancer.
[0122] Reference to a tumor or cancer as a "Stage 0," "Stage I,"
"Stage II," "Stage III," or "Stage IV", and various sub-stages
within this classification, indicates classification of the tumor
or cancer using the Overall Stage Grouping or Roman Numeral Staging
methods known in the art. Although the actual stage of the cancer
is dependent on the type of cancer, in general, a Stage 0 cancer is
an in situ lesion, a Stage I cancer is small localized tumor, a
Stage II and III cancer is a local advanced tumor which exhibits
involvement of the local lymph nodes, and a Stage IV cancer
represents metastatic cancer. The specific stages for each type of
tumor is known to the skilled clinician.
[0123] The term "metastatic breast cancer" means the state of
breast cancer where the cancer cells are transmitted from the
original site to one or more sites elsewhere in the body, by the
blood vessels or lymphatics, to form one or more secondary tumors
in one or more organs besides the breast.
[0124] An "advanced" cancer is one which has spread outside the
site or organ of origin, either by local invasion or metastasis.
Accordingly, the term "advanced" cancer includes both locally
advanced and metastatic disease.
[0125] A "refractory" cancer is one which progresses even though an
anti-tumor agent, such as a chemotherapy, is being administered to
the cancer patient. An example of a refractory cancer is one which
is platinum refractory.
[0126] A "recurrent" cancer is one which has regrown, either at the
initial site or at a distant site, after a response to initial
therapy, such as surgery.
[0127] A "locally recurrent" cancer is cancer that returns after
treatment in the same place as a previously treated cancer.
[0128] An"operable" or"resectable" cancer is cancer which is
confined to the primary organ and suitable for surgery
(resection).
[0129] A "non-resectable" or "unresectable" cancer is not able to
be removed (resected) by surgery.
[0130] The terms "HER2-positive" and "HER2 expressing" are used
herein interchangeably. A "HER2-positive" cancer comprises cancer
cells which have higher than normal levels of HER2. Examples of
HER2-positive cancer include HER2-positive breast cancer and
HER2-positive gastric cancer. Optionally, HER2-positive is a HER2
overexpressing cancer, and in certain embodiments the HER-2
positive cancer has an immunohistochemistry (IHC) score of 2+ or 3+
and/or an in situ hybridization (ISH) amplification ratio
.gtoreq.2.0.
[0131] In situ hybridization (ISH) determines the number of her2
copies using a DNA probe coupled to a fluorescent, chromogenic, or
silver detection system (ie, FISH, CISH, or SISH), or a combination
of CISH and SISH systems (bright-field double ISH (BDISH) or
dual-hapten, dual-color ISH (DDISH)). ISH may be conducted using a
single probe to enumerate her2 copies per nucleus only or as a
dual-probe technique where hybridization of a chromosome 17
centromere probe (chromosome enumeration probe 17, CEP17) allows
determination of the her2:CEP17 ratio. The two-probe approach may
be performed as a dual-color technique, with cohybridization of the
two probes on the same slide, or as a monochrome assay where each
probe is used on sequential slides. The her2:CEP17 ratio is
sometimes regarded as a better reflection of her2 amplification
status than mean her2 copy number, as the latter is also dependent
on other parameters, such as the mitotic index of the tumor,
section thickness, nuclear truncation effects, and abnormal
chromosome copy number (aneusomy). The phrase "in situ
hybridization (ISH) amplification ratio .gtoreq.2.0" refers to
her2:CEP17 ratio .gtoreq.2.0. For further details see, e.g. Sauter
G, et al. Guidelines for human epidermal growth factor receptor 2
testing: biologic and methodologic considerations. J Clin Oncol
2009; 27:1323-1333, and the review article by Hanna et al. Modern
Pathology (2014) 27, 4-18.
[0132] Herein, a "patient" or "subject" is a human patient. The
patient may be a "cancer patient," i.e. one who is suffering or at
risk for suffering from one or more symptoms of cancer, in
particular gastric or breast cancer.
[0133] A "patient population" refers to a group of cancer patients.
Such populations can be used to demonstrate statistically
significant efficacy and/or safety of a drug, such as
Pertuzumab.
[0134] A "relapsed" patient is one who has signs or symptoms of
cancer after remission. Optionally, the patient has relapsed after
adjuvant or neoadjuvant therapy.
[0135] A cancer or biological sample which "displays HER
expression, amplification, or activation" is one which, in a
diagnostic test, expresses (including overexpresses) a HER
receptor, has amplified HER gene, and/or otherwise demonstrates
activation or phosphorylation of a HER receptor.
[0136] The term "synergistic" as used herein refers to a
therapeutic combination which is more effective than the additive
effects of the two or more single agents. A determination of a
synergistic interaction between an anti-Her2 antibody-dug
conjugate, such as trastuzumab-MCC-DM1, and a selective Bcl-2
inhibitor may be based on the results obtained from the assays
described herein, or in other assay systems known in the art,
utilizing a standard programs for quantifying synergism,
additivism, and antagonism among anticancer agents. The program
preferably utilized is that described by Chou and Talalay, in "New
Avenues in Developmental Cancer Chemotherapy," Academic Press,
1987, Chapter 2. Combination Index (CI) values less than 0.8
indicate synergy, values greater than 1.2 indicate antagonism and
values between 0.8 to 1.2 indicate additive effects. The
combination therapy may provide "synergy" and prove "synergistic",
i.e., the effect achieved when the active ingredients used together
is greater than the sum of the effects that results from using the
compounds separately. A synergistic effect may be attained when the
active ingredients are: (1) co-formulated and administered or
delivered simultaneously in a combined, unit dosage formulation;
(2) delivered by alternation or in parallel as separate
formulations; or (3) by some other regimen. When delivered in
alternation therapy, a synergistic effect may be attained when the
compounds are administered or delivered sequentially, e.g., by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e., serially in time, whereas in
combination therapy, effective dosages of two or more active
ingredients are administered together.
[0137] "Neoadjuvant therapy" or"preoperative therapy" herein refers
to therapy given prior to surgery.
[0138] The goal of neoadjuvant therapy is to provide immediate
systemic treatment, potentially eradicating micrometastases that
would otherwise proliferate if the standard sequence of surgery
followed by systemic therapy were followed. Neoadjuvant therapy may
also help to reduce tumor size thereby allowing complete resection
of initially unresectable tumors or preserving portions of the
organ and its functions. Furthermore, neoadjuvant therapy permits
an in vivo assessment of drug efficacy, which may guide the choice
of subsequent treatments.
[0139] "Adjuvant therapy" herein refers to therapy given after
definitive surgery, where no evidence of residual disease can be
detected, so as to reduce the risk of disease recurrence. The goal
of adjuvant therapy is to prevent recurrence of the cancer, and
therefore to reduce the chance of cancer-related death. Adjuvant
therapy herein specifically excludes neoadjuvant therapy.
[0140] "Definitive surgery" is used as that term is used within the
medical community. Definitive surgery includes, for example,
procedures, surgical or otherwise, that result in removal or
resection of the tumor, including those that result in the removal
or resection of all grossly visible tumor. Definitive surgery
includes, for example, complete or curative resection or complete
gross resection of the tumor. Definitive surgery includes
procedures that occur in one or more stages, and includes, for
example, multi-stage surgical procedures where one or more surgical
or other procedures are performed prior to resection of the tumor.
Definitive surgery includes procedures to remove or resect the
tumor including involved organs, parts of organs and tissues, as
well as surrounding organs, such as lymph nodes, parts of organs,
or tissues. Removal may be incomplete such that tumor cells might
remain even though undetected.
[0141] "Survival" refers to the patient remaining alive, and
includes disease free survival (DFS), progression free survival
(PFS) and overall survival (OS). Survival can be estimated by the
Kaplan-Meier method, and any differences in survival are computed
using the stratified log-rank test.
[0142] "Progression-Free Survival" (PFS) is the time from the first
day of treatment to documented disease progression (including
isolated CNS progression) or death from any cause on study,
whichever occurs first.
[0143] "Disease free survival (DFS)" refers to the patient
remaining alive, without return of the cancer, for a defined period
of time such as about 1 year, about 2 years, about 3 years, about 4
years, about 5 years, about 10 years, etc., from initiation of
treatment or from initial diagnosis. In one aspect of the
invention, DFS is analyzed according to the intent-to-treat
principle, i.e., patients are evaluated on the basis of their
assigned therapy. The events used in the analysis of DFS can
include local, regional and distant recurrence of cancer,
occurrence of secondary cancer, and death from any cause in
patients without a prior event (e.g, breast cancer recurrence or
second primary cancer).
[0144] "Overall survival" refers to the patient remaining alive for
a defined period of time, such as about 1 year, about 2 years,
about 3 years, about 4 years, about 5 years, about 10 years, etc.,
from initiation of treatment or from initial diagnosis. In the
studies underlying the invention the event used for survival
analysis was death from any cause.
[0145] By "extending survival" is meant increasing DFS and/or OS in
a treated patient relative to an untreated patient, or relative to
a control treatment protocol. Survival is monitored for at least
about six months, or at least about 1 year, or at least about 2
years, or at least about 3 years, or at least about 4 years, or at
least about 5 years, or at least about 10 years, etc., following
the initiation of treatment or following the initial diagnosis.
[0146] "Hazard ratio" in survival analysis is a summary of the
difference between two survival curves, representing the reduction
in the risk of death on treatment compared to control, over a
period of follow-up. Hazard ratio is a statistical definition for
rates of events. For the purpose of the invention, hazard ratio is
defined as representing the probability of an event in the
experimental arm divided by the probability of an event in the
control arm at any specific point in time.
[0147] By "monotherapy" is meant a therapeutic regimen that
includes only a single therapeutic agent for the treatment of the
cancer or tumor during the course of the treatment period.
[0148] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the growth, development
or spread of a hyperproliferative condition, such as cancer. For
purposes of this invention, beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0149] The term "a method of treating" or its equivalent, when
applied to, for example, cancer refers to a procedure or course of
action that is designed to reduce or eliminate the number of cancer
cells in a patient, or to alleviate the symptoms of a cancer. "A
method of treating" cancer or another proliferative disorder does
not necessarily mean that the cancer cells or other disorder will,
in fact, be eliminated, that the number of cells or disorder will,
in fact, be reduced, or that the symptoms of a cancer or other
disorder will, in fact, be alleviated. Often, a method of treating
cancer will be performed even with a low likelihood of success, but
which, given the medical history and estimated survival expectancy
of a patient, is nevertheless deemed to induce an overall
beneficial course of action.
[0150] The terms "co-administration" or "co-administering" refer to
the administration of the anti-HER2 antibody-drug conjugate and the
selective Bcl-2 inhibitor as two separate formulations. The
co-administration can be simultaneous or sequential in cither
order. In one further embodiment, there is a time period while both
(or all) active agents simultaneously exert their biological
activities. The anti-HER2 antibody-drug conjugate and the selective
Bcl-2 inhibitor are co-administered either simultaneously or
sequentially (e.g. via an intravenous (i.v.) through a continuous
infusion (one for the antibody-drug conjugate and eventually one
for the Bcl-2 inhibitor; or the Bcl-2 inhibitor is administered
orally). When both therapeutic agents are co-administered
sequentially the agents are administered in two separate
administrations that are separated by a "specific period of time".
The term specific period of time is meant anywhere from 1 hour to
15 days. For example, one of the agents can be administered within
about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, 2 or 1 hour from the administration of the other
agent, and, in one embodiment, the specific period time is 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 day, or 24, 23, 22, 21, 20, 19,
18,17,16,15,14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
hour.
[0151] The term "simultaneously" means at the same time or within a
short period of time, usually less than 1 hour.
[0152] A drug that is administered "concurrently" with one or more
other drugs is administered during the same treatment cycle, on the
same day of treatment as the one or more other drugs, and,
optionally, at the same time as the one or more other drugs. For
instance, for cancer therapies given every 3 weeks, the
concurrently administered drugs are each administered on day-1 of a
3-week cycle.
[0153] A dosing period as used herein is meant a period of time,
during which each therapeutic agent has been administered at least
once. A dosing cycle is usually about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 days.
[0154] In certain embodiments, a dosing cycle is for 21 days.
[0155] In certain embodiments of a method of treating cancer in a
patient as provided herein, the method comprises administering the
anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor
for one or more dosing cycles to the patient. In one embodiment,
the one or more dosing cycles each last for at least one week. In
another embodiment, the one or more dosing cycles are each for at
least two weeks, three weeks, four weeks, five weeks, six weeks,
seven weeks, eight weeks, nine weeks, or for more than nine weeks.
In one embodiment, each dosing cycle is three weeks.
[0156] In a preferred embodiment, the antibody-drug conjugate is
administered as an intravenous (i.v.) infusion every three weeks
(21-day cycle).
[0157] In another preferred embodiment, the antibody-drug conjugate
is KADCYLA.RTM. (ado-trastuzumab emtansine), which is administered
as a 3.6 mg/kg i.v. infusion every 3 weeks (21-day cycle).
[0158] In some embodiments of the method of treatment provided
herein, the selective Bcl-2 inhibitor is
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide (GDC-0199). In certain
embodiments, the amount of GDC-0199 administered to the patient per
dose is increased during the first dosing cycle from initial
amounts of between 10 mg to 80 mg to final amounts of between 190
mg to 400 mg. In certain embodiments, the amount of GDC-0199 per
dose administered to the patients begins with 50 mg or 100 mg, and
is increased to 300 mg per dose. In some embodiments, the amount of
GDC-0199 in the initial doses administered to the patient can, for
example, be between 20 mg to 60 mg (e.g., 20 mg, 25 mg, 30 mg, 35
mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg doses), followed by dose
amounts of 100 mg, 200 mg, 300 mg or more of GDC-0199.
[0159] An "adverse event" is any unfavorable and unintended sign,
symptom, or disease temporally associated with the use of an
investigational (medicinal) product or other protocol-imposed
intervention, regardless of attribution; and includes: adverse
events (AEs) not previously observed in the patient that emerge
during the protocol-specified AE reporting period, including signs
or symptoms associated with breast cancer that were not present
before the AE reporting period; complications that occur as a
result of protocol-mandated interventions (e.g., invasive
procedures such as biopsies); if applicable, AEs that occur before
assignment of study treatment associated with medication washout,
no treatment run-in, or other protocol-mandated intervention;
Preexisting medical conditions (other than the condition being
studied) judged by the investigator to have worsened in severity or
frequency or changed in character during the protocol-specified AE
reporting period.
[0160] It is self-evident that the antibody-drug conjugates are
administered to the patient in a "therapeutically effective amount"
(or simply "effective amount") which is the amount of the
respective compound or combination that will elicit the biological
or medical response of a tissue, system, animal or human that is
being sought by the researcher, veterinarian, medical doctor or
other clinician. The administration of an effective amount of a
therapeutically agent can be a single administration or split dose
administration. "split dose administration" is meant an effective
amount is a split into multiple doses, preferably 2, and
administered within 1 or 2 days. For example, if 100 mg of a
selective Bcl-2 inhibitor is deemed effective, it can be
administered in one 100 mg administration or two 50 mg
administrations. Split dose administration is sometimes desirable
at the beginning of a dosing period to reduce side effects. When an
effective amount is administered in split dosing, it is still
considered one administration of an effective amount. For example,
when 100 mg is the effective amount of a selective Bcl-2 inhibitor
and that amount is administered in two 50 mg doses over a period of
time, e.g. 2 days, only one effective amount is administered during
that period of time.
[0161] A "fixed" or "flat" dose of a therapeutic agent herein
refers to a dose that is administered to a human patient without
regard for the weight (WT) or body surface area (BSA) of the
patient. The fixed or flat dose is therefore not provided as a
mg/kg dose or a mg/m2 dose, but rather as an absolute amount of the
therapeutic agent.
[0162] A "loading" dose herein generally comprises an initial dose
of a therapeutic agent administered to a patient, and is followed
by one or more maintenance dose(s) thereof. Generally, a single
loading dose is administered, but multiple loading doses are
contemplated herein. Usually, the amount of loading dose(s)
administered exceeds the amount of the maintenance dose(s)
administered and/or the loading dose(s) are administered more
frequently than the maintenance dose(s), so as to achieve the
desired steady-state concentration of the therapeutic agent earlier
than can be achieved with the maintenance dose(s).
[0163] A "maintenance" dose herein refers to one or more doses of a
therapeutic agent administered to the patient over a treatment
period. Usually, the maintenance doses are administered at spaced
treatment intervals, such as approximately every week,
approximately every 2 weeks, approximately every 3 weeks, or
approximately every 4 weeks, preferably every 3 weeks.
[0164] "Infusion" or "infusing" refers to the introduction of a
drug-containing solution into the body through a vein for
therapeutic purposes. Generally, this is achieved via an
intravenous (IV) bag.
[0165] An "intravenous bag" or "IV bag" is a bag that can hold a
solution which can be administered via the vein of a patient. In
one embodiment, the solution is a saline solution (e.g. about 0.9%
or about 0.45% NaCl). Optionally, the IV bag is formed from
polyolefin or polyvinyl chloride.
[0166] In the context of this invention, additional other
cytotoxic, chemotherapeutic or anti-cancer agents, or compounds
that enhance the effects of such agents (e.g. cytokines) may be
used in the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor
combination treatment of HER2 expressing cancer. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended. Preferably the anti-HER2 antibody-drug
conjugate and Bcl-2 inhibitor combination treatment is used without
such additional cytotoxic, chemotherapeutic or anti-cancer agents,
or compounds that enhance the effects of such agents.
[0167] Such agents include, for example: alkylating agents or
agents with an alkylating action, such as cyclophosphamidc (CTX;
e.g. CYTOXAN.RTM.), chlorambucil (CHL; e.g. LEUKERAN.RTM.),
cisplatin (CisP; e.g. PLATINOL.RTM.) busulfan (e.g. MYLERAN.RTM.),
melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine
(TEM), mitomycin C, and the like; anti-metabolites, such as
methotrexate (MTX), etoposide (VP16; e.g. VEPESID.RTM.),
6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),
5-fluorouracil (5-FU), capecitabine (e.g. XELODA.RTM.), dacarbazine
(DTIC), and the like; antibiotics, such as actinomycin D,
doxorubicin (DXR; e.g. ADRIAMYCIN.RTM.), daunorubicin (daunomycin),
bleomycin, mithramycin and the like; alkaloids, such as vinca
alkaloids such as vincristine (VCR), vinblastine, and the like; and
other antitumor agents, such as paclitaxel (e.g. TAXOL.RTM.) and
paclitaxel derivatives, the cytostatic agents, glucocorticoids such
as dexamethasone (DEX; e.g. DECADRON.RTM.) and corticosteroids such
as prednisone, nucleoside enzyme inhibitors such as hydroxyurea,
amino acid depleting enzymes such as asparaginase, leucovorin and
other folic acid derivatives, and similar, diverse antitumor
agents.
[0168] The following agents may also be used as additional agents:
arnifostine (e.g. ETHYOL.RTM.), dactinomycin, mechlorethamine
(nitrogen mustard), streptozocin, cyclophosphamide, lomustine
(CCNU), doxorubicin lipo (e.g. DOXIL.RTM.), gemcitabine (e.g.
GEMZAR.RTM.), daunorubicin lipo (e.g. DAUNOXOME.RTM.),
procarbazine, mitomycin, docetaxel (e.g. TAXOTERE.RTM.),
aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin,
CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38),
floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon
beta, interferon alpha, mitoxantrone, topotecan, leuprolide,
megestrol, melphalan, mercaptopurine, plicamycin, mitotane,
pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen,
teniposide, testolactone, thioguanine, thiotepa, uracil mustard,
vinorelbine, chlorambucil. Preferably the type II anti-CD20
antibody and Bcl-2 inhibitor combination treatment is used without
such additional agents.
[0169] The use of the cytotoxic and anticancer agents described
above as well as antiproliferative target-specific anticancer drugs
like protein kinase inhibitors in chemotherapeutic regimens is
generally well characterized in the cancer therapy arts, and their
use herein falls under the same considerations for monitoring
tolerance and effectiveness and for controlling administration
routes and dosages, with some adjustments. For example, the actual
dosages of the cytotoxic agents may vary depending upon the
patient's cultured cell response determined by using histoculture
methods. Generally, the dosage will be reduced compared to the
amount used in the absence of additional other agents.
[0170] Typical dosages of an effective cytotoxic agent can be in
the ranges recommended by the manufacturer, and where indicated by
in vitro responses or responses in animal models, can be reduced by
up to about one order of magnitude concentration or amount. Thus,
the actual dosage will depend upon the judgment of the physician,
the condition of the patient, and the effectiveness of the
therapeutic method based on the in vitro responsiveness of the
primary cultured malignant cells or histocultured tissue sample, or
the responses observed in the appropriate animal models.
[0171] In the context of this invention, an effective amount of
ionizing radiation may be carried out and/or a radiopharmaceutical
may be used in addition to the anti-HER2 antibody-drug conjugate
and Bcl-2 inhibitor combination treatment. The source of radiation
can be either external or internal to the patient being treated.
When the source is external to the patient, the therapy is known as
external beam radiation therapy (EBRT). When the source of
radiation is internal to the patient, the treatment is called
brachytherapy (BT). Radioactive atoms for use in the context of
this invention can be selected from the group including, but not
limited to, radium, cesium-137, iridium-192, americium-241,
gold-198, cobalt-57, copper-67, technetium-99, iodine-123,
iodine-131, and indium-111. Is also possible to label the antibody
with such radioactive isotopes. Preferably the type II anti-CD20
antibody and Bcl-2 inhibitor combination treatment is used without
such ionizing radiation.
[0172] Radiation therapy is a standard treatment for controlling
unresectable or inoperable tumors and/or tumor metastases. Improved
results have been seen when radiation therapy has been combined
with chemotherapy. Radiation therapy is based on the principle that
high-dose radiation delivered to a target area will result in the
death of reproductive cells in both tumor and normal tissues. The
radiation dosage regimen is generally defined in terms of radiation
absorbed dose (Gy), time and fractionation, and must be carefully
defined by the oncologist. The amount of radiation a patient
receives will depend on various considerations, but the two most
important are the location of the tumor in relation to other
critical structures or organs of the body, and the extent to which
the tumor has spread. A typical course of treatment for a patient
undergoing radiation therapy will be a treatment schedule over a 1
to 6 week period, with a total dose of between 10 and 80 Gy
administered to the patient in a single daily fraction of about 1.8
to 2.0 Gy, 5 days a week. In a preferred embodiment of this
invention there is synergy when tumors in human patients are
treated with the combination treatment of the invention and
radiation. In other words, the inhibition of tumor growth by means
of the agents comprising the combination of the invention is
enhanced when combined with radiation, optionally with additional
chemotherapeutic or anticancer agents. Parameters of adjuvant
radiation therapies are, for example, contained in WO 99/60023.
[0173] The anti-HER2 antibody-drug conjugates are administered to a
patient according to known methods, by intravenous administration
as a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, or intrathecal routes. Intravenous
or subcutaneous administration of the antibodies is preferred.
[0174] The Bcl-2 inhibitors are administered to a patient according
to known methods, e.g. by intravenous administration as a bolus or
by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, or peroral routes. Intravenous,
subcutaneous or oral administration of the Bcl-2 inhibitors is
preferred.
[0175] As used herein, a "pharmaceutically acceptable carrier" is
intended to include any and all material compatible with
pharmaceutical administration including solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and other materials and compounds
compatible with pharmaceutical administration. Except insofar as
any conventional media or agent is incompatible with the active
compound, use thereof in the compositions of the invention is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
[0176] A "vial" is a container suitable for holding a liquid or
lyophilized preparation. In one embodiment, the vial is a
single-use vial, e.g. a 20-cc single-use vial with a stopper.
[0177] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0178] Trastuzumab-MCC-DM1 (T-DM1: KADCYLA.RTM., ado-trastuzumab
emtansine)
[0179] The present invention includes therapeutic combinations
comprising trastuzumab-MCC-DM1 which has the structure:
##STR00002##
[0180] where Tr is trastuzumab, and p is an integer from 1 to 8.
The drug to antibody ratio or drug loading is represented by p in
the above structure of trastuzumab-MCC-DM1. The drug loading value
p is 1 to 8. Trastuzumab-MCC-DM1 includes all mixtures of variously
loaded and attached antibody-drug conjugates where 1, 2, 3, 4, 5,
6, 7, and 8 drug moieties are covalently attached to the antibody
trastuzumab.
[0181] Trastuzumab is produced by a mammalian cell (Chinese Hamster
Ovary, CHO) suspension culture. The HER2 (or c-erbB2)
proto-oncogene encodes a transmembrane receptor protein of 185 kDa,
which is structurally related to the epidermal growth factor
receptor. HER2 protein overexpression is observed in 25%30% of
primary breast cancers and can be determined using an
immunohistochemistry based assessment of fixed tumor blocks (Press
M F, et al (1993) Cancer Res 53:4960-70. Trastuzumab is an antibody
that has antigen binding residues of, or derived from, the murine
4D5 antibody (ATCC CRL 10463, deposited with American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the
Budapest Treaty on May 24, 1990). Exemplary humanized 4D5
antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,
huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN.RTM.)
as in U.S. Pat. No. 5,821,337.
[0182] The antibody-drug conjugate, trastuzumab-MCC-DM1, comprises
a maytansinoid drug moiety DM1 (U.S. Pat. Nos. 5,208,020;
6,441,163) and may be prepared from ansamitocin fermentation
products (U.S. Pat. No. 6,790,954; US 2005/0170475).
[0183] Selective Bcl-2 Inhibitors
[0184] In a preferred embodiment, the selective Bcl-2 inhibitor of
the present invention is
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide. (a.k.a. ABT-199 or
GDC-O 199), a Bcl-2 inhibitor of formula I, which is described in
International Publication No. WO2010/0138588 and in US publication
NO. US2010/0305122, which are incorporated by reference herein.
##STR00003##
[0185] Other selective Bcl-2 inhibitors include, for example,
Oblimersen, SPC-2996, RTA-402, Gossypol, AT-101, Obatoclax
mesylate, A-371191, A-385358, A-438744, ABT-737, ABT-263, AT-101,
BL-11, BL-193, GX-15003, 2-Methoxyanitimycin A.sub.3, HA-14-1,
KF-67544, Purpurogallin, TP-TW-37, YC-137 and Z-24, described e.g.
in Zhai, D., et al., Cell Death and Differentiation 13 (2006)
1419-1421.
[0186] Pharmaceutical Compositions
[0187] Pharmaceutical compositions or formulations of the present
invention include combinations of trastuzumab-MCC-DM1, a selective
Bcl-2 inhibitor, and one or more pharmaceutically acceptable
carrier, glidant, diluent, or excipient.
[0188] Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors of the
present invention may exist in unsolvated as well as solvated forms
with pharmaceutically acceptable solvents such as water, ethanol,
and the like, and it is intended that the invention embrace both
solvated and unsolvated forms.
[0189] Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors of the
present invention may also exist in different tautomeric forms, and
all such forms are embraced within the scope of the invention. The
term "tautomer" or "tautomeric form" refers to structural isomers
of different energies which are interconvertible via a low energy
barrier. For example, proton tautomers (also known as prototropic
tautomers) include interconversions via migration of a proton, such
as keto-enol and imine-enamine isomerizations. Valence tautomers
include interconversions by reorganization of some of the bonding
electrons.
[0190] Pharmaceutical compositions encompass both the bulk
composition and individual dosage units comprised of more than one
(e.g., two) pharmaceutically active agents including
trastuzumab-MCC-DM1 and a selective Bcl-2 inhibitor selected from
the lists of the additional agents described herein, along with any
pharmaceutically inactive excipients, diluents, carriers, or
glidants. The bulk composition and each individual dosage unit can
contain fixed amounts of the aforesaid pharmaceutically active
agents. The bulk composition is material that has not yet been
formed into individual dosage units. An illustrative dosage unit is
an oral dosage unit such as tablets, pills, capsules, and the like.
Similarly, the herein-described method of treating a patient by
administering a pharmaceutical composition of the present invention
is also intended to encompass the administration of the bulk
composition and individual dosage units.
[0191] Pharmaceutical compositions also embrace
isotopically-labeled compounds of the present invention which are
identical to those recited herein, but for the fact that one or
more atoms are replaced by an atom having an atomic mass or mass
number different from the atomic mass or mass number usually found
in nature. All isotopes of any particular atom or element as
specified are contemplated within the scope of the compounds of the
invention, and their uses. Exemplary isotopes that can be
incorporated into compounds of the invention include isotopes of
hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine,
chlorine and iodine, such as .sup.2H, .sup.3H, .sup.11C, .sup.13C,
.sup.14C, .sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O,
.sup.32P, .sup.33P, .sup.35S, .sup.18F, .sup.36Cl, .sup.123I and
.sup.125I. Certain isotopically-labeled compounds of the present
invention (e.g., those labeled with .sup.3H and .sup.14C) are
useful in compound and/or substrate tissue distribution assays.
Tritiated (.sup.3H) and carbon-14 (.sup.14C) isotopes are useful
for their ease of preparation and detectability. Further,
substitution with heavier isotopes such as deuterium (.sup.2H) may
afford certain therapeutic advantages resulting from greater
metabolic stability (e.g., increased in vivo half-life or reduced
dosage requirements) and hence may be preferred in some
circumstances. Positron emitting isotopes such as .sup.15O,
.sup.13N, .sup.11C and .sup.18F are useful for positron emission
tomography (PET) studies to examine substrate receptor occupancy.
Isotopically labeled compounds of the present invention can
generally be prepared by following procedures analogous to those
disclosed in the Schemes and/or in the Examples herein below, by
substituting an isotopically labeled reagent for a non-isotopically
labeled reagent.
[0192] Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors may be
formulated in accordance with standard pharmaceutical practice for
use in a therapeutic combination for therapeutic treatment
(including prophylactic treatment) of hyperproliferative disorders
in mammals including humans. The invention provides a
pharmaceutical composition comprising trastuzumab-MCC-DM1 in
association with one or more pharmaceutically acceptable carrier,
glidant, diluent, or excipient.
[0193] Suitable carriers, diluents and excipients are well known to
those skilled in the art and include materials such as
carbohydrates, waxes, water soluble and/or swellable polymers,
hydrophilic or hydrophobic materials, gelatin, oils, solvents,
water and the like. The particular carrier, diluent or excipient
used will depend upon the means and purpose for which the compound
of the present invention is being applied. Solvents are generally
selected based on solvents recognized by persons skilled in the art
as safe (GRAS) to be administered to a mammal. In general, safe
solvents are non-toxic aqueous solvents such as water and other
non-toxic solvents that are soluble or miscible in water. Suitable
aqueous solvents include water, ethanol, propylene glycol,
polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixtures
thereof. The formulations may also include one or more buffers,
stabilizing agents, surfactants, wetting agents, lubricating
agents, emulsifiers, suspending agents, preservatives,
antioxidants, opaquing agents, glidants, processing aids,
colorants, sweeteners, perfuming agents, flavoring agents and other
known additives to provide an elegant presentation of the drug
(i.e., a compound of the present invention or pharmaceutical
composition thereof) or aid in the manufacturing of the
pharmaceutical product (i.e., medicament).
[0194] The formulations may be prepared using conventional
dissolution and mixing procedures. For example, the bulk drug
substance (i.e., compound of the present invention or stabilized
form of the compound (e.g., complex with a cyclodextrin derivative
or other known complexation agent) is dissolved in a suitable
solvent in the presence of one or more of the excipients described
above. The compound of the present invention is typically
formulated into pharmaceutical dosage forms to provide an easily
controllable dosage of the drug and to enable patient compliance
with the prescribed regimen.
[0195] The pharmaceutical composition (or formulation) for
application may be packaged in a variety of ways depending upon the
method used for administering the drug. Generally, an article for
distribution includes a container having deposited therein the
pharmaceutical formulation in an appropriate form.
[0196] Suitable containers are well known to those skilled in the
art and include materials such as bottles (plastic and glass),
sachets, ampoules, plastic bags, metal cylinders, and the like. The
container may also include a tamper-proof assemblage to prevent
indiscreet access to the contents of the package. In addition, the
container has deposited thereon a label that describes the contents
of the container. The label may also include appropriate
warnings.
[0197] Pharmaceutical formulations of the compounds of the present
invention may be prepared for various routes and types of
administration with pharmaceutically acceptable diluents, carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(1995) 18th edition, Mack Publ. Co., Easton, Pa.), in the form of a
lyophilized formulation, milled powder, or an aqueous solution.
Formulation may be conducted by mixing at ambient temperature at
the appropriate pH, and at the desired degree of purity, with
physiologically acceptable carriers, i.e., carriers that are
non-toxic to recipients at the dosages and concentrations employed.
The pH of the formulation depends mainly on the particular use and
the concentration of compound, but may range from about 3 to about
8.
[0198] The pharmaceutical formulation is preferably sterile. In
particular, formulations to be used for in vivo administration must
be sterile. Such sterilization is readily accomplished by
filtration through sterile filtration membranes.
[0199] The pharmaceutical formulation ordinarily can be stored as a
solid composition, a lyophilized formulation or as an aqueous
solution.
[0200] The pharmaceutical formulations of the invention will be
dosed and administered in a fashion, i.e., amounts, concentrations,
schedules, course, vehicles and route of administration, consistent
with good medical practice. Factors for consideration in this
context include the particular disorder being treated, the
particular mammal being treated, the clinical condition of the
individual patient, the cause of the disorder, the site of delivery
of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "therapeutically effective amount" of the compound to be
administered will be governed by such considerations, and is the
minimum amount necessary to prevent, ameliorate, or treat the
coagulation factor mediated disorder. Such amount is preferably
below the amount that is toxic to the host or renders the host
significantly more susceptible to bleeding.
[0201] Acceptable diluents, carriers, excipients and stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl, ethanol, or benzylalcohol; alkyl parabens
such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., including Tween 80,
PLURONICS.TM. or polyethylene glycol (PEG), including PEG400. The
active pharmaceutical ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
18th edition, (1995) Mack Publ. Co., Easton, Pa.
[0202] The pharmaceutical formulations include those suitable for
the administration routes detailed herein. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. Techniques
and formulations generally are found in Remington's Pharmaceutical
Sciences 18.sup.th Ed. (1995) Mack Publishing Co., Easton, Pa. Such
methods include the step of bringing into association the active
ingredient with the carrier which constitutes one or more accessory
ingredients. In general the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then,
if necessary, shaping the product.
[0203] As a general proposition, the initial pharmaceutically
effective amount of trastuzumab-MCC-DM1 administered per dose will
be in the range of about 0.01-100 mg/kg, namely about 0.1 to 20
mg/kg of patient body weight per day, with the typical initial
range of compound used being 0.3 to 15 mg/kg/day.
[0204] In a preferred embodiment, trastuzumab-MCC-DM1 is formulated
as a lyophilized powder in single-use vials containing 100 mg per
vial or 160 mg per vial, and is administered at a dose of 3.6 mg/kg
as an intravenous infusion every 3 weeks.
[0205] Pharmaceutical compositions of the anti-Bcl-2 active agent
alone, e.g. the Bcl-2 inhibitor, depend on their pharmaceutical
properties; e.g. for small chemical compounds such as e.g. ABT-737,
ABT-199 or ABT-263, one formulation could be e.g. the
following:
a) Tablet Formulation (Wet Granulation):
TABLE-US-00001 [0206] Item Ingredients mg/tablet 1. Compound of
formula (I) 5 25 100 500 2. Lactose Anhydrous DTG 125 105 30 150 3.
Sta-Rx 1500 6 6 6 30 4. Microcrystalline Cellulose 30 30 30 150 5.
Magnesium Stearate 1 1 1 1 Total 167 167 167 831
Manufacturing Procedure:
[0207] 1. Mix items 1, 2, 3 and 4 and granulate with purified
water. 2. Dry the granules at 50.degree. C. 3. Pass the granules
through suitable milling equipment. 4. Add item 5 and mix for three
minutes; compress on a suitable press.
b) Capsule Formulation:
TABLE-US-00002 [0208] Item Ingredients mg/capsule 1. Compound of
formula (I) 5 25 100 500 2. Hydrous Lactose 159 123 148 -- 3. Corn
Starch 25 35 40 70 4. Talc 10 15 10 25 5. Magnesium Stearate 1 2 2
5 Total 200 200 300 600
Manufacturing Procedure:
[0209] 1. Mix items 1, 2 and 3 in a suitable mixer for 30 minutes.
2. Add items 4 and 5 and mix for 3 minutes. 3. Fill into a suitable
capsule.
[0210] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interracial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0211] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0212] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0213] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
EXAMPLES
Example 1
[0214] Expression of Bcl-2 Family Pro-Survival Molecules in
T-DM1-Resistant Breast Cancer Cells
[0215] Preparation of T-DM1 Resistant Cell Lines
[0216] Initially, KPL-4 and BT-474M1 cells were made resistant to
T-DM1 by continuous culture in the presence of T-DM1, starting at a
very low concentration of 10 ng/mL, which was gradually increased
to 2 .mu.g/mL. The cells derived are the "resistant pools" which
were maintained in culture in 2 .mu.g/mL T-DM1. To derive stably
resistant clones, each pool (KPL-4 and BT-474M1) was subjected to
single cell sorting and cloning. Clones were maintained without
T-DM1 such that clones that had stable resistance in the absence of
T-DM1 could be identified.
[0217] TaqMan Analysis
[0218] Total RNA was prepared using Qiagen RNeasy Mini kit. Genomic
DNA was removed by DNase I. Gene expression was quantified using
real time quantitative PCR (qPCR or TaqMan). TaqMan One-Step
Universal Master Mix (Applied Biosystems) was used for all
reactions. The reaction was performed in a standard 96-well plate
format with ABI 7500 Real-Time qPCR System. 100 ng total RNA was
used as template in each reaction. For data analysis, raw Ct was
normalized to house-keeping gene HP1BP3.
[0219] The results of TaqMan analysis are presented in FIG. 1A. The
figure shows that Bcl-2 mRNA expression was increased in T-DM1
resistant KPL-4 and T-DM1 resistant BT-474M1 cell lines (normalized
to the housekeeping gene HP1BP3) relative to the parent,
non-resistant KPL-4 and BT-474M1 cells.
[0220] Western blot analysis was performed as follows: Cells were
lysed in corrected FLAG elution buffer (CFEB) (19.17 mM Tris (pH
7.5), 916.7 .mu.M MgCl2, 92.5 mM NaCl and 0.1% Triton X-100) with
protease and phosphatase inhibitors (Roche); in some cases 6 M urea
was added. Cleared lysates were quantitated and equal amounts of
proteins were reduced, alkylated, separated by SDS-PAGE, and
transferred onto PVDF membranes (Invitrogen) following standard
procedures. Western blotting was performed as recommended by the
respective antibody manufacturers. Western blot analysis shown in
FIG. 1B confirms that Bcl-2 is overexpressed in T-DM1 resistant
KPL-4 and BT-474 cell lines.
Example 2
[0221] Cell Proliferation Assay--Parental and T-DM1-Resistant KPL-4
Breast Cancer Cell Lines
[0222] The cell proliferation assay was performed for 3 days using
Cell-Titer Glo reagent. KPL-4 parental breast cancer cells and
KPL-4 T-DM1-resistant breast cancer cells, prepared as described in
Example 1, were treated with T-DM1, GDC-0199 or a combination of
T-DM1 and GDC-0199 at fixed ratios. Synergy was analyzed using the
Chou and Talalay Drug Combination Dose-Effect Analysis with
CalcuSyn software in order to obtain a Combination Index "CI" (Chou
and Talalay (1984) Adv. Enzyme Regul. 22:27-55). CI values of less
than 1 indicate synergy, while CI=1 indicated additivity.
[0223] Assay conditions: Cells were maintained in Ham's F-12: high
glucose DMEM (1:1) supplemented with 10% heat-inactivated fetal
bovine serum and 2 mM L-glutamine. Cells were plated in 96-well
plates (4000 cells per well for KPL-4 parental cells; 8000 cells
per well for KPL-4 T-DM1 resistant cells) and allowed to adhere
overnight at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. Medium was then removed and replaced by fresh culture
medium containing either T-DM1, GDC-0199, the combination of both.
Cell Titer-Glo (Promega Corp.) was added to the wells at 3 days
after drug administration and the luminescent signal was measured
using EnVision Multilabel Plate Reader (PerkinElmer). Combination
Index (C.I.) values were generated using CalcuSyn software
(Biosoft, Inc)
[0224] As shown in FIG. 2, the combination of T-DM1 and GDC-0199
has a synergistic anti-proliferative effect in KPL-4
T-DM1-resistant breast cancer cells.
Example 3
[0225] Caspase 3/7 Activation Luminescence Assay--Parental and
T-DM1-Resistant KPL-4 Breast Cancer Cell Lines
[0226] As noted before, the Bcl-2 family of proteins regulates
programmed cell death triggered by developmental cues and in
response to multiple stress signals. When activated, they can
permeabilize the outer membrane of mitochondria and release
pro-apoptogenic factors (e.g. cytochrome C) needed to activate the
caspases that dismantle the cell (Wang, K., Genes and Development
15 (2001) 2922-2933; (Adams, 2003 supra); Green, D. R., and
Kroemer, G., Science 305 (2004) 626-629). Thus, activation of
caspases, such as caspases 3 and 7, indicates induction of
apoptosis.
[0227] In the present experiment, caspase 3/7 activation
luminescence assay was performed using the Caspase-Glo.RTM. reagent
(Promega) essentially following manufacturer's instructions. Assays
were performed in the same manner as the viability assays except
that drug incubation times were 24 hours and Caspase-Glo 3/7
(Promega) was used to measure apoptosis.
[0228] As shown in FIG. 3, right panel, T-DM1-resistant KPL-4
(HER2+) breast cancer cells were re-sensitized to T-DM1 when
treated for 24 hours with a combination of T-DM1+GDC-0199, as shown
by increased apoptosis (increased caspase 3/7 activation). The same
combination showed no effect in parental cells. It is noted that
the results were assessed 24 hours after treatment, which is too
early for apoptosis induced by T-DM1 alone in the parental cell
line (left panel).
[0229] FIGS. 4A and 4B presents the results of a caspase 3/7
activation luminescence in vitro apoptosis assay measuring
activation of caspases 3 and 7 in KPL-4 T-DM1-resistant human
breast cancer cells relative to parental cells using different
concentrations of T-DM1 and GDC-0199, respectively. Increased
apoptosis is observed in KPL-4 T-DM1 resistant cells upon the
addition of increasing concentrations of GDC-0199 (1, 2.5 or 5
.mu.M) to either 0.1 or 1 .mu.g/mL T-DM1. In contrast, T-DM1
induces robust apoptosis in KPL-4 parental cells which is not
further enhanced by the addition of GDC-199.
[0230] FIG. 5A presents the results of a caspase 3/7 activation
luminescence in vitro apoptosis assay measuring activation of
caspases 3 and 7 in Clone #17 T-DM1-resistant KPL-4 human breast
cancer cell line treated with T-DM1, GDC-0199 or T-DM1+GDC-0199.
Similar to observations with the KPL-4 T-DM1 resistant pool of
cells, induction of apoptosis in Clone #17 was increased upon the
addition of increasing concentrations of GDC-0199.
[0231] FIGS. 6A and 6B show the results of a caspase 3/7 activation
luminescence in vitro apoptosis assay measuring activation of
caspases 3 and 7 in Clone #8 T-DM1 resistant KPL-4 human breast
cell line treated with 0.1 .mu.g/mL and 1 .mu.g/mL T-DM1
concentrations, respectively, alone or in combination with the
indicated concentrations of GDC-0199.
[0232] As shown in FIGS. 4A, 4B, 5A, 6A and 6B, the results
obtained with different clones of T-DM1-resistant KPL-4 breast
cancer cell lines confirm the enhanced proapoptotic activity of the
T-DM-1+GDC-0199 combination at various concentrations.
Example 4
[0233] Xenograft Studies--KPL-4 T-DM1-Resistant Breast Cancer Cell
Lines
[0234] For all xenograft studies, three million T-DM1-resistant
KPL-4 breast cancer cells were implanted in #2/3 mammary fat pads
of female SCID-beige mice. When tumors reached a volume of
approximately 200 mm.sup.3, mice were randomized into treatment
groups (n=10 mice pre group): 5 mg/kg T-DM1 q3w, 100 mg/kg GDC-0199
qd, combination of the two or vehicle. The results of these
xenograft studies for xenografts of various clones of the
T-DM1-resistant KPL-4 breast cancer cells are shown in FIGS. 5B and
6C. The results indicate enhanced anti-tumor effect when T-DM1 and
GDC-0199 were used in combination, relative to single agent
activity.
Example 5
[0235] IHC Studies--T-DM1-Resistant KPL-4 Xenograft Tumors
[0236] FFPE (formalin-fixed paraffin-embedded) xenograft tumors
were sectioned for analysis of Bcl-2 and HER2 (ErbB2) expression by
immunohistochemistry (IHC), using DAB detection method. Bcl-2
antibody SP66 was obtained from Ventana. Human tonsil sections
served as Bcl-2 positive controls. Anti-HER2 antibody 4D5 was
obtained from Ventana. Human breast cancer cell lines served as
positive controls (SK-BR-3 as 3+; MDA-MB-361 as 2+; MBA-MB-231 as
negative).
[0237] FIG. 7A shows the expression of Bcl-2 in formalin-fixed
paraffin-embedded T-DM1-resistant KPL-4 xenograft tumor samples
(Clones #8 and #17) determined by immunohistochemistry (IHC), using
DAB detection method, as described above.
[0238] FIG. 7B shows the expression of HER2 (ErbB2) in
formalin-fixed paraffin-embedded T-DM1-resistant KPL-4 xenograft
tumor samples (Clones #8 and #17) determined by
immunohistochemistry (IHC), using DAB detection method, as
described above.
[0239] Anti-Bcl-2 antibody results: Vehicle groups of each clone
showed similar low frequency of Bcl-2 reactive cells, most often
located at the perimeter of tumor lobules (not shown). The
frequency and intensity of the Bcl-2 signal at the tumor lobule
margins in the T-DM1 treated groups was increased or not
changed.
[0240] Anti-HER2 antibody results: Vehicle and T-DM I treated
tumors in all clones showed very high frequency of HER2 3+IHC. In
some T-DM1 treated tumors, there were regions of weaker HER2
staining (clone #17), most often adjacent to the stromal bands
surrounding the tumor lobules (not shown).
[0241] The Bcl-2 IHC results demonstrate that Bcl-2 expression is
maintained in the T-DM1 resistant clones #8 and #17 when grown as
xenograft tumors (FIG. 7A; see also Example 6 which shows very
little Bcl-2expression in KPL-4 parental cells by Western blot).
Bcl-2 expression in T-DM1-treated clone #17 is higher than the
corresponding vehicle control. FIG. 7B depicts HER2 expression as
assessed by IHC. In contrast to the relatively lower HER2
expression observed in cells grown in vitro, clones #8 and #17, in
both vehicle and T-DM1-treated tumors, show high HER2 expression at
the 2+ and 3+level. All clone #8 tumors were determined to be
85-95% HER2+ or 3+, with a very low frequency of 2+ or 1+tumor
cells. Clone #17 tumors were more variable, with 35-75% HER2
3+cells and 20-65% cells HER2 2+ in the vehicle group.
Example 6
[0242] Xenograft Studies--KPL-4 T-DM1-Resistant Breast Tumors
[0243] FIG. 8 shows Western blot expression data of Bcl-2, HER2,
Bcl-xL and Pgp in KPL-4 T-DM1-resistant clone #17 xenograft tumors
treated with GDC-0199, T-DM1 or T-DM-1+GDC-0199. (The three digit
numbers above lanes 4-19 indicated individual xenograft tumors.)
The expression data show that HER2 and Bcl-2 expression are
maintained in all groups, as compared to the corresponding cells
grown in vitro in cell culture.
Example 7
[0244] Caspase 3/7 Luminescence and Fluorescence Activation
Assays--T-DM1-Sensitive Breast Cancer Cell Lines
[0245] Caspase 3/7 activation luminescence assay was performed as
described in Example 3.
[0246] The caspase 3 activation fluorescence in vitro apoptosis
assay was performed using IncuCyte.TM. reagents and equipment to
measure caspase activation over time (kinetic analysis) essentially
following manufacturing instructions.
[0247] FIG. 9A presents results of a caspase 3/7 luminescence in
vitro apoptosis assay, testing the effect of five separate
concentrations of GDC-0199 (.mu.M) in combination with 9 different
concentrations of T-DM1 on caspase activity in HER2+MDA-MB-361
breast cancer cells, which are sensitive to T-DM1 (naive). The
results demonstrate caspases 3 and 7 activation with T-DM1 which is
enhanced in a dose-dependent manner with increasing concentrations
of GDC-0199.
[0248] FIG. 9B presents the results of a caspase 3 fluorescence in
vitro apoptosis assay, testing the effect of three different
concentrations of GDC-0199 (0.63 .mu.M, 1.25 .mu.M, 2.5 .mu.M),
alone and in combination with T-DM1 (0.1 .mu.g/mL), on caspase
activity in HER2+T-DM1 sensitive (naive) MDA-MB-361 breast cancer
cells. The results demonstrate that GDC-0199 enhances caspase
activation above that induced by T-DM1 alone in a dose- and
time-dependent manner, and therefore results in enhanced apoptosis
with all combinations.
[0249] FIG. 10A presents the results of a caspase 3/7 luminescence
in vitro apoptosis assay, testing the effect of five separate
concentrations of GDC-0199 (.mu.M) in combination with 9 different
concentrations of T-DM1 on caspase activity in T-DM1 naive
HER2+HCC1569 breast cancer cells. The results demonstrate that
T-DM1 alone does not induce apoptosis but addition of GDC-0199
results in enhanced caspase activity and hence enhanced apoptosis
in all combinations.
[0250] FIG. 10B presents the results of a caspase 3 fluorescence in
vitro apoptosis assay, testing the effect of three different
concentrations of GDC-0199 (0.63 .mu.M, 1.25 .mu.M, 2.5 .mu.M),
alone and in combination with T-DM1 (0.1 .mu.g/mL), on caspase
activity in HER2+HCC1569 breast cancer cells. The results
demonstrate that GDC-0199 enhances caspase activation above that
induced by T-DM1 alone in a dose- and time-dependent manner, and
therefore results in enhanced apoptosis with all combinations.
[0251] These results show that the T-DM1/GDC-0199 combination is
also effective in T-DM1 naive (i.e. not T-DM1 resistant) cell
lines.
Example 8
[0252] Xenograft Studies--TDM-1-Sensitive (Naive) MDA-MB-361 Breast
Tumors
[0253] Ten million MDA-MB-361 breast cancer cells were implanted
into the right mammary fat pad of female NOD/SCID mice one day
after implantation of 60-day release 17p-estradiol pellets. When
tumors reached a volume of approximately 200-300 mm.sup.3, mice
were randomized into treatment groups (n=10 mice per group) and
administered T-DM1 (1, 3, or 7 mg/kg i.v. once), GDC-0199 (100
mg/kg qd.times.21) or a combination of T-DM1 and GDC-0199 as shown
in FIG. 11. The results indicate enhanced anti-tumor activity of
GDC-0199 with 7 mg/kg T-DM1, relative to single agent activity.
Example 9
[0254] Western Analysis: Effects of T-DM1+/-GDC-0199 on Bcl-2
Family Member Proteins in HER2+Breast Cancer Cell Lines
[0255] The effect of treatment with T-DM1 (1.25 .mu.g/mL) alone or
in combination with GDC-0199 (1.25 .mu.M) was studied on various
T-DM1 naive HER2+breast cancer cell lines. The results are shown in
FIG. 12. Four out of the eight HER2+breast cancer cell lines tested
(BT-474, HCC1569, MDA-361 and ZR-75-30) expressed Bcl-2; all eight
breast cancer cell lines expressed the other Bcl-2 family members
assessed-Bcl-xL and Mcl-. Three of the cell lines (BT-474, MDA_361
and ZR-75-30) showed phosphorylation of Bcl-2 after T-DM1
treatment, a known effect of exposure to anti-mitotic agents such
as T-DM1. As shown in FIGS. 9A, 9B, 10A and 10B, MDA-MB-361 and
HCC1569 showed enhanced apoptosis when treated with a combination
of T-DM1 and GDC-0199.
[0256] T-DM1 (KADCYLA.RTM.) exhibits significant clinical benefits
in the treatment of cancer for patients, such as breast cancer
patients, who have progressed on prior HER2-targeted therapies,
such as on treatment with trastuzumab (HERCEPTIN.RTM.). The U.S.
Food and Drug Administration approved KADCYLA.RTM. (ado-trastuzumab
emtansine), for the treatment of patients with HER2-positive,
metastatic breast cancer who previously received treatment with
trastuzumab and a taxane. The data presented here demonstrate that
combination treatment with a Bcl (e.g. Bcl-2) inhibitor and T-DM1
significantly improves the efficacy of T-DM1 administered as a
single agent. The results also demonstrate that such combination
treatment with T-DM I and a Bcl-2 inhibitor is effective both in
the treatment of T-DM1 sensitive (naive) HER2 positive cancers
(e.g. breast cancers) and HER2 positive cancers (e.g. breast
cancers) resistant to treatment with T-DM1.
[0257] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and examples for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
21214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 2102449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly
Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly
* * * * *