Combination therapy of anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor

Abstract

The present invention relates to a combination therapy involving an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for treating patients suffering from cancer, particularly cancers expressing HER-2.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 6, 2016, application number 201680040184.1 (international application number PCT / US2016 / 041184), and name “Combination therapy of anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor”.

[0002] Sequence Listing

[0003] This application contains a sequence listing submitted electronically in ASCII format, and the entire contents of which are incorporated herein by reference. The ASCII copy created on June 28, 2016 is named GNE-0416-WO.txt and is 6,298 bytes in size. Technical Field

[0004] The present invention relates to a combination therapy involving an anti-HER2 antibody-drug conjugate and a Bcl-2 inhibitor for treating cancer. In a specific embodiment, the present invention relates to the use of trastuzumab-MCC-DM1 (trastuzumab emtansine); ) and a selective Bcl-2 inhibitor to treat HER2-positive cancers, such as HER2-positive breast cancer or gastric cancer. Background Art

[0005] Anti-HER2 antibody-drug conjugates

[0006] 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 about 20% of human breast cancer (hereinafter referred to as HER2-positive breast cancer), and is related to 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 fixed tumor block assessment based on immunohistochemistry (Press MF, et al (1993) Cancer Res 53: 4960-70).

[0007] Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1κ monoclonal antibody that is a humanized form of the murine anti-HER2 antibody (4D5) that selectively binds to the extracellular domain of HER2 with high affinity (Kd=5 nM) in a cell-based assay (US5677171; US5821337; US6054297; US6165464; US6339142; US6407213; US6639055; US6719971; US6800738; US7074404; 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 to inhibit the proliferation of human tumor cells that overexpress HER2 in both in vitro assays and in animals (Hudziaket 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 MD, 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).

[0008] It was approved in 1998 for the treatment of patients with metastatic breast cancer that overexpresses HER2 who have been treated with a wide range of existing anticancer therapies (Baselga et al, (1996) J. Clin. Oncol. 14:737-744) and has been used in more than 300,000 patients (Slamon DJ, et al. N Engl J Med 2001;344:783-92; Vogel CL, et al. J Clin Oncol 2002;20:719-26; Marty M, et al. J Clin Oncol 2005;23:4265-74; Romond EH, et al. TN Engl J Med 2005;353:1673-84; Piccart-Gebhart MJ, 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, FDA approved (Trastuzumab, Genentech Inc.) is indicated as part of a regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of patients with HER2-positive, node-positive breast cancer.

[0009] An alternative approach to antibody-targeted therapy is to use antibodies to specifically deliver cytotoxic drugs to cancer cells that express an antigen. Antibody-drug conjugates, or ADCs, are monoclonal antibodies that have been conjugated to highly potent cytotoxic agents. ADCs represent a new approach to confer tumor selectivity to systemically administered anti-tumor therapeutics. ADCs are designed to focus the delivery of highly potent cytotoxic agents to tumor cells, taking advantage of tumor-specific and / or overexpressed surface antigens. The potential of this approach is to create a more favorable therapeutic window for these drugs than can be achieved by administering them as free drugs.

[0010] Maytansinoids are derivatives of the antimitotic drug maytansine that bind to microtubules in a manner similar to vinca alkaloid drugs (Issell BF 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 LI, Howie GA, et al (1975) Science 189 (4207): 1002-1005.3; Cassady JM, Chan KK, Floss HG. (2004) ChemPharm Bull 52 (1): 1-26.4). The related plant ester maytansine has been studied in about 800 patients as a chemotherapeutic agent at a dose of 2.0 mg / m2 once every 3 weeks as a single dose or for 3 consecutive days (Issell BF, Crooke ST. (1978) Maytansine. Cancer Treat Rev 5: 199-207). Despite preclinical activity, the activity of maytansine in the clinic is modest at doses that can be safely delivered. Dose-limiting toxicity (DLT) is gastrointestinal toxicity, including nausea, vomiting and diarrhea (usually followed by constipation). These toxicities are dose-dependent, but not schedule-dependent. Peripheral neuropathy (mainly sensory) has been reported and is most pronounced in patients with preexisting neuropathy. Subclinical transient increases in liver transaminases, alkaline phosphatase and total bilirubin have been reported. Constitutive toxicities including weakness, drowsiness, irritability and insomnia are common. Less common toxicities include infusion site phlebitis and mild bone marrow suppression. Due to the narrow therapeutic window, further development of the drug was abandoned in the 1980s.

[0011] Trastuzumab-MCC-DM1 (T-DM1, trastuzumab emtansine conjugate, ado-trastuzumab emtansine conjugate, ), a novel antibody-drug conjugate (ADC) for the treatment of HER2-positive breast cancer, consists of the cytotoxic agent DM1 (a thiol-containing maytansinoid anti-microtubule agent) conjugated to trastuzumab at the lysine side chain via an MCC linker, with an average drug loading (drug / antibody ratio) of 3.5. After binding to HER2 expressed on tumor cells, T-DM1 undergoes receptor-mediated internalization, leading to the intracellular release of DM1-containing cytotoxic catabolites and subsequent cell death.

[0012] 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. DLT (dose-limiting toxicity) consisted of transient thrombocytopenia in patients treated with 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). The same study also showed that administration of 2.4 mg / kg per week was also well tolerated and had antitumor activity (Beeram (2012) Cancer 118 (23): 5733-5740).

[0013] A phase II study (TDM4374g) showed that T-DM1 administered at 3.6 mg / kg q3w had single-agent antitumor activity in a heavily pretreated patient population with HER2-positive metastatic breast cancer (Krop (2012) 30(26):3234-3241). A phase III study (TDM4370g) showed that T-DM1 administered at 3.6 mg / kg q3w significantly prolonged progression-free survival and overall survival with less toxicity compared with treatment with lapatinib + 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).

[0014] On February 22, 2013, the U.S. Food and Drug Administration approved the ado-trastuzumab emtansine conjugate under the trade name Marketed for the treatment of patients with HER2-positive metastatic breast cancer who have previously received trastuzumab and a taxane.

[0015] Bcl-2 inhibitors

[0016] Bcl-2 family proteins regulate programmed cell death triggered by developmental cues and in response to multiple stress signals (Cory. S., and Adams, JM, Nature Reviews Cancer 2 (2002) 647-656; Adams, Genes und Development 17 (2003) 2481-2495; Danial, NN, and Korsmeyer, SJ, Cell 116 (2004) 205-219). While Bcl-2 itself and several close relatives (Bcl-xL, Bcl-W, Mcl-1 and Al) that carry three or four conserved Bcl-2 homology (BH) regions promote cell survival, apoptosis is driven by two other subfamilies. The initial signal for cell death is transmitted by a different group of BH3-only proteins, including Bad, Bid, Bim, Puma and Noxa, which usually have only a small BH3 interaction domain (Huang and Strasser, Cell 103 (2000) 839-842). However, the multidomain proteins Bax or Bak containing BH1-BH3 are essential for cell death (Cheng, et al., Molecular Cell 8 (2001) 705-711; Wei, MC, et al., Science 292 (2001) 727-730; Zong, WX, et al., Genes and Development 15 148 (2001) 1-1486). When activated, they can penetrate the outer membrane of mitochondria and release pro-apoptotic factors (such as cytochrome C) required to activate caspases that disassemble the cell (Wang, K., Genes and Development 15 (2001) 2922-2933; (Adams, 2003 supra); Green, DR, and Kroemer, G., Science 305 (2004) 626-629).

[0017] The interaction between the three members of the Bcl-2 family determines whether a cell lives or dies. When BH3-only proteins are activated, for example, in response to DNA damage, they can bind to the groove on their pro-activation related substances via their BH3 domains (Sattler, et al., Science 275 (1997) 983-986). However, how BH3-only proteins and Bcl-2-like proteins control the activation of Bax and Bak is still not well understood (Adams, 2003 supra). Most of the attention is focused on Bax. This soluble monomeric protein (Hsu, YT, et al., Journal of Biological Chemistry 272 (1997) 13289-13834; Wolter, KG, et al., Journal of Cell Biology 139 (1997) 1281-92) usually has a membrane targeting domain inserted into its groove, which may be responsible for its cytoplasmic 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, as reviewed in Lucken-Ardjomande, S., and Martinou, JC, J Cell Sci 118 (2005) 473-483, but their physiological relevance remains to be determined. Alternatively, Bax can be activated by direct binding to certain BH3-only domain proteins (Lucken-Ardjomande, S., and Martinou, JC, 2005 supra), best documented as a truncated form of Bid, tBid (Wei, MC, 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, PF, 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, ZN, et al., Cell 74 (1993) 609-619) has been problematic because Bcl-2 is membrane-bound and Bax is cytosolic, and their interaction appears to be highly dependent on the detergent used for cell lysis (Hsu, YT, and Youle, 1997 supra). Nevertheless, it is recognized that the BH3 region of Bax can mediate binding to 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 Bcl-2 prevents Bax oligomerization, even though heterodimers cannot be detected (Mikhailov, V., et al., Journal of Biological Chemistry 276 (2001) 18361-18374). Therefore, it remains uncertain whether activin directly or indirectly limits Bax activation.

[0018] Although Bax and Bak appear to be functionally equivalent in most cases (Lindsten, T., et al., Molecular Cell 6 (2000) 1389-1399; Wei, MC, et al., 2001 supra), substantial differences in their regulation are expected from their unique localization in healthy cells. Unlike Bax (which is mainly cytosolic), Bak is located in a complex on the outer membrane of mitochondria and on the endoplasmic reticulum of healthy cells (Wei, MC, et al., 2000 supra; Zong, WX, et al., Journal of Cell Biology 162 (2003) 59-69). Nevertheless, upon receiving cytotoxic signals, both Bax and Bak change conformation and Bax translocates to the organelle membrane, where both Bax and Bak then form associative homo-oligomers, leading to membrane permeabilization (Hsu, YT, et al., PNAS 94 (1997) 3668-3672; Wolter, KG, 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, MC, et al., 2001 supra; Mikhailov, V., et al., Journal of Biological Chemistry 278 (2003) 5367-5376).

[0019] There are a variety of Bcl-2 inhibitors, all of which have the same property of inhibiting the pro-activating members of the Bcl-2 family of proteins and are therefore promising candidates for the treatment of cancer. Such Bcl-2 inhibitors are, for example, 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 A3, HA-14-1, KF-67544, Purpurogallin, TP-TW-37, YC-137 and Z-24, and are described, for example, in Zhai, D., et al., Cell Death and Differentiation 13 (2006) 1419-1421.

[0020] The link between other Bcl-2 family proteins and cancer is also well established and well documented (Strasser, A. 2011 EMBO J. 30, 3667-3683), and inhibitors of other Bcl family members are also known. L Selective inhibitors A-1155463 and A-1331852 are described, for example, in Leverson et al., Science Translational Medicine Vol 7, Issue 279 279ra40. L Selective benzothiazole hydrazone inhibitors are disclosed in Sleebs et al., J. Med. Chem. 2013, 56, 5514-5540. For other Bcl-X LInhibitors are described, for example, in Koehler et al., ACS Med. Chem. Lett. 2014, 5, 662-667; and Tao et al, ACS Med. Chem. Lett. 2014, 5, 1088-10. MCI-1 inhibitors and their use 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., Bioorganic & Medicinal Chemistry Letters 24 (2014) 1484-1488; Abulwerdi et al., Mol Cancer Ther 2014; 13:565-5; Abulwerdi 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. al., Pharmacology & Therapeutics 145 (2015) 76-84. Mcl-1 / Bcl-xL dual inhibitors were disclosed by Tanaka et al., J. Med. Chem. 2013, 56, 9635-9645. Summary of the invention

[0021] In one aspect, the invention relates to a method of treating cancer in a human in need thereof, comprising administering to the human a therapeutic amount of an anti-HER2 antibody-drug conjugate and an inhibitor of a Bcl family protein.

[0022] In another aspect, the invention relates to a method of treating cancer in a human in need thereof, comprising administering to such human a therapeutic amount of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor.

[0023] In one embodiment, the cancer is a HER2-positive cancer.

[0024] In another embodiment, the cancer is HER2-positive breast cancer or gastric cancer.

[0025] 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 of ≥2.0 (her2:CEP17 in situ hybridization (ISH) amplification ratio).

[0026] In another embodiment, the HER2-positive cancer, such as breast cancer or gastric cancer, is resistant to treatment with the anti-HER2 antibody-drug conjugate administered as a single agent.

[0027] In another embodiment, the HER2-positive cancer, such as breast cancer or gastric cancer, is sensitive to treatment with the 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 patients who have not tolerated treatment with the anti-HER2 antibody-drug conjugate.

[0028] In various embodiments, the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor exhibit synergistic activity, including, but not limited to, synergistic activity in HER2-positive cancers (such as breast cancer or gastric cancer) that are resistant to treatment with the anti-HER2 antibody-drug conjugate administered as a single agent.

[0029] In all embodiments, the anti-HER2 antibody-drug conjugate can be, for example, trastuzumab-MCC-DM1.

[0030] In all embodiments, the selective Bcl-2 inhibitor can be, for example, 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0031] In another aspect, the invention relates to a method of treating HER-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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0032] In one embodiment, the cancer is HER2-positive breast cancer or gastric cancer.

[0033] 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 of ≥2.0 (her2:CEP17 in situ hybridization (ISH) amplification ratio).

[0034] In 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.

[0035] In another 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 a combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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 who has not tolerated treatment with trastuzumab-MCC-DM1.

[0036] In another 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 patients who have not tolerated treatment with the anti-HER2 antibody-drug conjugate. In another embodiment, trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof show synergistic activity, including, but not limited to, synergistic activity in HER2-positive cancers such as breast cancer or gastric cancer.

[0037] In another embodiment, trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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.

[0038] In another embodiment, trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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.

[0039] In another embodiment, trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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.

[0040] In another aspect, the present invention relates to 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 treating cancer.

[0041] 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.

[0042] In another aspect, the present invention relates to 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 treating cancer.

[0043] In one embodiment, the present invention relates to 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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer.

[0044] In all embodiments, the cancer may be a HER2-positive cancer.

[0045] In all embodiments, the cancer may be, for example, breast cancer or gastric cancer.

[0046] 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.

[0047] 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 trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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 used to treat patients who have not tolerated treatment with trastuzumab-MCC-DM1.

[0048] In another aspect, the present invention relates to 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 treating cancer.

[0049] 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.

[0050] In another aspect, the invention relates to a combination of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for use in treating cancer.

[0051] In one embodiment, the combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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 used to treat cancer.

[0052] In all combinations, the cancer can be, for example, a HER2-positive cancer, such as HER2-positive breast cancer or gastric cancer.

[0053] In a specific embodiment, the cancer, such as breast cancer or gastric cancer, may be resistant to treatment with the anti-HER2 antibody-drug conjugate or trastuzumab-MCC-DM1 when administered as a single agent.

[0054] In another embodiment, the HER2-positive cancer, such as breast cancer or gastric cancer, may be sensitive to treatment with an anti-HER2 antibody-drug conjugate, such as trastuzumab-MCC-DM1, when administered as a single agent, and the combination can be used to treat patients who have not tolerated treatment with an anti-HER2 antibody-drug conjugate, such as trastuzumab-MCC-DM1.

[0055] In another aspect, the present invention relates to a method for diagnosing that a HER2-positive tumor is resistant to treatment with a HER2 antibody-drug conjugate, comprising determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to the expression level in a control sample, and diagnosing that the cancer is resistant to treatment with the anti-HER2 antibody-drug conjugate when the expression level in the tumor sample is at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher than the expression level in the control sample.

[0056] In another aspect, the present invention relates to a method for diagnosing that a HER2-positive tumor is susceptible to treatment with a HER2 antibody-drug conjugate, comprising determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to the expression level in a control sample, and diagnosing that the cancer is susceptible to treatment with the anti-HER2 antibody-drug conjugate when the expression level in the tumor sample is at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher than the expression level in the control sample.

[0057] In another aspect, the present invention relates to a method for diagnosing that a subject having a HER2-positive tumor is resistant or susceptible to treatment with an anti-HER2 antibody-drug conjugate, comprising (i) obtaining a tumor sample from the subject, (ii) measuring the expression level of a Bcl-2 gene or its product in the tumor sample relative to the expression level in a control sample, and (iii) diagnosing that the tumor is resistant to treatment with an anti-HER2 antibody-drug conjugate when the Bcl-2 expression level measured in the tumor sample is at least 2 times, or at least 3 times, or at least 4 times, or at least 5 times higher than the expression level in the control sample, or diagnosing that the tumor is susceptible to treatment with an anti-HER2 antibody-drug conjugate when the Bcl-2 expression level measured in the tumor sample is less than 2 times, or less than 3 times, or less than 4 times, or less than 5 times higher than the expression level in the control sample.

[0058] In one embodiment, the subject is a human patient.

[0059] In another embodiment, the control sample is a tumor sample of the same cell type that is not resistant to treatment with the anti-HER2 antibody-drug conjugate.

[0060] In another embodiment, the tumor is breast cancer or gastric cancer.

[0061] In another embodiment, the tumor sample is a formalin-fixed, paraffin-embedded tumor sample.

[0062] In all embodiments, the diagnostic method may further comprise the step of measuring the expression level of the HER2 gene or its product in the tumor sample.

[0063] In all embodiments, the diagnostic method may further include 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 times, or at least 3 times, or at least 4 times, or at least 5 times higher than the expression level in the control sample.

[0064] In all embodiments, the diagnostic method may further comprise the step of treating the patient with an anti-HER2 antibody-drug conjugate when the expression level of Bcl-2 measured in the tumor sample is less than 2-fold lower than the expression level in the control.

[0065] In one embodiment, the anti-HER2 antibody-drug conjugate is trastuzumab-MCC-DM1.

[0066] In another embodiment, the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0067] In another aspect, the present invention relates to a kit for in vitro diagnosis or prognosis of HER2-positive tumors resistant to treatment with an anti-HER2 antibody-drug conjugate in a biological sample obtained from a patient, comprising a specific binding partner for the Bcl-2 gene or its expression product.

[0068] In one embodiment, the binding partner is an anti-Bcl-2 antibody

[0069] In another embodiment, the binding partner is a nucleic acid that hybridizes to the Bcl-2 gene.

[0070] In another aspect, the invention relates to a kit comprising an anti-HER2 antibody drug conjugate and a selective Bcl-2 inhibitor for use in combination treatment of a patient having a HER2-expressing cancer.

[0071] In one embodiment of the invention, the kit further comprises a pharmaceutically acceptable carrier. The kit may further comprise a sterile diluent, which is preferably stored in a separate additional container. The kit may further comprise a package insert, which comprises printed instructions for use of the combination therapy as a method for expressing HER2 cancers such as HER2-expressing breast cancer or gastric cancer.

[0072] As in other aspects, the HER2-expressing cancer can be, for example, breast cancer or gastric cancer, and in various embodiments, the anti-HER2 antibody drug conjugate can be trastuzumab-MCC-DM1 and / or the selective Bcl-2 inhibitor can be 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0073] In a specific embodiment, the HER2-expressing cancer to be treated, such as breast cancer or gastric cancer, is resistant to treatment with an anti-HER2 antibody-drug conjugate or trastuzumab-MCC-DM1 when administered as a single agent.

[0074] In another embodiment, the HER2-expressing cancer, such as breast cancer or gastric cancer, may be sensitive to treatment with an anti-HER2 antibody-drug conjugate (e.g., trastuzumab-MCC-DM1) when administered as a single agent, and the combination can be used to treat patients who are resistant to treatment with an anti-HER2 antibody-drug conjugate (e.g., trastuzumab-MCC-DM1).

[0075] The present invention also includes the following items:

[0076] 1. A method of treating cancer in a human in need thereof, comprising administering to the human an effective amount of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor.

[0077] 2. The method of claim 1 , wherein the cancer is a HER2-positive cancer.

[0078] 3. The method of claim 2, wherein the cancer is HER2-positive breast cancer or gastric cancer.

[0079] 4. The method of claim 3, wherein the HER2-positive breast cancer or gastric cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification rate of ≥2.0.

[0080] 5. The method of any one of items 1 to 4, wherein the HER2-positive cancer is resistant to treatment with the anti-HER2 antibody-drug conjugate administered as a single agent.

[0081] 6. The method of any one of items 1 to 4, wherein the HER2-positive cancer is sensitive to treatment with the anti-HER2 antibody-drug conjugate administered as a single agent.

[0082] 7. The method of claim 5 or claim 6, wherein the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor show synergistic activity.

[0083] 8. The method of any one of items 1 to 7, wherein the anti-HER2 antibody-drug conjugate is trastuzumab-MCC-DM1.

[0084] 9. The method of any one of items 1 to 8, wherein the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0085] 10. The method of any one of items 1 to 9, wherein the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor are administered as a combined preparation or are administered alternately.

[0086] 11. A method of treating HER-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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0087] 12. The method of claim 11, wherein the cancer is HER2-positive breast cancer or gastric cancer.

[0088] 13. The method of claim 12, wherein the HER2-positive breast cancer or gastric cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification rate of ≥2.0.

[0089] 14. The method of any one of items 11 to 13, wherein the HER2-positive cancer is resistant to treatment with trastuzumab-MCC-DM1 administered as a single agent.

[0090] 15. The method of any one of items 11 to 13, wherein the HER2-positive cancer is sensitive to treatment with the anti-HER2 antibody-drug conjugate administered as a single agent.

[0091] 16. The method of any one of items 11 to 15, wherein the trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof show synergistic activity.

[0092] 17. The method of any one of items 11 to 16, wherein the trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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.

[0093] 18. The method of claim 17, wherein the trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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 as a combined formulation or alternately.

[0094] 19. The method of claim 18, wherein the trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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.

[0095] 20. The method of claim 18, wherein the trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-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 continuously.

[0096] 21. Use of a combination of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor in the preparation of a medicament for treating cancer.

[0097] 22. 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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer.

[0098] 23. A combination of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for use in treating cancer.

[0099] 24. A combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof for use in treating cancer.

[0100] 25. The combination of item 24, which is a pharmaceutical composition.

[0101] 26. The use of item 21 or 22, or the combination of item 23 or 25, wherein the cancer is a HER2-positive cancer.

[0102] 27. The use or combination of claim 26, wherein the cancer is HER2-positive breast cancer or gastric cancer.

[0103] 28. The use or combination of claim 26, wherein the cancer is resistant to treatment with the anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1 when administered as a single agent.

[0104] 29. The use or combination of claim 26, wherein the cancer is sensitive to treatment with the anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1 when administered as a single agent.

[0105] 30. A kit comprising a combination of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for treating a cancer having HER2 expression.

[0106] 31. The kit of claim 30, wherein the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor are in the form of separate preparations.

[0107] 32. The kit of claim 30, wherein the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor are in the form of the same formulation.

[0108] 33. The kit of any one of items 30 to 32, further comprising a package insert directing administration of the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor to a human having a cancer that expresses HER2.

[0109] 34. The kit of claim 33, wherein the cancer is resistant to treatment with the anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1 when administered as a single agent.

[0110] 35. The kit of claim 33, wherein the cancer is sensitive to treatment with the anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1 when administered as a single agent.

[0111] 36. A method for diagnosing that a HER2-positive tumor is resistant to treatment with a HER2 antibody-drug conjugate, comprising determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to the expression level in a control sample, and diagnosing that the cancer is resistant to treatment with the anti-HER2 antibody-drug conjugate when the expression level in the tumor sample is at least 2 times higher than the expression level in the control sample.

[0112] 37. A method for diagnosing that a HER2-positive tumor is susceptible to treatment with a HER2 antibody-drug conjugate, comprising determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to the expression level in a control sample, and diagnosing that the cancer is susceptible to treatment with the anti-HER2 antibody-drug conjugate when the expression level in the tumor sample is at least 2 times higher than the expression level in the control sample.

[0113] 38. A method for diagnosing that a subject having a HER2-positive tumor is resistant or susceptible to treatment with an anti-HER2 antibody-drug conjugate, comprising (i) obtaining a tumor sample from the subject, (ii) measuring the expression level of a Bcl-2 gene or its product in the tumor sample relative to the expression level in a control sample, and (iii) diagnosing that the tumor is resistant to treatment with an anti-HER2 antibody-drug conjugate when the Bcl-2 expression level measured in the tumor sample is at least 2 times higher than the expression level in the control sample, or diagnosing that the tumor is susceptible to treatment with an anti-HER2 antibody-drug conjugate when the Bcl-2 expression level measured in the tumor sample is less than 2 times higher than the expression level in the control sample.

[0114] 39. The method of claim 38, wherein the subject is a human patient.

[0115] 40. The method of any one of items 36 to 39, wherein the control sample is a tumor sample of the same cell type that is not resistant to treatment with the anti-HER2 antibody-drug conjugate.

[0116] 41. The method of any one of items 36 to 39, wherein the tumor is breast cancer or gastric cancer.

[0117] 42. The method of any one of items 36 to 39, wherein the tumor sample is a formalin-fixed, paraffin-embedded tumor sample.

[0118] 43. The method of any one of items 36 to 42, further comprising a step of measuring the expression level of the HER2 gene or its product in the tumor sample.

[0119] 44. The method of any one of items 36 to 43, further comprising the 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 times higher than the expression level in the control sample.

[0120] 45. The method of any one of items 36 to 43, further comprising the step of treating the patient with an anti-HER2 antibody-drug conjugate when the expression level of Bcl-2 measured in the tumor sample is less than 2-fold lower than the expression level in the control.

[0121] 46. ​​The method of claim 44 or 45, wherein the anti-HER2 antibody-drug conjugate is trastuzumab-MCC-DM1.

[0122] 47. The method of item 44 or 46, wherein the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0123] 48. A kit for in vitro diagnosis or prognosis of HER2-positive tumors resistant to treatment with an anti-HER2 antibody-drug conjugate in a biological sample obtained from a patient, comprising a specific binding partner for the Bcl-2 gene or an expression product thereof.

[0124] 49. The polymer of claim 48, wherein the binding partner is an anti-Bcl-2 antibody.

[0125] 50. The polymer of claim 49, wherein the binding partner is a nucleic acid that hybridizes with the Bcl-2 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1A and 1B Shown is the expression of Bcl-2 family pro-activating molecules in T-DM1 resistant KPL-4 and BT-474M1 human breast cancer cells (HER2 positive) relative to parental cells. Figure 1A mRNA expression assessed by TaqMan qRT-PCR analysis is shown; Figure 1B Protein expression analyzed by Western blot is shown.

[0127] Figure 2 Shown are the results of a cell viability assay, showing that the T-DM1 + ​​GDC-0199 combination was synergistic in KPL-4 T-DM1 resistant human breast cancer cells, whereas no synergy was observed in KPL-4 parental cells.

[0128] Figure 3 Shown are the results of a caspase activation assay measuring activation of caspases 3 and 7. The results show that T-DM1 resistant KPL-4 human breast cancer cells were again sensitized to T-DM1 in the presence of GDC-0199 at 24 hours of drug treatment, whereas there was no effect of T-DM1 + / - GDC-0199 in KPL-4 parental cells at 24 hours.

[0129] Figure 4A and 4BShown are 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. Results show a dose-dependent increase in caspase 3 and 7 activation with the addition of GDC-0199 to T-DM1, with minimal effects of T-DM1 alone. In KPL-4 parental cells, T-DM1 induced robust activation of caspases 3 and 7, with minimal increases upon addition of GDC-0199.

[0130] Figure 5A Shown are the results of a caspase activation assay measuring the activation of caspases 3 and 7 in clone #17 T-DM1-resistant KPL-4 human breast cancer cell line treated for 48 hours with 1 ug / mL T-DM1 alone or in combination with the indicated doses of GDC-0199. The results show a dose-dependent increase in caspase 3 and 7 activation with the addition of GDC-0199 to T-DM1, with minimal effect of T-DM1 alone.

[0131] Figure 5B Shown are the effects of T-DM1 (5 mg / kg administered once daily), GDC-0199 (100 mg / kg qd x 21), or their combination on tumor growth (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 where single agent treatment was inactive.

[0132] Fig. 6A and 6B Shown are the results of a caspase activation assay measuring the activation of caspases 3 and 7 at 0.1 μg / mL and 1 μg / mL T-DM1 concentrations, respectively, in the clone #8 T-DM1-resistant KPL-4 human breast cell line, alone or in combination with the indicated concentrations of GDC-0199.

[0133] Figure 6C Shown are the effects of T-DM1 (5 mg / kg q3w x 2), GDC-0199 (100 mg / kg qd x 21), or the combination on tumor growth (measured by tumor volume) of clone #8 T-DM1-resistant KPL-4 human breast cancer xenografts in SCID beige mice.

[0134] Fig. 7A Shown is 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 the DAB detection method.

[0135] Figure 7BShown is 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 the DAB detection method.

[0136] Figure 8 Shown is protein expression measured by Western blot analysis of Bcl-2 and HER2 in clone #17KPL-4T-DM1 resistant xenograft tumors treated with GDC-0199, T-DM1 or T-DM-1 + GDC-0199.

[0137] Fig. 9A Results of a luminescent in vitro apoptosis assay for caspase 3 / 7 activation are shown, testing the effects of 5 different concentrations of GDC-0199 (μM) 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 show that enhanced apoptosis was greater than T-DM1 alone using all tested combinations.

[0138] Fig. 9B Results of a kinetic caspase 3 / 7 activation fluorescence in vitro apoptosis assay are shown, testing the effects of 3 different concentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 μM) alone or in combination with T-DM1 (0.1 μg / mL) in HER2+ MDA-MB-361 breast cancer cells. The results show that enhanced caspase activation was greater than T-DM1 alone using all tested combinations.

[0139] Fig. 10A The results of the caspase 3 / 7 activated luminescent in vitro apoptosis assay are shown, testing the effect of 5 different concentrations of GDC-0199 (μM) and 9 different concentrations in HER2+HCC1569 breast cancer cells (T-DM1 resistant cells) treated for 24 hours. The results show that T-DM1 alone did not induce apoptosis, but apoptosis was enhanced using all tested combinations.

[0140] Fig. 10B The results of a kinetic caspase 3 / 7 fluorescence in vitro apoptosis assay are shown, testing the effects of 3 different concentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 μM) alone and in combination with T-DM1 (0.1 μg / mL) in HER2+ HCC1569 breast cancer cells. The results show that the combination treatment dose-dependently enhances caspase activation.

[0141] Fig.11The effects of T-DM1 (1 mg / kg, 3 mg / kg, 7 mg / kg, iv q3wx1) and GDC-0199 (100 mg / kg, po, qdx 21) alone or in combination on tumor growth in the HER2+ MDA-MB-361 breast cancer xenograft model are shown. Significant tumor growth delay was observed using GDC-0199 in combination with 7 mg / kg T-DM1.

[0142] Fig.12 Shown is Western blot analysis of Bcl-2 family members (total and phospho-Bcl-2 and -Bcl-xL, total Mcl-1 and Bim) by T-DM1 (with or without GDC-0199) in HER2+ breast cancer cell lines BT-474, EFM192A, KPL-4, HCC1569, HCC1954, MDA-361, UACC-812, ZR-75-30.

[0143] Fig.13 The amino acid sequence of the trastuzumab light chain (SEQ ID NO: 1) is shown.

[0144] Fig.14 The amino acid sequence of the trastuzumab heavy chain (SEQ ID NO: 2) is shown. DETAILED DESCRIPTION

[0145] Certain embodiments of the present invention will now be described in detail, examples of which are illustrated in the accompanying structures and formulas. When describing the present invention in conjunction with the listed embodiments, it should be understood that the embodiments are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to include all optional forms, modifications and equivalent forms that may be included within the scope of the present invention as defined in the claims. Those skilled in the art will recognize those methods and materials that are similar or equivalent to the methods and materials described in this application and can be used to implement the present invention. The present invention is by no means limited to the described methods and materials.

[0146] All references cited in this application are fully incorporated into this application by reference. If one or more of the references, patents and similar materials introduced are different or contradictory from this application (including but not limited to defined terms, term usage, described technology, etc.), this application shall prevail.

[0147] definition

[0148] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0149] The term "monoclonal antibody" used in the present application refers to the antibody obtained from a substantially homogeneous antibody population, that is, the single antibody comprising the population is identical and / or combines the same epitope, except for possible variant antibodies, such as mutations occurring during the production of naturally occurring mutations or monoclonal antibody preparations, and such variants are usually present in small amounts. Compared with polyclonal antibody preparations that usually comprise different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristic of the antibody obtained from a substantially homogeneous antibody population, and is not interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals, wherein the transgenic animals comprise all or part of human immunoglobulin loci, such as such methods and other exemplary methods described in the present application for preparing monoclonal antibodies.

[0150] The anti-HER2 antibody used in the antibody-drug conjugate of the present application is a monoclonal antibody.

[0151] The term "chimeric antibody" refers to a monoclonal antibody comprising a variable region (i.e., a 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 technology. Chimeric antibodies comprising mouse variable regions and human constant regions are particularly preferred. Such mouse / human chimeric antibodies are products of immunoglobulin genes expressed by DNA segments encoding mouse 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 the class or subclass of the original antibody. Such "chimeric" antibodies are also referred to as "class switching antibodies". Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques well known in the art. See, for example, Morrison, SL, et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

[0152] The term "humanized antibody" refers to an antibody whose framework or "complementarity determining region" (CDR) has been modified to contain CDRs of an immunoglobulin with different specificity than the parent immunoglobulin. In a preferred embodiment, mouse CDRs are transplanted into the framework region of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those that represent sequences that recognize the antigens described above for chimeric antibodies and bifunctional antibodies.

[0153] The term "human antibody" 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 art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Pharmacol. 5 (2001) 368-374) and can be produced by a variety of techniques, including phage display. Based on this technology, human antibodies against a variety of targets can be produced. Examples of human antibodies are described, for example, in Kellermann, SA, et al., Curr Opin Biotechnol. 13 (2002) 593-597. For the use of phage display technology to produce and select human antibodies, see, for example, Winter et al., Ann Review Immunol, 1994, 12: 433-455; for methods for producing fully human antibodies from transgenic mice and phage display platforms, see, for example, Lonberg, Current Opinion Immunol, 2008, 20 (4): 450-459.

[0154] The term "recombinant human antibody" used herein is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells such as NS0 or CHO cells or from animals (e.g., mice) transgenic for human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in a rearranged form.

[0155] As used herein, "specific binding" or "specifically binds to" refers to binding that is sufficiently selective for a target to distinguish it from binding to an unwanted or nonspecific target. In one embodiment, an antibody that specifically binds to a target has a binding affinity (Kd) for the target of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In another embodiment, KD is 10 -10 mol / l or lower (e.g. 10 -12 Binding affinity is determined using standard binding assays, such as Scatchard plot analysis of cells expressing the target antigen.

[0156] The term "nucleic acid molecule" used in this application is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, it is double-stranded DNA.

[0157] The "constant domains" are not involved directly in binding the antibody to an antigen, but are involved in effector functions (ADCC, complement fixation and CDC).

[0158] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies 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 th ed., WH Freeman and Co., page 91 (2007).).

[0159] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain whose sequence is highly variable ("complementarity determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs of the present application include:

[0160] (a) hypervariable loops present 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));

[0161] (b) CDRs present 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));

[0162] (c) antigenic contacts present 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

[0163] (d) a combination of (a), (b), and / or (c), comprising 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).

[0164] The term "anti-HER2 antibody" of the present invention refers to an antibody that specifically binds to the HER2 antigen.

[0165] The term "trastuzumab" as defined in this application, and "huMAb4D5-8" are used interchangeably. Such antibodies preferably comprise Fig.13 (SEQ ID NO: 1) and Fig.14 The light chain and heavy chain amino acid sequences are shown in (SEQ ID NO. 2).

[0166] "Epitope 4D5" or "4D5 epitope" or "4D5" is the region in the extracellular domain of HER2 to which antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. The epitope is close to the transmembrane domain of HER2 and within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, a conventional cross-blocking assay as described in, for example, 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 an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region of about residue 529 to about residue 625, inclusive, of HER2).

[0167] "Epitope 2C4" or "2C4 epitope" is the region in the extracellular domain of HER2 to which antibody 2C4 binds. To screen for antibodies that bind to the 2C4 epitope, a conventional cross-blocking assay is performed. The conventional cross-blocking assay 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 an 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)).

[0168] The terms "T-DM1", "trastuzumab-MCC-DM1", "ado-trastuzumab emtansine conjugate", "trastuzumab emtansine conjugate" and "trastuzumab emtansine conjugate" are defined in this application. Used interchangeably and refer to trastuzumab linked to the maytansinoid drug moiety DM1 via the linker moiety MCC, including all mixtures of various loaded and linked antibody-drug conjugates wherein 1, 2, 3, 4, 5, 6, 7 and 8 drug moieties are covalently linked to the antibody trastuzumab (US 7097840; US 2005 / 0276812; US 2005 / 0166993).

[0169] The term "Bcl-2" used in the present application refers to the Bcl-2 protein (Swiss Prot ID No. P10415), which is a member of the Bcl-2 family of proteins (Cory, S., and Adams, JM, Nature Reviews Cancer 2 (2002) 647-656; Adams, Genes und Development 17 (2003) 2481-2495; Danial, NN, and Korsmeyer, SJ, Cell 116 (2004) 205-219; Petros, AM, Biochim Biophys Acta 1644 (2004) 83-94).

[0170] The term "selective Bcl-2 inhibitor" as used herein refers to polypeptides and small molecules that inhibit the activation 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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide (also known as ABT-199 or GDC-0199) or a pharmaceutically acceptable salt thereof, which is a Bcl-2 inhibitor of Formula I, described in International Publication No. WO2010 / 0138588 and U.S. Publication No. US2010 / 0305122, which are incorporated herein by reference.

[0171]

[0172] 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide

[0173] Formula I.

[0174] The "anti-tumor agent" of the present application refers to a drug used to treat cancer. Non-limiting examples of the anti-tumor agent of the present application include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, anti-hormone compounds, cytokines, EGFR targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and antibodies, cytotoxic agents, antibodies that induce apoptosis, COX inhibitors, farnesyl transferase inhibitors, antibodies that bind to oncofetal protein CA 125, HER2 vaccines, Raf or ras inhibitors, liposomal doxorubicin, topotecan, taxanes, dual tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib and bevacizumab.

[0175] "Chemotherapy" is the use of chemical therapeutic agents that can be used to treat cancer.

[0176] "Chemotherapeutic agents" are compounds that can be used to treat cancer, regardless of their mechanism of action. Chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plantalkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizing drugs, and kinase inhibitors. Examples of chemotherapeutic agents include: erlotinib ( Genentech / OSI Pharm.), docetaxel ( Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine ( 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 ( Bristol-Myers Squibb Oncology, Princeton, NJ), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentaazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No.85622-93-1, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethylamine, ), doxorubicin Akti-1 / 2, HPPD and rapamycin.

[0177] Other examples of chemotherapeutic agents include: oxaliplatin ( Sanofi), bortezomib ( Millennium Pharm.), sunitinib (sutent) ( SU11248, Pfizer), letrozole ( Novartis), imatinib mesylate ( 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 ( AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, Wyeth), lapatinib ( GSK572016, GlaxoSmith Kline), lonafarnib (SARASAR TM, SCH 66336, Schering Plough), sorafenib ( BAY43-9006, Bayer Labs), gefitinib ( AstraZeneca), irinotecan ( CPT-11, Pfizer), tipifarnib (ZARNESTRA TM , Johnson & Johnson), ABRAXANE TM (Cremophor-free), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus ( Wyeth), pazopanib (GlaxoSmithKline), canfosfamide ( Telik, thiotepa, and cyclosphosphamide Alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and methylamelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenephosphoramide sulfide, and thiamethoxam; ylenethiophosphoramide) and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, naphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma 1I, calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186); anthracycline antibiotics (dynemicin), dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromophores enediyne antibiotic chromophores. chromophore), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, ditoxin 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 acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, doxycycline; antimetabolites 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, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folicacid replenishers, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone;Mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A, bacillus sporin A, and anguidine); urethane; 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 Novantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine ( Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above substances.

[0178] The terms "cancer" and "cancerous" refer to or describe the physiological condition of mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include breast cancer, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), stomach cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. In a preferred embodiment, the cancer is breast cancer. In another preferred embodiment, the cancer is stomach cancer.

[0179] The term "stage 0," "stage I," "stage II," "stage III," or "stage IV," and the various substages within this classification, indicates the classification of the tumor or cancer using the Overall Stage Grouping or Roman Numeral Staging method known in the art. Although the actual cancer stage depends on the type of cancer, in general, stage 0 cancers are in situ lesions, stage I cancers are small localized tumors, stage II and stage III cancers are locally advanced tumors that exhibit involvement of local lymph nodes, and stage IV cancers represent metastatic cancers. The specific stage of each type of tumor is known to the skilled clinician.

[0180] The term "metastatic breast cancer" refers to a state of breast cancer in which cancer cells travel from the original site via blood or lymphatic vessels to one or more sites elsewhere in the body to form one or more secondary tumors in one or more organs other than the breast.

[0181] "Advanced" cancer is one that has spread outside the original site or organ, either by local invasion or metastasis. Thus, the term "advanced" cancer includes both locally advanced and metastatic disease.

[0182] A "refractory" cancer is one that progresses even though anti-tumor agents, such as chemotherapy, are administered to the cancer patient. An example of a refractory cancer is a platinum-refractory cancer.

[0183] A "recurrent" cancer is a cancer that grows again at the original site or at a distant site after responding to initial treatment, such as surgery.

[0184] "Locally recurrent" cancer is cancer that has come back after treatment in the same site as a previously treated cancer.

[0185] An "operable" or "resectable" cancer is one that is confined to the organ of origin and is amenable to surgery (resection).

[0186] "Unresectable" or "unresectable" cancer cannot be removed (cut) by surgery.

[0187] The terms "HER2-positive" and "expressing HER2" are used interchangeably in this application. "HER2-positive" cancers include cancer cells with higher than normal levels of HER2. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, HER2-positive is a cancer that overexpresses HER2, and in certain embodiments, a HER-2-positive cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification rate ≥ 2.0.

[0188] In situ hybridization (ISH) determines the number of her2 copies using DNA probes coupled to fluorescent, chromogenic or silver detection systems (i.e., FISH, CISH or SISH) or a combination of CISH and SISH systems (bright field dual ISH (BDISH) or dual hapten, dual color ISH (DDISH)). ISH can be performed using a single probe to count only her2 copies per nucleus, or as a dual probe technique, in which hybridization of the chromosome 17 centromere probe (chromosome enumeration probe 17, CEP17) allows determination of the her2:CEP17 ratio. The dual probe method can be performed as a dual color technique, with two probes co-hybridized on the same slide, or as a single color assay, in which each probe is used on a continuous slide. The her2:CEP17 ratio is sometimes considered to be a better reflection of the her2 amplification status than the average her2 copy number, because the latter also depends on other parameters, such as the mitotic index of the tumor, the thickness of the section, the nuclear truncation effect, and the abnormal chromosome copy number (aneuploidy). The phrase "in situ hybridization (ISH) amplification ratio ≥ 2.0" refers to a her2:CEP17 ratio ≥ 2.0. For more information, see 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.

[0189] A "patient" or "subject" of the present application is a human patient. The patient may be a "cancer patient", ie a person suffering from or at risk of suffering from one or more symptoms of cancer, in particular gastric cancer or breast cancer.

[0190] A "patient population" refers to a group of cancer patients. Such a population can be used to demonstrate that a drug, such as Pertuzumab, has statistically significant efficacy and / or safety.

[0191] A "relapsed" patient is one who has signs or symptoms of cancer after remission. Optionally, the patient relapses after adjuvant or neoadjuvant therapy.

[0192] A cancer or biological sample that "exhibits HER expression, amplification or activation" is a cancer or biological sample that expresses (including overexpresses) a HER receptor, has an amplified HER gene, and / or otherwise demonstrates activation or phosphorylation of a HER receptor in a diagnostic test.

[0193] The term "synergy" as used herein refers to a therapeutic combination that is more effective than the additive effects of two or more single agents. The determination of the synergistic interaction between anti-Her2 antibody-drug conjugates such as trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors can be based on the results obtained from the assays described herein, or in other assay systems known in the art, using standard procedures for quantifying synergy, additive effects and antagonism between anticancer agents. The preferred procedure used is the procedure 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 ​​from 0.8 to 1.2 indicate additive effects. Combination therapy can provide "synergy" and prove to be "synergistic", that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. A synergistic effect may be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered as separate formulations in alternation or in parallel; or (3) by other regimens. When delivered in alternation therapy, a synergistic effect may be obtained when the compounds are administered or delivered sequentially, for example by different injections in separate syringes. Generally, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., serially in time, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0194] "Neoadjuvant therapy" or "preoperative therapy" herein refers to therapy given before surgery. The goal of neoadjuvant therapy is to provide immediate systemic therapy that may eradicate micrometastases that would otherwise proliferate if the standard sequence of surgery followed by systemic therapy was followed. Neoadjuvant therapy can also help reduce tumor size, allowing complete resection of an initially unresectable tumor or preservation of some organs and their function. In addition, neoadjuvant therapy allows in vivo evaluation of drug efficacy, which can guide the selection of subsequent treatments.

[0195] "Adjuvant therapy" as used herein refers to therapy given after definitive surgery in which no evidence of residual disease can be detected, thereby reducing the risk of disease recurrence. The goal of adjuvant therapy is to prevent cancer recurrence and, therefore, reduce the chance of cancer-related death. Adjuvant therapy as used herein specifically excludes neoadjuvant therapy.

[0196] "Definitive surgery" is used as a term used in the medical community. Definitive surgery includes, for example, procedures, surgeries or other procedures that result in the removal or excision of a tumor, including those that result in the removal or excision of all visibly visible tumors. Definitive surgery includes, for example, complete or radical excision of a tumor or total complete excision. Definitive surgery includes procedures that occur in one or more stages, and includes, for example, multi-stage surgical procedures in which one or more surgeries or other procedures are performed prior to excision of the tumor. Definitive surgery includes procedures that remove or excise a tumor, including involved organs, parts of organs and tissues, and surrounding organs, such as lymph nodes, parts of organs or tissues. Removal may be incomplete, such that tumor cells may remain even if they are not detected.

[0197] "Survival" means that the patient is still 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 calculated using a stratified log-rank test.

[0198] "Progression-free survival" (PFS) was the time from the first day of treatment to documented disease progression (including isolated CNS progression) or death from any cause on study, whichever occurred first.

[0199] "Disease-free survival (DFS)" refers to a patient who is still alive, without recurrence of cancer, for a determined 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 the initial treatment or from the initial diagnosis. In one aspect of the invention, DFS is analyzed according to the intention-to-treat principle, i.e., patients are evaluated according to their designated therapy. Events used in the analysis of DFS may include local, regional, and distant cancer recurrence, the occurrence of secondary cancers, and death from any cause in patients without prior events (e.g., breast cancer recurrence or second primary cancer).

[0200] "Overall survival" means that the patient is still alive, from the initial treatment or from the initial diagnosis for a certain period of time, such as about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc. In the present study, the event used for survival analysis is death from any cause.

[0201] "Prolonged survival" refers to an increase in DFS and / or OS in treated patients relative to untreated patients, or relative to a control treatment regimen. 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., after initial treatment or after initial diagnosis.

[0202] The "hazard ratio" in survival analysis is a summary of the difference between two survival curves, representing the reduced risk of death with treatment compared to control over a period of follow-up. The hazard ratio is a statistical definition of the rate of occurrence of an event. For the purposes of this invention, the hazard ratio is defined as representing the probability of an event in the experimental group divided by the probability of an event in the control group at any particular time point.

[0203] "Monotherapy" refers to a treatment regimen that includes only a single therapeutic agent for treating a cancer or tumor during the course of treatment.

[0204] The terms "treatment" and "treatment" refer to therapeutic treatments and preventive or prophylactic measures, wherein the purpose is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the growth, development or spread of hyperproliferative diseases such as cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease, a stabilized (i.e., non-worsening) state of the disease, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. If not receiving treatment, "treatment" also means prolonging survival compared to the expected survival. Patients in need of treatment include those who already have the disease or condition, as well as those who are prone to have the disease or condition, or those in which the disease or condition is to be prevented.

[0205] When applied to, for example, cancer, the term "treatment method" or its equivalent refers to an operation or course of action designed to reduce or eliminate the number of cancer cells or alleviate the symptoms of cancer in a patient. A "treatment method" for cancer or another proliferative disease does not necessarily mean that the cancer cells or other condition will actually be eliminated, that the number of cells will actually be reduced or the condition will be alleviated, or that the symptoms of the cancer or other condition will actually be alleviated. Generally, a method of treating cancer is performed even if there is a small chance of success, but it is still believed to result in an overall beneficial course of action in view of the patient's medical history and estimated expected survival.

[0206] The term "co-administration" or "co-administration" refers to the administration of an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor as two separate formulations. Co-administration can be performed simultaneously or sequentially in any order. In another embodiment, there is a time period during which both (or all) active agents exert their biological activities simultaneously. The anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor can be co-administered simultaneously or sequentially (e.g., intravenously (iv) by continuous infusion (one for the antibody-drug conjugate and the last for the Bcl-2 inhibitor; or oral administration of the Bcl-2 inhibitor). When the two therapeutic agents are co-administered sequentially, the agents are administered in two separate administrations separated by a "specific time period". The term specific time period refers to any time from 1 hour to 15 days. For example, one agent may be administered at about 15, 14, 13, 12, 11, 10, 9, or more of the time that the other agent is administered. , 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, and in one embodiment the specific time period 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.

[0207] The term "simultaneously" means at the same time or within a short period of time, usually less than 1 hour.

[0208] A drug that is administered "concurrently" with one or more other drugs is administered during the same treatment cycle, on the same day as the one or more other drugs, and optionally at the same time as the one or more other drugs. For example, for a cancer therapy given once every 3 weeks, the concurrently administered drugs are each administered on Day 1 of a 3-week cycle.

[0209] The administration period used in this application refers to a period of time during which each therapeutic agent has been administered at least once. The administration cycle is generally 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.

[0210] In certain embodiments, the dosing cycle is 21 days.

[0211] In certain embodiments of the method for treating cancer in patients provided herein, the method includes administering an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor to the patient for one or more dosing cycles. In one embodiment, one or more dosing cycles each last for at least one week. In another embodiment, one or more dosing cycles each last for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks or more than nine weeks. In one embodiment, each dosing cycle is three weeks.

[0212] In a preferred embodiment, the antibody-drug conjugate is administered as an intravenous (iv) infusion once every three weeks (21 day cycle).

[0213] In another preferred embodiment, the antibody-drug conjugate is (ado-trastuzumab emtansine conjugate), which is administered as a 3.6 mg / kg iv infusion every 3 weeks (21-day cycle).

[0214] In some embodiments of the treatment methods provided herein, the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide (GDC-0199). In certain embodiments, during the first dosing cycle, the amount of GDC-0199 administered to the patient per dose is increased from an initial amount of 10 mg to 80 mg to a final amount of 190 mg to 400 mg. In certain embodiments, the amount of GDC-0199 administered to the patient per dose starts at 50 mg or 100 mg and is increased to 300 mg per dose. In some embodiments, the amount of GDC-0199 in the initial dose administered to the patient can be, for example, 20 mg to 60 mg (e.g., a 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, or 60 mg dose), followed by doses of 100 mg, 200 mg, 300 mg, or more, of GDC-0199.

[0215] An "adverse event" is any unfavorable and undesirable sign, symptom, or disease, regardless of attribution, associated with the use of an investigational (medicinal) product or other protocol-mandated intervention; and includes: an adverse event (AE) not previously observed in a patient occurring during the protocol-specified AE reporting period, including signs or symptoms related to breast cancer that were not present prior to the AE reporting period; a complication occurring as a result of a protocol-authorized intervention (e.g., an invasive procedure such as a biopsy); if applicable, an AE occurring prior to assignment of study treatment associated with a washout, run-in, or other protocol-authorized intervention; a worsening in severity or frequency or a change in nature of a pre-existing condition (other than a study condition) as determined by the investigator during the protocol-specified AE reporting period.

[0216] It goes without saying that the antibody-drug conjugate is administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the corresponding compound or combination that elicits the biological or medical response of a tissue, system, animal or human that the researcher, veterinarian, doctor or other clinician is looking for. The administration of an effective amount of a therapeutic agent can be a single administration or divided dose administration. "Divided dose administration" means that the effective amount is divided into multiple doses, preferably 2, and administered within 1 or 2 days. For example, if 100 mg of a selective Bcl-2 inhibitor is considered to be effective, it can be administered in a single 100 mg administration or two 50 mg administrations. Sometimes divided dose administration is required at the beginning of the administration period to reduce side effects. When an effective amount is administered in divided administration, it is still considered to be an effective amount administered once. For example, when 100 mg is an effective amount of a selective Bcl-2 inhibitor and the amount is administered in two 50 mg doses over a period of time (e.g., 2 days), only one effective amount is administered during this period.

[0217] A "fixed" or "flat" dose of a therapeutic agent herein refers to a dose that is administered to a human patient regardless of the patient's weight (WT) or body surface area (BSA). Thus, a fixed dose is not expressed in mg / kg doses or mg / m 2 Doses are not provided, but rather absolute amounts of therapeutic agents are provided.

[0218] The "loading" dose of the present application generally comprises an initial dose of a therapeutic agent administered to a patient, and is followed by one or more maintenance doses thereof. Typically, a single loading dose is administered, but the present application encompasses multiple loading doses. Typically, the amount of the loading dose administered exceeds the amount of the maintenance dose administered and / or the loading dose is administered more frequently than the maintenance dose, to achieve the desired steady-state concentration of the therapeutic agent earlier than the desired steady-state concentration of the therapeutic agent that can be achieved with the maintenance dose.

[0219] The "maintenance" dose of the present application refers to one or more doses of therapeutic agents administered to a patient during the treatment period. Typically, the maintenance dose is administered at intervals of treatment, such as about every week, about every 2 weeks, about every 3 weeks, or about every 4 weeks, preferably every 3 weeks.

[0220] "Infusion" or "infusion" refers to the introduction of a drug-containing solution into the body through a vein for therapeutic purposes. Typically, this is accomplished through an intravenous (IV) bag.

[0221] An "intravenous bag" or "IV bag" is a bag that can hold a solution that can be administered through a patient's vein. 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 of a polyolefin or polyvinyl chloride.

[0222] In the context of the present invention, additional other cytotoxic agents, chemotherapeutic agents or anticancer agents or compounds that enhance the effects of these agents (e.g., cytokines) can be used in the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor combination therapy of cancers expressing HER2. Such molecules are suitable for being present in a combined form in an amount effective for the intended purpose. Preferably, anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor combination therapy are used without such additional cytotoxic agents, chemotherapeutic agents or anticancer agents, or compounds that enhance the effects of these agents.

[0223] Such agents include, for example, alkylating agents or agents with alkylating effects, such as cyclophosphamide (CTX; e.g. ), chlorambucil (CHL; e.g. ), cisplatin (CisP; e.g. ), busulfan (e.g. ), melphalan, carmustine (BCNU), streptozotocin, trimethoprim-sulfamethoxazole (TEM), mitomycin C, etc.; antimetabolites, such as methotrexate (MTX), etoposide (VP16; e.g. ), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g. ), dacarbazine (DTIC), etc.; antibiotics such as actinomycin D, doxorubicin (DXR; for example ), daunorubicin (daunomycin), bleomycin, mithramycin, etc.; alkaloids, such as vinca alkaloids, such as vincristine (VCR), vinblastine, etc.; and other anti-tumor agents, such as paclitaxel (e.g. ) and paclitaxel derivatives, cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g. ) and corticosteroids such as prednisone, nucleosidase inhibitors such as hydroxyurea, amino acid consuming enzymes such as asparaginase, folinic acid and other folic acid derivatives, and similar anti-tumor agents. The following agents can also be used as additional agents: arnifostine (e.g. ), dactinomycin, mechlorethamine (nitrogen mustard), streptozotocin, cyclophosphamide, lomustine (CCNU), liposomal doxorubicin (e.g. ), gemcitabine (e.g. ), daunorubicin liposomes (e.g. ), procarbazine, mitomycin, docetaxel (e.g. ), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT11 (irinotecan), 10-hydroxy7-ethylcamptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprorelin, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil. Preferably, the combination therapy of type II anti-CD20 antibody and Bcl-2 inhibitor is used without the use of the above-mentioned additional agents.

[0224] The use of the above-mentioned cytotoxic and anticancer agents and antiproliferative target-specific anticancer drugs such as protein kinase inhibitors in chemotherapy regimens is generally well characterized in the field of cancer treatment, and their use in this application belongs to the same considerations of monitoring tolerance and effectiveness and controlling the route of administration and dosage, with some adjustments. For example, the actual dose of the cytotoxic agent may vary depending on the cultured cell response of the patient determined by using tissue culture methods. Typically, the dose will be reduced compared to the amount used in the absence of additional other agents.

[0225] Typical dosages of effective cytotoxic agents may be within the range recommended by the manufacturer and may be lowered by up to about an order of magnitude in concentration or amount, where indicated by in vitro responses or responses in animal models. Therefore, the actual dosage will depend on the physician's judgment, the patient's condition, and the effectiveness of the treatment approach based on the in vitro responsiveness of primary cultured malignant cells or tissue samples in tissue culture or the responses observed in appropriate animal models.

[0226] In the context of the present invention, in addition to the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor combination therapy, an effective amount of ionizing radiation and / or radiopharmaceuticals can be used. The radiation source can be outside or inside the patient to be treated. When the source is outside the patient, the therapy is called external beam radiation therapy (EBRT). When the radiation source is located inside the patient, the therapy is called brachytherapy (BT). The radioactive atoms used in the context of the present invention can be selected from, 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. Antibodies can also be labeled with such radioisotopes. It is preferred to use type II anti-CD20 antibodies and Bcl-2 inhibitor combination therapy without using such ionizing radiation.

[0227] Radiation therapy is the standard treatment for controlling tumors and / or tumor metastases that cannot be removed or operated on. When radiation therapy is combined with chemotherapy, improved results have been seen. Radiation therapy is based on the principle that high-dose radiation delivered to the target area can cause the death of germ cells in tumors and normal tissues. The radiation dose regimen is usually defined according to the radiation absorbed dose (Gy), time and fractionation, and must be carefully defined by an oncologist. The amount of radiation received by the patient depends on various considerations, but the two most important are the location of the tumor relative to other key structures or organs of the body and the extent to which the tumor has spread. The typical course of treatment for a patient undergoing radiation therapy will be a 1 to 6 week treatment schedule with a total dose of 10 to 80Gy, administered to the patient in a single daily portion of about 1.8 to 2.0Gy, 5 days a week. In a preferred embodiment of the present invention, there is a synergistic effect when treating a tumor of a human patient with the combination therapy of the present invention and radiation therapy. In other words, when combined with radiation, tumor growth is inhibited by means of an agent comprising the combination of the present invention, optionally in combination with another chemotherapeutic agent or anticancer agent. The parameters of adjuvant radiation therapy are, for example, included in WO 99 / 60023.

[0228] The anti-HER2 antibody-drug conjugate is administered by intravenous administration as a bolus or by continuous infusion over a period of time according to known methods, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial or intrathecal routes. The antibody is preferably administered intravenously or subcutaneously.

[0229] The Bcl-2 inhibitor is administered by intravenous administration as a bolus or by continuous infusion over a period of time according to known methods, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal or oral routes. Preferably, the Bcl-2 inhibitor is administered intravenously, subcutaneously or orally.

[0230] As used herein, "pharmaceutically acceptable carrier" is intended to include any and all materials compatible with drug administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other materials and compounds compatible with drug administration. Except in the case where any conventional media or agents are incompatible with the active compound, their use in the compositions of the present invention is also included. Supplementary active compounds may also be incorporated into the composition.

[0231] A "vial" is a container suitable for holding a liquid or lyophilized formulation. In one embodiment, the vial is a single-use vial, such as a 20-cc single-use vial with a stopper.

[0232] 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.

[0233] Trastuzumab-MCC-DM1 (T-DM1; ado-trastuzumab emtansine conjugate)

[0234] The present invention includes a therapeutic combination product comprising trastuzumab-MCC-DM1 having the following structure:

[0235]

[0236] Wherein Tr is trastuzumab and p is an integer from 1 to 8. In the above structure of trastuzumab-MCC-DM1, the ratio of drug to antibody or drug loading is represented by p. Trastuzumab-MCC-DM1 includes all mixtures of various loaded and linked antibody-drug conjugates, wherein 1, 2, 3, 4, 5, 6, 7 and 8 drug moieties are covalently linked to the antibody trastuzumab.

[0237] Trastuzumab is produced by mammalian cell (Chinese hamster ovary, CHO) suspension culture. HER2 (or c-erbB2) proto-oncogene encodes a transmembrane receptor protein of 185kDa, which is structurally related to epidermal growth factor receptor. HER2 protein overexpression is observed in 25%-30% of primary breast cancers, and can be determined using fixed tumor block assessment based on immunohistochemistry (Press MF, et al (1993) Cancer Res 53: 4960-70). Trastuzumab is an antibody with antigen binding residues of mouse 4D5 antibody or derived therefrom (ATCC CRL 10463, deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, on May 24, 1990 according to the Budapest Treaty). Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 as described in US Pat. No. 5,821,337

[0238] The antibody-drug conjugate trastuzumab-MCC-DM1 contains the maytansinoid drug moiety DM1 (US5208020; US6441163) and can be prepared from ansamitocin fermentation products (US6790954; US2005 / 0170475).

[0239] Selective Bcl-2 inhibitors

[0240] 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-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide (also known as ABT-199 or GDC-0199), which is a Bcl-2 inhibitor of Formula I, described in International Publication No. WO2010 / 0138588 and U.S. Publication No. US2010 / 0305122, which are incorporated herein by reference.

[0241]

[0242] 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide

[0243] Formula I

[0244] 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 (navitoclax), AT-101, BL-11, BL-193, GX-15-003, 2-methoxyantimycin A3, HA-14-1, KF-67544, erythrogallol, TP-TW-37, YC-137 and Z-24, described, for example, in Zhai, D., et al., Cell Death and Differentiation 13 (2006) 1419-1421.

[0245] Pharmaceutical composition

[0246] The pharmaceutical composition or formulation of the present invention comprises a combination of trastuzumab-MCC-DM1, a selective Bcl-2 inhibitor and one or more pharmaceutically acceptable carriers, glidants, diluents or excipients.

[0247] The 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 the present invention is intended to include both solvated and unsolvated forms.

[0248] Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors of the present invention may also exist in different tautomeric forms, and all of these forms are included within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0249] Pharmaceutical compositions include bulk compositions and single dosage units containing one or more (e.g., two) pharmaceutically active agents (including trastuzumab-MCC-DM1 and a selective Bcl-2 inhibitor selected from the additional agents described herein), as well as any pharmaceutically inactive excipients, diluents, carriers, or glidants. Bulk compositions and each single dosage unit may contain a fixed amount of the above-mentioned pharmaceutically active agents. Bulk compositions are substances that have not yet been formed into single dosage units. Illustrative dosage units are oral dosage units, such as tablets, pills, capsules, etc. Similarly, the methods described herein for treating patients by administering the pharmaceutical compositions of the present invention are also intended to cover the administration of bulk compositions and single dosage units.

[0250] Pharmaceutical compositions also include isotopically labeled compounds of the invention, which are identical to those described herein except that one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. All isotopes of any particular atom or element specified are encompassed within the scope of the compounds of the invention and their uses. Exemplary isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 32 P. 33 P. 35 S. 18 F. 36 Cl, 123 I and 125 I. Certain isotopically labeled compounds of the invention (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 ( 14 C) isotopes are useful because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium ( 2 H) substitution may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and thus may be preferred in some circumstances. Positron emitting isotopes such as 15 O. 13 N. 11 C and 18F can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the invention can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0251] Trastuzumab-MCC-DM1 and a selective Bcl-2 inhibitor can be formulated according to standard pharmaceutical practice for therapeutic treatment (including prophylactic treatment) of hyperproliferative disorders in mammals (including humans) in therapeutic combination. The present invention provides a pharmaceutical composition comprising trastuzumab-MCC-DM1 and one or more pharmaceutically acceptable carriers, glidants, diluents or excipients.

[0252] 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 water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. The specific carrier, diluent or excipient used will depend on the means and purpose of the application of the compound of the present invention. The solvent is generally selected based on a (GRAS) solvent that is recognized by those skilled in the art as safe for mammalian administration. Typically, a safe solvent is a non-toxic aqueous solvent, such as water and other non-toxic solvents that are soluble in water or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300), etc. and mixtures thereof. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, aromatics, flavoring agents, and other known additives that provide an elegant presentation of a drug (i.e., a compound of the present invention or its pharmaceutical composition) or contribute to the manufacture of a drug (i.e., a drug).

[0253] The formulation can be prepared using conventional dissolution and mixing operations. For example, bulk drug substance (i.e., a compound of the invention or a stabilized form of the compound (e.g., a complex with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients. The compounds of the invention are usually formulated into pharmaceutical dosage forms to provide a drug that is easy to control dosage and allows patients to comply with the prescribed treatment regimen.

[0254] The pharmaceutical composition (or preparation) for administration can be packaged in a variety of ways, depending on the method used to administer the drug. Typically, the article for distribution includes a container in which the pharmaceutical preparation has been deposited in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic bottles and glass bottles), pouches, ampoules, plastic bags, metal cylinders, etc. The container may also include an anti-tampering device to prevent incautious access to the package contents. In addition, a label describing the container contents has been deposited on the container. The label may also include appropriate warnings.

[0255] Pharmaceutical formulations of the compounds of the invention can be prepared with pharmaceutically acceptable diluents, carriers, excipients or stabilizers for various routes and types of administration (Remington's Pharmaceutical Sciences (1995) 18th edition, Mack Publ. Co., Easton, PA) in the form of lyophilized preparations, ground powders or aqueous solutions. The formulation can be made by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dose and concentration used) at ambient temperature at a suitable pH and with the desired purity. The pH of the formulation depends primarily on the specific use and concentration of the compound, but can be in the range of about 3 to about 8.

[0256] Pharmaceutical preparations are preferably sterile. Specifically, preparations for in vivo administration must be sterile. This sterilization can be easily accomplished by filtration through a sterile filtration membrane.

[0257] Pharmaceutical formulations can typically be stored as solid compositions, lyophilized formulations, or as aqueous solutions.

[0258] The pharmaceutical preparations of the present invention will be administered and applied in a certain manner (i.e., amount, concentration, schedule, course of treatment, vehicle, and route of administration) consistent with good medical practice. Factors considered in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of administration, the schedule of administration, and other factors known to the physician. The "therapeutically effective amount" of the compound to be administered will be subject to these considerations and is the minimum amount required to prevent, improve, or treat a coagulation factor-mediated condition. This amount is preferably less than an amount that is toxic to the host or that makes the host significantly more susceptible to bleeding.

[0259] Acceptable diluents, carriers, excipients and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, ethanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 1,000 mmol / l) 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 counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM , including Tween 80, PLURONICS TM Or polyethylene glycol (PEG), including PEG400. Active pharmaceutical ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co., Easton, PA.

[0260] Pharmaceutical formulations include those suitable for the routes of administration detailed herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. Techniques and formulations generally are described in Remington's Pharmaceutical Sciences 18 th Ed. (1995) Mack Publishing Co., Easton, PA. These methods include the step of combining the active ingredient with a carrier that constitutes one or more auxiliary ingredients. Generally, the formulation is prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0261] As a general suggestion, the initial pharmaceutically effective amount of trastuzumab-MCC-DM1 administered per dose will be in the range of about 0.01-100 mg / kg, ie, about 0.1-20 mg / kg patient body weight / day, with a typical starting range of 0.3-15 mg / kg / day of the compound used.

[0262] In a preferred embodiment, trastuzumab-MCC-DM1 is formulated as a lyophilized powder in a single-use vial containing 100 mg / vial or 160 mg / vial and is administered as an intravenous infusion once every 3 weeks at a dose of 3.6 mg / kg.

[0263] Pharmaceutical compositions of anti-Bcl-2 active agents (e.g., Bcl-2 inhibitors) used alone depend on their pharmaceutical properties; for example, for small compounds such as ABT-737, ABT-199 or ABT-263, a formulation may be, for example, the following:

[0264] a) Tablet preparation (wet granulation):

[0265]

[0266] Preparation operation:

[0267] 1. Mix items 1, 2, 3 and 4 and granulate with purified water.

[0268] 2. Dry the granules at 50°C.

[0269] 3. Pass the granules through suitable grinding equipment.

[0270] 4. Add item 5 and mix for three minutes; compress on a suitable tablet press.

[0271] b) Capsule preparation:

[0272]

[0273] Preparation operation:

[0274] 1. Items 1, 2 and 3 were mixed in a suitable mixer for 30 minutes.

[0275] 2. Add items 4 and 5 and mix for 3 minutes.

[0276] 3. Fill into suitable capsules.

[0277] The active ingredient can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0278] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0279] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0280] 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 should be understood that modifications may be made to the described procedures without departing from the spirit of the present invention.

[0281] Example

[0282] Example 1

[0283] Expression of Bcl-2 family pro-activating molecules in T-DM1-resistant breast cancer cells

[0284] Preparation of T-DM1-resistant cell lines

[0285] 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 and gradually increasing to 2 μg / mL. The derived cells are "resistance pools" maintained in culture in 2 μg / mL T-DM1. To obtain stable resistant clones, each pool (KPL-4 and BT-474M1) was single cell sorted and cloned. Clones were kept free of T-DM1 so that clones with stable resistance in the absence of T-DM1 could be identified.

[0286] TaqMan Assays

[0287] Total RNA was prepared using the 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. Reactions were performed using an ABI 7500 real-time quantitative PCR system in a standard 96-well plate format. 100 ng of total RNA was used as a template in each reaction. For data analysis, the original Ct was normalized to the housekeeping gene HP1BP3.

[0288] The results of TaqMan analysis are shown in Figure 1A The figure shows increased Bcl-2 mRNA expression in T-DM1 resistant KPL-4 and T-DM1 resistant BT-474M1 cell lines (normalized to the housekeeping gene HP1BP3) relative to parental, non-resistant KPL-4 and BT-474M1 cells.

[0289] Western blot analysis was performed as follows: cells were lysed in a calibrated FLAG elution buffer (CFEB) (19.17 mM Tris (pH 7.5), 916.7 μM MgCl2, 92.5 mM NaCl, and 0.1% Triton X-100) with protease and phosphatase inhibitors (Roche); 6 M urea was added in some cases. Cleared lysates were quantified and equal amounts of protein were reduced, alkylated, separated by SDS-PAGE, and transferred to PVDF membranes (Invitrogen) according to standard procedures. Western blot analysis was performed according to the recommendations of the respective antibody manufacturers. Figure 1B Western blot analysis shown confirms that Bcl-2 is overexpressed in T-DM1-resistant KPL-4 and BT-474 cell lines.

[0290] Example 2

[0291] Cell proliferation assay - parental and T-DM1-resistant KPL-4 breast cancer cell lines

[0292] Cell proliferation assays were 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 a fixed ratio. To obtain the combination index "CI" (Chou and Talalay (1984) Adv. Enzyme Regul. 22: 27-55), synergy was analyzed using Chou and Talalay drug combination dose-effect analysis with CalcuSyn software. A CI value less than 1 indicates synergy, while CI = 1 indicates additivity.

[0293] Measurement conditions :The cells were maintained in Ham's F-12: high glucose DMEM (1: 1) supplemented with 10% heat-inactivated fetal bovine serum and 2mM L-glutamine. The cells were plated in 96-well plates (4000 cells per well for KPL-4 parental cells; 8000 cells per well for KPL-4T-DM1 resistant cells) and allowed to adhere overnight at 37°C in a humid atmosphere of 5% CO2. The culture medium was then removed and replaced with fresh culture medium containing T-DM1, GDC-0199 or a combination of both. Cell Titer-Glo (Promega Corp.) was added to the wells 3 days after drug administration, and the luminescent signal was measured using an EnVision multi-label microplate reader (PerkinElmer). The combination index (CI) value was generated using CalcuSyn software (Biosoft, Inc).

[0294] like Figure 2 As shown, the combination of T-DM1 and GDC-0199 has a synergistic antiproliferative effect in KPL-4T-DM1-resistant breast cancer cells.

[0295] Example 3

[0296] Caspase 3 / 7 Activation Luminescent Assay - Parental and T-DM1-Resistant KPL-4 Breast Cancer Cell Lines

[0297] As mentioned above, Bcl-2 family proteins regulate programmed cell death triggered by developmental cues and respond to multiple stress signals. When activated, they can permeabilize the outer membrane of mitochondria and release pro-apoptotic factors (e.g., cytochrome C) required for activating caspases that disassemble cells (Wang, K., Genes and Development 15 (2001) 2922-2933; (Adams, 2003 supra); Green, DR, and Kroemer, G., Science 305 (2004) 626-629). Therefore, activation of caspases such as caspase 3 and 7 indicates induction of apoptosis.

[0298] In this experiment, Caspase- Caspase 3 / 7 activation luminescence assay was performed using ELISA kit (Promega). The assay was performed in the same manner as the viability assay, except that the drug incubation time was 24 hours and Caspase-Glo 3 / 7 (Promega) was used to measure apoptosis.

[0299] like Figure 3 As shown in the right figure, when treated with the combination of T-DM1 + ​​GDC-0199 for 24 hours, T-DM1-resistant KPL-4 (HER2+) breast cancer cells are again sensitive to T-DM1, as shown by increased apoptosis (increased caspase 3 / 7 activation). The same combination had no effect on parental cells. It is worth noting that the results were evaluated 24 hours after treatment, which is too early for T-DM1 alone to induce apoptosis in the parental cell line (left figure).

[0300] Figure 4A and 4B Results of a caspase 3 / 7 activation luminescent in vitro apoptosis assay are shown, which measures activation of caspase 3 and 7 relative to parental cells in KPL-4T-DM1-resistant human breast cancer cells using different concentrations of T-DM1 and GDC-0199, respectively. Increased apoptosis was observed in KPL-4T-DM1-resistant cells after adding increasing concentrations of GDC-0199 (1, 2.5, or 5 μM) to 0.1 or 1 μg / mL T-DM1. In contrast, T-DM1 induced strong apoptosis in KPL-4 parental cells, which was not further enhanced by the addition of GDC-0199.

[0301] Figure 5AShown are the results of a caspase 3 / 7 activation luminescent 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 the results observed with the KPL-4 T-DM1 resistant cell pool, induction of apoptosis in clone #17 increased following the addition of increasing concentrations of GDC-0199.

[0302] Fig. 6A and 6B Shown are the results of a caspase 3 / 7 activation luminescent 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 μg / mL and 1 μg / mL T-DM1 concentrations alone or in combination with the indicated concentrations of GDC-0199, respectively.

[0303] like Figure 4A , 4B As shown in Figures 5A, 6A and 6B, the results obtained with different clones of the T-DM1-resistant KPL-4 breast cancer cell line demonstrated that various concentrations of the T-DM-1+GDC-0199 combination enhanced the pro-apoptotic activity.

[0304] Example 4

[0305] Xenograft studies - KPL-4T-DM1 anti-breast cancer cell line

[0306] For all xenograft studies, 3 million T-DM1-resistant KPL-4 breast cancer cells were implanted into the #2 / 3 mammary fat pad of female SCID-beige mice. When tumors reached approximately 200 mm 3 At 1:1 volume, mice were randomized into treatment groups (n=10 mice / group): 5 mg / kg T-DM1 q3w, 100 mg / kg GDC-0199qd, a combination of both, or vehicle. The results of these xenograft studies for xenografts of various clones of T-DM1-resistant KPL-4 breast cancer cells are shown in Figure 5B and 6C The results showed that when T-DM1 and GDC-0199 were used in combination, the anti-tumor effect was enhanced relative to the activity of the single agents.

[0307] Example 5

[0308] IHC studies - T-DM1-resistant KPL-4 xenograft tumors

[0309] FFPE (formalin fixed paraffin embedded) xenograft tumor sections were analyzed for Bcl-2 and HER2 (ErbB2) expression by immunohistochemistry (IHC) using the DAB detection method. Bcl-2 antibody SP66 was obtained from Ventana. Human tonsil sections were used as Bcl-2 positive controls. Anti-HER2 antibody 4D5 was obtained from Ventana. Human breast cancer cell lines were used as positive controls (SK-BR-3 as 3+; MDA-MB-361 as 2+; MBA-MB-231 as negative control).

[0310] Fig. 7A Shown is 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 the DAB detection method as described above.

[0311] Figure 7B Shown is 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 the DAB detection method as described above.

[0312] Anti-Bcl-2 antibody results: The vehicle group of each clone showed similar low frequencies of Bcl-2 reactive cells, most often located at the periphery of the tumor lobules (not shown). The frequency and intensity of Bcl-2 signals at the edge of tumor lobules increased or did not change in the T-DM1 treated groups.

[0313] Anti-HER2 antibody results: Vehicle and T-DM1 treated tumors showed very high frequency of HER2 3+ IHC in all clones. In some T-DM1 treated tumors, there were areas of weaker HER2 staining (clone #17), most often adjacent to the stromal bands surrounding the tumor lobules (not shown).

[0314] Bcl-2 IHC results showed that Bcl-2 expression was maintained in T-DM1-resistant clones #8 and #17 when grown as xenograft tumors ( Fig. 7A ; see also Example 6, which shows minimal Bcl-2 expression by Western blot in KPL-4 parental cells). Bcl-2 expression in clone #17 treated with T-DM1 was higher than the corresponding vehicle control. Figure 7BHER2 expression assessed by IHC is depicted. In contrast to the relatively low HER2 expression observed in cells grown in vitro, clones #8 and #17 showed high HER2 expression at 2+ and 3+ levels in both vehicle and T-DM1 treated tumors. All clone #8 tumors were determined to be 85-95% HER2+ or 3+, with very low frequencies of 2+ or 1+ tumor cells. Clone #17 tumors were more variable, with 35-75% HER2 3+ cells and 20-65% HER2 2+ cells in the vehicle group.

[0315] Example 6

[0316] Xenograft studies - KPL-4T-DM1-resistant breast tumors

[0317] Figure 8 Western blot expression data for 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 are shown. (The three digits above lanes 4-19 represent individual xenograft tumors). The expression data show that all groups maintained HER2 and Bcl-2 expression compared to the corresponding cells grown in vitro in cell culture.

[0318] Example 7

[0319] Caspase 3 / 7 Luminescent and Fluorescent Activation Assay - T-DM1 Sensitive Breast Cancer Cell Line The caspase 3 / 7 activation luminescent assay was performed as described in Example 3.

[0320] Using IncuCyte TM Reagents and Equipment The caspase 3 activation fluorescent in vitro apoptosis assay was performed to measure caspase activation over time (kinetic analysis) essentially following the manufacturer's instructions.

[0321] Fig. 9A The results of the caspase 3 / 7 luminescent in vitro apoptosis assay are shown, testing the effects of 5 different concentrations of GDC-0199 (μM) in combination with 9 different concentrations of T-DM1 on caspase activity in HER2+MDA-MB-361 breast cancer cells (sensitive to T-DM1 (not tolerant)). The results show that activation of caspases 3 and 7 with T-DM1 is enhanced in a dose-dependent manner as the concentration of GDC-0199 increases.

[0322] Fig. 9BThe results of the caspase 3 fluorescence in vitro apoptosis assay are shown, and the effects of 3 different concentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 μM) on caspase activity in T-DM1-sensitive (untolerant) MDA-MB-361 breast cancer cells were tested alone or in combination with T-DM1 (0.1 μg / mL). The results show that GDC-0199 enhances the above-mentioned caspase activation induced by T-DM1 alone in a dose- and time-dependent manner, and thus leads to enhanced apoptosis using all combinations.

[0323] Fig. 10A Results of a caspase 3 / 7 luminescent in vitro apoptosis assay are shown, testing the effects of 5 different concentrations of GDC-0199 (μM) with 9 different concentrations of T-DM1 on caspase activity in T-DM1-naive HER2+ HCC1569 breast cancer cells. The results show that T-DM1 alone does not induce apoptosis, but the addition of GDC-0199 results in enhanced caspase activity and, therefore, apoptosis in all combinations.

[0324] Fig. 10B The results of the caspase 3 fluorescence in vitro apoptosis assay are shown, and the effects of 3 different concentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 μM) on caspase activity in HER2+HCC1569 breast cancer cells were tested alone or in combination with T-DM1 (0.1 μg / mL). The results show that GDC-0199 enhances the above caspase activation induced by T-DM1 alone in a dose- and time-dependent manner, and thus leads to enhanced apoptosis using all combinations.

[0325] These results show that the T-DM1 / GDC-0199 combination is also effective in T-DM1-naive (ie, non-T-DM1-resistant) cell lines.

[0326] Example 8

[0327] Xenograft Studies - TDM-1 Sensitive (Non-Tolerized) MDA-MB-361 Breast Tumors

[0328] One day after implantation of the 60-day 17β-estradiol-releasing pellet, 10 million MDA-MB-361 breast cancer cells were implanted into the right mammary fat pad of female NOD / SCID mice. When tumors reached approximately 200-300 mm 3 When the volume of 100 mg / kg was reached, mice were randomly divided into treatment groups (n=10 mice / group) and administered T-DM1 (1, 3 or 7 mg / kg intravenously once a day (ivonce)), GDC-0199 (100 mg / kg qd x 21), or a combination of T-DM1 and GDC-0199, as Fig.11 The results showed that GDC-0199 and 7 mg / kg T-DM1 had enhanced antitumor activity compared to the activity of single agents.

[0329] Example 9

[0330] Western analysis: Effects of T-DM1+ / -GDC-0199 on Bcl-2 family member proteins in HER2+ breast cancer cell lines

[0331] The effects of T-DM1 (1.25 μg / mL) treatment alone or in combination with GDC-0199 (1.25 μM) on various T-DM1-naïve HER2+ breast cancer cell lines were investigated. Fig.12 As shown. Four 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 evaluated, Bcl-xL and Mcl-1. Three cell lines (BT-474, MDA_361, and ZR-75-30) showed phosphorylation of Bcl-2 after T-DM1 treatment, which is a known effect of exposure to anti-mitotic agents such as T-DM1. Fig. 9A , 9B As shown in Figures 10A and 10B, MDA-MB-361 and HCC1569 showed enhanced apoptosis when treated with the combination of T-DM1 and GDC-0199.

[0332] T-DM1 Demonstrated clear clinical benefit in treating cancer in patients, such as in HER2-targeted therapies (such as trastuzumab) Approved by the U.S. Food and Drug Administration for breast cancer that has progressed before. (ado-trastuzumab emtansine conjugate) to treat HER2-positive metastatic breast cancer patients who had previously received treatment with trastuzumab and paclitaxel. The data shown in the present application show that combined 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 show that this combination treatment with T-DM1 and a Bcl-2 inhibitor is effective in treating both T-DM1-sensitive (non-tolerant) HER2-positive cancers (e.g., breast cancer) and HER2-positive cancers (e.g., breast cancer) that are resistant to treatment with T-DM1.

[0333] All publications and patent applications cited in this specification are incorporated herein by reference, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent to those skilled in the art based on the teachings of the present invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. Sequence Listing <110> F. HOFFMANN-LA ROCHE AG <120> Combination therapy of anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor <130> GNE-0416-WO <140> <141> <150> 62 / 189,610 <151> 2015-07-07 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly

Claims

1. Use of a specific binding partner of a Bcl-2 gene or a specific binding partner of an expression product of a Bcl-2 gene in the preparation of a kit for diagnosing that a HER2-positive tumor is resistant to treatment with trastuzumab-MCC-DM1 (T-DM1), wherein the diagnosis comprises: (i) determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to a control sample, and (ii) when the expression level in the tumor sample is at least 2 times higher than the expression level in the control sample, diagnosing that the cancer is resistant to treatment with the T-DM1, and wherein the tumor is breast cancer, The specific binding partner of the Bcl-2 gene is a nucleic acid that hybridizes with the Bcl-2 gene, and The specific binding partner of the expression product of the Bcl-2 gene is an anti-Bcl-2 antibody.

2. Use of a specific binding partner of a Bcl-2 gene or a specific binding partner of an expression product of a Bcl-2 gene in the preparation of a kit for diagnosing susceptibility of a HER2-positive tumor to treatment with trastuzumab-MCC-DM1 (T-DM1), wherein the diagnosis comprises: (i) determining the expression level of a Bcl-2 gene or its product in a tumor sample obtained from a patient with HER2-positive cancer relative to a control sample, and (ii) when the expression level in the tumor sample is less than 2 times higher than the expression level in the control sample, diagnosing that the cancer is susceptible to treatment with the T-DM1, and wherein the tumor is breast cancer, The specific binding partner of the Bcl-2 gene is a nucleic acid that hybridizes with the Bcl-2 gene, and The specific binding partner of the expression product of the Bcl-2 gene is an anti-Bcl-2 antibody.

3. Use of a specific binding partner of a Bcl-2 gene or a specific binding partner of an expression product of a Bcl-2 gene in the preparation of a kit for diagnosing whether a subject with a HER2-positive tumor is resistant or susceptible to treatment with trastuzumab-MCC-DM1 (T-DM1), wherein the diagnosis comprises: (i) obtaining a tumor sample from the subject, (ii) measuring the expression level of the Bcl-2 gene or its product in the tumor sample relative to a control sample, and (iii) diagnosing the tumor as resistant to treatment with T-DM1 when the Bcl-2 expression level measured in the tumor sample is at least 2-fold higher than the expression level in the control sample, or diagnosing the tumor as susceptible to treatment with T-DM1 when the Bcl-2 expression level measured in the tumor sample is less than 2-fold higher than the expression level in the control sample, and wherein the tumor is breast cancer, The specific binding partner of the Bcl-2 gene is a nucleic acid that hybridizes with the Bcl-2 gene, and The specific binding partner of the expression product of the Bcl-2 gene is an anti-Bcl-2 antibody.

4. The use of claim 3, wherein the subject is a human patient.

5. The use according to any one of claims 1 to 4, wherein the control sample is a tumor sample of the same cell type that is not resistant to treatment with the T-DM1.

6. The use according to any one of claims 1 to 4, wherein the tumor sample is a formalin-fixed, paraffin-embedded tumor sample.

7. The use according to any one of claims 1 to 4, wherein the diagnosis further comprises measuring the expression level of the HER2 gene or its product in the tumor sample.

8. The use of any one of claims 1 to 4, wherein when the expression level of Bcl-2 measured in the tumor sample is at least 2-fold higher than the expression level in the control sample, the kit further comprises T-DM1 and a selective Bcl-2 inhibitor for treating the subject.

9. The use of claim 8, wherein the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

10. The use of any one of claims 1 to 4, wherein when the expression level of Bcl-2 measured in the tumor sample is less than 2 times higher than the expression level in the control, the kit further comprises T-DM1 for treating the subject.

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