MÉTODO PARA O PROGNÓSTICO DE UM INDIVÍDUO COM UM CÂNCER DE MAMA HER2 POSITIVO COMO SENDO RESISTENTE OU SUSCEPTÍVEL AO TRATAMENTO COM TRASTUZUMAB-MCCDM1 E KIT PARA O PROGNÓSTICO IN VITRO DE UM CÂNCER DE MAMA HER2 POSITIVO RESISTENTE OU SUSCEPTÍVEL AO TRATAMENTO COM TRASTUZUMAB-MCC-DM1 EM UMA AMOSTRA BIOLÓGICA

Description

1 / 72 METHOD FOR PROGNOSING AN INDIVIDUAL WITH HER2-POSITIVE BREAST CANCER AS BEING RESISTANT OR SUSCEPTIBLE TO TREATMENT WITH TRASTUZUMAB-MCCDM1 AND KIT FOR IN VITRO PROGNOSIS OF HER2-POSITIVE BREAST CANCER RESISTANT OR SUSCEPTIBLE TO TREATMENT WITH TRASTUZUMAB-MCC-DM1 IN A BIOLOGICAL SAMPLE Separated from BR112017028012-4, filed on 06 / 07 / 2016. SEQUENCE LISTING

[001] This request contains a Sequence Listing, which was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The said ASCII copy, created on June 28, 2016, is named GNE-0416-WO.txt and is 6,298 bytes in size. TECHNICAL FIELD

[002] The present invention relates to a combination therapy involving an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for the treatment of cancer. In a particular embodiment, the invention relates to methods of using trastuzumab-MCC-DM1 (trastuzumab emtansine, KADCYLA®) and a selective Bcl-2 inhibitor for the treatment of HER2-positive cancer, such as HER2-positive breast cancer or gastric cancer. BACKGROUND OF THE INVENTION Antibody-drug conjugates against HER2

[003] The HER2 tyrosine kinase receptor (ErbB2) is a member of the epidermal growth factor receptor (EGFR) family of transmembrane receptors. HER2 overexpression is observed in approximately 20% of breast cancers (hereinafter referred to as HER2-positive breast cancer) and is implicated in the aggressive growth and poor clinical outcomes associated with these cancers. Petition 870260055232, dated 08 / 06 / 2026, page 12 / 222 2 / 72 tumors (Slamon et al., (1987) Science 235: 177-182). HER2 protein overexpression can be determined using an immunohistochemical-based analysis of fixed tumor blocks (Press MF, et al (1993) Cancer Res 53: 4960-70).

[004] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, HuMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived, IgG1 kappa, monoclonal antibody that is a humanized version of a murine anti-HER2 antibody (4D5) that selectively binds with high affinity in a cell-based assay (Kd = 5 nM) to the extracellular domain of HER2 (US 5,677,171; US ​​5,821,337; US 6,054,297; US 6,165,464; US 6,339,142; US 6,407,213; US 6,639,055; US 6,719,971; US ​​6,800,738; US 7,074,404; Coussens et al., (1985) Science 230: 1132-9; Slamon et al., (1989) Science 244: 707-12; Slamon et al., (2001) New Engl. J. Med. 344: 783-792). Trastuzumab has been shown, in animals and in vitro assays, to inhibit the proliferation of human tumor cells that overexpress HER2 (Hudziak et 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).

[005] HERCEPTIN® was approved in 1998 for the treatment of patients with metastatic breast cancers that overexpress HER2 (Baselga et al., (1996) J. Clin. Oncol. 14: 737-744) who had received extensive prior anticancer therapy, and has since been Petition 870260055232, dated 08 / 06 / 2026, page 13 / 222 3 / 72 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]. doxorubicin, Cyclophosphamide and paclitaxel for the adjuvant treatment of patients with HER2-positive, nodule-positive breast cancer.

[006] An alternative approach to antibody-targeted therapy is to use antibodies to deliver cytotoxic drugs specifically to cancer cells that express the antigen. Antibody-drug conjugates, or ADCs, are monoclonal antibodies to which highly potent cytotoxic agents have been conjugated. ADCs represent a novel approach to conferring tumor selectivity in systemically administered antitumor therapy. Using surface antigens that are tumor-specific and / or overexpressed, ADCs are designed to target the delivery of highly potent cytotoxic agents to tumor cells. The potential of this approach is to create a more favorable therapeutic window for these agents than would occur with their administration in the form of free drugs.

[007] Maitansinoids, derived from the anti-mitotic drug maytansine, 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 maytansinoid containing thiol derived from the naturally occurring ansamitocine P3 ester (Remillard S, Rebhun LI, Howie GA, et al., (1975) Science 189(4207): 1002-1005.3; Cassady JM, Chan KK, Floss HG. Petition 870260055232, dated 08 / 06 / 2026, page 14 / 222 4 / 72 (2004) Chem Pharm Bull 52(1): 1-26.4). The plant-related ester, maytansine, was studied as a chemotherapeutic agent in approximately 800 patients, administered at a dose of 2.0 mg / m2 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, maytansine's clinical activity was modest at doses that could be safely administered. Dose-limiting toxicity (DLT) was gastrointestinal, consisting of nausea, vomiting, and diarrhea (frequently followed by constipation). These toxic effects were dose-dependent but not schedule-dependent. A pre-existing neuropathy (predominantly sensory) was reported and was more evident in patients with pre-existing neuropathy. Transient subclinical elevations of liver transaminase, alkaline phosphatase, and total bilirubin have been reported.Constitutive toxicities, including weakness, lethargy, dysphoria, and insomnia, were common. Less common toxicities included local infusion phlebitis and mild myelosuppression. Further development of the drug was abandoned in the 1980s due to the narrow therapeutic window.

[008] Trastuzumab-MCC-DM1 (T-DM1, trastuzumab entansine, trastuzumab entansine ado, KADCYLA®), a novel antibody-drug conjugate (ADC) for the treatment of HER2-positive breast cancer, is composed of the cytotoxic agent DM1 (a thiol containing an anti-microtubule maytansinoid agent) conjugated to trastuzumab lysine side chains via an MCC ligand, with an average drug loading (drug-to-antibody ratio) of 3.5. After binding to HER2-expressed tumor cells, T-DM1 undergoes receptor-mediated internalization, resulting in the intracellular release of DM1-containing cytotoxic catabolites and subsequent cell death. Petition 870260055232, dated 08 / 06 / 2026, p. 15 / 222 5 / 72

[009] 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. The 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). This same study also showed that weekly dosing with 2.4 mg / kg was also well tolerated and had antitumor activity. (Beeram (2012) Cancer 118(23): 5733-5740.)

[0010] A phase II study (TDM4374g) demonstrated that TDM1, administered at 3.6 mg / kg q3w, had single-agent antitumor activity in a population of pre-treated patients with HER2-positive metastatic breast cancer. (Krop (2012) 30(26): 3234-3241.A phase III study (TDM4370g) demonstrated that T-DM1, administered at 3.6 mg / kg q3w, significantly prolonged progression-free survival and overall survival with less toxicity compared to treatment with lapatinib plus capecitabine in patients with HER2-positive advanced breast cancer previously treated with trastuzumab and taxane. (Verma (2012) New England Journal of Medicine 367: 1783-1791.).

[0011] The US Food and Drug Administration approved trastuzumab emtansine, marketed under the trade name KADCYLA®, on February 22, 2013, for the treatment of HER2-positive metastatic breast cancer patients who have received prior treatment with trastuzumab and taxane. Bcl-2 inhibitors

[0012] The Bcl-2 protein family regulates 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 and Development Petition 870260055232, dated 08 / 06 / 2026, page 16 / 222 6 / 72 (2003) 2481-2495; Denial, NN, and Korsmeyer, SJ, Cell 116 (2004) 205-219). Considering that cell survival is promoted through Bcl-2 itself and several nearby proteins (Bcl-xL, Bcl-W, Mcl1, and Al), which have three or four conserved Bcl-2 homology regions (BH), apoptosis is driven by two other subfamilies. The initial signal for cell death is transmitted through the diverse group of BH3-only proteins, including Bad, Bid, Bim, Puma, and Noxa, which have in common only the small BH3 interaction domain (Huang and Strasser, Cell 116 (2000) 839-842). However, Bax or Bak, multidomain proteins containing BH1-BH3, are required to lead to 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 permeabilize the outer mitochondrial membrane and release pro-apoptogenic factors (e.g., cytochrome C) needed to activate caspases that dismantle the cell (Wang, K., Genes and Development 15 (2001) 2922-2933; (Adams, 2003 supra), Green, DR, and Kroemer, G., Science 305 (2004) 626-629).

[0013] The interactions between members of these three factions of the Bcl-2 family determine whether a cell lives or dies. When only BH3 proteins have been activated, for example, in response to DNA damage, they can bind through their BH3 domain to a groove in their pro-survivor relatives (Sattler, et al., Science 275 (1997) 983-986). How only BH3 and Bcl-2 type proteins control the activation of Bax and Bak, however, remains poorly understood (Adams, 2003, supra). Most attention has been 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) generally has its membrane target domain inserted in its groove, probably leading Petition 870260055232, dated 08 / 06 / 2026, page 17 / 222 7 / 72 taking into account its cytosolic location (Nechushtan, A., et al., EMBO Journal 18 (1999) 2330-2341; Suzuki, et al., Cell 103 (2000) 645-654; Schinzel, A., et al., J Cell Bio1 164 (2004) 1021-1032). Several unrelated peptides / proteins have been proposed to modulate Bax activity, reviewed in Lucken-Ardjomande, S., and Martinou, JC, J Cell Sci 118 (2005) 473-483, but their physiological relevance remains to be established. Alternatively, Bax can be activated through direct involvement only via certain BH3 proteins (Lucken-Ardjomande, S., and Martinou, JC, 2005 supra), the best documented being 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 older model, in which Bcl-2 directly involves Bax (Oltvai, ZN, et al., Cell 74 (1993) 609-619) has become problematic because Bcl-2 is membrane-bound while Bax is cytosolic, and their interaction appears highly dependent on the detergents used for cell lysis (Hsu, YT, and Youle, 1997 supra). However, it is well established that the BH3 region of Bax can mediate association with Bcl-2 (Zha, H. and Reed, J., Journal of Biological Chemistry 272 (1997) 31482-88; Wang, K., et al., Molecular und Cellular Biology 18 (1998) 6083-6089) and that Bcl-2 prevents the oligomerization of Bax, even if no heterodimers are detected (Mikhailov, V., et al., Journal of Biological Chemistry 276 (2001) 18361-18374). Thus, it remains uncertain whether pro-survival proteins restrict Bax activation directly or indirectly.

[0014] Although Bax and Bak appear in most circumstances to be functionally equivalent (Lindsten, T., et al., Molecular Cell 6 (2000) 1389-1399; Wei, MC, et al., 2001 supra), substantial differences Petition 870260055232, dated 08 / 06 / 2026, p. 18 / 222 8 / 72 cial changes in its regulation are expected from its distinct location in healthy cells. Unlike Bax, which is largely cytosolic, Bak resides in complexes in the outer membrane of mitochondria and in the endoplasmic reticulum of healthy cells (Wei, MC, et al., 2000 supra, Zong, WX, et al., Journal of Ce11 Biology 162 (2003) 59-69). However, upon receiving cytotoxic signals, Bax and Bak change conformation, and Bax translocates to organelle membranes, where both Bax and Bak then form homo-oligomers that can associate, 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; 5367-5376).

[0015] Several Bcl-2 inhibitors exist on the membrane, all possessing the same property of inhibiting prosurvivor members of the Bcl-2 protein family and, therefore, are promising candidates for cancer treatment. 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.

[0016] The link between other proteins of the Bcl-2 family and cancer is also well established and widely documented (Strasser, A. 2011 EMBO J. 30, 3667-3683), and inhibitors of other members of the Bcl family are also known. The selective Bcl-XL inhibitors A-1155463 and A-1331852 are described, for example, in Leverson et al., Science Translational Medicine Vol. 7, Issue 279 279ra40. Inhibition Petition 870260055232, dated 08 / 06 / 2026, p. 19 / 222 Selective benzothiazole hydrazone BcI-Xl inhibitors are reported in Sleebs et al., J. Med. Chem. 2013, 56, 5514-5540. For descriptions of other BcI-Xl inhibitors, see, for example, Koehler et al., ACS Med. Chem. Lett. 2014, 5, 662-667; and Tao et al., ACS Med. Chem. Lett. 2014, 5, 1088-10. MCl-1 inhibitors and their uses as cancer therapies 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., Pharmacology & Therapeutics 145 (2015) 76-84. Dual Mcl-1 / Bcl-xL inhibitors are disclosed by Tanaka et al., J. Med. Chem. 2013, 56, 9635-9645. SUMMARY OF THE INVENTION

[0017] In one aspect, the invention relates to a method for treating cancer in a human being in need thereof comprising administering to such human being an effective amount of an anti-HER2 antibody-drug conjugate and an inhibitor of a BcI family protein.

[0018] In another aspect, the invention relates to a method for treating cancer in a human being in need thereof, characterized by comprising administering to such human being an effective amount of an anti-HER2 antibody-drug conjugate and a selective BcI-2 inhibitor.

[0019] In one modality, cancer is cancer positive for HER2.

[0020] In another modality, the cancer is HER2-positive breast cancer or gastric cancer.

[0021] In yet another modality, HER2 breast cancer Petition 870260055232, dated 08 / 06 / 2026, page 20 / 222 10 / 72 positive or gastric cancer has an immunohistochemistry (IHC) score for HER2 of 2+ or 3+ and / or an in situ hybridization amplification rate (ISH) (her2: CEP17 in situ hybridization amplification rate (ISH) of >2.0).

[0022] In an additional modality, cancer positive for HER2-positive cancers, such as breast cancer or gastric cancer, are resistant to treatment with an anti-HER2 antibody-drug conjugate administered as a single agent.

[0023] In yet another modality, cancer positive for HER2-positive cancers, such as breast cancer or gastric cancer, are responsive to treatment with an anti-HER2 antibody-drug conjugate administered as a single agent, and the combination of the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor can be administered to a single patient for treatment with the anti-HER2 antibody-drug conjugate.

[0024] In a different embodiment, the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor show synergistic activity, including, but not limited to, synergistic activity in a HER2-positive cancer, such as breast cancer or gastric cancer, that is resistant to treatment with an anti-HER2 antibody-drug conjugate administered as a single agent.

[0025] In all modalities, the anti-HER2 antibody-drug conjugate may, for example, be trastuzumab-MCC-DM1.

[0026] In all embodiments, the selective Bcl-2 inhibitor may, for example, be 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro4-((tetrahydro-2H-pyran-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0027] In another aspect, the invention relates to a method for treating HER2-positive cancer in a human being that Petition 870260055232, dated 08 / 06 / 2026, page 21 / 222 11 / 72 of it requires understanding the administration to humans of an effective amount of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0028] In one modality, the cancer is HER2-positive breast cancer or gastric cancer.

[0029] In another modality, HER2-positive breast cancer or gastric cancer has an immunohistochemistry (IHC) score for HER2 of 2+ or 3+ and / or an in situ hybridization amplification rate (ISH) (her2: CEP17 in situ hybridization amplification rate (ISH) of >2.0.

[0030] In another additional modality, cancer positive for HER2-resistant cancers, such as breast cancer or gastric cancer, are resistant to treatment with trastuzumab-MCC-DM1 administered as a single agent.

[0031] In an additional modality, cancer positive for HER2-positive cancers, such as breast cancer or gastric cancer, are sensitive to treatment with trastuzumab-MCC-DM1 administered as a single agent, and the combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof may be administered to a single patient for treatment with trastuzumab-MCC-DM1.

[0032] In yet another modality, cancer positive for HER2-positive cancers, such as breast cancer or gastric cancer, are responsive to treatment with an anti-HER2 antibody-drug conjugate administered as a single agent and with combination antibody-drug combinations. Petition 870260055232, dated 08 / 06 / 2026, page 22 / 222 12 / 72 anti-HER2 drug and the selective Bcl-2 inhibitor can be administered to a single patient for treatment with the anti-HER2 antibody-drug conjugate. In another embodiment, trastuzumab-MCCDM1 and 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro2H-pyran-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof show synergistic activity, including, but not limited to, synergistic activity in a HER2-positive cancer, such as breast cancer or gastric cancer.

[0033] In yet another modality, trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro2H-pyrano-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof are co-administered.

[0034] In another embodiment, trastuzumab-MCC-DM1 and 2-(1Hpyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyrano-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof are administered concomitantly.

[0035] In yet another modality, trastuzumab-MCC-DM1 and the 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro2H-pyrano-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof are administered consecutively.

[0036] In another aspect, the 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 the treatment of cancer.

[0037] In one embodiment, the Bcl family protein is a Bcl-2-like protein, such as Mcl-1, Bcl-xl, Bcl-w (BCL2L2) or Bcl Petition 870260055232, dated 08 / 06 / 2026, page 23 / 222 13 / 72 xs, preferably Mcl-1 or Bcl-xl.

[0038] In another aspect, the 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 the treatment of cancer.

[0039] In one embodiment, the invention relates to the use of a combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of cancer.

[0040] In all modalities, the cancer can be HER2-positive cancer.

[0041] In all forms, cancer can, for example, be breast cancer or gastric cancer.

[0042] In all modalities, HER2-positive cancer, such as breast cancer or gastric cancer, may be resistant to treatment with trastuzumab-MCC-DM1 administered as a single agent.

[0043] In all modalities, HER2-positive cancer, such as breast cancer or gastric cancer, may be responsive to treatment with trastuzumab-MCC-DM1 administered as a single agent, and the combination of trastuzumab-MCC-DM1 and 2-(1H-pyrrolo[2,3b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof may be used to treat a single patient for treatment with trastuzumab-MCC-DM1.

[0044] In another aspect, the invention relates to the use of a Petition 870260055232, dated 08 / 06 / 2026, page 24 / 222 14 / 72 combination of an anti-HER2 antibody-drug conjugate and an inhibitor of a Bcl family protein in the preparation of a drug for the treatment of cancer.

[0045] In one embodiment, the Bcl family protein is a Bcl-2-like protein, such as Mcl-1, Bcl-xl, Bcl-w (BCL2L2) or Bclxs, preferably Mcl-1 or Bcl-xl.

[0046] 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 the treatment of cancer.

[0047] In one embodiment, the combination of trastuzumab-MCCDM1 and 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro2H-pyran-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof is for use in the treatment of cancer.

[0048] In all combinations, the cancer can, for example, be HER2-positive cancer, such as HER2-positive breast cancer or gastric cancer.

[0049] In one particular embodiment, the cancer, 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.

[0050] In another embodiment, HER2-positive cancer, such as breast cancer or gastric cancer, may be responsive to treatment with the anti-HER2 antibody-drug conjugate, for example, trastuzumab-MCC-DM1, when administered as a single agent, and the combination may be used to treat a single patient for treatment with the anti-HER2 antibody-drug conjugate, for example, trastuzumab-MCC-DM1.

[0051] In another aspect, the invention relates to a method for diagnosing a treatment-resistant HER2-positive tumor. Petition 870260055232, dated 08 / 06 / 2026, page 25 / 222 15 / 72 with an anti-HER2 antibody-drug conjugate, characterized by comprising the determination of the expression level of the 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 diagnosis of said cancer as resistant to treatment with said anti-HER2 antibody-drug conjugate when the expression level in said tumor sample is at least 2 times, or at least 3 times, or at least 4 times, or at least 5 times higher than the expression level in said control sample.

[0052] In a further aspect, the invention relates to a method for diagnosing a HER2-positive tumor amenable to treatment with an anti-HER2 antibody-drug conjugate, characterized by comprising determining the expression level of the 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 said cancer as amenable to treatment with said anti-HER2 antibody-drug conjugate when the expression level in said tumor sample is less than 2 times, or at least 3 or at least 4 times, or at least 5 times greater than the expression level in said control sample.

[0053] In a further aspect, the invention relates to a method for diagnosing a subject with a HER2-positive tumor as being resistant or susceptible to treatment with an anti-HER2 antibody-drug conjugate, characterized by comprising (i) obtaining a tumor sample from said subject, (ii) measuring the expression level of the Bcl-2 gene or its product in said tumor sample relative to a control sample, and (iii) diagnosing said tumor as being resistant to treatment with a Petition 870260055232, dated 08 / 06 / 2026, p. 26 / 222 16 / 72 antibody-drug conjugate anti-HER2 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, or diagnose the tumor as being susceptible to treatment with an antibody-drug conjugate anti-HER2 when the measured expression level of Bcl-2 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.

[0054] In one modality, the subject is a human patient.

[0055] In another embodiment, the control sample is a tumor sample of the same cell type that is not resistant to treatment with the aforementioned anti-HER2 antibody-drug conjugate.

[0056] In yet another scenario, the tumor is breast cancer or gastric cancer.

[0057] In an additional embodiment, the tumor sample is a formalin-fixed, paraffin-embedded tumor sample.

[0058] In all modalities, the diagnostic method may also include a step measuring the expression level of the HER2 gene or its product in the tumor sample.

[0059] In all modalities, the diagnostic method may also include a treatment step of 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 4 times, or at least 5 times greater than the expression level in the aforementioned control sample.

[0060] In all modalities, the diagnostic method may additionally include the treatment stage for the patient in question. Petition 870260055232, dated 08 / 06 / 2026, page 27 / 222 17 / 72 entity with an anti-HER2 antibody-drug conjugate when the measured expression level of Bcl-2 in the tumor sample is less than 2 times higher than the expression level in the control sample.

[0061] In one embodiment, the antiHER2 antibody-drug conjugate is trastuzumab-MCC-DM1.

[0062] In another embodiment, the selective Bcl-2 inhibitor is 2-(1 Hpyrrolo[2,3-b ]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2 H-pyran-4yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0063] In another aspect, the invention relates to a kit for the in vitro diagnosis or prognosis of a HER2-positive tumor resistant to treatment with an anti-HER2 antibody-drug conjugate in a biological sample obtained from a patient, characterized by comprising a binding partner specific for the Bcl-2 gene or its expression product.

[0064] In one embodiment, the binding partner is an anti-Bcl-2 antibody.

[0065] In another embodiment, the linking partner is a nucleic acid that hybridizes with the Bcl-2 gene.

[0066] In a further aspect, the invention relates to a kit comprising an anti-HER2 antibody drug conjugate and a selective Bcl-2 inhibitor for the combined treatment of a patient with a cancer that expresses HER2.

[0067] In one embodiment of the present invention, the kit further comprises a pharmaceutically acceptable vehicle. The kit may also include a sterile diluent, which is preferably stored in a separate additional container. The kit may also include a package insert comprising printed instructions directing the use of the combined treatment as a method for an expression. Petition 870260055232, dated 08 / 06 / 2026, page 28 / 222 18 / 72 of HER2-positive cancers, such as HER2-positive breast cancer or gastric cancer.

[0068] As in other aspects, the expression of cancer HER2 can, for example, be breast cancer or gastric cancer, and in several modalities the anti-HER2 drug-antibody conjugate can be trastuzumab-MCC-DM1 and / or the selective Bcl-2 inhibitor can be 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro2H-pyran-4-yl)methylamine)phenylsulfonyl)benzamide or a pharmaceutically acceptable salt thereof.

[0069] In a particular form, the expression cancer of HER2-negative cancers being treated, such as breast cancer or gastric cancer, are resistant to treatment with an anti-HER2 antibody-drug conjugate or trastuzumab-MCC-DM1 when administered as a single agent.

[0070] In another embodiment, HER2-expressing cancer, such as breast cancer or gastric cancer, may be responsive to treatment with the anti-HER2 antibody-drug conjugate, for example, trastuzumab-MCC-DM1, when administered as a single agent, and the combination may be used to treat a single patient for treatment with the anti-HER2 antibody-drug conjugate, for example, trastuzumab-MCC-DM1. BRIEF DESCRIPTION OF THE FIGURES

[0071] FIGS. 1A and 1B show the expression of Bcl-2 family survival molecules in T-DM1-resistant human breast cancer cells KPL-4 and BT-474M1 (HER2-positive) compared to mother cells. FIG. 1A shows mRNA expression assessed by TaqMan qRT-PCR analysis; FIG. 1B shows protein expression by Western blot analysis.

[0072] FIG. 2 presents the results of a feasibility test. Petition 870260055232, dated 08 / 06 / 2026, page 29 / 222 19 / 72 Cellular data showing that the T-DM1 + ​​GDC-0199 combination has a synergistic effect on T-DM1-resistant KPL-4 human breast cancer cells, while no synergism is observed in KPL-4 matrix cells.

[0073] FIG. 3 presents the results of a caspase activation assay measuring the activation of caspases 3 and 7. The results show that within 24 hours of drug treatment, T-DM1-resistant human KPL-4 breast cancer cells are re-sensitized to T-DM1 in the presence of GDC-0199, whereas within 24 hours, there is no effect of T-DM1 + / - GDC-0199 on KPL-4 stem cells.

[0074] FIGS. 4A and 4B present the results of a caspase activation assay measuring the activation of caspases 3 and 7 in T-DM1-resistant human KPL-4 breast cancer cells relative to stem cells after 48 hours of drug treatment. The results show dose-dependent increases in caspase 3 and 7 activation with the addition of GDC-0199 to T-DM1, with minimal effect from T-DM1 alone. In KPL-4 stem cells, T-DM1 induces robust caspase 3 and 7 activation with minimal increase upon the addition of GDC-0199.

[0075] FIG. 5A presents the results of a caspase activation assay measuring the activation of caspases 3 and 7 in the T-DM1-resistant KPL-4 human breast cancer cell line in Clone #17 treated with 1 pg / mL of T-DM1 alone or in combination with the indicated doses of GDC-0199 for 48 hours. The results show dose-dependent increases in caspase 3 and 7 activation with the addition of GDC-0199 to T-DM1, with minimal effect from TDM1 alone.

[0076] FIG. 5B shows the effect of T-DM1 (5 mg / kg administered once), GDC-0199 (100 mg / kg qd x 21) or the combination on tumor growth (as measured by tumor volume) of xeno Petition 870260055232, dated 08 / 06 / 2026, page 30 / 222 20 / 72 transplants of T-DM1-resistant KPL-4 human breast cancer cells from Clone #17 into SCID beige mice. Treatment with the combination drug resulted in tumor stasis, with no single-agent treatment activity.

[0077] FIGS. 6A and 6B show the results of a caspase activation assay measuring the activation of caspases 3 and 7 in the T-DM1-resistant KPL-4 human breast cancer cell line in Clone #8 treated with concentrations of 0.1 pg / mL and 1 pg / mL of TDM1, respectively, alone or in combination with the indicated concentrations of GDC-0199.

[0078] FIG. 6C shows the effect of T-DM1 (5 mg / kg q3w x 2), GDC-0199 (100 mg / kg qd x 21) or the combination in tumor growth (as measured by tumor volume) of T-DM1-resistant KPL-4 human breast cancer cell xenotransplants from Clone #8 in SCID beige mice.

[0079] FIG. 7A shows the expression of Bcl-2 in formalin-fixed paraffin-embedded KPL-4 T-DM1 xenograft tumor samples (Clones #8 and #17) determined by immunohistochemistry (IHC) using the DAB detection method.

[0080] FIG. 7B shows the expression of HER2 (ErbB2) in formalin-fixed paraffin-embedded T-DM1-resistant KPL-4 xenograft tumor samples (Clones #8 and #17) determined by immunohistochemistry (IHC) using the DAB detection method.

[0081] FIG. 8 shows the protein expression as measured by Western blot assay analysis of Bcl-2 and HER2 in T-DM1-resistant KPL-4 xenotransplant tumors in Clone #17, treated with GDC-0199, T-DM1 or T-DM-1 + GDC-0199.

[0082] FIG. 9A presents the results of an in vitro apoptosis assay of activation of a luminescent caspase 3 / 7 by means of Petition 870260055232, dated 08 / 06 / 2026, page 31 / 222 21 / 72 test of the effect of five separate concentrations of GDC-0199 (μM) in combination with 9 different concentrations of T-DM1 after 24 hours of treatment on HER2+ MDA-MB361 breast cancer cells (naive NT-DM1 cells). The results demonstrate enhanced apoptosis greater than T-DM1 alone with all combinations tested.

[0083] FIG. 9B presents the results of an in vitro fluorescent apoptosis assay of caspase 3 / 7 kinetic activation, testing the effect of three 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+ MDA-MB-361 breast cancer cells. The results demonstrate enhanced caspase activation greater than T-DM1 alone with all combinations tested.

[0084] FIG. 10A presents the results of an in vitro apoptosis assay of activation of a luminescent caspase 3 / 7 by testing the effect of five separate concentrations of GDC-0199 (μM) in combination with 9 different concentrations for 24 hours of treatment in HER2+ HCC1569 breast cancer cells (naive NT-DM1 cells). The results do not demonstrate induction of apoptosis by T-DM1 alone, but enhanced apoptosis with all combinations tested.

[0085] FIG. 10B presents the results of an in vitro caspase 3 / 7 kinetic fluorescent apoptosis assay, testing the effect of three 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), on HER2+HCC 1569 breast cancer cells. The results demonstrate dose-dependent increased caspase activation with combined treatment.

[0086] FIG. 11 shows the effect of T-DM1 (1 mg / kg, 3 mg / kg, 7 mg / kg, iv q3wx1) and GDC-0199 (100 mg / kg, po, qd x 21), individually. Petition 870260055232, dated 08 / 06 / 2026, page 32 / 222 22 / 72 or in combination, in tumor growth in the HER2+ MDA-MB-361 breast cancer xenotransplantation model. A significant delay in tumor growth was observed with GDC-0199 combined with 7 mg / kg of T-DM1.

[0087] FIG. 12 shows the Western blot analysis of the effects of T-DM1, with or without GDC-0199, on members of the Bcl-2 family (total and phospho-Bcl-2 and -Bcl-xL, total Mcl-1 and Bim) in the cell lines BT-474, EFM192A, KPL-4, HCC1569, HCC1954, MDA-361, UACC-812, HER2+ breast cancer ZR-75-30.

[0088] FIG. 13 shows the trastuzumab light chain amino acid sequence (SEQ ID NO: 1).

[0089] FIG. 14 shows the amino acid sequence of trastuzumab heavy chain (SEQ ID NO: 2). DETAILED DESCRIPTION OF THE INVENTION

[0090] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. Although the invention will be described in conjunction with the listed embodiments, it will be understood that it is not intended to limit the invention to the embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention, as defined by the claims. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0091] All references cited throughout the description are expressly incorporated herein by reference in their entirety. In the event that one or more of the incorporated literature, patents and similar materials differ from or contradict this application, including but not limited to Petition 870260055232, dated 08 / 06 / 2026, page 33 / 222 23 / 72 limited to the defined terms, use of term, techniques described or similar, this application controls. DEFINITIONS

[0092] The term antibody in this document is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., biospecific antibodies), and antibody fragments for as long as they exhibit the desired antigen-binding activity.

[0093] The term monoclonal antibody, as used in this document, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or mutations originating during the production of a monoclonal antibody preparation, these variants generally being present in smaller quantities. On the other hand, polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen.Thus, the monoclonal modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any specific method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the immune loci. Petition 870260055232, dated 08 / 06 / 2026, page 34 / 222 24 / 72 human globulin. These methods and other examples for producing monoclonal antibodies are described in this document.

[0094] The anti-HER2 antibody used in the antibody-drug conjugates in this document is a monoclonal antibody.

[0095] 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, generally prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred. Such murine / human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of chimeric antibodies encompassed by the present invention are those in which the class or subclass has been modified or altered from that of the original antibody.Such chimeric antibodies are also referred to as class-switched antibodies. The methods for producing chimeric antibodies involve conventional recombinant DNA techniques and gene transfection now 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.

[0096] The term humanized antibody refers to antibodies in which the structure or complementarity-determining regions (CDRs) have been modified to comprise the CDR of an immunoglobulin with different specificity compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted onto the structure region of a human antibody to prepare Petition 870260055232, dated 08 / 06 / 2026, page 35 / 222 25 / 72 rare the 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 preferential CDRs correspond to those that represent sequences that recognize the antigens mentioned above for chimeric and bifunctional antibodies.

[0097] The term human antibody, as used in this document, is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of 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 methods, including phage display. Based on this technology, human antibodies can be produced against a wide variety of targets. 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 for the production and selection of human antibodies, see, for example, Winter et al., Ann Review Immunol, 1994, 12: 433-455; and for the production of fully human antibodies from transgenic mice and phage display platforms, see, for example, Lonberg, Current Opinion Immunol, 2008, 20 (4): 450-459.

[0098] The term recombinant human antibody, as used in this document, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as, for example, antibodies isolated from a host cell such as an NS0 or CHO cell or from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies possess variable regions and Petition 870260055232, dated 08 / 06 / 2026, page 36 / 222 26 / 72 constants derived from human immunoglobulin sequences in a rearranged form.

[0099] As used in this document, specifically binding or specifically binds to refers to binding that is selective enough for a target to distinguish it from binding to unwanted or nonspecific targets. In one embodiment, an antibody that specifically binds to a target will have a binding affinity for the target (Kd) 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 less, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M). In yet another embodiment, the Kd is 10⁻¹⁰ mol / L or less (e.g., 10⁻¹² mol / L). Binding affinity is determined with a standard binding assay, such as Scatchard plot analysis in cells expressing the target antigen.

[00100] The term nucleic acid molecule, as used in this document, is intended to include both DNA and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded. In one embodiment, it is double-stranded DNA.

[00101] Constant domains are not directly involved in antibody binding to an antigen, but are involved in effector functions (ADCC, complement binding and CDC).

[00102] The term variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in antibody binding to the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, each domain comprising four conserved structure regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).]

[00103] In this document, the term hypervariable region or HVR Petition 870260055232, dated 08 / 06 / 2026, page 37 / 222 27 / 72 refers to each of the regions of an antibody variable domain that are hypervariable in sequence (complementarity-determining regions or CDRs) and / or form structurally defined loops (hypervariable loops) and / or contain antigen-contact residues (antigen contacts). Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Examples of HVRs in this document include:

[00104] (a) hypervariable loops that occur 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));

[00105] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[00106] (c) antigen contacts that occur 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

[00107] (d) combinations of (a), (b) and / or (c) including 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).

[00108] The term anti-HER2 antibody according to the invention is an antibody that binds specifically to the HER2 antigen.

[00109] As defined in this document, the terms trastuzumab, HERCEPTIN® and huMAb4D5-8 are used interchangeably. Such an antibody preferably comprises the light and heavy chain amino acid sequences shown in FIG. 13 (SEQ ID NO: 1) and FIG. 14 (SEQ ID NO: 2), respectively.

[00110] The 4D5 epitope, 4D5 epitope or 4D5 is the region in the extracellular domain of HER2 to which the 4D5 antibody (ATCC CRL) binds. Petition 870260055232, dated 08 / 06 / 2026, p. 38 / 222 28 / 72 10463) and trastuzumab bind. This epitope is located near the transmembrane domain of HER2 and within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, a routine cross-blocking assay, as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues from approximately residue 529 to residue 625 inclusive of HER2).

[00111] The 2C4 epitope or 2C4 epitope is the region in the extracellular domain of HER2 to which the 2C4 antibody binds. To screen for antibodies that bind to the 2C4 epitope, a routine cross-blocking assay, as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be done to assess whether the antibody binds to the 2C4 epitope of HER2. The 2C4 epitope comprises domain II residues 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)).

[00112] As defined herein, the terms T-DM1, trastuzumab MCC-DM1, emtansine-trastuzumab, trastuzumab emtansine, and KADCYLA® are used interchangeably and refer to trastuzumab linked via the MCC linker moiety to the DM1 maytansinoid drug moiety, including all variously loaded and attached antibody-drug conjugate mixtures where drug moieties 1, 2, 3, 4, 5, 6, 7, and 8 are covalently linked to the trastuzumab antibody (US 7097840; US 2005 / 0276812; US 2005 / 0166993). Petition 870260055232, dated 08 / 06 / 2026, p. 39 / 222 29 / 72

[00113] The term Bcl-2, as used in this document, refers to the protein Bcl-2 (ID Prot Swiss No. P10415), a member of the Bcl-2 protein family (Cory, S. and Adams, JM, Nature Reviews Cancer 2 (2002) 647-656; Adams, Genes und Development 17 (2003) 24812495; Danial, NN, and Korsmeyer, SJ, Cell 116 (2004) 205-219; Petros, AM, Biochim Biophys Acta 1644 (2004) 83-94).

[00114] The term selective Bcl-2 inhibitor, as used in this document, refers to polypeptides and small molecules that inhibit survival members of the Bcl-2 protein family. Preferably, the selective Bcl-2 inhibitor is 2-(1 / - / -pyrrolo[2,3-b]pyridine5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl>)- / \ / -(3-nitro-4-((tetrahydro-2 / - / -pyrano-4yl)methylamine)phenylsulfonyl)benzamide, (also known as ABT199 or GDC-0199), or a pharmaceutically acceptable salt thereof, a Bcl-2 inhibitor of formula I, which is described in International Publication No. WO2010 / 0138588 and US Publication No. US2010 / 0305122, which are incorporated herein by reference. Formula 1 2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4dimethylcyclohexane-1-enyl)methyl)piperazine-1-yl)- / \ / -(3-nitro-4-((tetrahydro-2 / - / -pyran-4-yl)methylamine)phenylsulfonyl)benzamide

[00115] Here, an antitumor agent refers to a drug used to treat cancer. Examples of antitumor agents Petition 870260055232, dated 08 / 06 / 2026, page 40 / 222 Non-limiting applications of the present invention include chemotherapy agents, HER dimerization inhibitors, their HER antibodies, antibodies directed against tumor-associated antigens, anti-hormonal compounds, cytokines, EGFR-targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and antibodies, cytotoxic agents, apoptosis-inducing antibodies, COX inhibitors, farnesyl transferase inhibitors, antibodies that bind to the 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.

[00116] Chemotherapy is the use of a chemotherapeutic agent useful in the treatment of cancer.

[00117] A chemotherapeutic agent is a chemical compound useful in the treatment of cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, antimitotic plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0] nona-2,7,9-triene-9-carboxamide, CAS No.85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®) and doxorubicin (ADRIAMYCIN®). Petition 870260055232, dated 08 / 06 / 2026, p. 41 / 222 31 / 72 Akti-1 / 2, HPPD and rapamycin.

[00118] Further examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, 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), XL147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plow), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (without Cremophor),albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorammbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), Pazopanib (GlaxoSmithKline), campphosphamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; Ethyleneimines and methylamylamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bulatacin and bulatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, Petition 870260055232, dated 08 / 06 / 2026, p. 42 / 222 32 / 72 KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembin, fenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enedine antibiotics (e.g., calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186); dynemycin, dynemycin A; bisphosphonates, such as clodronate; a esperamycin;as well as neocarzinostatin chromophore and related chromoprotein enedin antibiotic chromophores), aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, cromomycins, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and desoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, chelamicin, rodorrubicin, streptonigrin, Streptozocin, tubercidine, ubenimex, zinostatin, zorrubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate; pruine analogues, such as fludarabine, 6-mercaptopurine, tiamiprine, thioguanine;Pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; enyluracil; amsacri; Petition 870260055232, dated 08 / 06 / 2026, page 43 / 222 33 / 72 na; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornitrine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxine; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; Trichothecenes (T-2 toxin, verracurine A, roridine A and anguidin); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®);novantron; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, 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.

[00119] The terms cancer and cancerous refer to or describe the physiological condition in mammals that is typically characterized by the unregulated growth of cells. A tumor comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoid diseases. More specific examples of such cancers include breast cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, and squamous cell carcinoma of the lung, peritoneal cancer Petition 870260055232, dated 08 / 06 / 2026, page 44 / 222 34 / 72 nio, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. In a preferred embodiment, the cancer is breast cancer. In another preferred embodiment, the cancer is gastric cancer.

[00120] Reference to a tumor or cancer as stage 0, stage I, stage II, stage III, or stage IV, and various substages within that classification, indicates the classification of the tumor or cancer using global stage grouping methods or Roman numeral stage grouping methods known in the art. Although the actual stage of the cancer depends on the type of cancer, generally, a stage 0 cancer is an in situ lesion, a stage I cancer is a small, localized tumor, a stage II and III cancer is a locally advanced tumor with local lymph node involvement, and a stage IV cancer represents metastatic cancer. The specific stages for each type of tumor are known to specialist clinicians.

[00121] The term metastatic breast cancer means the state of breast cancer in which cancer cells are transmitted from the site of origin to one or more sites elsewhere in the body via blood or lymphatic vessels, to form one or more secondary tumors in one or more organs other than the breast.

[00122] An advanced cancer is one that has spread outside the site or organ of origin, through local invasion or metastasis. Therefore, the term advanced cancer includes locally advanced or metastatic diseases. Petition 870260055232, dated 08 / 06 / 2026, page 45 / 222 35 / 72

[00123] A refractory cancer is one that progresses despite an antitumor agent, such as chemotherapy, being administered to the cancer patient. An example of a refractory cancer is one that is refractory to platinum.

[00124] A recurrent cancer is one that has grown back, either at the original site or at a distant site, after a response to initial therapy, such as surgery.

[00125] A locally recurrent cancer is a cancer that returns after treatment in the same place as a previously treated cancer.

[00126] An operable or extirpable cancer is a cancer that is confined to the primary organ and suitable for surgery (excision).

[00127] An inoperable or non-extirpable cancer is one that cannot be removed (extirpated) by surgery.

[00128] The terms HER2-positive and HER2-expressing are used interchangeably in this document. The term HER2-positive cancer encompasses cancer cells that have higher than normal levels of HER2. Examples of HER2-positive cancer include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, HER2-positive is a HER2-overexpressing cancer, and in modalities, HER2-positive cancer has an immunohistochemical (IHC) score of 2+ or 3+ and / or an in situ hybridization amplification ratio (ISH) >2.0.

[00129] In situ hybridization (ISH) determines the number of her2 copies using a DNA probe coupled to a fluorescent, chromogenic, or silver detection system (i.e., FISH, CISH, or SISH), or a combination of CISH and SISH systems (dual bright-field ISH (BDISH) or double-color, double hapten ISH (DDISH)). ISH can be conducted using a single probe to enumerate her2 copies. Petition 870260055232, dated 08 / 06 / 2026, p. 46 / 222 36 / 72 splices per nucleus alone or as a dual-probe technique, where hybridization of a centromeric chromosome 17 probe (chromosome enumeration probe 17, CEP17) allows determination of the her2:CEP17 ratio. The dual-probe approach can be performed as a two-color technique, with co-hybridization of the two probes on the same slide, or as a monochromatic assay where probe is used on sequential slides. The her2:CEP17 ratio is sometimes considered a better reflection of her2 amplification status than the average number of her2 copies, since the latter is also dependent on other parameters such as tumor mitotic index, section thickness, nuclear truncation effects, and abnormal chromosome copy number (aneusomy). The phrase hybridization amplification rate (ISH) >2.0 in situ refers to a her2:CEP17 ratio >2.0. For more details, see, for example, Sauter G, et al.Guidelines for the human epidermal growth factor receptor 2 test: biological and methodological considerations. J Clin Oncol 2009; 27: 1323-1333, and the review article by Hanna et al. Modern Pathology (2014) 27, 4-18.

[00130] Here, a patient or individual is a human patient. The patient may be a cancer patient, that is, someone who suffers from or is at risk of suffering from one or more symptoms of cancer, in a specific breast or gastric cancer.

[00131] A patient population refers to a group of patients with cancer. Such populations can be used to demonstrate statistically significant efficacy and / or safety of a drug, such as pertuzumab.

[00132] A patient with relapse is one who presents with signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[00133] A biological or cancer sample that presents a Petition 870260055232, dated 08 / 06 / 2026, page 47 / 222 37 / 72 HER expression, amplification, or activation is defined as the expression (including overexpression) of an HER receptor in a diagnostic test, or the amplification of the HER gene, and / or the demonstration of activation or phosphorylation of an HER receptor.

[00134] The term synergistic, as used in this document, refers to a therapeutic combination that is more effective than the additive effects of the two or more individual agents. The determination of a synergistic interaction between an anti-Her2 antibody-drug conjugate, such as trastuzumab-MCC-DM1, and a selective Bcl2 inhibitor can be based on the results obtained from the assays described in this document, or on other assay systems known in the art, using a standard program to quantify synergism, additivism, and antagonism between anticancer agents. The program preferably used is that described by Chou and Talalay, in New Avenues in Developmental Cancer Chemotherapy, Academic Press, 1987, Chapter 2. Combination Index (CI) values ​​less than 0.8 indicate synergy, values ​​greater than 1.2 indicate antagonism, and values ​​between 0.8 and 1.2 indicate additive effects.Combination therapy can provide synergy and prove to be synergistic, that is: the effect obtained when the active ingredients are used together is greater than the sum of the effects resulting from the use of the compounds separately. A synergistic effect can be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit-dose formulation; (2) administered by alternation or in parallel as separate formulations; or (3) by some other regimen. When administered in alternation therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each ingredient... Petition 870260055232, dated 08 / 06 / 2026, page 48 / 222 38 / 72 active ingredient is administered sequentially, that is, in a series over time, while in combination therapy the effective doses of two or more active ingredients are administered together.

[00135] Neoadjuvant therapy or preoperative therapy refers, in this document, to therapy provided before surgery. The goal of neoadjuvant therapy is to provide immediate systemic treatment, potentially eradicating micrometastases that would proliferate if the conventional sequence of surgery followed by systemic therapy were continued. Neoadjuvant therapy can also help reduce tumor size, allowing for complete excision of initially inextirpable tumors or preserving portions of the organ and its functions. Furthermore, neoadjuvant therapy allows for an in vivo evaluation of drug efficacy, which can guide the choice of subsequent treatments.

[00136] Adjuvant therapy here refers to therapy given after definitive surgery, where no evidence of residual disease can be detected, in order to reduce 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 specifically excludes neoadjuvant therapy here.

[00137] Definitive surgery is used as the term is used within the medical community. Definitive surgery includes, for example, surgical or other procedures that result in the removal or extirpation of the tumor, including those that result in the removal or extirpation of the entire widely visible tumor. Definitive surgery includes, for example, complete or curative extirpation or complete gross extirpation of the tumor. Definitive surgery includes procedures that occur in one or more stages and includes, for example, multi-stage surgical procedures where one or more surgical or other procedures are performed before the extirpation of the tumor. Surgery Petition 870260055232, dated 08 / 06 / 2026, p. 49 / 222 39 / 72 definitive includes procedures to remove or excise the tumor, including the organs involved, parts of organs and tissues, as well as adjacent organs such as lymph nodes, parts of organs or tissues. Removal may be incomplete, so that tumor cells may still remain undetected.

[00138] Survival refers to the patient remaining alive and includes disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). Survival can be estimated using the Kaplan-Meier method, and any differences in survival are calculated using the stratified log-rank test.

[00139] Progression-free survival (PFS) is the time from the first day of treatment to documented disease progression (including isolated CNS progression) or death from any cause under study, whichever occurs first.

[00140] Disease-free survival (DFS) refers to a patient remaining alive, without recurrence of cancer, for a defined period of time, for example: approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 10 years, etc., from the start of treatment or initial diagnosis. In one aspect of the invention, DFS is analyzed according to the intent-to-treat principle, i.e., patients are evaluated based on the assigned therapy. Events used in the DFS analysis may include local, regional, and distant cancer recurrence, the occurrence of secondary cancer, and death from any cause in patients without a preceding event (e.g., recurrence of breast cancer or a second primary cancer).

[00141] Overall survival refers to the patient remaining alive for a defined period of time, such as approximately one year, approximately two years, approximately three years, approximately four years, approximately five years, approximately ten years, etc., from the start of treatment or from the diagnosis. Petition 870260055232, dated 08 / 06 / 2026, p. 50 / 222 40 / 72 initial tico. In the studies underlying the invention, the event used for survival analysis was death due to any cause.

[00142] Prolonged survival is defined as increasing DFS and / or OS in a treated patient relative to an untreated patient or a control treatment protocol. Survival is monitored for at least about six months or at least about 1 year or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., after the start of treatment or after the initial diagnosis.

[00143] Hazard ratio in a survival analysis is a summary of the difference between two progression-free survival curves, representing the reduction in the risk of death in treatment compared to the control group, during a follow-up period. Hazard ratio is a statistical definition for event rates. For the purpose of the invention, 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 specific time point.

[00144] Monotherapy is understood to be a therapeutic regimen that includes a single therapeutic agent for the treatment of cancer or a tumor during the treatment period.

[00145] The terms treat and treatment refer to therapeutic and prophylactic treatment or preventive measures, the objective of which is to prevent or reduce an undesirable pathological change or disorder, such as the growth, development, or spread of a hyperproliferative condition, such as cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom alleviation, reduction in disease severity, stabilized disease status (i.e., no worsening), and delayed progression. Petition 870260055232, dated 08 / 06 / 2026, page 51 / 222 41 / 72 or slowing of disease progression, improvement or palliation of disease status, and remission (partial or total), detectable or undetectable. Treatment can also mean prolonged survival compared to the survival expected without treatment. Those who need treatment include those who already have the condition or disorder, as well as those likely to have the condition or disorder or those in whom the condition or disorder should be prevented.

[00146] The term a treatment method or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action that is designed to reduce or eliminate the number of cancerous cells in a patient or to alleviate the symptoms of a cancer. A treatment method for cancer or another proliferative disorder does not necessarily mean that the cancerous cells or other disorder will in fact be eliminated, that the number of cells or disorder will in fact be reduced, or that the symptoms of a cancer or other disorder will in fact be alleviated. Frequently, a cancer treatment method will be performed even with a low probability of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless induced to result in a generally beneficial course of action.

[00147] The terms co-administration or joint administration refer to the administration of the antiHER2 antibody-drug conjugate and the selective Bcl-2 inhibitor as two separate formulations. Co-administration can be simultaneous or sequential in any order. In another embodiment, there is a period of time while both (or all) active agents simultaneously exert their biological activities. The antiHER2 antibody-drug conjugate and the selective Bcl-2 inhibitor are co-administered simultaneously or sequentially (e.g., intravenously (iv) at Petition 870260055232, dated 08 / 06 / 2026, p. 52 / 222 42 / 72 via a continuous infusion (one for the antibody-drug conjugate and possibly one for the Bcl-2 inhibitor, or the Bcl-2 inhibitor is administered orally). When both therapeutic agents are co-administered sequentially, the agents are administered in two separate administrations that are separated by a specific time period. The term specific time period means 1 to 15 days. For example, one of the agents may be administered within approximately 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour from the administration of the other agent, and in one embodiment, the specific 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.

[00148] The term simultaneously means at the same time or within a short period of time, usually less than 1 hour.

[00149] A drug that is administered concurrently with one or more other drugs is administered during the same treatment cycle, on the same treatment day as one or more other drugs, and optionally, at the same time as one or more other drugs. For example, for cancer therapies given every 3 weeks, the concurrently administered drugs are each given on day 1 of a 3-week cycle.

[00150] A dosing period as used in this document means a period of time during which each therapeutic agent was administered at least once. A dosing cycle is typically 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.

[00151] In certain modalities, a dosing cycle is 21 days.

[00152] In certain modalities of a cancer treatment method in a patient, as provided in this document, the method Petition 870260055232, dated 08 / 06 / 2026, p. 53 / 222 43 / 72 comprises administering the anti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor for one or more dosing cycles to the patient. In one embodiment, the one or more dosing cycles last at least one week. In another embodiment, the one or more dosing cycles last respectively 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.

[00153] In a preferred embodiment, the antibody-drug conjugate is administered as an intravenous (iv) infusion every three weeks (21-day cycle).

[00154] In another preferred embodiment, the antibody-drug conjugate is KADCYLA® (emtansine ado-trastuzumab), which is administered as an IV infusion of 3.6 mg / kg every 3 weeks (21-day cycle).

[00155] In some embodiments of the treatment method provided in this document, the selective Bcl-2 inhibitor is 2-(1H-pyrrolo[2,3b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4yl)methylamine)phenylsulfonyl)benzamide (GDC-0199). In certain embodiments, the amount of GDC-0199 per dose administered to the patient is increased during the first dosing cycle from initial amounts between 10 mg and 80 mg to final amounts between 190 mg and 400 mg. In certain formulations, the amount of GDC-0199 per dose administered to patients 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 doses administered to the patient may, for example, be between doses of 20 mg to 60 mg (e.g., 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg), followed by dose amounts of 100 mg, 200 mg, 300 mg or more of GDC-0199. (Reference 870260055232, dated 08 / 06 / 2026, page 54 / 222). 44 / 72 0199.

[00156] An adverse event is any unintended and unfavorable sign, symptom, or illness temporarily associated with the use of an investigational product (medicine) or other protocol-mandated intervention, regardless of attribution; and includes: adverse effects (AEs) not previously observed in the patient that arise during the protocol-specified AE reporting period, including signs or symptoms associated with breast cancer that were not present before the AE reporting period; complications that occur as a result of protocol-mandated interventions; pre-existing medical conditions (other than the condition being studied) judged by the investigator to have worsened in severity or frequency or changed in characteristics during the protocol-specified AE reporting period.

[00157] It is evident that antibody-drug conjugates are administered to the patient in a therapeutically effective amount (or simply effective amount), which is the amount of the respective compound or combination that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, physician, or other clinician. Administration of an effective amount of a therapeutic agent may be a single administration or a split-dose administration. Split-dose administration means that an effective amount is divided into multiple doses, preferably 2, and administered within 1 or 2 days. For example, if 100 mg of a selective Bcl2 inhibitor is considered effective, it may be administered as one 100 mg administration or two 50 mg administrations. Split-dose administration is sometimes desirable at the beginning of a dosing period to reduce side effects.When an effective amount is administered in divided doses, it is still considered one. Petition 870260055232, dated 08 / 06 / 2026, page 55 / 222 45 / 72 administration of an effective amount. For example, when 100 mg is the effective amount of a selective Bcl-2 inhibitor and this amount is administered in two 50 mg doses over a period of time, for example, 2 days, only one effective amount is administered during that time period.

[00158] A fixed or flat dose of a therapeutic agent in this document refers to a dose that is administered to a human patient without regard to the patient's weight (WT) or body surface area (BSA). The fixed or flat dose is therefore not given as a mg / kg or mg / m2 dose, but as an absolute quantity of the therapeutic agent.

[00159] A loading dose, in this document, comprises an initial dose of a therapeutic agent administered to a patient and is followed by one or more doses thereof. Generally, a single loading dose is administered, but multiple loading doses are contemplated herein. Typically, the amount of loading dose(s) administered exceeds the amount of maintenance dose(s) administered and / or the loading dose(s) is / are administered more frequently than the maintenance dose(s) in order to achieve the desired steady-state concentration of the therapeutic agent sooner than can be achieved with the maintenance dose(s).

[00160] Dose maintenance refers to one or more doses of a therapeutic agent administered to the patient over a period of treatment. Generally, maintenance doses are administered at spaced treatment intervals, such as approximately every week, approximately every 2 weeks, approximately every 3 weeks, or approximately every 4 weeks, preferably every 3 weeks.

[00161] Infusion or to infuse refers to the introduction of a solution containing a drug into the body through a vein for therapeutic purposes. Petition 870260055232, dated 08 / 06 / 2026, page 56 / 222 46 / 72 rapêuticos. Generally, this is achieved through an intravenous (IV) bag.

[00162] An intravenous bag or IV bag is a bag that can hold a solution that can be administered through the 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 made of polyolefin or polyvinyl chloride.

[00163] In the context of this invention, other additional cytotoxic, chemotherapeutic or anticancer agents, or compounds that enhance the effects of such agents (e.g., cytokines), may be used in the anti-HER2 antibody-drug conjugate and in the Bcl-2 inhibitor combination treatment of HER2-expressing cancer. Such molecules are duly present in combination in amounts that are effective for the intended purpose. Preferably, the anti-HER2 antibody-drug conjugate and the Bcl-2 inhibitor combination treatment are used without such additional cytotoxic, chemotherapeutic or anticancer agents or compounds that enhance the effects of such agents.

[00164] These agents include, for example: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; for example, CYTOXAN®), chlorambucil (CHL; for example, LEUKERAN®), cisplatin (CisP; for example, PLATINOL®), busulfan (for example, MYLERAN®), melphalan, streptozotocin, carmustine (BCNU), triethylenemelamine (TEM), mitomycin C and the like; antimetabolites, such as methotrexate (MTX), etoposide (VP16; for example, VEPESID®), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (for example, XELODA®), dacarbazine (DTIC) and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; for example, ADRIAMYCIN®), daunorubicin (daunomycin), ble Petition 870260055232, dated 08 / 06 / 2026, page 57 / 222 47 / 72 omycin, mithramycin and similar substances; alkaloids, such as vinca alkaloids like vincristine (VCR), vinblastine and similar substances; and other antitumor agents, such as paclitaxel (e.g., TAXOL®) and paclitaxel derivatives, cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g., DECADRON®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid-destroying enzymes such as asparaginase, leucovorin and other folic acid derivatives and similar substances, various antitumor agents.The following agents may also be used as add-on agents: arnifostine (e.g., ETHYOL®), dactinomycin, mechlormethine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g., DOXIL®), gemcitabine (e.g., GEMZAR®), daunorubicin lipo (e.g., DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g., TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil.Preferably, combination treatment with type II anti-CD20 antibody and Bcl-2 inhibitor is used without such additional agents.

[00165] The use of the cytotoxic and anticancer agents described above, as well as target-specific antiproliferative anticancer drugs, such as protein kinase inhibitors in chemotherapeutic regimens, is generally well characterized in cancer therapy techniques, and its use in this document encompasses the same considerations for monitoring tolerance and efficacy and for controlling routes of administration and dosages, with some adjustments. For example, the dosages Petition 870260055232, dated 08 / 06 / 2026, page 58 / 222 48 / 72 actual genes of cytotoxic agents may vary depending on the patient's cultured cell response determined through the use of histoculture methods. Generally, the dosage will be reduced compared to the amount used in the absence of other additional agents.

[00166] Typical dosages of an effective cytotoxic agent may be within the ranges recommended by the manufacturer, and where indicated by in vitro responses or responses in animal models, may be reduced by up to approximately an order of magnitude in concentration or quantity. Thus, the actual dosage will depend on the physician's judgment, the patient's condition, and the effectiveness of the therapeutic method based on the in vitro responsiveness of the primary malignant cell sample and histocultures, or the responses observed in appropriate animal models.

[00167] In the context of this invention, an effective amount of ionizing radiation can be delivered and / or a radiopharmaceutical can be used in addition to the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitor treatment. The radiation source can be external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT). The radioactive atoms for use in the context of this invention can be selected from the group including, but not limited to, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111. It is also possible to label the antibody with these radioactive isotopes. Preferably, the combination treatment with type II anti-CD20 antibody and Bcl-2 inhibitor is used without such ionizing radiation.

[00168] Radiotherapy is a standard treatment for controlling non-resistant or inoperable tumors and / or tumor metastases. Re Petition 870260055232, dated 08 / 06 / 2026, page 59 / 222 49 / 72 improved results were observed when radiotherapy was combined with chemotherapy. Radiotherapy is based on the principle that high-dose radiation administered to a target area will result in the death of reproductive cells in both tumor and normal tissues. The radiation dosage regimen is generally defined in terms of absorbed radiation dose (Gy), time, and fractionation, and must be carefully determined by the oncologist. The amount of radiation a patient receives will depend on several considerations, but the two most important are the location of the tumor relative to other critical structures or organs of the body and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiation therapy will be a treatment schedule over a period of 1 to 6 weeks, with a total dose between 10 and 80 Gy administered to the patient in a single daily fraction of approximately 1.8 to 2.0 Gy, 5 days a week.In a preferred embodiment of this invention, synergy exists when tumors in human patients are treated with the combination treatment of the invention and radiation. In other words, the inhibition of tumor growth by means of the agents comprising the combination of the invention is increased when combined with radiation, optionally with additional chemotherapeutic or anticancer agents. The parameters of adjuvant radiation therapies are, for example, contained in WO 99 / 60023.

[00169] Anti-HER2 antibody-drug conjugates are administered to a patient according to known methods, by intravenous bolus administration or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial or intrathecal routes. Intravenous or subcutaneous administration of antibodies is preferred. Petition 870260055232, dated 08 / 06 / 2026, page 60 / 222 50 / 72

[00170] Bcl-2 inhibitors are administered to a patient according to known methods, for example, by intravenous bolus administration or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerosternal, subcutaneous, intra-articular, intrasynovial, intrathecal or perioral routes. Intravenous, subcutaneous or oral administration of Bcl-2 inhibitors is preferred.

[00171] As used in this document, pharmaceutically acceptable carrier is intended to include any and all materials compatible with pharmaceutical administration, including solvents, dispersing media, coatings, antibacterial and antifungal agents, absorption retardants and isotonics, and other materials and compounds compatible with pharmaceutical administration. Except to the extent that any conventional medium or agent is incompatible with the active compound, its use in the compositions of the invention is contemplated. Additional active compounds may also be incorporated into the compositions.

[00172] A vial is a container suitable for storing a lyophilized liquid or preparation. In one embodiment, the vial is a disposable vial, for example, a 20-cc disposable vial with a stopper.

[00173] The term package insert is used to refer to the instructions commonly included in commercial packaging of therapeutic products, which contain information about indications, use, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic products. Trastuzumab-MCC-DM1 (T-DM1; KADCYLA®, emtansine adotrastuzumab)

[00174] The present invention includes therapeutic combinations comprising trastuzumab-MCC-DM1 having the following structure: Petition 870260055232, dated 08 / 06 / 2026, p. 61 / 222 51 / 72 N--Tr HP Formula II

[00175] where Tr is trastuzumab and p is an integer from 1 to 8. The drug-to-antibody ratio or drug loading is represented by p in the structure above for trastuzumab-MCC-DM1. The drug loading value p is from 1 to 8. Trastuzumab-MCC-DM1 includes all mixtures of variously loaded and attached antibody-drug conjugates in which 1, 2, 3, 4, 5, 6, 7 and 8 drug moieties are covalently linked to the trastuzumab antibody.

[00176] Trastuzumab is produced by a mammalian cell suspension culture (Chinese Hamster Ovary, CHO). The HER2 proto-oncogene (or c-erbB2) encodes a 185kDa transmembrane receptor protein, which is structurally related to the epidermal growth factor receptor. Overexpression of the HER2 protein is observed in 25%-30% of primary breast cancers and can be determined using an immunohistochemical-based analysis of fixed tumor blocks (Press MF, et al (1993) Cancer Res 53: 4960-70). Trastuzumab is an antibody that has antigen-binding residues of, or is derived from, the murine 4D5 antibody (ATCC CRL 10463, deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the Budapest Treaty on May 24, 1990). Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (HERCEPTIN®) as per US document 5,821,337.

[00177] The antibody-drug conjugate, trastuzumab-MCC-DM1, Petition 870260055232, dated 08 / 06 / 2026, p. 62 / 222 52 / 72 comprises a moiety of the DM1 maytansinoid drug (US 5,208,020; US 6,441,163) and may be prepared from fermentation products containing ansamitocine (US 6,790,954; US 2005 / 0170475). Selective Bcl-2 inhibitors

[00178] In a preferred embodiment, the selective Bcl-2 inhibitor of the present invention is 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4((tetrahydro-2H-pyrano-4-yl)methylamino)phenylsulfonyl)benzamide, (also known as ABT-199 or GDC-0199), a Bcl-2 inhibitor of formula I, which is described in International Publication No. WO2010 / 0138588 and U.S. Publication No. US2010 / 0305122, which are incorporated herein by reference. 2-(1H-pyrrolo[2,3-b]pyridine-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4dimethylcyclohexane-1-enyl)methyl)piperazine-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamine)phenylsulfonyl)benzamide Formula I

[00179] Other selective Bcl-2 inhibitors include, for example, Oblimersen, SPC-2996, RTA-402, Gossypol, AT-101, Obatoclax mesylate, A-371191, A-385358, A-438744, ABT-737, ABT-263, AT-101, BL. [Reference to document number 870260055232, dated 06 / 08 / 2026, p. 63 / 222] 53 / 72 11, BL-193, GX-15-003, 2-methoxyantimycin A3, HA-14-1, KF-67544, Purpurogallin, TP-TW-37, YC-137 and Z-24, described, for example, in Zhai, D., et al., Cell Death and Differentiation 13 (2006) 1419 -1421. PHARMACEUTICAL COMPOSITIONS

[00180] The pharmaceutical compositions or formulations of the present invention include combinations of trastuzumab-MCC-DM1, a selective Bcl-2 inhibitor, and one or more pharmaceutically acceptable carriers, glands, diluents or excipients.

[00181] Trastuzumab-MCC-DM1 and the selective Bcl2 inhibitors of the present invention may exist in non-solvated and solvated forms with pharmaceutically acceptable solvents, such as water, ethanol and the like, and it is intended that the invention covers both solvated and non-solvated forms.

[00182] Trastuzumab-MCC-DM1 and the selective Bcl2 inhibitors of the present invention may also exist in different tautomeric forms, and all such forms are included within the scope of the invention. The term tautomer or tautomeric form refers to structural isomers of different energies that are interconvertible across a low-energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions through the migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by the rearrangement of some of the bonding electrons.

[00183] Pharmaceutical compositions encompass both the bulk composition and individual dosage units comprising more than one (e.g., two) pharmaceutically active agents including trastuzumab-MCC-DM1 and a selective Bcl-2 inhibitor selected from the lists of additional agents described in this document, together with any excipients, diluents, trans Petition 870260055232, dated 08 / 06 / 2026, p. 64 / 222 54 / 72 pharmaceutically inactive carriers or sliders. The bulk composition and each individual dosage unit may contain fixed amounts of the pharmaceutically active agents mentioned above. The bulk composition is material that has not yet been formed into individual dosage units. An illustrative dosage unit is an oral dosage unit such as tablets, pills, capsules, and the like. Similarly, the method described in this document for treating a patient by administering a pharmaceutical composition of the present invention also aims to encompass the administration of the bulk composition and the individual dosage units.

[00184] Pharmaceutical compositions also encompass isotopically labeled compounds of the present invention, which are identical to those reported in this document, except that one or more atoms are replaced by an atom with an atomic mass or mass number different from the atomic mass or mass number generally found in nature. All isotopes of any specific atom or element, as specified, are contemplated within the scope of the compounds of the invention, and their uses. Exemplary isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 32P, 33P, 35S, 18F, 36Cl, 123I, and 125I. Certain isotopically labeled compounds of the present invention (for example, those labeled 3H or 14C) are useful in tissue distribution assays of the compounds and / or the substrate.Tritiated (3H) and carbon-14 (14C) isotopes are useful because of their ease of preparation and detectability. Further substitution with heavier isotopes, such as deuterium (2H), may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., longer half-life). Petition 870260055232, dated 08 / 06 / 2026, page 65 / 222 55 / 72 in vivo or reduced dosage requirements) and, consequently, may be preferable in some circumstances. Positron-emitting isotopes, such as 15O, 13N, 11C, and 18F, are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy. The isotopically labeled compounds of the present invention can generally be prepared by the following procedures analogous to those disclosed in the Schemes and / or Examples in this document below, replacing an isotopically labeled reagent with a non-isotopically labeled reagent.

[00185] Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors can be formulated according to standard pharmaceutical practice for use in a therapeutic combination for the therapeutic treatment (including prophylactic treatment) of hyperproliferative disorders in mammals, including humans. The invention provides a pharmaceutical composition comprising trastuzumab-MCC-DM1 in association with one or more pharmaceutically acceptable carriers, glidants, diluents, or excipients.

[00186] Suitable carriers, diluents, and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, expandable and / or water-soluble polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The specific carrier, diluent, or excipient used will depend on the means and purposes for which the compound of the present invention is being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe (GRAS) for administration to a mammal. In general, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents Petition 870260055232, dated 08 / 06 / 2026, page 66 / 222 56 / 72 of these include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc., and mixtures thereof. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, fragrance agents, flavoring agents, and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or a pharmaceutical composition thereof) or to aid in the manufacture of the pharmaceutical product (i.e., a medicament).

[00187] The formulations can be prepared using conventional dissolution and mixing procedures. For example, the bulk drug substance (i.e., the compound of the present invention or a stabilized form of the compound (e.g., 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 excipients described above. The compound of the present invention is typically formulated in pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to allow patient compliance with the prescribed regimen.

[00188] The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending on the method used to administer the drug. Generally, an article for distribution includes a container holding the pharmaceutical formulation in the appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-evident seal assembly to prevent indiscreet access to the contents of the package. In addition Petition 870260055232, dated 08 / 06 / 2026, page 67 / 222 57 / 72 In addition, the container has a label describing the contents of the container. The label may also include appropriate warnings.

[00189] Pharmaceutical formulations of the compounds of the present invention can be prepared by various routes and types of administration with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1995) 18th edition, Mack Publ. Co., Easton, PA), in the form of a lyophilized formulation, ground powder, or an aqueous solution. The formulation can be carried out by mixing at room temperature at the appropriate pH, and to the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are not toxic to receptors at the dosages and concentrations employed. The pH of the formulation depends mainly on the specific use and concentration of the compound, but can vary from about 3 to about 8.

[00190] The pharmaceutical formulation is preferably sterile. In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily achieved by filtration through sterile filtration membranes.

[00191] Pharmaceutical formulations can commonly be stored as a solid composition, a lyophilized formulation, or as an aqueous solution.

[00192] The pharmaceutical formulations of the invention will be dosed and administered in a manner, i.e., quantities, concentrations, times, course, vehicles and route of administration, in accordance with good medical practice. Factors for consideration in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of distribution of the agent, the method of administration, the administration schedule, and other factors known to medical professionals. The therapeutically effective amount of the compound to be Petition 870260055232, dated 08 / 06 / 2026, page 68 / 222 The 58 / 72 dose administered will be governed by such considerations, and is the minimum amount necessary to prevent, improve, or treat the coagulation factor-mediated disorder. This amount is preferably below the amount that is toxic to the host or that makes the host significantly more susceptible to bleeding.

[00193] Acceptable diluents, carriers, excipients and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, ethanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and mcresol); low molecular weight polypeptides (less than about 10 residues); proteins such as albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents, such as EDTA;Sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, including Tween 80, PLURONICS™, or polyethylene glycol (PEG), including PEG400. The active pharmaceutical ingredients may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethicone) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Petition 870260055232, dated 08 / 06 / 2026, page 69 / 222 59 / 72 These techniques are described in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co., Easton, PA.

[00194] Pharmaceutical formulations include those suitable for the routes of administration detailed in this document. Formulations may conveniently be presented in unit dosage form and may be prepared according to any of the known methods of pharmaceutical art. Techniques and formulations in general are found in Remington's Pharmaceutical Sciences 18th Ed. (1995) Mack Publishing Co., Easton, PA. Such methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. In general, formulations are prepared by bringing into association, uniformly and intimately, the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, molding the product.

[00195] As a general proposition, the initial therapeutically effective amount of trastuzumab-MCC-DM1 administered per dose will be in the range of about 0.01-100 mg / kg, alternatively about 0.1 to 20 mg / kg of the patient's body weight per day, with the typical initial range of the compound used being 0.3 to 15 mg / kg / day.

[00196] In a preferred embodiment, trastuzumab-MCC-DM1 is formulated as a lyophilized spray agent in single-use vials containing 100 mg per vial or 160 mg per vial and is administered at a dose of 3.6 mg / kg as an intravenous infusion every 3 weeks.

[00197] The pharmaceutical compositions of the isolated anti-Bcl-2 active agent, for example, the Bcl-2 inhibitor, depend on its pharmaceutical properties; for example, for small chemical compounds such as, for example, ABT-737, ABT-199 or ABT-263, a formulation could be, for example, as follows: Petition 870260055232, dated 08 / 06 / 2026, page 70 / 222 60 / 72 a) Tablet formulation (wetted granulation): Item Ingredients mg / tablet 1. Formula Compound (I) 5 25 100 500 2. Anhydrous Lactose DTG 125 105 30 150 3. Sta-Rx 1500 6 6 6 30 4. Microcrystalline Cellulose 30 30 30 150 5. Magnesium Stearate 1 1 1 1 Total 167 167 167 831 Manufacturing procedure: 1. Mix items 1, 2, 3, and 4 and grind with purified water. 2. Dry the granules at 50 °C. 3. Pass the granules through suitable milling equipment. 4. Add item 5 and mix for three minutes; compact in a suitable press. b) Capsule formulation: Item Ingredients (mg / capsule) 1. Formula Compound (I) 5 25 100 500 2. Hydrated Lactose 159 123 148 — 3. Corn Starch 25 35 40 70 4. Talc 10 15 10 25 5. Magnesium Stearate 1 2 2 5 Total 200 200 300 600 Manufacturing procedure: 1. Mix items 1, 2, and 3 in a suitable blender for 30 minutes. 2. Add items 4 and 5 and mix for 3 minutes. Fill into a suitable capsule.

[00198] The active ingredients can also be trapped in microcapsules prepared, for example, by coacervation techniques or by means of inter-racial polymerization, for example, hydroxymethylcellulose. Petition 870260055232, dated 08 / 06 / 2026, page 71 / 222 61 / 72 loose or gelatinous microcapsules and poly(methylmethacrylate) microcapsules, respectively, 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 16th edition, Osol, A. Ed. (1980).

[00199] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in which the matrices are in the form of molded articles, for example, 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), L-glutamic acid and gamma-ethyl-L-glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of a lactic acid glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[00200] Formulations to be used for in vivo administration must be sterile. This is readily achieved by filtration through sterile filtration membranes.

[00201] The following examples, sequence listings and figures are provided to aid understanding of the present invention, the true scope of which is defined in the appended claims. It is understood that modifications may be made to the procedures presented without departing from the spirit of the invention. EXAMPLES Example 1 Expression of Bcl-2 family survival molecules in T-DM1 resistant breast cancer cells Petition 870260055232, dated 08 / 06 / 2026, page 72 / 222 62 / 72 Preparation of T-DM1 resistant cell lines

[00202] Initially, KPL-4 and BT-474M1 cells were made resistant to T-DM1 by continuous culture in the presence of T-DM1, starting at a very low concentration of 10 ng / mL, which was gradually increased to 2 μg / mL. The resulting cells are the resistant groups that were maintained in culture at 2 μg / mL of TDM1. To obtain stably resistant clones, each group (KPL4 and BT-474M1) was subjected to single-cell screening and cloning. The clones were maintained without T-DM1 so that clones possessing stable resistance in the absence of T-DM1 could be identified. TaqMan Analysis

[00203] Total RNA was prepared using the Qiagen RNeasy Mini kit. Genomic DNA was removed using DNase I. Gene expression was quantified using quantitative real-time PCR (qPCR or TaqMan). TaqMan One-Step Universal Master Mix (Applied Biosystems) was used for all reactions. The reaction was performed in a standard 96-well plate format with an eABI 7500 Real-Time qPCR system. 100 ng of total RNA was used as a template in each reaction. For data analysis, the crude Ct was normalized to the constructive HP1BP3 gene.

[00204] TaqMan analysis results are presented in FIG. 1A. The figure shows that Bcl-2 mRNA expression increased in T-DM1-resistant KPL-4 and T-DM1-resistant BT-474M1 cell lines (normalized for the constitutive gene HP1BP3) compared to non-resistant KPL-4 and BT-474M1 parent cells.

[00205] Western blot analysis was performed as follows: cells were lysed in corrected FLAG elution buffer (CFEB) (19.17 mM Tris (pH 7.5), 916.7 μM MgCl2, 92.5 mM NaCl and 0.1% Triton X-100) with protease and phosphatase inhibitors (Roche); in some cases, 6 M urea was added. The eliminated lysates were Petition 870260055232, dated 08 / 06 / 2026, page 73 / 222 63 / 72 quantified and equal amounts of proteins were reduced, alkylated, separated by SDS-PAGE, and transferred to PVDF membranes (Invitrogen) following standard procedures. Western blot was performed as recommended by the respective antibody manufacturers. The Western blot test analysis shown in FIG. 1B confirms that Bcl-2 is overexpressed in T-DM1 and BT-474 resistant KPL-4 cell lines. Example 2 Cell proliferation assay - T-DM1 resistant KPL-4 breast cancer cell matrices and lines.

[00206] The cell proliferation assay was performed over 3 days using the Cell-Titer Glo reagent. KPL-4 matrix breast cancer cells and KPL-4 T-DM1-resistant breast cancer cells, prepared as described in Example 1, were treated with T-DM1, GDC-0199, or a combination of T-DM1 and GDC-0199 at fixed ratios. Synergy was analyzed using Chou and Talalay's Dose-Effect Analysis of Combination Drugs with CalcuSyn software to obtain a Combination Index CI (Chou and Talalay (1984) Adv. Enzyme Regul. 22: 27-55). CI values ​​less than 1 indicate synergy, while CI = 1 indicated additivity.

[00207] Assay conditions: Cells were maintained in Ham F-12: high-glucose DMEM (1:1) supplemented with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine. Cells were plated in 96-well plates (4000 cells per well for KPL-4 stem cells, 8000 cells per well for T-DM1-resistant KPL-4 cells) and left to adhere overnight at 37°C in a humidified 5% CO2 atmosphere. The 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. Petition 870260055232, dated 08 / 06 / 2026, page 74 / 222 64 / 72 co and the luminescent signal was measured using an EnVision Multilabel Plate Reader (PerkinElmer). Combination Index (CI) values ​​were generated using CalcuSyn software (Biosoft, Inc.)

[00208] As shown in FIG. 2, the combination of T-DM1 and GDC-0199 has a synergistic antiproliferative effect on T-DM1-resistant KPL-4 breast cancer cells. Example 3 Caspase 3 / 7 activation luminescence assay - T-DM1 resistant KPL-4 breast cancer matrices and cell lines

[00209] As noted earlier, the Bcl2 protein family regulates programmed cell death triggered by developmental cues and in response to multiple stress signals. When activated, they can permeabilize the outer mitochondrial membrane and release pro-apoptogenic factors (e.g., cytochrome C) necessary to activate caspases that dismantle the cell (Wang, K., Genes and Development 15 (2001) 2922-2933; (Adams, 2003 supra), Green, DR, and Kroemer, G., Science 305 (2004) 626-629). Thus, activation of caspases, such as caspases 3 and 7, indicates induction of apoptosis.

[00210] In the present experiment, the caspase 3 / 7 activation luminescence assay was performed using CaspaseGlo® Reagent (Promega) essentially following the manufacturer's instructions. The assays were performed in the same way as the viability assays, except that the drug incubation periods were 24 hours and Caspase-Glo 3 / 7 (Promega) was used to measure apoptosis.

[00211] As shown in FIG. 3, right panel, T-DM1-resistant (HER2+) KPL-4 breast cancer cells were resensitized to T-DM1 when treated for 24 hours with a combination of T-DM1 + ​​GDC-0199, as shown by the increase in apoptosis (increased caspase 3 / 7 activation). The same Petition 870260055232, dated 08 / 06 / 2026, page 75 / 222 The 65 / 72 combination showed no effect on the matrix cells. Note that the results were evaluated 24 hours after treatment, which is too early for apoptosis induced by T-DM1 alone in the matrix cell line (left panel).

[00212] FIGS. 4A and 4B present the results of an in vitro caspase 3 / 7 activation apoptosis luminescence assay measuring the activation of caspases 3 and 7 in T-DM1-resistant human KPL-4 breast cancer cells relative to stem cells using different concentrations of T-DM1 and GDC-0199, respectively. Increased apoptosis is observed in T-DM1-resistant KPL-4 cells after the addition of increasing concentrations of GDC-0199 (1, 2.5, or 5 μM) to 0.1 or 1 μg / mL of T-DM1. In contrast, T-DM1 induces robust apoptosis in KPL-4 stem cells, which is not enhanced by the addition of GDC-0199.

[00213] FIG. 5A presents the results of an in vitro caspase 3 / 7 activation luminescence apoptosis assay measuring the activation of caspases 3 and 7 in the T-DM1-resistant KPL-4 human breast cancer cell line in Clone #17 treated with TDM1, GDC-0199, or T-DM1 + ​​GDC-0199. Similar to observations with the T-DM1-resistant KPL-4 cell group, apoptosis induction in Clone #17 was enhanced after the addition of increasing concentrations of GDC-0199.

[00214] FIGS. 6A and 6B show the results of an in vitro caspase 3 / 7 activation luminescence apoptosis assay measuring the activation of caspases 3 and 7 in Clone #8 in the T-DM1-resistant KPL-4 human breast cancer cell line treated with concentrations of 0.1 pg / mL and 1 pg / mL of T-DM1, respectively, alone or in combination with the indicated concentrations of GDC-0199.

[00215] As shown in FIGS. 4A, 4B, 5A, 6A and 6B, the results Petition 870260055232, dated 08 / 06 / 2026, page 76 / 222 66 / 72 of those obtained with different clones of T-DM1-resistant KPL-4 breast cancer cell lines confirm the enhanced proapoptotic activity of the T-DM-1 + GDC-0199 combination at various concentrations. Example 4 Xenotransplantation studies - T-DM1 resistant KPL-4 breast cancer cell lines

[00216] For all xenotransplantation studies, three million T-DM1-resistant KPL-4 breast cancer cells were implanted into mammary fat pads of #2 / 3 of female SCID-beige mice. When the tumors reached a volume of approximately 200 mm3, the mice were randomized into treatment groups (n=10 pre-group mice): 5 mg / kg TDM1 q3w, 100 mg / kg GDC-0199 qd, combination of the two, or vehicle. The results of these xenotransplantation studies for xenotransplants of various clones of TDM1-resistant KPL-4 breast cancer cells are shown in FIGS. 5B and 6C. The results indicate an improved antitumor effect when T-DM1 and GDC-0199 were used in combination, compared to the single-agent activity. Example 5 IHC studies - TDM1-resistant KPL-4 xenotransplant tumors

[00217] FFPE xenotransplant tumors (embedded in formalin-fixed paraffin) were segmented for analysis of Bcl-2 and HER2 (ErbB2) expression by immunohistochemistry (IHC) using the DAB detection method. The Bcl-2 SP66 antibody was obtained from Ventana. Human tonsil sections served as positive controls for Bcl-2. The anti-HER2 4D5 antibody was obtained from Ventana. Human breast cancer cell lines served as positive controls (SK-BR-3 as 3+; MDAPetition 870260055232, 08 / 06 / 2026, p. 77 / 222) 67 / 72 MB-361 as 2+; MBA-MB-231 as negative).

[00218] FIG. 7A shows the expression of Bcl-2 in formalin-fixed paraffin-embedded KPL-4 T-DM1 xenograft tumor samples (Clones #8 and #17) determined by immunohistochemistry (IHC) using the DAB detection method, as described above.

[00219] FIG. 7B shows the expression of HER2 (ErbB2) in formalin-fixed paraffin-embedded T-DM1-resistant KPL-4 xenograft tumor samples (Clones #8 and #17) determined by immunohistochemistry (IHC) using the DAB detection method, as described above.

[00220] Results of anti-Bcl-2 antibodies: the vehicle groups of each clone showed a low frequency of similar Bcl-2 reactive cells, located more frequently at the perimeter of the tumor lobules (not shown). The frequency and intensity of the Bcl-2 signal at the margins of the tumor lobule in the T-DM1 treated groups were increased or unchanged.

[00221] Anti-HER2 antibody results: vehicle-treated tumors and T-DM1 in all clones showed a high frequency of HER2 with 3+ IHC. In some tumors treated with T-DM1, there were weaker staining regions with HER2 (clone #17), mostly adjacent to the stromal bands surrounding the tumor lobules (not shown).

[00222] IHC results for Bcl-2 demonstrate that Bcl-2 expression is maintained in T-DM1-resistant clones #8 and #17 when grown as xenotransplant tumors (Figure 7A; see also Example 6 which shows very little Bcl-2 expression in KPL-4 stem cells by Western blot assay). Bcl-2 expression in T-DM1-treated clone #17 is higher than the corresponding vehicle control. FIG. 7B represents the expression of Petition 870260055232, dated 08 / 06 / 2026, p. 78 / 222 68 / 72 HER2 as assessed by IHC. In contrast to the relatively low HER2 expression observed in cultured cells in vitro, clones #8 and #17, in both vehicle- and T-DM1-treated tumors, exhibit high HER2 expression at levels 2+ and 3+. All tumors from clone #8 were determined to be 85-95% HER2+ or 3+, with a very low frequency of 2+ or 1+ tumor cells. Tumors from clone #17 were more variable, with 35-75% HER2 3+ cells and 20-65% HER2 2+ cells in the vehicle group. Example 6 Xenotransplantation studies - TDM1-resistant KPL-4 breast tumors

[00223] FIG. 8 shows Western blot expression data for Bcl-2, HER2, Bcl-xL, and Pgp in T-DM1-resistant KPL-4 xenograft tumors from clones #17 treated with GDC-0199, TDM1, or TDM-1 + GDC-0199. (Three-digit numbers above ranges 4-19 indicated individual xenograft tumors.) Expression data show that HER2 and Bcl-2 expression is maintained across all groups compared to corresponding cells cultured in vitro in cell culture. Example 7 Caspase 3 / 7 luminescence and fluorescence activation assays - T-DM1 sensitive breast cancer cell lines

[00224] The caspase 3 / 7 activation luminescence assay was performed as described in Example 3.

[00225] The in vitro caspase 3 activation fluorescence apoptosis assay was performed using IncuCyte™ reagents and equipment to measure caspase activation over time (kinetic analysis) essentially following the manufacturer's instructions.

[00226] FIG. 9A presents results from an apoptosis assay. Petition 870260055232, dated 08 / 06 / 2026, page 79 / 222 69 / 72 of caspase 3 / 7 luminescence in vitro, test of the effect of five separate 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, which are sensitive to T-DM1 (naive). The results demonstrate the activation of caspases 3 and 7 with T-DM1, which is enhanced in a dose-dependent manner with increasing concentrations of GDC-0199.

[00227] FIG. 9B presents the results of a caspase 3 fluorescent in vitro apoptosis assay, testing the effect of three 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), on caspase activity in sensitive MDA-MB-361 breast cancer cells (naive HER2+ T-DM1). The results demonstrate that GDC-0199 increases caspase activation above that induced by TDM1 alone in a dose- and period-dependent manner, therefore resulting in enhanced apoptosis with all combinations.

[00228] FIG. 10A presents results from an in vitro caspase 3 / 7 luminescence apoptosis assay, testing the effect of five separate concentrations of GDC-0199 ^M) in combination with 9 different concentrations of T-DM1 on caspase activity in naive HER2+ HCC1569 breast cancer cells on caspase activity in T-DM1. The results demonstrate that T-DM1 alone does not induce apoptosis, but the addition of GDC-0199 results in enhanced caspase activity and therefore increases apoptosis in all combinations.

[00229] FIG. 10B presents the results of an in vitro caspase 3 fluorescent apoptosis assay, testing the effect of three 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), on caspase activity in HER2+ HCC1569 breast cancer cells. The results demonstrate that GDC-0199 increases caspase activation. Petition 870260055232, dated 08 / 06 / 2026, page 80 / 222 70 / 72 above that induced by T-DM1 alone in a dose- and period-dependent manner, therefore resulting in enhanced apoptosis with all combinations.

[00230] These results show that the T-DM1 / GDC0199 combination is also effective in naive (i.e., non-resistant to T-DM1) T-DM1 cell lines. Example 8 Xenotransplantation studies - TDM-1 sensitive MDA-MB-361 breast tumors (naive)

[00231] Ten million MDA-MB-361 breast cancer cells were implanted into the right mammary fat pad of female NOD / SCID mice one day after implantation of 60-day-release 17β estradiol pellets. When tumors reached a volume of approximately 200-300 mm3, mice were randomized into treatment groups (n = 10 mice per group) and administered with T-DM1 (1, 3, or 7 mg / kg iv once), GDC0199 (100 mg / kg qd x 21), or a combination of T-DM1 and GDC-0199 as shown in FIG. 11. The results indicate an improved antitumor activity of GDC-0199 with 7 mg / kg of T-DM1, compared to the single-agent activity. Example 9 Western blot analysis: effects of T-DM1 + / - GDC-0199 on Bcl-2 family member proteins in HER2+ breast cancer cell lines

[00232] The effect of treatment with T-DM1 (1.25 μg / mL) alone or in combination with GDC-0199 (1.25 μM) was studied in several T-DM1-naive HER2+ breast cancer cell lines. The results are shown in FIG. 12. Four of the eight HER2+ breast cancer cell lines tested (BT-474, HCC1569, MDA-361, and ZR-75-30) expressed Bcl-2; all eight cell lines Petition 870260055232, dated 08 / 06 / 2026, page 81 / 222 71 / 72 breast cancer cells expressed the other evaluated Bcl-2 family members - Bcl-xL and Mcl-1. Three of the cell lines (BT-474, MDA_361, and ZR-75-30) showed Bcl-2 phosphorylation after treatment with TDM1, a known effect of exposure to antifungal agents such as T-DM1. As shown in FIGS. 9A, 9B, 10A, and 10B, MDA-MB361 and HCC1569 showed improved apoptosis when treated with a combination of T-DM1 and GDC-0199.

[00233] T-DM1 (KADCYLA®) exhibits significant clinical benefits in cancer treatment for patients, such as breast cancer patients, who have progressed on prior HER2-targeted treatments, such as treatment with trastuzumab (HERCEPTIN®). The US Food and Drug Administration has approved KADCYLA® (adotrastuzumab emtansine) for the treatment of HER2-positive, metastatic breast cancer patients who have received prior treatment with trastuzumab and taxane. The data presented here demonstrate that combination therapy with a Bcl inhibitor (e.g., Bcl-2) and T-DM1 significantly improves the efficacy of T-DM1 administered as a single agent. The results also demonstrate that this combination treatment with T-DM1 and a Bcl-2 inhibitor is effective in treating both T-DM1-sensitive (naive) HER2-positive cancers (e.g., breast cancers) and T-DM1-resistant HER2-positive cancers (e.g., breast cancers).

[00234] All publications and patent applications cited in this descriptive report are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated for incorporation by reference. Although the invention disclosed above has been described in detail by way of illustration and example for the sake of clarity, it will be readily apparent to those skilled in the art. Petition 870260055232, dated 08 / 06 / 2026, p. 82 / 222 72 / 72 In light of the teachings of this invention, certain alterations and modifications may be made without departing from the spirit or scope of the appended claims. Petition 870260055232, dated 08 / 06 / 2026, p. 83 / 222

Claims

1 / 2 CLAIMS 1. A method for predicting whether an individual with HER2-positive breast cancer is resistant or susceptible to treatment with trastuzumab-MCC-DM1, characterized in that it comprises: (i) measuring the expression level of the Bcl-2 gene or its product in a tumor sample relative to a control sample, and (ii) predicting said tumor as resistant to treatment with trastuzumab-MCC-DM1 when the measured expression level of Bcl-2 in said tumor sample is at least 2 times higher than the expression level in said control sample, or predicting said tumor as susceptible to treatment with trastuzumab-MCC-DM1 when the measured expression level of Bcl-2 in said tumor sample is less than 2 times higher than the expression level in said control sample.

2. Method, according to claim 1, characterized in that said control sample is a tumor sample of the same cell type that is not resistant to treatment with said trastuzumab-MCC-DM1.

3. Method according to claim 1, characterized in that the tumor sample is a formalin-fixed, paraffin-embedded tumor sample.

4. Method, according to claim 1, characterized in that it further comprises the step of measuring the expression level of the HER2 gene or its product in said tumor sample.

5. Kit for the in vitro prognosis of a HER2-positive breast cancer as being resistant or susceptible to treatment with trastuzumab-MCC-DM1 in a biological sample, as defined in the method of claim 1, characterized in that it comprises Petition 870260055232, dated 08 / 06 / 2026, page 84 / 222 2 / 2 and provides a specific binding partner for the Bcl-2 gene or its expression product.

6. Kit according to claim 5, characterized in that said binding partner is an anti-Bcl-2 antibody. Petition 870260055232, dated 08 / 06 / 2026, p. 85 / 222

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