Treatment of HER2-positive cancer

Abstract

In one aspect, the present invention provides a method for treating or alleviating the effects of HER2-positive cancer in a subject. In some embodiments, the method comprises administering a combination therapy comprising an anti-HER2 antibody and tucatinib. In some embodiments, the method further comprises administering a chemotherapeutic agent (e.g., an antimetabolite) to the subject. Pharmaceutical compositions and kits are also provided herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 491,872, filed April 28, 2017. The contents of these priority documents and all other references disclosed herein are incorporated herein by reference in their entirety for all purposes. Background Art

[0003] Cancer is a disease that places a huge burden on health care and has a significant impact on society in the United States and around the world. In the United States alone, it is estimated that more than 1.6 million people were diagnosed with new cases of cancer in 2016 and approximately 600,000 people died from cancer. Cancer is an extremely heterogeneous disease, with tumors arising from nearly every cell type in the body and associated with many environmental and genetic risk factors. Furthermore, cancer strikes people of all ages and all racial, cultural, and socioeconomic groups.

[0004] Cancer is often the result of mutations that may occur in many genes that play a role in a large number of cellular processes. In many instances, cancer cells carry mutations in genes that control processes such as cell growth, division, differentiation, or interaction with the extracellular environment. For example, many cancers involve mutations that increase the activity of HER2, a cell surface receptor that promotes cell growth and differentiation.

[0005] In many cases, tumors are resistant to specific cancer therapies or are initially sensitive to specific therapies but subsequently become resistant. The generation of drug resistance is usually the result of mutations that change the activity of cellular components (e.g., mutations that cause constitutive activity of signal transduction molecules) or cause changes in gene expression (e.g., mutations that cause cell signal transduction receptors such as HER2 to express increased expression). In some cases, drug resistance occurs or is produced simultaneously with the appearance of mutations that transform cancer into a more aggressive (e.g., metastatic) form. Metastatic cancers are generally associated with a worse prognosis than non-metastatic cancers.

[0006] The MOUNTAINEER clinical trial (ClinicalTrials.gov identifier NCT03043313) is testing the efficacy of a combination of tucatinib and trastuzumab for the treatment of patients with HER2-positive metastatic CRC.

[0007] Cancer characterized by overexpression of HER2 (referred to as HER2-positive cancer) is usually associated with a poor prognosis and is resistant to many standard therapies. Therefore, there is a need for novel therapies effective for treating cancers such as HER2-positive cancers or metastatic HER2-positive cancers. The present invention meets this need and also provides other advantages. SUMMARY OF THE INVENTION

[0009] In some aspects, the present invention provides a method for treating or alleviating the effects of HER2-positive cancer in a subject, the method comprising administering a combination of an anti-HER2 antibody and tucatinib and a chemotherapeutic agent, thereby treating HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, gallbladder cancer, breast cancer, and combinations thereof. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is an unresectable, locally advanced cancer.

[0010] In some embodiments, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine, margetuximab, and combinations thereof. In some cases, the anti-HER2 antibody is trastuzumab. In some cases, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab. In some embodiments, the administration of the anti-HER2 antibody is performed before, during, or after the administration of tucatinib.

[0011] In some embodiments, the cancer comprises cells having a wild-type KRAS exon 2 genotype. In some embodiments, the cancer comprises cells having a wild-type NRAS genotype. In some embodiments, the cancer comprises cells having a wild-type BRAF genotype. In some embodiments, the subject suffers from a cancer that is refractory to standard of care including cixutumumab or panitumumab.

[0012] In some embodiments, the subject produces a tumor growth inhibition (TGI) index of at least about 85%. In some cases, the subject produces a TGI index of about 100%. In some embodiments, the combination of anti-HER2 antibody and tucatinib is synergistic. In some embodiments, the subject produces a TGI index that is greater than the TGI index observed when anti-HER2 antibody or tucatinib is used alone.

[0013] In some embodiments, the dose of Tucatinib is about 3-7 mg / kg of the subject's body weight, twice daily. In some embodiments, the dose of Tucatinib is about 300 mg, twice daily. In some embodiments, the dose of anti-HER2 antibody is about 6 mg-8 mg / kg of the subject's body weight, once every three weeks. In some embodiments, the dose of anti-HER2 antibody is about 600 mg, once every three weeks. In some embodiments, Tucatinib or anti-HER2 antibody is administered orally, intravenously, or subcutaneously.

[0014] In some embodiments, the method further comprises administering a chemotherapeutic agent (e.g., an antimetabolite, such as capecitabine). In some embodiments, the antimetabolite is selected from the group consisting of capecitabine, carmofur, doxidluridine, fluorouracil, tegafur, and combinations thereof. In some embodiments, the antimetabolite is capecitabine. In some embodiments, the dose of capecitabine is about 1,000 mg / m 2 In some embodiments, the chemotherapeutic agent is administered orally. In some embodiments, capecitabine is administered as a 150 mg or 500 mg tablet.

[0015] In other aspects, the present invention provides a pharmaceutical composition comprising an anti-HER2 antibody, tucatinib and a pharmaceutically acceptable carrier. In some embodiments, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine, magotuximab and combinations thereof. In some cases, the anti-HER2 antibody is trastuzumab. In some cases, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimetabolite. In some embodiments, the antimetabolite is capecitabine.

[0016] In other aspects, the present invention provides a kit for treating or alleviating the effects of HER2-positive cancer in a subject, the kit comprising a pharmaceutical composition of the present invention. In some embodiments, the kit comprises instructions for use. In some embodiments, the kit comprises one or more reagents.

[0017] Other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and accompanying drawings.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1HER2 amplification has been shown to occur in a variety of cancers (Yan et al. Cancer Metastasis Rev. (2015) 34: 157-164).

[0020] Figure 2A and 2B A relationship between HER2 status and survival in non-small cell lung cancer (NSCLC) was shown (Journal of Thoracic Oncology. Vol. 3, No. 5, May 2008). Figure 2A The impact of HER2 status on overall survival (OS) is shown. Figure 2B The impact of HER2 status on progression-free survival (PFS) is shown.

[0021] Figures 3A-3C The combination of tucatinib and trastuzumab was shown to be active in a HER2-amplified colorectal cancer (CRC) patient-derived xenograft (PDX) model. Data are presented as group mean + / - SEM. Figure 3A Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the CTG-0121 CRC PDX model. Figure 3B Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the CTG-0784 CRC PDX model. Figure 3C Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the CTG-0383 CRC PDX model.

[0022] Figure 4A and 4B The combination of tucatinib and trastuzumab was shown to be active in a HER2-amplified esophageal cancer patient-derived xenograft (PDX) model. Data are presented as group mean + / - SEM. Figure 4A Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the CTG-0137 esophageal cancer PDX model. Figure 4B Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the CTG-0138 esophageal cancer PDX model.

[0023] Figures 5A-5C The combination of tucatinib and trastuzumab was shown to be active in a HER2-positive gastric cancer patient-derived xenograft (PDX) model. Data are presented as group mean + / - SD. Figure 5A Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the GXA 3038 gastric cancer PDX model. Figure 5B Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the GXA 3039 gastric cancer PDX model. Figure 5C Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the GXA 3054 gastric cancer PDX model.

[0024] Figure 6 The combination of tucatinib and trastuzumab was shown to be active in the CTG-0927 HER2-positive cholangiocarcinoma patient-derived xenograft (PDX) model. Data are presented as mean + / - SEM.

[0025] Fig. 7A and 7B The combination of tucatinib and trastuzumab was shown to be active in a HER2-positive non-small cell lung cancer (NSCLC) model. Data are presented as group mean + / - SEM. Fig. 7A Shown are the effects of tucatinib and trastuzumab, alone or in combination, on tumor growth in the Calu-3 NSCLC xenograft model. Figure 7B Shown are the effects of tucatinib and trastuzumab (alone or in combination) on tumor growth in the NCI-H2170 NSCLC xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Introduction

[0028] HER2 gene amplification occurs in many different cancers. For example, Figure 1 The prevalence of HER2-positive cancers in a study (Yan et al. Cancer Metastasis Rev. (2015) 234: 157-164) in which 37,992 samples were analyzed was summarized. In this study, samples were analyzed in a central laboratory and tumor HER2 status was determined using immunohistochemistry (IHC). If the IHC score was 3+, the sample was determined to be HER2-positive. Figure 1 Several cancers represented in this study responded to tucatinib in investigator-initiated trials and are approved for treatment with anti-HER2 therapy.

[0029] In colorectal cancer (CRC) (about 130,000 patients diagnosed each year), HER2 amplification is present in about 3.5% of all cases, and in about 6-10% of cases, the tumor has a wild-type genotype for KRAS, NRAS, and BRAF. As a treatment method, for example, targeted HER2 treatment of CRC has been validated by the results of the HERACLES trial, which evaluated the effect of a combination of the anti-HER2 antibody trastuzumab and the tyrosine kinase inhibitor lapatinib. In the HERACLES trial, 914 patients with metastatic CRC (with a wild-type KRAS genotype in exon 2 (codons 12 and 13)) were screened—48 of these patients (5%) had tumors that were HER2-positive. In this study, an objective response rate (ORR) of 30% was observed (1 patient had a complete response and 27 patients had a partial response). In addition, 12 of the 27 patients (44%) had stable disease. All patients had been previously treated with the antibodies cetuximab or panitumumab, with an ORR of 0%. In addition, a combination of the anti-HER2 antibodies trastuzumab and pertuzumab was shown to be active in this patient population. An ORR of 38% and a clinical benefit rate of 54% were observed, with a median time to progression of 5.6 months for all patients.

[0030] In esophageal cancer, with approximately 16,980 new cases diagnosed each year (notably, this rate is 20 to 30 times higher in China), the incidence of HER2-positive tumors is approximately 20%. Similar to CRC, targeting HER2 is a validated treatment approach in gastric and esophageal cancers. In the TOGA trial, which evaluated the effect of trastuzumab in combination with cisplatin or a fluoropyrimidine compared with chemotherapy alone, the combination therapy resulted in an increase in overall survival of 2.7 months (13.8 months vs. 11.1 months, hazard ratio 0.74 (95% CI 0.60-0.91, p=0.0046)). In addition, the GATSBY trial evaluated ado-trastuzumab emtansine (also known as T-DM1) versus a taxane in patients who exhibited disease progression during or after first-line fluoropyrimidine plus platinum therapy (with or without a HER2-targeted agent).

[0031] In non-small cell lung cancer (NSCLC) (about 200,000 new cases are expected to be diagnosed in 2017), HER2 amplification occurs in approximately 3% of tumors. A trend toward decreased overall survival and progression-free survival has been observed in HER2-positive NSCLC patients treated with standard chemotherapy (Figure 2), but clinical trials have not yet focused on HER2 3+ / FISH+ patients, and there is no suitable algorithm for the specific treatment of HER2-positive NSCLC (Cancer (2004) 104: 2149-2155; Annals of Oncology (2004) 15: 19-27). In addition, HER2 amplification can serve as an acquired resistance mechanism to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Up to 12% of EGFR-mutant NSCLC tumors have HER2 amplification (which occurs independently of the EGFR T790M mutation); this patient population is unlikely to respond to HER2-selective therapy.

[0032] The present invention is based (in part) on the observation that the combination of the small molecule TKI tucatinib and the anti-HER2 antibody trastuzumab resulted in tumor regression in the BT-474 HER2-amplified breast tumor xenograft model, and HER2 amplification is present in many cancers, as described above. Non-clinical data have been validated by the activity of tucatinib and trastuzumab observed in the ONT-380-005 doublet study for the treatment of HER2-positive metastatic breast cancer. The present invention is also based (in part) on the finding that the combination of tucatinib and trastuzumab effectively inhibits tumor growth in several other HER2-positive tumor xenograft models, including CRC, esophageal cancer, gastric cancer, cholangiocarcinoma, and NSCLC.

[0033] II. Definitions

[0034] Unless otherwise specified, the meaning of all technical and scientific terms used herein is consistent with the common understanding of those of ordinary skill in the art to which the invention belongs. In addition, any method or material similar or equivalent to the methods or materials described herein may be used in the practice of the present invention. For the purpose of understanding the present invention, the following terms are defined.

[0035] As used herein, the terms "a", "an", or "the" include not only aspects of one construct, but also aspects of more than one construct. For example, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural references. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes one or more agents known to those skilled in the art, and so forth.

[0036] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error for a measured amount, taking into account the nature or precision of the measurement. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to "about X" specifically refers to at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" is intended to teach and provide written description support for a claim limitation of, for example, "0.98X."

[0037] Alternatively, in biological systems, the terms "about" and "approximately" can mean a value within a certain order of magnitude, preferably within 5 times of a given value, and more preferably within 2 times of a given value. Unless otherwise indicated, the numbers given herein are approximate values, indicating that the term "about" or "approximately" can be inferred without explicit explanation.

[0038] When "about" is used at the beginning of a numerical range, it applies to both ends of the range. Thus, "about 5-20%" is equivalent to "about 5 to about 20%". When "about" is used for the first value of a group of values, it applies to all values ​​in the group. Thus, "about 7, 9 or 11 mg / kg" is equivalent to "about 7%, about 9% or about 11 mg / kg".

[0039] The term "or" as used herein should generally be understood in a non-closed manner. For example, a claim referring to "a composition comprising A or B" will generally mean a composition comprising A and B on one hand. However, "or" should be understood to exclude those aspects that cannot be combined if there is no contradiction (e.g., the composition pH is 9-10 or 7-8).

[0040] The group "A or B" is generally equivalent to the group "selected from the group consisting of A and B".

[0041] The terms "comprising" as used herein should generally be understood as not excluding other ingredients. For example, a claim referring to "a composition comprising A" will cover compositions containing A and B; A, B and C; A, B, C and D; A, B, C, D and E; etc.

[0042] As used herein, the terms "subject", "individual" and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, rats, apes, humans, farm animals, competitive animals and pets, and also encompass tissues, cells and their progeny of biological entities obtained by in vivo or in vitro culture.

[0043] As used herein, the term "therapeutically effective amount" includes a dose sufficient to produce the desired result relative to a specified disorder, condition, or mental state. The desired result may include a subjective or objective improvement in the recipient of the dose. For example, an effective amount of an anti-HER2 antibody and a combination of tucatinib include an amount sufficient to alleviate signs, symptoms, effects, or causes of cancer (e.g., colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, or gallbladder cancer). For another example, an effective amount of an anti-HER2 antibody and a combination of tucatinib include an amount sufficient to alleviate signs, symptoms, effects, or causes of metastatic or HER2-positive cancer. For another example, an effective amount of an anti-HER2 antibody and a combination of tucatinib include an amount sufficient to prevent the development of cancer.

[0044] Therefore, a therapeutically effective amount can be an amount that slows, reverses or prevents tumor growth, increases survival time, or inhibits tumor progression or metastasis. In addition, for example, an effective amount of anti-HER2 antibodies and tucatinib includes an amount sufficient to cause substantial improvement in a subject with cancer when administered to a subject. The effective amount can vary with the following: the type and stage of the cancer being treated, the type and concentration of one or more administered compositions (e.g., containing anti-HER2 antibodies and tucatinib), and the amount of other drugs administered in the same manner.

[0045] For the purposes of the present invention, the therapeutically effective amount is determined by those considerations known to those skilled in the art. This amount must effectively achieve the desired therapeutic effect in a subject suffering from cancer. The therapeutically effective amount depends in particular on the type and severity of the disease to be treated and the treatment regimen. The therapeutically effective amount is usually determined in a properly designed clinical trial (e.g., a dose range study), and those skilled in the art will know how to properly conduct such tests to determine the therapeutically effective amount. As is generally known, the therapeutically effective amount depends on various factors, including the distribution profile of the in vivo therapeutic agent (e.g., a combination of anti-HER2 antibodies and tucatinib) or the composition, the relationship between various pharmacological parameters (e.g., half-life in vivo) and unwanted side effects, and other factors, such as age and gender.

[0046] The term "survival" or "survival time" refers to the length of time after the diagnosis of a disease or the start or completion of a specific course of treatment for a disease (e.g., cancer). The term "overall survival" includes such clinical endpoints, which describe patients who have survived a certain period of time after being diagnosed with a disease (e.g., cancer) or treating a disease (e.g., cancer). The term "disease-free survival" includes such a length of time after treating a specific disease (e.g., cancer), during which the patient survives and has no signs of disease (e.g., no known recurrence). In certain embodiments, disease-free survival is a clinical parameter for evaluating the efficacy of a specific therapy, typically measured in units of 1 or 5 years. The term "progression-free survival (PFS)" includes such a length of time during or after treating a specific disease (e.g., cancer), during which the patient lives with the disease but without additional symptoms of the disease. In some embodiments, PFS is assessed as central nervous system (CNS) PFS or non-CNS PFS. In some embodiments, survival is expressed as a median or mean value.

[0047] The term "treatment / treatment" as used herein includes, but is not limited to, methods and operations that produce beneficial changes in the health status of the recipient (e.g., the cancer status of the patient). The change can be subjective or objective and can be associated with features such as signs or symptoms of the cancer being treated. For example, if the patient notices a reduction in pain, then a successful treatment of pain has occurred. For example, if a reduction in the amount of swelling has occurred, then a beneficial treatment of inflammation has occurred. Similarly, if a clinician notices objective changes, such as a reduction / reduction in the number of cancer cells, cancer cell growth, cancer tumor size, or cancer cell antibodies to cancer drugs, then the treatment of cancer is also considered to be effective. The term also includes preventing the deterioration of the patient's status. Treatment / treatment as used herein also includes administering a combination of anti-HER2 antibodies and Tucatinib to a patient suffering from cancer (e.g., colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, or gallbladder cancer).

[0048] The terms "administer" and "administering" include oral administration to a subject, topical contact, administration by suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intrathecal, intranasal (e.g., inhalation, nasal spray or drops) or subcutaneous administration, or implantation of a sustained-release device, e.g., a small osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other modes of delivery include, but are not limited to, the use of liposome preparations, intravenous infusions, transdermal patches, and the like. Those skilled in the art will be aware of other methods for administering a therapeutically effective amount of an anti-HER2 antibody of the present invention and a combination of tucatinib for preventing or alleviating one or more symptoms associated with cancer.

[0049] The term "co-administration / co-administration" as used herein includes sequential or simultaneous administration of two or more structurally different compounds. For example, two or more structurally different pharmaceutically active compounds can be co-administered by administering a pharmaceutical composition suitable for oral administration, the pharmaceutical composition containing two or more structurally different active pharmaceutically active compounds. For another example, two or more structurally different compounds can be co-administered by administering one compound and then another compound. Two or more structurally different compounds may include anti-HER2 antibodies and Tucatinib. In some cases, the co-administered compounds are administered by the same route. In other cases, the co-administered compounds are administered via different routes. For example, one compound can be administered orally, while other compounds can be administered via intravenous, intramuscular, subcutaneous or intraperitoneal injection (e.g., sequentially or simultaneously). Compounds or compositions that can be administered simultaneously or sequentially so that anti-HER2 antibodies and Tucatinib are simultaneously present in an object or cell at an effective concentration.

[0050] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that assists in administering a cell, an organism, or an active agent to an object. "Pharmaceutically acceptable carrier" refers to such a carrier or excipient that can be included in the composition of the present invention and will not cause significant adverse toxicological effects to the object. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's solution, ordinary sucrose, ordinary glucose, adhesives, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings and coloring agents, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delay agents, etc. The carrier can also include such a substance that is used to provide formulation stability, sterility and isotonicity (e.g., antimicrobial preservatives, antioxidants, chelating agents, and buffers), for preventing the effects of microorganisms (e.g., antibacterial and antifungal agents, such as benzoic acid, chlorobutanol, phenol, sorbic acid, etc.) or for providing a flavor that the formulation can be eaten, etc. In some cases, a carrier is an agent that assists in delivering a small molecule drug or antibody to a target cell or tissue. It will be appreciated by those skilled in the art that other pharmaceutically acceptable carriers may be used in the present invention.

[0051] The term "cancer" as used herein is intended to include members of a class of diseases characterized by uncontrolled growth of abnormal cells. The term includes cancers of any stage and grade, including advanced, recurrent, pre-metastatic and post-metastatic cancers. The term also includes HER2-positive cancers. Drug resistance or multi-drug resistant cancers are also included. Cancers suitable for treatment according to the methods of the present invention include colorectal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), gallbladder cancer (e.g., bile duct cancer, gallbladder cancer), bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small intestine cancer, head and neck cancer, uterine cancer, breast cancer and cervical cancer. In some cases, unknown primary cancers are suitable, particularly if they are HER2-positive. In some embodiments, cancer has metastasized (e.g., metastasized to the brain). "Tumor" as used herein includes one or more cancer cells. The term does not exclude combinations of cancers.

[0052] In the context of cancer, the term "stage" refers to a classification of the extent of cancer. Factors considered when staging cancer include, but are not limited to, tumor size, tumor invasion of nearby tissues, and whether the tumor has metastasized to other sites. The specific criteria and parameters used to distinguish one stage from another can vary depending on the type of cancer. For example, cancer staging is used to help determine prognosis or identify the most appropriate treatment options.

[0053] A non-limiting example of a cancer staging system is referred to as the "TNM" system. In the TNM system, "T" indicates the size and extent of the main tumor, "N" indicates the number of nearby lymph nodes to which the cancer has spread, and "M" indicates whether the cancer has metastasized. "TX" represents a main tumor that cannot be measured, "T0" represents a main tumor that cannot be found, and "T1", "T2", "T3" and "T4" represent the size or extent of the main tumor, wherein the larger number corresponds to a larger tumor or a tumor that has grown into nearby tissue. "NX" represents that the cancer in the nearby lymph nodes cannot be measured, "N0" represents that there is no cancer in the nearby lymph nodes, and "N1", "N2", "N3" and "N4" represent the location and number of lymph nodes to which the cancer has spread, wherein the larger number corresponds to a larger number of lymph nodes containing cancer. "MX" represents a metastasis that cannot be measured, "M0" represents that no metastasis has occurred, and "M1" represents that the cancer has metastasized to other parts of the body.

[0054] As another non-limiting example of a cancer staging system, cancer is classified or graded as having one of the following five stages: "Stage 0," "Stage I," "Stage II," "Stage III," or "Stage IV." Stage 0 indicates that abnormal cells are present, but have not spread to nearby tissues. This is also commonly referred to as carcinoma in situ (CIS). CIS is not cancer, but can subsequently develop into cancer. Stages I, II, and III indicate the presence of cancer. Higher numbers correspond to larger tumor sizes or the tumor has expanded to nearby tissues. Stage IV indicates that the cancer has metastasized. Those skilled in the art will be familiar with the different cancer staging systems and will be able to easily apply or interpret them.

[0055] The term "HER2" (also known as HER2 / neu, ERBB2, CD340, receptor tyrosine protein kinase erbB-2, proto-oncogene Neu, and human epidermal growth factor receptor 2) refers to members of the receptor tyrosine kinase human epidermal growth factor receptor (HER / EGFR / ERBB) family. Amplification or overexpression of HER2 plays a significant role in the development and progression of certain aggressive cancer types, including colorectal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), gallbladder cancer (e.g., cholangiocarcinoma, gallbladder cancer), bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small intestine cancer, head and neck cancer, uterine cancer, cervical cancer and breast cancer. Non-limiting examples of HER2 nucleotide sequences are shown in GenBank reference numbers NP_001005862, NP_001289936, NP_001289937, NP_001289938, and NP_004448. Non-limiting examples of HER2 peptide sequences are shown in GenBank reference numbers NP_001005862, NP_001276865, NP_001276866, NP_001276867, and NP_004439.

[0056] When HER2 is amplified or overexpressed in or on a cell, the cell is referred to as being "HER2 positive." The level of HER2 amplification or overexpression in a HER2 positive cell is typically expressed as a score ranging from 0-3 (i.e., HER2 0, HER2 1+, HER2 2+, or HER2 3+), with higher scores corresponding to a higher degree of expression.

[0057] The term "tucatinib" is also referred to as ONT-380 and ARRY-380, which refers to a small molecule tyrosine kinase inhibitor that inhibits or blocks HER2 activation. Tucatinib has the following structure:

[0058]

[0059] The term "anti-HER2 antibody" refers to an antibody that binds to the HER2 protein. Anti-HER2 antibodies used to treat cancer are generally monoclonal, although the term does not exclude polyclonal antibodies. Anti-HER2 antibodies inhibit HER2 activation or downstream signal transduction by a variety of mechanisms. As non-limiting examples, anti-HER2 antibodies can prevent ligand binding, receptor activation or receptor signal propagation, resulting in a decrease in HER2 expressed or localized in cells, inhibiting HER2 cleavage, or inducing antibody-mediated cytotoxicity. Non-limiting examples of anti-HER2 antibodies suitable for use in the methods and compositions of the present invention include trastuzumab, pertuzumab, adotrastuzumab emtansine (also referred to as T-DM1), magotuximab and combinations thereof.

[0060] The term "chemotherapeutic agent" refers to a group of compounds that can be used to treat or alleviate cancer and its symptoms. In some embodiments, chemotherapeutic agents include alkylating antineoplastic drugs (e.g., nitrogen mustards such as methoxyphosphamide, ifosfamide, melphalan, chlorambucil and cyclophosphamide; alkyl sulfonates such as busulfan; nitrosoureas such as streptozotocin, carmustine and lomotine; triazines such as dacarbazine and temozolomide; and ethylimines such as thiotepa and octreotamide), antimetabolites (see below), antitumor antibiotics (e.g., anthracyclines such as daunorubicin, doxorubicin, epirubicin, ida ... rubicin and valrubicin; bleomycin; mitomycin C, mitoxantrone, and actinomycin) aromatase inhibitors (e.g., steroid inhibitors such as exemestane; and nonsteroidal inhibitors such as anastrozole and letrozole), kinase inhibitors (e.g., tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, lapatinib, nilotinib, sunitinib, and sorafenib; and, for example, bosunitinib, noratinib inib), vatalanib and toceranib), mTor inhibitors (e.g., rapamycin and its analogs, such as temsirolimus, everolimus and dafolimus); PIcK / mTOR dual inhibitors; and ATP-competitive mTOR inhibitors (e.g., sapanisertib), retinoids (e.g., tretinoin, alitretinoin, bexarotene and isotretinoin), topoisomerase inhibitors (e.g., doxorubicin, etoposide, teniposide, mitoxantrone, novobiocin, merbaron, aclatubicin, camptothecin, camptothecin prodrugs or derivatives, such as irinotecan and topothecan), and plant alkaloids (e.g., vinca alkaloids vinblastine, vinorelbine, vincristine and vindesine; taxanes, such as docetaxel and paclitaxel).

[0061] The term "antimetabolite" refers to a group of compounds that can be used to treat cancer. Antimetabolites are generally structurally similar to compounds in common metabolism (such as folic acid, purines or pyrimidines), which enables them to interfere with metabolic processes that incorporate structurally similar compounds. For example, the antimetabolite 5-fluorouracil ("fluorouracil") interferes with the metabolic pathway that incorporates the compound uracil. In some embodiments, antimetabolites include pyrimidine antagonists such as capecitabine, cytarabine, decitabine, fluorouracil, and gemcitabine; purine antagonists such as fludarabine and 6-mercaptopurine; and folic acid antagonists such as methotrexate and pemetrexed. In some embodiments, antimetabolites include carmofur, cytarabine, doxifluridine, floxuridine, fluorouracil, fludarabine, gemcitabine, hydroxyurea, 6-mercaptopurine, methotrexate, pemetrexed, and flurbiprofen. In some embodiments, antimetabolites (eg, fluoropyrimidines) include capecitabine, cabofur, doxifluridine, 5-fluorouracil, and tegafur (preferably capecitabine).

[0062] The term "capecitabine" refers to a prodrug of fluorouracil having the following structure:

[0063]

[0064] Capecitabine undergoes hydrolysis in the liver and tissues to form fluorouracil, the active ingredient. Fluorouracil is a fluorinated pyrimidine antimetabolite that inhibits thymidylate synthase, blocking the methylation of deoxyuridylate to thymidylate, interfering with DNA and (to a lesser extent) RNA synthesis.

[0065] The term "tumor growth inhibition (TGI) index" refers to a value that is used to indicate the extent to which an agent (e.g., tucatinib, anti-HER2 antibody, or a combination thereof) inhibits tumor growth when compared to an untreated control. The TGI index at a specific time point (e.g., a specific number of days into an experiment or clinical trial) is calculated according to the following formula:

[0066]

[0067] Wherein, "Tx Day 0" means the first day after treatment is given (i.e., the first day of administration of the experimental therapy or the control therapy (e.g., vehicle only)), and "Tx Day X" means X number of days after Day 0. Typically, the average volume of the treated or control group is used. As a non-limiting example, in such an experiment, i.e., study day 0 corresponds to "Tx Day 0", and the TGI index is calculated on study day 28 (i.e., "Tx Day 28"), if the average tumor volume on study day 0 in both groups is 250 mm 3 , while the average tumor volume in the experimental and control groups was 125 mm 3 and 750mm 3, then the TGI index on day 28 is 125%.

[0068] As used herein, the term "synergy" or "synergy" refers to the observed results of producing such effects (e.g., inhibition of tumor growth, prolonged survival) when a combination of components or agents (e.g., a combination of tucatinib and an anti-HER2 antibody) is administered, which is greater than the effect that would be expected based on the cumulative performance or effects of the individual components. In some embodiments, synergy is determined by performing a Bliss analysis (see, e.g., Foucquier et al. Pharmacol. Res. Perspect. (2015) 3(3): e00149, incorporated herein by reference in its entirety for all purposes). The Bliss Independence model assumes that drug efficacy is the result of a probabilistic process and that the drugs act completely independently (i.e., the drugs complement each other (e.g., the drugs have different sites of action), but each contributes to a common result). According to the Bliss Independence model, the expected effect of the combination of two drugs is calculated using the following formula:

[0069] E AB =E A +E B -E A ×E B ,

[0070] Where E A and E B represent the effects of drugs A and B, respectively, and E AB represents the effect of the combination of drugs A and B. When the observed effect of the combination is higher than the expected effect E AB The combination of two drugs is considered synergistic when the observed effect of the combination is equal to E AB The combination of two drugs is considered additive when . Alternatively, when the observed effect of the combination is less than E AB The combination of two drugs is considered antagonistic.

[0071] The observed effect of a drug combination can be based on, for example, a TGI index of an object or a population of objects, tumor size (e.g., volume, mass), an absolute change in tumor size (e.g., volume, mass) between two or more time points (e.g., between the first day of treatment and a specific number of days after the first treatment), a rate of change in tumor size (e.g., volume, mass), or survival time. When the TGI index is used as a measured value for the observed effect of a drug combination, the TGI index can be determined at one or more time points. When the TGI index is determined at two or more time points, in some cases, the average or median of multiple TGI indices can be used as a measured value for the observed effect. In addition, the TGI index can be determined in a single object or a population of objects. When the TGI index is determined in a population, the average or median TGI index in the population (e.g., at one or more time points) can be used as a measured value for the observed effect. When tumor size or tumor growth rate is used as a measured value for the observed effect, the tumor size or tumor growth rate can be measured in an object or a population of objects. In some cases, the mean or median tumor size or tumor growth rate is determined for one subject at two or more time points, or in a population of subjects at one or more time points. When survival time is measured in a population, the mean or median survival time can be used as a measure of the observed effect.

[0072] The combination effect E can be calculated using a single dose or multiple doses of the drug. AB , the drugs that make up the combination (e.g., tucatinib and an anti-HER2 antibody). In some embodiments, the predicted combination effect E is calculated using only a single dose of each of drugs A and B (e.g., tucatinib and an anti-HER2 antibody). AB , and E A and E B The values ​​are based on the observed effects when the various drugs are administered as single agents. A and E B The value of E is based on the observed effects of drugs A and B when given as a single agent. A and E B The TGI index can be based on, for example, the TGI index of a subject or population of subjects in each treatment group, tumor size (e.g., volume, mass) measured at one or more time points, the absolute change in tumor size (e.g., volume, mass) between two or more time points (e.g., between the first day of treatment administration and a specified number of days after the first administration of treatment), the rate of change in tumor size (e.g., volume, mass) between two or more time points (e.g., between the first day of treatment administration and a specified number of days after the first administration of treatment), or survival time.

[0073] When using TGI index as the measured value of observed effect, TGI index can be determined at one or more time points.When TGI index is determined at two or more time points, in some cases, mean value or median value can be used as the measured value of observed effect.In addition, TGI index can be determined in a single object or a subject population in each treatment group.When TGI index is determined in a subject population, the average or median TGI index in each population (for example, at one or more time points) can be used as the measured value of observed effect.When tumor size or tumor growth rate is used as the measured value of observed effect, tumor size or tumor growth rate can be measured in the object or subject population in each treatment group.In some cases, at two or more time points for an object, or at one or more time points in a subject population, determine average or median tumor size or tumor growth rate.When measuring survival time in a population, average or median survival time can be used as the measured value of observed effect.

[0074] In some embodiments, a series of doses are used to calculate the predicted combined effect E AB (That is, the effects of each drug when administered as a single agent are observed at multiple doses, and the observed effects at multiple doses are used to determine the predicted combined effect at a particular dose). As a non-limiting example, one may use E calculated according to the formula A and E B Calculate the value of E AB :

[0075]

[0076]

[0077] Among them, E A最大 and E B最大 are the maximum effects of drugs A and B, respectively. 50 and B 50 are the half-maximal effects of drug A and B, respectively, a and b are the administered doses of drug A and B, respectively, and p and q are coefficients derived from the shapes of the dose-response curves of drug A and B, respectively (see, e.g., Foucquier et al. Pharmacol. Res. Perspect. (2015) 3(3): e00149).

[0078] In some embodiments, a combination of two or more drugs is considered synergistic when it produces an observed TGI index that is higher than the predicted TGI index for the drug combination (e.g., when the predicted TGI index is based on the assumption that the drugs will produce a combined effect, the class and effect are additive). In some cases, the combination is considered synergistic when the observed TGI index is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% higher than the predicted TGI index for the drug combination.

[0079] In some embodiments, tumor growth rate (e.g., rate of change in tumor size (e.g., volume, mass)) is used to determine whether a drug combination is synergistic (e.g., if the drug combination produces an additive effect, when the tumor growth rate is slower than expected, then the drug combination is synergistic). In some embodiments, survival time is used to determine whether a drug combination is synergistic (e.g., if the drug combination produces an additive effect, when the survival time of a subject or a population of subjects is longer than expected, then the drug combination is synergistic).

[0080] The term "KRAS" refers to the gene encoding the KRAS GTPase. The KRAS gene is also referred to as V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, K-Ras, C-Ki-RAS, K-Ras2, KRAS2 and transforming protein p21. In humans, KRAS is located on chromosome 12 and comprises 4 coding exons and 5' non-coding exons. KRAS is a member of the Ras subfamily of GTPases and is primarily associated with regulating cell growth and differentiation. Specifically, KRAS transmits signals from the cell surface (e.g., from activated HER2 receptors) to the nucleus via the RAS / MAPK pathway. In some cases, mutations in KRAS have been identified, particularly activated mutations (e.g., mutations that cause constitutively active GTP-bound states and activate downstream proliferation signal transduction pathways), and the mutations are associated with a weak response to anti-HER2 therapy. Mutations in KRAS are present in approximately 35%-45% of human CRCs, and specifically, codons 12 and 13 (found in exon 2) are mutation hotspots, with approximately 95% of KRAS mutations located in one of these two codons. Common KRAS mutations present in CRC include G12D, G12A, G12R, G12C, G12S, G12V, and G13D. Non-limiting examples of KRAS mRNA sequences are shown in GenBank reference codes NM_004985→NP_004976 and NM_033360→NP_203524.

[0081] The term "NRAS" refers to the gene encoding the NRAS GTPase. The NRAS gene is also referred to as a neuroblastoma Ras viral oncogene homolog, N-Ras, NRAS1, CMNS and ALPS4. In humans, KRAS is located at chromosome 1 and comprises 7 exons. NRAS is a member of the GTPase Ras subfamily and is related to regulating cell growth and differentiation. Specifically, NRAS transmits signals from the cell surface (e.g., from activated HER2 receptors) to the nucleus via the RAS / MAPK pathway. In some cases, NRAS activated mutations (e.g., mutations that cause constitutively active GTP-bound states and activate downstream proliferation signal transduction pathways) are associated with weak responses to anti-HER2 therapy. Mutations identified in colorectal cancer include I263T, S310F, A466T, R678Q, L755S, V777L, V842I, R868W and N1219S. A non-limiting example of a NRAS mRNA sequence is shown in GenBank Reference No. NM_002524→NP_002515.

[0082] The term "BRAF" refers to the gene encoding B-Raf serine / threonine kinase. The BRAF gene is also referred to as proto-oncogene B-Raf, v-Raf mouse sarcoma viral oncogene homolog B, B-RAF1, BRAF1, NS7, B-Raf and RAFB1. In humans, BRAF is located on chromosome 7. B-Raf is a member of the kinase Raf family and is involved in regulating cell growth and differentiation. Specifically, B-Raf transmits signals from the cell surface (e.g., from activated HER2 receptors) to the nucleus via the RAS / MAPK pathway. Mutations in BRAF are related to the development of certain cancers and, in some cases, are associated with a weak response to anti-HER2 therapy. V600E BRAF mutations have been identified in colorectal cancer. Other BRAF mutations that have been identified include R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, and A727V. Non-limiting examples of NRAS mRNA sequences are shown in GenBank reference number NM_004333→NP_004324.

[0083] III. Description of Embodiments

[0084] A. Methods for treating and alleviating cancer

[0085] In one aspect, the present invention provides a method for treating or alleviating the effects of cancer (e.g., colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, gallbladder cancer, breast cancer, or a combination thereof) in a subject, the method comprising administering a combination of an anti-HERE2 antibody and Tucatinib to a subject. In some preferred embodiments, the method further comprises administering a chemotherapeutic agent (e.g., an antimetabolite, such as capecitabine). In some preferred embodiments, the cancer is a HER2-positive (e.g., HER2 1+, 2+, or 3+) cancer. In some embodiments, the cancer is a metastatic cancer. In some cases, the cancer is a HER2-positive metastatic cancer. In some embodiments, the cancer is an unresectable, locally advanced cancer.

[0086] Anti-HER2 antibodies suitable for treating or alleviating cancer according to the methods of the present invention include, but are not limited to, trastuzumab, pertuzumab, ado-trastuzumab emtansine, magotuximab, and combinations thereof. In some cases, anti-HER2 antibodies include tocilizumab. In some cases, anti-HER2 antibodies include a combination of trastuzumab and pertuzumab.

[0087] The method of the present invention is applicable to the prevention or treatment of any number of cancers, including various solid tumors, particularly HER2 positive metastatic cancers. In some embodiments, the type of cancer treated or alleviated is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), gallbladder cancer (e.g., bile duct cancer, gallbladder cancer), bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small intestine cancer, head and neck cancer, uterine cancer, breast cancer and cervical cancer. In some cases, the method is suitable for treating unknown primary HER2 positive cancers. In some embodiments, the cancer treated or alleviated is selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, breast cancer and gallbladder cancer. In some preferred embodiments, cancer is breast cancer.

[0088] In some embodiments, cancer is advanced cancer. In some embodiments, cancer is drug-resistant cancer (for example, the cancer is resistant to cetuximab or panitumumab). In some cases, cancer is multi-drug resistant cancer. In some embodiments, the subject suffers from such cancer, and the cancer has resistance, recurrence or refractory to one or more drugs or therapies, and the one or more drugs or therapies are the standard of care for the cancer being treated. In some cases, the subject suffers from such cancer, and the cancer is resistant, recurrence or refractory to the standard of care including cetuximab or panitumumab. In some embodiments, the subject has been treated with fluoropyrimidine (for example, 5-fluorouracil, capecitabine) oxaliplatin, irinotecan or anti-VEGF antibodies (bevacizumab, ramucirumab, ziv-aflibercept) before, or such treatment is disabled in the subject.

[0089] In some embodiments, the dose of Tucatinib is about 0.1 mg-10 mg / kg of the subject's body weight (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / kg of the subject's body weight). In some embodiments, the dose of Tucatinib is about 2-8 mg / kg of the subject's body weight (e.g., about 3-7; about 4-7; about 2.5-6; about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8). In some embodiments, the dose of tucatinib is about 10 mg-100 mg / kg body weight of the subject (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / kg body weight of the subject). In specific embodiments, the dose of Tucatinib is about 1 mg-50 mg / kg of subject body weight (e.g., 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / kg of subject body weight). In some instances, the dose of Tucatinib is about 50 mg / kg of subject body weight.

[0090] In some embodiments, the dose of tucatinib comprises 1 mg-100 mg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg) of tucatinib. In some embodiments, the dose of tucatinib comprises about 100 mg-1,000 mg (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, In some embodiments, the dose of Tucatinib is about 150, 200, 250, 300, 350, 400, 450 or 500 mg (e.g., when administered twice daily). In a specific embodiment, the dose of Tucatinib is about 300 mg (e.g., when administered twice daily).

[0091] In some embodiments, the dose of tucatinib comprises at least about 1,000 mg-10,000 mg (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 0, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200 , 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700 , 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more mg) of tucatinib.

[0092] In some embodiments, the dose of Tucatinib comprises a therapeutically effective amount of Tucatinib. In some embodiments, the dose of Tucatinib comprises less than a therapeutically effective amount of Tucatinib (e.g., when multiple doses are administered to achieve a desired clinical or therapeutic effect).

[0093] In some embodiments, the dose of the anti-HER2 antibody is about 0.1 mg-10 mg / kg body weight of the subject (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / kg body weight of the subject). In some embodiments, the dose of the anti-HER2 antibody is about 10 mg-100 mg / kg body weight of the subject (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / kg body weight of the subject). In some embodiments, the dose of the anti-HER2 antibody is at least about 100 mg-500 mg / kg of the subject's body weight (e.g., at least about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more mg / kg of the subject's body weight). In some cases, the dose of the anti-HER2 antibody is about 6 mg / kg of the subject's body weight. In other cases, the dose of the anti-HER2 antibody is about 8 mg / kg of the subject's body weight. In some cases, the dose of the anti-HER2 antibody is about 2 mg / kg of the subject's body weight. In other cases, the dose of the anti-HER2 antibody is about 20 mg / kg of the subject's body weight. In some embodiments, an initial loading dose of 8 mg / kg is administered, followed by a subsequent dose of 6 mg / kg.

[0094] In some embodiments, the dose of anti-HER2 antibody comprises 1 mg-100 mg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg) of anti-HER2 antibody. In some embodiments, the dose of the anti-HER2 antibody comprises about 100 mg-1,000 mg (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, or 1,000 mg) of an anti-HER2 antibody.

[0095] In a specific embodiment, the dose of the anti-HER2 antibody comprises about 100 mg-400 mg (e.g., about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 or 400 mg) of anti-HER2 antibody. As a non-limiting example, when a dose of 6 mg / kg is used, the dose for a 50 kg subject is about 300 mg. As another non-limiting example, when a dose of 8 mg / kg is used, the dose for a 50 kg subject is about 400 mg.

[0096] In some embodiments, the dose of the anti-HER2 antibody comprises at least about 1,000 mg-10,000 mg (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,0 ... 700、2,800、2,900、3,000、3,100、3,200、3,300、3,400、3,500、3,600、3,700、3,800、3,900、4,000、4,100、4,200、4,300、4,400、4,500、4,600、4,700、4,800、4,900、5,000、5,100、5,200 , 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7 , 800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more mg) of an anti-HER2 antibody.

[0097] In some embodiments, the dosage of anti-HER2 antibody comprises a therapeutically effective amount of anti-HER2 antibody. In some embodiments, the dosage of anti-HER2 antibody comprises an anti-HER2 antibody less than a therapeutically effective amount (e.g., when multiple doses are given to achieve a desired clinical or therapeutic effect).

[0098] In some embodiments, the dose of antimetabolite (eg, capecitabine) is about 100 mg-2,000 mg / mm 2The amount of the drug may be about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, or 2,000 mg / mm 2 In some cases, the dose of the antimetabolite is about 1,000 mg / mm 2 In some cases, the dose of the antimetabolite is about 1,250 mg / mm 2 The surface area of ​​the object.

[0099] In some embodiments, the dose of antimetabolite (capecitabine) comprises about 100 mg-4.000 mg (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, In some embodiments, the dosage of capecitabine is about 150, 300, 450, 500, 600, 650, 750, 800, 900, 950, 1000, or 1100 mg. In some embodiments, the dosage of capecitabine is in a 150 or 500 mg tablet.

[0100] In some embodiments, the dose of antimetabolite (capecitabine) comprises a therapeutically effective amount of antimetabolite. In some embodiments, the dose of antimetabolite comprises less than a therapeutically effective amount of antimetabolite (e.g., when multiple doses are administered to achieve a desired clinical or therapeutic effect).

[0101] For example, data from animal studies (e.g., rodents and monkeys) can be used in formulating a range of dosage for use in humans. The dosage of the compounds of the invention lies preferably within a range of circulating concentrations that include the ED with little or no toxicity. 50The dosage may vary within this range depending on the dosage form and route of administration. For any composition used in the methods of the invention (e.g., a combination comprising tucatinib, an anti-HER2 antibody, or capecitabine), the therapeutically effective amount can be estimated initially from cell culture assays. The dosage can be formulated in animal models to achieve results including IC 50 The concentration of the test compound that achieves half-maximal inhibition of symptoms can be used to determine the circulating plasma concentration range of the compound as determined by cell culture. Such information can be used to more accurately determine the human dose. For example, levels in plasma can be measured by high performance liquid chromatography (HPLC).

[0102] It will be further understood that the appropriate dosage of the composition (e.g., a combination comprising tucatinib, an anti-HER2 antibody, or capecitabine) depends on the potency of the composition and the desired effect to be achieved. For example, when one or more of these compositions are administered to a mammal, a physician, veterinarian, or researcher first prescribes a relatively low dose, then increases the dose until an appropriate response is obtained. In addition, it will be understood that the specific dosage level for any particular subject depends on a variety of factors, including the activity of the specific composition employed; the subject's age, weight, general health, sex, and diet; administration time; administration route; excretion rate and pattern; the effect of any drug combination; and the activity or expression level to be regulated.

[0103] In certain embodiments, a combination of Tucatinib, an anti-HER2 antibody or antimetabolite (e.g., capecitabine) is administered to a subject. When a combination of Tucatinib, an anti-HER2 antibody or antimetabolite is administered to a subject together, Tucatinib, an anti-HER2 antibody or antimetabolite can be administered simultaneously or sequentially. In some embodiments, an anti-HER2 antibody or antimetabolite is administered during administration of Tucatinib. In some embodiments, an anti-HER2 antibody or antimetabolite is administered before administration of Tucatinib. In some embodiments, an anti-HER2 antibody or antimetabolite is administered after administration of Tucatinib. Tucatinib and capecitabine can be administered together or sequentially (e.g., Tucatinib can be administered before or after capecitabine).

[0104] In some embodiments, tucatinib and anti-HER2 antibodies or antimetabolites are administered simultaneously. In some embodiments, tucatinib and anti-HER2 antibodies or antimetabolites are not administered simultaneously, but are administered the same number of times per day, or the same number of times per week, or the same number of times per month (e.g., both are administered once a day, twice a day, once a week, twice a week, etc.). In some embodiments, tucatinib and anti-HER2 antibodies or antimetabolites are administered with different dosage regimens. As a non-limiting example, tucatinib is administered once a day, while anti-HER2 antibodies are administered twice a day, and vice versa. As another non-limiting example, tucatinib is administered once a day, while anti-HER2 antibodies are administered once every 2, 3, 4, 5, 6 or more days, and vice versa. It will also be understood by those skilled in the art that certain factors may affect the dosage and time required for effective treatment of the subject, including, but not limited to, the severity of the malignant condition or disease, previous treatment, the age or general health of the subject, and other diseases present. Furthermore, treating a subject with a therapeutically effective amount of a composition (eg, a combination comprising tucatinib and an anti-HER2 antibody) can include a single treatment or, preferably, can include a series of treatments.

[0105] Optimal dosage, toxicity, and therapeutic efficacy of a composition administered according to the methods of the invention (e.g., a combination comprising tucatinib and an anti-HER2 antibody) may vary depending on the relative potency of the combination administered and may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (a dose lethal to 50% of the population) or ED 50 (the dose that has therapeutic efficacy in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 Agents with large therapeutic indices are preferred. Although agents that exhibit toxic side effects may be used, care should be taken in designing delivery systems that target such agents to the site of affected tissue to minimize potential damage to normal cells, thereby reducing side effects.

[0106] The optimal dosage regimen can be calculated from the measured values ​​of the active ingredients accumulated in the subject's body. Typically, the dosage is about 1 ng to about 1,000 mg / kg body weight and can be administered once or more daily, weekly, monthly or annually. One of ordinary skill in the art can easily determine the optimal dosage, administration method and repetition rate. One of ordinary skill in the art can determine the optimal dosage of a combination of tucatinib, anti-HER2 antibodies or capecitabine given to a person according to the established regimens provided in the art and herein.

[0107] Regardless of whether Tucatinib, anti-HER2 antibodies or antimetabolites are administered simultaneously or sequentially, Tucatinib, anti-HER2 antibodies or antimetabolites can be administered at any dose described herein. In some embodiments, the dose of Tucatinib, anti-HER2 antibodies or antimetabolites is a therapeutically effective amount. In some embodiments, the dose of Tucatinib is a therapeutically effective amount, and the dose of anti-HER2 antibodies or antimetabolites is less than the therapeutically effective amount (i.e., one or more subsequent doses of anti-HER2 antibodies or antimetabolites are administered, so that the therapeutically effective amount is delivered to the subject). In some embodiments, the dose of anti-HER2 antibodies or antimetabolites is a therapeutically effective amount, and the dose of Tucatinib is less than the therapeutically effective amount (i.e., one or more subsequent doses of Tucatinib are administered, so that the therapeutically effective amount is delivered to the subject). In some instances, the dose of tucatinib is about 150, 200, 250 or 300 mg (e.g., when administered twice daily), the dose of the anti-HER2 antibody is about 2 mg, 6 mg or 8 mg / kg of subject body weight (e.g., when administered once daily or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days), or the dose of the antimetabolite is about 1,000 mg / mm 2 In other cases, the dose of tucatinib is about 150, 200, 250 or 300 mg (e.g., when administered twice daily), the dose of the anti-HER2 antibody is about 600 mg (e.g., when administered once daily or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days), or the dose of the antimetabolite is about 1,000 mg / mm 2 Subject body surface area (eg, when given twice daily).

[0108] When tucatinib, anti-HER2 antibody or antimetabolite are co-administered to a subject, they can be administered by the same route or by different routes. As a non-limiting example, tucatinib or antimetabolite can be administered orally, while the anti-HER2 antibody can be administered intravenously, intramuscularly, subcutaneously or intraperitoneally simultaneously.

[0109] For sequential co-administration, Tucatinib can be administered before anti-HER2 antibodies, and vice versa. In some embodiments, Tucatinib and anti-HER2 antibodies or antioxidants are administered by the same route, but the administration of Tucatinib and anti-HER2 antibodies or capecitabine is separated by the same amount of time. In some embodiments, Tucatinib and anti-HER2 antibodies or antioxidants are administered by different routes, and the administration of Tucatinib and anti-HER2 antibodies or antioxidants is separated by the same amount of time. As a non-limiting example, Tucatinib is administered orally, and anti-HER2 antibodies are subsequently administered by another route (e.g., intravenous, intramuscular, subcutaneous or intraperitoneal) after a period of time. In addition, antimetabolites can be administered orally before or after Tucatinib or anti-HER2 antibodies.

[0110] For sequential co-administration, those skilled in the art will be able to readily determine the appropriate time between administration of tucatinib and the other one or more agents (i.e., anti-HER2 antibodies or antimetabolites). In some embodiments, the administration of tucatinib and the other one or more agents is separated by 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more minutes. In some embodiments, the interval between administration of tucatinib and the other one or more agents is 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 or more hours. In some embodiments, the interval between administration of tucatinib and the other one or more agents is 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 or more days. In some embodiments, the interval between administration of tucatinib and the other one or more agents is 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 or more months.

[0111] In some embodiments, tucatinib and the other one or more agents (ie, anti-HERE antibodies or anti-metabolites) are administered 1, 2, 3, 4, 5 or more times daily. In some embodiments, tucatinib and the other one or more agents (ie, anti-HERE antibodies or chemotherapeutic agents) are administered 1, 2, 3, 4, 5, 6, 7 or more times per week. In some embodiments, tucatinib and the other one or more agents are administered 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, 30 or more times per month.

[0112] In some embodiments, tucatinib and the other one or more agents are administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, tucatinib and the other one or more agents are administered once every 1, 2, 3, 4 or more weeks. In some embodiments, tucatinib and the other one or more agents are administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

[0113] Following successful treatment, it may be necessary for the subject to undergo maintenance therapy to prevent recurrence of cancer (eg, colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, gallbladder cancer, breast cancer, or a combination thereof).

[0114] Determination of a therapeutically effective amount is within the skill of the art, particularly in light of the detailed disclosure of the present invention. Typically, an effective or therapeutically effective amount of a composition (e.g., a combination comprising tucatinib and one or more other agents) is determined by first administering a low dose or a small amount of the composition, and then incrementally increasing the dose or dosage administered until the desired effect is observed in the treated subject with minimal or no toxic side effects.

[0115] The composition (e.g., a combination of tucatinib and one or more other agents) is administered in a single or multiple doses, depending on the dose and frequency required and tolerated by the patient. In any case, the composition should provide an amount of compound sufficient to effectively treat the patient. Generally, the dose is sufficient to prevent, treat or alleviate the signs, symptoms or effects of the disease without causing unacceptable toxicity to the patient.

[0116] In some embodiments, treating a subject includes inhibiting cancer (e.g., colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, biliary cancer, breast cancer, or a combination thereof) cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell growth, reducing or eliminating one or more signs or symptoms of cancer, reducing the size (e.g., volume) of a cancer tumor, reducing the number of cancer tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy or other cell death, increasing the survival time of a subject, or enhancing the efficacy of another drug or therapy. In specific cases, the subject does not have cancer.

[0117] Tumor size (e.g., volume) can be measured using techniques including, but not limited to, X-ray imaging, computed tomography (CT) with or without contrast, magnetic resonance imaging (MRI) with or without contrast, positron emission tomography (PET), ultrasound, and other combinations. In some embodiments, the size or presence of tumor metastases (e.g., within the chest, abdomen, pelvis, or brain) is measured. Tumor sites can also be monitored using methods such as photography (e.g., skin photography), biopsy, bone imaging, laparoscopy, and endoscopy.

[0118] In some embodiments, the treated subject produces a tumor growth inhibition (TGI) index of about 10%-70% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%). Preferably, the treated subject produces a TGI index of at least about 70% (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). More preferably, the subject produces a TGI index of at least about 85% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). Even more preferably, the subject produces a TGI index of at least about 95% (e.g., about 95%, 96%, 97%, 98%, 99% or 100%). Most preferably, the treated subjects produce a TGI index of about 100% or more (e.g., about 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or more).

[0119] In a specific embodiment, the subject produces a TGI index that is higher than the TGI index observed when using tucatinib and one or more other agents alone. In some cases, the subject produces a TGI index that is higher than the TGI index observed when using tucatinib alone. In other cases, the subject produces a TGI index that is higher than the TGI index observed when using an anti-HER2 antibody alone. In some embodiments, the subject produces a TGI index that is higher than the TGI index observed when using a chemotherapeutic agent (e.g., antimetabolites such as capecitabine) alone. In some embodiments, the treated subject develops a TGI index that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% higher than the TGI index observed with tucatinib, an anti-HER2 antibody or a chemotherapeutic agent alone.

[0120] In some embodiments, the combination of anti-HER2 antibody, tucatinib and chemotherapeutic agent (e.g., antimetabolite, such as capecitabine) is synergistic. In a specific embodiment, relative to the synergistic combination, the treated subject produces such a TGI index, which is higher than the TGI index that would be expected if the combination of tucatinib, anti-HER2 antibody and chemotherapeutic agent produces an additive effect. In some instances, when the combination of an anti-HER2 antibody, tucatinib, and a chemotherapeutic agent is administered, the observed TGI index is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% higher than the TGI index that would be expected if the combination of tucatinib, an anti-HER2 antibody, and a chemotherapeutic agent produced an additive effect.

[0121] In some embodiments, the HER2 status of the sample cells is determined. This determination can be performed before treatment (e.g., giving Tucatinib, anti-HER2 antibodies and chemotherapeutics) begins, during treatment, or after treatment has been completed. In some cases, it is determined that the HER2 status produces a decision to change therapy (e.g., switching to a different anti-HER2 antibody, adding another anti-HER2 antibody to the treatment regimen, suspending the use of anti-HER2 antibodies, Tucatinib or chemotherapeutics, suspending treatment completely, or switching to the method of the present invention by another treatment method).

[0122] In some embodiments, the sample cells are determined to be overexpressing or not overexpressing HER2. In specific embodiments, the cells are determined to be HER2 3+, HER2 2+, HER2 1+ or HER2 0 (i.e., HER is not overexpressed).

[0123] In some embodiments, the sample cells are cancer cells. In some cases, the sample cells are obtained from a subject with cancer. The sample cells can be obtained from a biopsy specimen, surgically removed, or obtained with a fine needle aspirate (FNA). In some embodiments, the sample cells are circulating tumor cells (CTCs).

[0124] HER2 expression can be compared with reference cells. In some embodiments, reference cells are non-cancerous cells obtained from the same object as the sample cells. In some embodiments, reference cells are non-cancerous cells obtained from different objects or subject populations. In some embodiments, measuring the expression of HER2 includes, for example, determining HER2 gene copy number or amplification, nucleic acid sequencing (for example, sequencing of genomic DNA or cDNA), measuring mRNA expression, measuring protein abundance, or a combination thereof. HER2 testing methods include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), display in situ hybridization (CISH), ELISA, and (for example, HER2 expression) RNA quantification, using techniques such as RT-PCR and microarray analysis.

[0125] In some embodiments, when HER2 is expressed with a higher level in sample cells compared to reference cells, it is determined that the sample cells are HER2 positive. In some embodiments, when HER2 is overexpressed at least about 1.5 times (for example, about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times or more times) compared to reference cells, it is determined that the cell is HER2 positive. In certain embodiments, a cell is determined to be HER2 positive when HER2 is overexpressed at least about 1.5-fold compared to a reference cell.

[0126] In some embodiments, the sample cells are determined to be HER2 positive when the FISH or VISH signal ratio is greater than 2. In some embodiments, the sample cells are determined to be HER2 positive when the HER2 gene copy number is greater than 6.

[0127] In some embodiments, the genotype of one or more genes in the sample cells is determined. In some cases, the sequence or genotype of KRAS, NRAS or BRAF is determined. Genotyping can be performed for the entire gene or only for part of the gene. In specific cases, only exons are genotyped. Genotyping can be performed before treatment (e.g., giving tucatinib, anti-HER2 antibodies and chemotherapeutics) begins, during the treatment plan, or after treatment has been completed. In some cases, genotyping produces a decision to change therapy (e.g., switching to a different anti-HER2 antibody, adding another anti-HER2 antibody to the treatment regimen, suspending the use of anti-HER2 antibodies, tucatinib or chemotherapeutics, suspending treatment completely, or switching to the method of the present invention by another treatment method).

[0128] In some embodiments, treatment is administered when the cancer comprises cells having a wild-type KRAS genotype. In some embodiments, the cancer comprises cells having a wild-type genotype in exon 2 of KRAS. In specific cases, the cancer comprises cells having a wild-type genotype in codon 12 or codon 13 of KRAS. In some embodiments, treatment is administered when the cancer comprises cells having a wild-type NRAS genotype. In some embodiments, treatment is administered when the cancer comprises cells having a wild-type BRAF genotype. In specific embodiments, treatment is administered when the cancer comprises cells having a wild-type genotype and a combination of KRAS, NRAS or BRAF. Cancer cells can be obtained by biopsy specimen, surgically removed, or obtained with fine needle aspirates (FNA). In some embodiments, the cancer cells are circulating tumor cells (CTCs).

[0129] In some aspects, the invention provides a method for treating or ameliorating the effects of HER2-positive cancer in a subject, the method comprising: administering a combination therapy comprising an anti-HER2 antibody and a combination of tucatinib and an antimetabolite, thereby treating the HER2-positive cancer.

[0130] In some aspects, the combination therapy also includes a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimetabolite. In some aspects, the antimetabolite is a member selected from the following: capecitabine, carmofur, doxidluridine, fluorouracil, tegafur and combinations thereof. In some aspects, the antimetabolite is capecitabine.

[0131] In some aspects, the cancer is selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer, gallbladder cancer, breast cancer, and combinations thereof. In some aspects, the cancer is an unresectable locally advanced cancer or a metastatic cancer. In some aspects, the cancer is breast cancer.

[0132] In some aspects, the antimetabolite is a member selected from capecitabine, carmofur, doxidluridine, fluorouracil, tegafur, and combinations thereof, wherein the subject has received prior treatment with trastuzumab, pertuzumab, and T-DM1. In some aspects, the antimetabolite is capecitabine.

[0133] In some aspects, the anti-HER2 antibody is a member selected from the group consisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine, magotuximab, and combinations thereof. In some aspects, the anti-HER2 antibody is trastuzumab. In some aspects, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab.

[0134] In some aspects, wherein the administration of the anti-HER2 antibody is before, during, or after the administration of tucatinib.

[0135] In some aspects, the cancer comprises cells having a wild-type KRAS exon 2 genotype. In some aspects, the cancer comprises cells having a wild-type NRAS genotype. In some aspects, the cancer comprises cells having a wild-type BRAF genotype.

[0136] In some aspects, the subject has a cancer that is relapsed or refractory to standard of care (eg, standard of care comprising cicillin or panitumumab).

[0137] In some aspects, the treated subject produces a tumor growth inhibition (TGI) index of at least about 85%. In some aspects, the treated subject produces a TGI index of about 100%.

[0138] In some aspects, the combination of anti-HER2 antibody and tucatinib is synergistic. In some aspects, the treated subject produces a TGI index that is higher than the TGI index observed when anti-HER2 antibody or tucatinib is used alone.

[0139] In some aspects, the dose of Tucatinib is about 3-7 mg / kg of subject body weight twice daily. In some aspects, the dose of Tucatinib is about 300 mg twice daily.

[0140] In some aspects, the dose of anti-HER2 antibody is about 6 mg-8 mg / kg subject body weight once every three weeks. In some aspects, the dose of anti-HER2 antibody is about 600 mg once every three weeks.

[0141] In some aspects, tucatinib or the anti-HER2 antibody is administered orally, intravenously, or subcutaneously (eg, orally).

[0142] In some aspects, the antimetabolite is administered orally.

[0143] In some aspects, the dose of the antimetabolite (e.g., capecitabine) is about 1,000 mg / m 2 Subjects' body surface area, twice daily.

[0144] In some aspects, the anti-HER2 antibody is administered intravenously or subcutaneously.

[0145] In some aspects, one or more therapeutic effects in a subject are improved relative to baseline after administration of the combination therapy. In some aspects, one or more therapeutic effects are selected from the group consisting of cancer-derived tumor size, objective response rate, duration of response, time to response, progression-free survival, and overall survival.

[0146] In some aspects, the size of a tumor derived from the cancer is reduced by at least about 10%. In some aspects, the size of the tumor is reduced by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% relative to the size of the tumor derived from the cancer prior to administration of the combination therapy.

[0147] In some embodiments, the objective response rate is at least about 20%. In some embodiments, the objective response rate is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%.

[0148] In some aspects, the subject exhibits progression-free survival of at least about 1 month after administration of the combination therapy. In some aspects, the subject exhibits progression-free survival of at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the combination therapy.

[0149] In some aspects, the subject exhibits an overall survival of at least about 1 month after administration of the combination therapy. In some aspects, the subject exhibits at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the combination therapy.

[0150] In some aspects, the duration of response to the antibody-drug conjugate after administration of the combination therapy is at least about 1 month. In some aspects, after administration of the combination therapy, the duration of response is at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0151] In some aspects, the subject has one or more adverse events and further receives an additional therapeutic agent to eliminate or reduce the severity of the one or more adverse events.

[0152] In some aspects, the subject is at risk of developing one or more adverse events and further receives an additional therapeutic agent to prevent or reduce the severity of the one or more adverse events.

[0153] In some aspects, one or more adverse events are grade 2 or higher adverse events. In some aspects, one or more adverse events are grade 3 or higher adverse events. In some aspects, one or more adverse events are serious adverse events.

[0154] In some aspects, the subject is a human.

[0155] In some aspects, the invention provides a method for treating HER2-positive cancer in a subject who has exhibited an adverse event after initiating treatment with a combination therapy comprising an anti-HER2 antibody and tucatinib at an initial dose level, the method comprising administering to the subject the combination therapy at a reduced dose level.

[0156] In some aspects, the combination therapy also includes a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimetabolite. In some aspects, the antimetabolite is selected from the group consisting of capecitabine, carmofur, doxifluridine, fluorouracil, fluridine, and combinations thereof. In some aspects, the antimetabolite is capecitabine.

[0157] In some aspects, one or more adverse events are grade 2 or higher adverse events. In some aspects, one or more adverse events are grade 3 or higher adverse events. In some aspects, the adverse event is liver toxicity. In some aspects, the adverse event is left ventricular dysfunction. In some aspects, the adverse event is a prolongation of the QTc interval.

[0158] In some aspects, the cancer is unresectable locally advanced cancer or metastatic cancer. In some aspects, the cancer is breast cancer.

[0159] In some aspects, the subject has received prior treatment with trastuzumab, pertuzumab, and T-DM1.

[0160] In some aspects, the initial dose level of Tucatinib is about 300 mg twice daily. In some aspects, the reduced dose level of Tucatinib is about 250 mg twice daily. In some aspects, the reduced dose level of Tucatinib is about 200 mg twice daily. In some aspects, the reduced dose level of Tucatinib is about 150 mg twice daily.

[0161] B. Pharmaceutical Compositions

[0162] In another aspect, the present invention provides a pharmaceutical composition comprising tucatinib, an anti-HER2 antibody and a pharmaceutically acceptable carrier. In some embodiments, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, adotrastuzumab emtansine, magotuximab and combinations thereof. In some cases, the anti-HER2 antibody is trastuzumab. In some cases, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent (e.g., antimetabolites such as capecitabine).

[0163] In some embodiments, Tucatinib is present at a concentration of about 0.1 nM-10 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.50.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 nM). In some embodiments, Tucatinib is present at a concentration of about 10 nM-100 nM (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nM). In some embodiments, tucatinib is present at a concentration of about 100 nM-1,000 nM (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nM). In some embodiments, tucatinib is administered at a concentration of at least about 1,000 nM to 10,000 nM (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2 ,800、2,900、3,000、3,100、3,200、3,300、3,400、3,500、3,600、3,700、3,800、3,900、4,000、4,100、4,200、4,300、4,400、4,500、4,600、4,700、4,800、4,900、5,000、5,100、5,200、5 ,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7 , 800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more nM).

[0164] In some embodiments, the anti-HER2 antibody is present at a concentration of about 0.1 nM-10 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.50.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 nM). In some embodiments, the anti-HER2 antibody is present at a concentration of about 10 nM-100 nM (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nM). In some embodiments, the anti-HER2 antibody is present at a concentration of about 100 nM-1,000 nM (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nM). In some embodiments, the anti-HER2 antibody is at least about 1,000 nM-10,000 nM (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 0, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200 , 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 700, 9,800, 9,900, 10,0 ...

[0165] In some embodiments, the chemotherapeutic agent (e.g., antimetabolite, such as capecitabine) is present at a concentration of about 0.1 nM-10 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 nM). In some embodiments, the antimetabolite is present at a concentration of about 10 nM-100 nM (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nM). In some embodiments, the chemotherapeutic agent (e.g., antimetabolite such as capecitabine) is present at a concentration of about 100 nM-1,000 nM (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nM). In some embodiments, the chemotherapeutic agent (e.g., antimetabolite such as capecitabine) is present in an amount of at least about 1,000 nM to 10,000 nM (e.g., at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 00、2,700、2,800、2,900、3,000、3,100、3,200、3,300、3,400、3,500、3,600、3,700、3,800、3,900、4,000、4,100、4,200、4,300、4,400、4,500、4,600、4,700、4,800、4,900、5,000、5,100、 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,7 00, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more nM).

[0166] The pharmaceutical compositions of the present invention can be prepared by any method known in the pharmaceutical field. Pharmaceutically acceptable carriers suitable for use in the present invention include any standard pharmaceutical carriers, buffers and excipients, including phosphate-buffered saline solutions, water and emulsions (such as oil / water or water / oil emulsions), and various types of wetting or adjuvants. Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, 19th edition, 1995). The preferred pharmaceutical carrier depends on the target mode of administration of the active agent.

[0167] The pharmaceutical composition of the present invention may include a drug (e.g., tucatinib, an anti-HER2 antibody or a chemotherapeutic agent) or any pharmaceutically acceptable salt thereof as an active ingredient and a combination of a pharmaceutically acceptable carrier or excipient or diluent. The pharmaceutical composition may optionally include other pharmaceutical ingredients.

[0168] Compositions (e.g., containing tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof) can be intimately mixed with suitable pharmaceutical carriers or excipients as active ingredients according to conventional pharmaceutical mixing techniques. Any carrier or excipient in the form of a formulation suitable for administration is contemplated for use with the compounds disclosed herein.

[0169] Pharmaceutical compositions include those suitable for oral, topical, parenteral, pulmonary, nasal or rectal administration. The most appropriate route of administration in any given case depends in part on the nature and severity of the cancer condition, and optionally the HER2 status or stage of the cancer.

[0170] Other pharmaceutical compositions include those suitable for systemic (e.g., enteral or parenteral) administration. Systemic administration includes oral, rectal, sublingual or sublabial administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other modes of delivery include, but are not limited to, using liposome formulations, intravenous infusion, transdermal patches, etc. In a specific embodiment, the pharmaceutical composition of the present disclosure can be administered intratumorally.

[0171] Compositions for pulmonary administration include, but are not limited to, dry powder compositions comprising a powder of a compound described herein (e.g., Tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof) or a salt thereof, and a powder of a suitable carrier or lubricant. Compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to those skilled in the art.

[0172] Compositions for systemic administration include, but are not limited to, dry powder compositions comprising a powder of a composition as described herein (e.g., tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof) and a suitable carrier or excipient. Compositions for systemic administration may be expressed as, but are not limited to, tablets, capsules, pills, syrups, solutions and suspensions.

[0173] In some embodiments, the composition (e.g., tucatinib, anti-HER2 antibody, chemotherapeutic agent or combination thereof) further comprises a pharmaceutical surfactant. In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, HPβCD, CD, glycerol, maltose, mannitol and saccharose.

[0174] The pharmaceutical compositions or drugs used in the present invention can be formulated using one or more physiologically acceptable carriers or excipients by standard techniques. Suitable pharmaceutical carriers are described herein and in Remington: The Science and Practice of Pharmacy, 21st edition, University of the Sciences in Philadelphia, LWW (Lippencott Williams & Wilkins) (2005).

[0175] The controlled release parenteral formulation of the composition (e.g., tucatinib, anti-HER2 antibodies, chemotherapeutics or combinations thereof) can be made into implants, oily injections or particle systems. An overview of delivery systems is provided in Banga, AJ, THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pennsylvania, (1995), which is incorporated herein by reference. Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles.

[0176] Polymers can be used for the controlled release of ions of the compositions of the present invention. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer R., Accounts Chem. Res., 26: 537-542 (1993)). For example, the block copolymer polaxamer 407 exists as a viscous but fluid liquid at low temperatures, but forms a semisolid gel at body temperature. It has been demonstrated that effective carriers for formulation and sustained delivery of recombinant interleukin 2 and urease (Johnston et al., Pharm. Res., 9: 425-434 (1992); and Pec et al., J. Parent. Sci. Tech., 44 (2): 58 65 (1990)). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm., 112: 215-224 (1994)). In another aspect, liposomes are used for drug targeting and controlled release of lipid-encapsulated drugs (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomique Publishing Co., Inc., Lancaster, Pa. (1993)). Many other systems for the controlled delivery of therapeutic proteins are known. See, e.g., U.S. Patent Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496, each of which is incorporated herein by reference.

[0177] For oral administration of tucatinib, or anti-HER2 antibodies, or a combination of chemotherapeutic agents, the pharmaceutical composition or drug can be in the form of, for example, tablets or capsules, which are prepared by conventional methods with pharmaceutically acceptable excipients. The present invention provides tablets and gelatin capsules, which contain tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof, or dry solid powders of these drugs, and (a) diluents or fillers, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, for example, silicon dioxide, talc, stearic acid, magnesium or calcium salts, metal stearates, colloidal silicon dioxide, hydrogenated vegetable oils. , corn starch, sodium benzoate, sodium acetate or polyethylene glycol; for tablets, (c) binders, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone or hydroxypropyl methylcellulose; if necessary, (d) disintegrants, for example, starch (for example, potato starch or sodium starch glycolate), glycolic acid, agar, alginic acid or its sodium salt or effervescent mixture; (e) wetting agents, for example, sodium lauryl sulfate, or (f) absorbents, colorants, flavorings and sweeteners.

[0178] Tablets may be film-coated or enteric-coated according to methods known in the art. Oral liquid preparations may be in the form of, for example, solutions, syrups or suspensions, or may be prepared as dry products for reconstitution with water or other suitable carriers prior to use. Such liquid preparations may be prepared in a conventional manner with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens, or sorbic acid). The preparation may also contain buffer salts, flavoring agents, colorants or sweeteners as appropriate. If desired, preparations for oral administration may be suitably formulated to provide controlled release of one or more active compounds.

[0179] Typical formulations for topical administration of tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof include ointments, salves, sprays, lotions and patches. However, pharmaceutical compositions can be formulated for any type of administration, for example, intradermal, subdermal, intravenous, intramuscular, subcutaneous, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection with a syringe or other device. Preparations for administration by inhalation (e.g., aerosol) or for oral or rectal administration are also contemplated.

[0180] Suitable formulations for transdermal administration include an effective amount of one or more compounds described herein, optionally with a carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to help penetrate the host skin. For example, a transdermal device is in the form of a bandage containing a backing member, a reservoir containing the compound and an optional carrier, an optional rate-controlling barrier for long-term delivery of the compound to the host skin at a controlled and predetermined rate, and a tool or means for fixing the device to the skin. Matrix transdermal formulations may also be used.

[0181] The compositions and formulations described herein (e.g., tucatinib, anti-HER2 antibodies, chemotherapeutic agents, or combinations thereof) can be formulated for parenteral administration by injection, for example, by push injection or continuous infusion. The formulations for injection can be in unit dosage form, for example, in ampoules or multidose containers, with added preservatives. The injectable composition is preferably an isotonic aqueous solution or suspension, and suppositories are preferably prepared from fat emulsions or suspensions. The composition may be sterilized or contain adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts or buffers for regulating osmotic pressure. Alternatively, one or more active ingredients may be in the form of a powder that is reconstituted with a suitable carrier such as sterile pyrogen-free water before use. In addition, it may also contain other therapeutically valuable substances. The compositions are prepared by conventional mixing, granulation or coating methods, respectively.

[0182] For administration by inhalation, the composition (e.g., containing tucatinib, anti-HER2 antibodies, chemotherapeutic agents or combinations thereof) can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., made of gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base (e.g., lactose or starch).

[0183] The compositions (e.g., containing tucatinib, an anti-HER2 antibody, a chemotherapeutic agent, or a combination thereof) can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0184] In addition, one or more active ingredients can be formulated as a depot preparation. Such long-acting preparations can be administered by (e.g., subcutaneous or intramuscular) implantation or intramuscular injection. Thus, for example, one or more compounds described herein can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or formulated into sparingly soluble derivatives, e.g., sparingly soluble salts.

[0185] In some aspects, the present invention provides a pharmaceutical composition comprising an anti-HER2 antibody, tucatinib, a chemotherapeutic agent (eg, an antimetabolite), and a pharmaceutically acceptable carrier.

[0186] In some aspects, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, ado-trastuzumab emtansine, magotuximab, and combinations thereof. In some aspects, the anti-HER2 antibody is trastuzumab. In some aspects, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab.

[0187] In some aspects, the chemotherapeutic agent is capecitabine.

[0188] C. Kit

[0189] In another aspect, the present invention provides a kit for treating or alleviating the effects of cancer in a subject, the kit comprising a pharmaceutical composition of the present invention (e.g., a pharmaceutical composition containing tucatinib, an anti-HER2 antibody, or a combination of chemotherapeutic agents). In some embodiments, the anti-HER2 antibody is trastuzumab, pertuzumab, adotrastuzumab emtansine, magotuximab, or a combination thereof. In some cases, the anti-HER2 antibody is trastuzumab. In some cases, the anti-HER2 antibody is a combination of trastuzumab and pertuzumab.

[0190] The kit is suitable for treating or alleviating the effects of many cancers, particularly HER2 positive or metastatic cancers. In some embodiments, the type of cancer treated or alleviated is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), gallbladder cancer (e.g., bile duct cancer, gallbladder cancer), bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small intestine cancer, head and neck cancer, uterine cancer, breast cancer and cervical cancer. In some cases, the kit is suitable for treating cancer of unknown primary, particularly if it is HER2 positive. In a specific embodiment, the cancer treated or alleviated is selected from the group consisting of colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer, bladder cancer and gallbladder cancer. In some embodiments, cancer is advanced cancer. In some embodiments, cancer is multidrug resistant cancer. In some cases, cancer is multidrug resistant cancer. In some embodiments, cancer is unresectable, locally advanced cancer. In some embodiments, cancer is metastatic cancer.

[0191] The materials and reagents for carrying out the various methods of the present invention can be provided in the kit to facilitate the execution of the method. As used herein, the term "kit" includes products that facilitate a process, test, analysis or manipulation. Specifically, the kit of the present invention can be used in many applications, including, for example, diagnosis, prediction, treatment, etc.

[0192] The kit may include chemical reagents and other components. In addition, the kit of the present invention may include, but is not limited to, instructions for the user of the kit, reagents and devices for administering a combination of tucatinib, anti-HER2 antibodies, antimetabolites, or pharmaceutical compositions thereof, sample tubes, supports, trays, racks, dishes, plates, solutions, buffers, or other chemical reagents. In some embodiments, the kit includes instructions, devices, or reagents for determining the genotype of a gene (e.g., KRAS, NRAS, BRAF) or determining HER2 expression in a sample. The kit of the present invention may also be packaged for convenient storage and safe transportation, for example, in a box with a lid.

[0193] In some aspects, the present invention describes a kit for treating or alleviating the effects of HER2-positive cancer in a subject, the kit comprising a pharmaceutical composition as described elsewhere herein. In some aspects, the kit further comprises instructions for use. In some aspects, the kit further comprises one or more reagents.

[0194] IV. Examples

[0195] The present invention will be described in more detail by specific examples. The examples provided below are only for illustrative purposes and are not intended to limit the present invention in any way. It is not difficult for those skilled in the art to find that there are multiple non-critical parameters that are variable or modifiable and obtain substantially the same or similar results.

[0196] The examples provided herein demonstrate that Tucatinib and Trastuzumab effectively inhibit tumor growth in many patient-derived xenograft (PDX) models. Specifically, Tucatinib and Trastuzumab effectively treat tumors derived from HER2-positive cancers, including colorectal cancer (CRC), esophageal cancer, gastric cancer, bile duct cancer, non-small cell lung cancer (NSCLC). In addition, the combination of Tucatinib and Trastuzumab is more effective in inhibiting tumor growth than any single drug. In a variety of tumors, when the two drugs are used in combination, a surprising synergistic effect is observed.

[0197] Example 1: Combination of Tucatinib and Trastuzumab in a Colorectal Cancer PDX Model

[0198] In this embodiment, the efficacy of Tucatinib and Trastuzumab was evaluated in a PDX model of HER2-positive CRC. Mice were subcutaneously inoculated with CTG-0121, CTG-0784 or CTG-0383 cells and then treated with Tucatinib, Trastuzumab or a combination of the two drugs (n=10 / group). Tucatinib was orally administered at a dose of 50 mg / kg for 28 days, twice a day (study days 0-27). Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg, once every 3 days. At the beginning of the study on day 0, 9 doses of Trastuzumab were administered. The vehicle-only group was used as a negative control.

[0199] like Figures 3A-3C As shown, Tucatinib and Trastuzumab inhibited tumor growth in all 3 CRC PDX models. In addition, when the combination of the two drugs was given, the inhibition of tumor growth was more significant than when any one drug was used alone. In the CTG-0121 model, Tucatinib, Trastuzumab and the combination of the two drugs produced 104%, 109% and 124% tumor growth inhibition (TGI) indexes on the 29th day of the study, respectively (Table 1). In the CTG-0784 model, Tucatinib, Trastuzumab and the combination of the two drugs produced 50%, 36% and 103% TGI indexes on the 29th day of the study, respectively. In the CTG-0383 model, Tucatinib, Trastuzumab and the combination of the two drugs produced 117%, 80% and 137% TGI indexes on the 29th day of the study, respectively. Surprisingly, when the combination of the two drugs was given to all 3 models, a synergistic effect was observed. Notably, the activity of the combination of tucatinib and trastuzumab in each HER2-positive CRC PDX model was comparable to that observed in HER2-positive breast cancer (BT-474).

[0200] Example 2: Combination of Tucatinib and Trastuzumab in an Esophageal Cancer PDX Model

[0201] In this example, the efficacy of Tucatinib and Trastuzumab was evaluated in a PDX model of HER2-positive esophageal cancer. Mice were subcutaneously inoculated with CTG-0137 or CTG-0138 cells and subsequently treated with Tucatinib, Trastuzumab, or a combination of the two drugs (n=10 / group). Tucatinib was orally administered at a dose of 50 mg / kg for 28 days, twice daily (study days 0-27). Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg, once every 3 days. At the beginning of the study on day 0, 9 doses of Trastuzumab were administered. The vehicle-only group served as a negative control.

[0202] In the CTG-0137 model, both tucatinib and trastuzumab inhibited tumor growth, demonstrating a TGI index of 49% and 55%, respectively, on study day 15 ( Figure 4Aand Table 1). Furthermore, when the two drugs were administered in combination, a synergistic effect was observed, resulting in a TGI index of 85%.

[0203] In the CTG-0138 model, when tucatinib was given as a single agent, it inhibited tumor growth, resulting in a 69% TGI index ( Figure 4B ). However, when tucatinib and trastuzumab were administered in combination, a synergistic effect was observed, resulting in a TGI index of 120% (Table 1).

[0204] Example 3: Combination of Tucatinib and Trastuzumab in Gastric Cancer PDX Model

[0205] In this example, the efficacy of Tucatinib and Trastuzumab was evaluated in a PDX model of HER2-positive gastric cancer. Mice were subcutaneously inoculated with GXA 3038, GXA 3039 or GXA 3054 cells and subsequently treated with Tucatinib, Trastuzumab or a combination of the two drugs (n=10 / group). Tucatinib was orally administered at a dose of 50 mg / kg for 28 days, twice daily (study days 0-27). Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg, once every 3 days. At the beginning of the study on day 0, 9 doses of Trastuzumab were administered. The vehicle-only group served as a negative control.

[0206] like Figures 5A-5C As shown, Tucatinib and Trastuzumab inhibit tumor growth in all 3 gastric cancer PDX models. In addition, when the combination of two drugs is given, the inhibition of tumor growth is more significant than when any one drug is used alone. In the GXA-3038 model, the combination of Tucatinib, Trastuzumab and two drugs produces 110%, 50% and 116% TGI index (Table 1) on the 28th day of the study. In the GXA-3039 model, the combination of Tucatinib, Trastuzumab and two drugs produces 48%, 38% and 103% TGI index on the 29th day of the study. In the GXA-3054 model, the combination of Tucatinib, Trastuzumab and two drugs produces 65%, 93% and 136% TGI index on the 17th day of the study. Surprisingly, when the combination of two drugs is given in all 3 models, a synergistic effect is observed.

[0207] Example 4: Combination of Tucatinib and Trastuzumab in a Cholangiocarcinoma PDX Model

[0208] In this example, the efficacy of Tucatinib and Trastuzumab was evaluated in a PDX model of HER2-positive cholangiocarcinoma. Mice were subcutaneously inoculated with CTG-0927 cells and subsequently treated with Tucatinib, Trastuzumab, or a combination of the two drugs (n=10 / group). Tucatinib was orally administered at a dose of 50 mg / kg for 28 days, twice daily (study days 0-27). Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg, once every 3 days. At the beginning of the study on day 0, 9 doses of Trastuzumab were administered. The vehicle-only group served as a negative control.

[0209] like Figure 6 As shown in Table 1, both tucatinib and trastuzumab inhibited tumor growth. In addition, when the combination of the two drugs was given, the inhibition of tumor growth was more significant than when either drug was used alone. On day 28 of the study, the TGI indexes of the tucatinib, trastuzumab, and combination therapy groups were 48%, 63%, and 86%, respectively.

[0210] Example 5: Combination of Tucatinib and Trastuzumab in NSCLC Model

[0211] In this example, the efficacy of tucatinib and trastuzumab was evaluated in two different models of HER2-positive NSCLC. For both studies, Calu-3 and NCI-H2170 cells were used, both of which express high levels of HER2, have gene amplification comparable to BT-474 breast cancer cells, and have previously demonstrated good in vitro responses to tucatinib.

[0212] Mice were inoculated subcutaneously with Calu-3 or NCI-H2170 cells and subsequently treated with tucatinib, trastuzumab, or a combination of the two drugs (n=10 / group). For the Calu-3 study, tucatinib was orally administered at a dose of 50 mg / kg for 21 days, twice daily, starting on study day 7. Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg, once every 3 days, starting on study day 7. Seven doses of trastuzumab were administered. A vehicle-only group served as a negative control. Three animals received a dose holiday (1 in the negative control group and 2 in the combination therapy group).

[0213] For the NCI-H2170 study, tucatinib was administered orally at a dose of 50 mg / kg twice daily for 21 days starting on study day 18. Trastuzumab was administered intraperitoneally at a dose of 20 mg / kg twice weekly starting on study day 18. A vehicle-only group served as a negative control.

[0214] like Fig. 7A and 7BAs shown in Table 1, both Tucatinib and Trastuzumab inhibited tumor growth in two NSCLC models. In addition, when the combination of the two drugs was given, the inhibition of tumor growth was more significant than when either drug was used alone. For the Calu-3 model, Tucatinib, Trastuzumab and the combination of the two drugs produced 63%, 86% and 100% tumor growth inhibition (TGI) indexes on the 28th day of the study, respectively. Surprisingly, a synergistic effect was observed in the combined therapy group. For the NCI-2170 model, Tucatinib, Trastuzumab and the combination of the two drugs produced 91%, 61% and 98% TGI indexes on the 39th day of the study, respectively.

[0215] Table 1: Summary of TGI Index

[0216]

[0217] Example 6: A Phase 2 Randomized, Double-Blind, Controlled Study of Tucatinib Versus Placebo in Combination with Capecitabine and Trastuzumab in Patients with Pre-treated Unresectable Locally Advanced or Metastatic HER2+ Breast Tumors

[0218] This example describes a double-blind study of tucatinib or placebo, in combination with capecitabine and trastuzumab, in patients with unresectable locally advanced or metastatic HER2+ breast cancer who had received prior treatment with trastuzumab, pertuzumab, and T-DM1.

[0219] Background and Principles

[0220] HER2+ breast cancer

[0221] Breast cancer is the most common form of cancer in women worldwide (1) and the second leading cause of cancer-related death in the United States (2). Approximately 20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2) (3, 4). HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. Tumors that overexpress HER2 are more aggressive and have historically been associated with poorer overall survival (OS) compared to HER2-negative cancers (5).

[0222] The introduction of HER2-targeted therapies using antibody-based therapies or small molecule tyrosine kinase inhibitors (TKIs) has led to significant and sustained improvements in disease-free survival (DFS), progression-free survival (PFS), and OS in adjuvant and metastatic settings (6-9). Trastuzumab, a humanized anti-HER2 antibody, remains the backbone of treatment in the adjuvant and first-line metastatic setting, usually in combination with a taxane. Anti-HER2 therapy combined with cytotoxic chemotherapy allows for simultaneous treatment with two drugs with different mechanisms of action, resulting in higher efficacy than either drug alone (6, 10, 11).

[0223] Although the results of early HER2+ breast cancer have improved, up to a quarter of all patients treated with anti-HER2 therapy relapse in the adjuvant setting. The development of new HER2-targeted therapies such as pertuzumab and T-DM1 (ado-trastuzumab emtansine or trastuzumab emtansine) for HER2+ breast cancer has led to a significant extension of the median survival of these patients; however, essentially all patients in metastatic settings will eventually progress. Treatment failure may be due to primary or acquired resistance to HER2 blockade (12-15). There is evidence that the efficacy of metastatic disease can be further improved by a combination of two different HER2-targeted antibodies or by using dual-targeted HER2 based on antibody-based therapies (such as trastuzumab and TKI) (8, 16). Specifically, the combination of small molecule TKIs with antibody-based therapies may be effective because it can help overcome resistance to antibody-mediated inhibition by utilizing alternative mechanisms of receptor inhibition. Lapatinib, a dual epidermal growth factor receptor (EGFR) / HER2 oral TKI, has been shown to have enhanced activity compared to lapatinib alone when used in combination with trastuzumab, even when given to patients who have previously progressed on prior trastuzumab-based therapy (17, 18). However, the use of lapatinib is limited by the drug’s anti-EGFR / human epidermal growth factor receptor 1 (HER1) activity, which results in toxicities such as rash, diarrhea, and fatigue. Therefore, there is a need for more selective small molecule inhibitors of HER2 that could be used in combination with other anti-HER2 therapies to improve clinical outcomes.

[0224] The current standard of care for patients with HER2+ metastatic disease includes pertuzumab plus trastuzumab and a taxane as first-line treatment for metastatic disease, followed by T-DM1 in the second line (4, 19). Treatment options for patients who progress following treatment with pertuzumab and T-DM1 remain relatively limited. Patients are typically treated with sequential anti-HER2 therapy (in the form of trastuzumab or lapatinib) combined with cytotoxic chemotherapy (such as capecitabine). HER2 therapy in combination with trastuzumab or lapatinib is also considered. However, no single regimen is considered standard of care in this setting, and better options are needed for these patients.

[0225] Brain metastases in HER2+ breast cancer

[0226] Perhaps the greatest unmet medical need in the post-trastuzumab era may be the treatment and prevention of brain metastases. Recent data suggest that the incidence of first recurrence in the brain is increasing in patients receiving trastuzumab-based adjuvant therapy (20), and approximately 30-50% of HER2+ patients with metastatic disease will develop brain metastases (20-22). The increased prevalence of brain metastases in patients with HER2+ breast cancer may be attributed to a variety of factors. First, HER2+ breast cancer appears to exhibit a tropism for the brain. Second, with better control of non-CN disease, patients may survive longer, making brain metastases a more critical clinical issue. Finally, the brain may represent a sanctuary for HER2+ disease because large molecules such as trastuzumab do not cross the blood-brain barrier (23).

[0227] Treatment options for brain metastases are limited. No specific systemic treatment options have been approved for brain metastases, and current treatment relies largely on the use of local therapies such as whole brain radiation therapy (WBRT), stereotactic radiotherapy (SRS), or surgery. Patients can also receive chemotherapy alone, or capecitabine and either lapatinib or trastuzumab, although brain response rates are generally not high (24, 25). The development of HER2-targeted systemic therapies with clinical benefit in brain and non-CNS sites of disease could improve clinical outcomes by improving overall PFS and OS and avoiding or delaying the use of radiation and its associated toxicities, including neurocognitive impairment.

[0228] Study Design

[0229] After all eligibility criteria were met, patients were randomized in a 2:1 ratio to receive either tucatinib or placebo in combination with capecitabine and trastuzumab. Approved trastuzumab biosimilars (intravenous or subcutaneous formulations) may also be studied as an alternative to trastuzumab.

[0230] Patients were randomized using a dynamic stratified randomization scheme. Rosenberger, William F. and John M. Lachin. "Chapter 7" Randomization in Clinical Trials Theory and Practice. Hoboken, NJ: John Wiley & Sons, 2016. Stratification factors included the presence or history of treated or untreated brain metastases (yes / no), Eastern Cooperative Oncology Group Performance Status (ECOG PS) (0 vs. 1), and world region (United States or Canada or rest of the world). Stratification for the presence of brain metastases was based on medical history and investigator assessment of screening contrast brain MRI. For stratification purposes and subsequent efficacy assessments, patients with prior brain metastases (treated or untreated) or definite presence of brain metastases on screening MRI were considered "yes." Patients without a prior history of brain metastases and suspicious for significant lesions on screening contrast MRI were also considered "yes" for stratification and follow-up purposes.

[0231] Each treatment was given in a 21-day cycle. Tucatinib (300 mg) or placebo was given orally (PO) twice daily (BID). If necessary, the dose of Tucatinib or placebo was reduced to 250 mg, 200 mg or even 150 mg PO BID to avoid side effects.

[0232] On days 1-14 of each 21-day cycle, 1000 mg / m 2 Capecitabine was given PO BID.

[0233] Trastuzumab is given as a loading dose of 8 mg / kg IV. Following the IV loading dose of trastuzumab, 6 mg / kg of trastuzumab is given every 21 days, except in special circumstances where it may be given weekly to compensate for a change in treatment regimen. No loading dose of trastuzumab is given to patients who have received trastuzumab within 4 weeks of starting the first cycle of the trial. These patients receive trastuzumab at 6 mg / kg for each cycle, including Cycle 1. Trastuzumab may also be given on a weekly basis, 2 mg / kg IV every 7 days, but only if trastuzumab infusions have been delayed and weekly infusions are required to resynchronize the cycle length to 21 days.

[0234] Alternatively, trastuzumab can be administered subcutaneously as a fixed dose of 600 mg every 3 weeks. Subcutaneous trastuzumab dosing does not require a loading dose nor the weekly schedule that is available for the IV formulation. Patients are allowed to transition from IV trastuzumab to subcutaneous trastuzumab.

[0235] Treatment was continued until unacceptable toxicity, disease progression, withdrawal of consent, or end of study. For patients with isolated progression in the brain and stable systemic disease, local therapies directed to the brain may be given.

[0236] Patients were evaluated for safety throughout the study. Safety assessments, including physical examinations, collection of AEs, and laboratory assessments, were performed at least every 3 weeks throughout the study treatment period and 30 days after the last dose of study drug. Cardiac ejection fraction was assessed by MUGA scan or ECHO at screening and every 12 weeks thereafter.

[0237] Laboratory evaluation includes the following tests: calcium, magnesium, inorganic phosphorus, uric acid, total protein, lactate dehydrogenase (LDH), albumin, blood urea nitrogen (BUN), creatinine, bicarbonate, glucose, potassium, chloride, and sodium. Liver function tests (LFTs) include the following: AST / SGOT, ALT / SGPT, total bilirubin, and alkaline phosphatase. Hematology panel includes the following tests: complete blood count (CBC) differential, hemoglobin, hematocrit (Hct), and platelets. Coagulation panel includes the following tests: INR, prothrombin time (PT), and aPTT. Urinalysis includes, but is not limited to, the following tests: color, appearance, pH, protein, glucose, ketones, and blood.

[0238] A contrast brain MRI was performed at baseline in all patients, regardless of prior history of brain metastases. Efficacy assessments included appropriate measurement of all known sites of metastatic or locally advanced unresectable disease (including at least the chest, abdomen, and pelvis) by high-quality spiral CT, PET / CT (if a high-quality CT scan was included), or MRI scans, as well as appropriate imaging of any other known sites of disease (e.g., skin lesion photography, bone imaging) at baseline and every 6 weeks for the first 24 weeks. Additional imaging such as nuclear medicine bone scans or other scans may be performed at the investigator's discretion. Treatment decisions were made based on the investigator's evaluation of the radiographic scans. All patients underwent a repeat brain contrast MRI within 30 days of the end of treatment, unless a brain contrast MRI had already been performed within 30 days or there was prior documentation of progression in the brain on study. If study treatment was discontinued for reasons other than disease progression, all reasonable efforts were made to assess and follow the patient's disease progression. All patients on study continued to be followed for OS after completion of study treatment.

[0239] For patients who were incidentally found to have undergone local therapy for brain metastases during the screening contrast brain MRI and then continued on study treatment, the performance of a repeat contrast MRI after completion of local therapy was as follows: For patients who received brain radiotherapy during the screening period, the original baseline contrast brain MRI served as a baseline for comparison for further response assessments. For patients who underwent surgical resection of brain metastases during the screening period, the postoperative contrast brain MRI served as the baseline.

[0240] Pharmacokinetic assessments of peak and trough levels of tucatinib and metabolite drug levels were performed. Blood samples were also collected for possible assessment of potential biomarkers of response, including circulating tumor DNA (ctDNA). Individual (patient) plasma tucatinib concentrations for each sampling time are listed; corresponding summary statistics for each sampling time are also calculated. Relative time profiles of plasma tucatinib are plotted for each subject (concentrations in log and linear scales); corresponding summary time plots are also constructed. The ratios of metabolite ONT-993 to parent drug tucatinib are listed and summarized for each sampling time.

[0241] Safety monitoring was performed in a blinded setting throughout the study. All relevant safety and efficacy data were reviewed regularly, including but not limited to deaths, discontinuations, dose reductions, adverse events, serious adverse events (SAEs), and cases of progressive disease within 6 weeks of study entry (blind and unblind).

[0242] Health-related quality of life and health care economics were assessed by using the EQ-5D-5L quality of life instrument and collecting data on health care resource utilization.

[0243] The primary efficacy endpoint was progression-free survival (PFS), which was defined as the time from randomization to centrally-reviewed recorded disease progression or death from any cause (whichever occurred earlier). For the primary endpoint of PFS, which was centrally assessed as a whole in the study, the two treatment groups were compared using the log-rank test. The p-value for this test was calculated using a rerandomization procedure to reflect the dynamic allocation used in randomization: known history of treated or untreated brain metastases (yes / no); ECOG PS (0 versus 1); and world region. All randomized patients were included in the primary analysis. Patients were treated as censored when they were last assessed for progression.

[0244] Secondary efficacy endpoints were progression-free survival in patients with brain metastases, duration of overall survival, objective response rate, clinical benefit rate, and duration of response (for patients who responded).

[0245] Experimental efficacy assessments were also performed using a bi-compartmental tumor assessment approach. In this analysis, progression of non-CNS disease was assessed according to the Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria (independent central assessment), and CNS disease was assessed according to the Response Assessment in NeuroOncology-Brain Metastases (RANO-BM) criteria. HER2 and other mutations were studied as possible response biomarkers, although descriptive subgroup analyses of primary and secondary endpoints were used.

[0246] Follow-up for PFS continued for 12 months after the last patient was randomized. Follow-up for OS continued until enough events were recorded to have 90% power to test the effect of treatment on OS. Because median survival in the control group may vary from 15 to 24 months, the primary analysis for OS was performed approximately 1-2 years after the primary analysis for PFS.

[0247] end

[0248] Primary End Point

[0249] PFS was defined as the time from randomization to centrally assessed documented disease progression (according to RECIST 1.1) or death from any cause, whichever occurred earlier.

[0250] Secondary End Points

[0251] Efficacy endpoints included: PFS in patients with brain metastases at baseline using RECIST 1.1 based on independent central assessment; OS; PFS defined as the time from randomization to investigator-assessed documented disease progression (per RECIST 1.1) or death from any cause, whichever occurred earlier; ORR based on independent central assessment (RECIST 1.1); DOR based on independent central assessment (RECIST 1.1); CBR based on independent central assessment (RECIST 1.1); and comparative health economics of tucatinib versus placebo.

[0252] Safety endpoints included: adverse events (AEs); clinical laboratory assessments; important signs and other relevant safety variables; dose maintenance frequency, dose reductions, and discontinuations for capecitabine; dose maintenance frequency, dose reductions, and discontinuations for tocatinib; and dose maintenance frequency, dose reductions, and discontinuations for trastuzumab.

[0253] Pharmacokinetic endpoints included plasma concentrations of tucatinib and metabolites.

[0254] Health economic and outcome endpoints included: cumulative incidence of health resource utilization, including, but not limited to, procedure time, length of stay, hospitalization, emergency department visit (ED visit), planned and unplanned provider visits, medication use, radiology, and other treatments and procedures; and health-related quality of life / health status using the EQ-5D-5L instrument.

[0255] Exemplary Endpoints

[0256] Exemplary endpoints include: PFS (according to RANO-BM using a dual-compartment tumor assessment approach (non-brain disease assessed according to RECIST 1.1 and CNS disease assessed according to RANO-BM)); non-CNS PFS according to RECIST 1.1 in patients who develop and locally treat first CNS progression and continue study treatment to achieve clinical benefit; ORR (by independent central assessment using the dual-compartment tumor assessment approach according to RANO-BM); duration of response (according to the RANO-BM dual-compartment tumor assessment approach by independent central assessment); time to brain progression (according to the RANO-BM dual-compartment tumor assessment approach by independent central assessment); CBR (according to the RANO-BM dual-compartment tumor assessment approach by independent central assessment); presence of HER2 mutations or other mutations as potential biomarkers of response; and time to intervention (surgery or radiation) for brain metastases.

[0257] Patient Opt-In and Opt-Out

[0258] Inclusion criteria

[0259] To be eligible for the study, patients had to meet the criteria described below.

[0260] (1) Patients must have histologically confirmed HER2+ breast cancer, with HER2+ defined by ISH or FISH or ICH methods. Prior to randomization, tissue blocks or sections must be submitted to confirm HER2 positivity (using ISH or FISH) by a central laboratory designated by the sponsor. Centrally confirmed HER2 results (one of IHC, ISH or FISH) from a previous study can be used to determine eligibility for this study with the sponsor's approval.

[0261] (2) Patients must have received prior treatment with trastuzumab, pertuzumab, and T-DM1.

[0262] (3) Patients must have unresectable locally advanced or metastatic breast cancer (confirmed by the investigator) after the last systemic treatment or be unable to tolerate the last systemic treatment.

[0263] (4) Patients must have measurable or non-measurable disease as assessed by RECIST 1.1.

[0264] (5) The patient must be at least 18 years of age at the time of consent.

[0265] (6) The patient's ECOG PS must be 0 or 1.

[0266] (7) In the opinion of the investigator, the patient must have a life expectancy of at least 6 months.

[0267] (8) Patients must have adequate liver function as defined by: total bilirubin ≤1.5X ULN, except for patients with known Gilbert's disease who may be included if conjugated bilirubin ≤1.5X ULN; and aminotransferases AST / SGOT and ALT / SGPT ≤2.5X ULN (≤5X ULN if liver metastases are present).

[0268] (9) Patients must have adequate baseline hematological parameters as defined by: ANC ≥ 1.5 x 10 3 / μL; platelet count ≥100 x 10 3 / μL (with medical supervisor approval, may include 75-100x 10 3 / μL); hemoglobin ≥9 g / dL; and in patients who had received a transfusion prior to study entry, transfusions had to have been given ≥14 days prior to starting treatment to establish adequate hematologic parameters independent of transfusion support.

[0269] (10) Patients must have a creatinine clearance ≥50 mL / min, calculated according to conventional guidelines, or in patients weighing ≤45 kg, serum creatinine should be within the conventional normal range.

[0270] (11) Patients must have an INR and aPTT ≤ 1.5X ULN unless taking medications known to alter INR and aPTT. Patients are prohibited from taking warfarin and other coumarin derivatives.

[0271] (12) Patients must have LVEF ≥ 50%, assessed by ECHO or MUGA scan, recorded within 4 weeks before the first dose of study treatment.

[0272] (13) If the patient is a female of childbearing potential, the patient must have a negative result on a serum pregnancy test performed within 7 days prior to the first dose of study treatment. Females are considered to be fertile (i.e., fertile) after menarche until after menopause unless permanently sterilized. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. Postmenopausal status is defined as the absence of menstruation for 12 months without other medical reasons.

[0273] (14) Females of childbearing potential (as defined above) and males with reproductive partners agree to use a highly effective method of birth control, i.e., a method that achieves a failure rate of less than 1% per year if used consistently and correctly. Such methods include: combined (estrogen and progestin) hormonal contraception (oral, intravaginal, or transdermal) associated with ovulation inhibition; progestin-only hormonal contraception (oral, injectable, or implantable) associated with ovulation inhibition; intrauterine devices; intrauterine hormone-releasing systems; bilateral tubal occlusion / ligation; vasectomy for partners or sexual abstinence. Male patients with reproductive partners must use barrier contraception. From the time the informed consent is signed until 7 months after the last dose of the study drug or investigational medicinal product, all study patients should use effective contraception as described above.

[0274] (15) Prior to commencing any research-related testing or procedures that are not standard of care for the patient's illness, the patient must provide signed informed consent in accordance with an IRB / IEC approved consent document.

[0275] (16) The patient must be willing and able to comply with the study procedures.

[0276] (17) For CNS inclusion, based on screening contrast brain MRI, patients must meet one of the following criteria: (i) no evidence of brain metastases; (ii) untreated brain metastases not requiring immediate local therapy (for patients with untreated CNS lesions >2.0 cm on screening contrast brain MRI, inclusion requires discussion with and approval of the medical supervisor); or (iii) previously treated brain metastases.

[0277] Brain metastases previously treated with local therapy may have been stable since treatment or have progressed from previous local CNS therapy, provided that, in the opinion of the investigator, there is no clinical indication for immediate retreatment with local therapy.

[0278] Patients treated with CNS local therapy for newly identified lesions found on contrast brain MRI performed during the screening period for this study were eligible for inclusion if all of the following criteria were met: time ≥21 days after the first dose of WBRT before treatment, time ≥7 days after the first dose of SRS before treatment, time ≥28 days after surgical resection; and the presence of additional sites of evaluable disease.

[0279] Records of any CNS treatments administered must be available to allow classification of target and nontarget lesions.

[0280] Exclusion criteria

[0281] Patients were excluded from the study for any of the reasons listed below.

[0282] (1) Patients had received lapatinib within 12 months of starting study treatment (except in cases where lapatinib was given for ≤21 days and discontinued due to reasons other than disease progression or severe toxicity); or had received other investigational HER2 / EGFR or HER2 TKIs at any time before.

[0283] (2) Patients have received capecitabine for metastatic disease (except in cases where capecitabine was given for ≤21 days and discontinued for reasons other than disease progression or severe toxicity). Patients who received adjuvant or neoadjuvant capecitabine at least 12 months prior to starting study treatment were eligible.

[0284] (3) The patient had a history of exposure to the following cumulative doses of anthracyclines: doxorubicin (>360 mg / m 2 ), epirubicin (>720mg / m 2 ), mitoxantrone (>120 mg / m 2 ), idarubicin (>90mg / m 2 ) or liposomal doxorubicin (eg, Doxil, Caelyx, Myocet) > 550 mg / m 2 ).

[0285] (4) The patient has a history of allergic reaction to trastuzumab, capecitabine, or compounds chemically or biologically similar to tucatinib, except for successfully controlled grade 1 or 2 infusion-related reactions to trastuzumab, or has a known hypersensitivity to any of the excipients in the study drug.

[0286] (5) Patients have received any systemic anticancer therapy (including hormonal therapy), non-CNS radiation, or experimental drugs within ≤ 3 weeks of the first dose of study treatment, or are currently participating in another interventional clinical trial. An exception to the washout of hormonal therapy is GnRH agonists for ovarian suppression in premenopausal women, which are permitted concomitant medications.

[0287] (6) Patients had any toxicity related to previous cancer treatment that had not recovered to ≤ Grade 1, with the exception of the following: alopecia and neuropathy (which must have recovered to ≤ Grade 2); and CHF (must have been ≤ Grade 1 in severity when present and must have resolved completely).

[0288] (7) Patients have clinically significant cardiopulmonary disease, such as: ventricular dysrhythm requiring treatment; uncontrolled hypertension (defined as persistent systolic blood pressure >150 mm Hg and / or diastolic blood pressure >100 mm Hg on antihypertensive medication); any history of symptomatic CHF; severe resting dyspnea (CTCAE grade 3 or higher) due to advanced malignancy or hypoxia requiring supplemental oxygen therapy; or conditions that may lead to drug-induced QT interval prolongation or torsade de pointes, such as congenital or acquired long QT syndrome, family history of sudden death, previous history of drug-induced QT prolongation, or current medications with a known and acceptable associated risk of QT prolongation (see the "Acceptable Association" row in Table 13 below).

[0289] (8) The patient has had a known myocardial infarction or unstable angina within 6 months prior to the first dose of study treatment.

[0290] (9) The patient is a known carrier of hepatitis B or hepatitis C or has other known chronic liver diseases.

[0291] (10) The patient is known to be HIV positive.

[0292] (11) The patient is pregnant, breastfeeding, or planning to become pregnant.

[0293] (12) Patients require treatment with warfarin or other coumarin derivatives (non-coumarin anticoagulants are allowed).

[0294] (13) The patient is unable to swallow pills or suffers from significant gastrointestinal disease that prevents adequate oral absorption of the drug.

[0295] (14) The patient has used a strong CYP3A4 inducer or inhibitor or a strong CYP2C8 inducer or inhibitor within 3 elimination half-lives of the inhibitor or inducer before the first dose of study treatment (see Tables 10 and 11 at the end of this example).

[0296] (15) The patient has known dihydropyrimidine dehydrogenase deficiency.

[0297] (16) The patient is unable to undergo brain contrast MRI for any reason.

[0298] (17) The patient has any other medical, social, or psychosocial factors that, in the opinion of the investigator, may affect safety or compliance with study procedures.

[0299] (18) Patients have evidence of need for systemic therapy within 2 years of starting study treatment for another malignancy.

[0300] For CNS exclusion, patients must not have any of the following criteria based on screening contrast brain MRI.

[0301] (19) Patients may not have any untreated brain lesions >2.0 cm in size unless discussed with the medical supervisor and approval for inclusion is obtained.

[0302] (20) Patients may not have been on a continuous systemic corticosteroid regimen of >2 mg total daily dexamethasone (or equivalent) for symptom control of brain metastases. However, patients on a chronic stable dose of ≤2 mg total daily dexamethasone (or equivalent) may be eligible for discussion and approval by a medical supervisor.

[0303] (21) Patients may not have any brain lesions that are considered to require immediate local treatment, including, but not limited to, lesions in anatomical sites where increase in size or possible treatment-related edema may pose a risk to the patient (e.g., brainstem lesions). Patients who have undergone local treatment for such lesions identified by screening contrast brain MRI may still be eligible for the study according to the criteria described in the CNS inclusion criteria above.

[0304] (22) Patients may not have known or concurrent LMDs recorded by the researchers.

[0305] (23) Patients may not have poorly controlled (>1 / week) general or complex partial seizures or demonstrate neurologic disease progression due to brain metastases despite CNS-directed therapy.

[0306] Criteria for discontinuation of study treatment

[0307] Subjects who discontinue the study will not be replaced. Patients may be withdrawn from study treatment due to any of the following: AEs, progressive disease, death, withdrawal of consent, loss to follow-up, physician decision due to clinical progression, physician decision (due to other factors), patient decision, protocol violation, termination by the sponsor, pregnancy during the trial or initiation of breastfeeding by the patient, or other appropriate criteria.

[0308] The reason for withdrawal from study treatment must be recorded in the patient's eCRF. Unless the patient withdraws consent for the study, complete the planned 30-day follow-up and long-term follow-up assessments. Patients are also followed for disease progression at least until a confirmed PFS event is observed. If an AE is the reason for withdrawal from study treatment, then record "Adverse Event" as the reason for treatment discontinuation, rather than physician decision or patient decision. Record treatment discontinuation due to the AE any time a patient experiences an AE that prevents the patient from restarting tucatinib, either at the investigator's discretion or due to a requirement for dose modification as described below (e.g., requirement for dose reduction to <150 mg BID tucatinib, maintenance of tucatinib for >6 weeks due to toxicity, or insufficient resolution of the AE to restart tucatinib). If an AE leads to discontinuation of study drug, then patients who discontinue tucatinib or placebo or both capecitabine and trastuzumab are recorded as having treatment discontinuation due to an "Adverse Event."

[0309] Because the primary study endpoint was defined as PFS as determined by central radiological assessment, every effort was made to confirm disease progression radiologically whenever possible. However, in cases where patients appeared to have progressive symptoms and signs of metastatic breast cancer, for whom radiological assessment was not possible or feasible, the investigators may remove patients from study treatment because of "physician decision regarding clinical progression." These patients were censored in the final analysis of the primary endpoint, so the use of this reason to remove such patients from study treatment was limited to cases in which the patient was clinically unsuitable for further radiological assessment and there was clinical confidence of cancer progression in the absence of radiological confirmation. Special consideration was given to ensure that other possible reasons, particularly AEs, were not more accurately described as reasons for discontinuation of study drug in these cases.

[0310] Long-term follow-up after discontinuation of study treatment continued until the patient withdrew from the study. The reason for withdrawal of the patient from the study could be any of the following: death, withdrawal of consent for follow-up, loss of follow-up, physician decision, termination of the study by the sponsor, or other appropriate reasons.

[0311] Dosage Considerations

[0312] Tables 2-7 provide guidance on dose modifications of tucatinib or placebo, capecitabine, and trastuzumab.

[0313] All AEs and laboratory abnormalities were assessed by the investigator for relationship to tocatinib or placebo, capecitabine, and trastuzumab, as applicable. AEs could be considered related to tocatinib or placebo alone, capecitabine alone, trastuzumab alone, 2 of the 3 drugs, all 3 drugs, or none of them. In cases where the relationship was unclear, discussion with the medical supervisor was conducted to maintain or adjust one or more study drugs. Dose adjustments (including maintenance dose, dose reduction, or discontinuation of drug) were made as described below.

[0314] If a drug delay of more than 6 weeks is required due to treatment-related toxicity, any study drug is discontinued unless a longer delay is approved by the medical supervisor. Study treatment is discontinued for patients who discontinue tucatinib or placebo.

[0315] Patients may discontinue capecitabine or trastuzumab due to toxicity and continue tucatinib or placebo in combination with capecitabine or trastuzumab, as appropriate. If capecitabine and trastuzumab are discontinued, patients should also discontinue study treatment with tucatinib or placebo.

[0316] Protocol-defined visits will continue as planned during the 21-day cycle, even during periods of dose hold or delay.

[0317] Capecitabine was administered only on days 1 to 14 of a cycle. No doses were given on days 15 to 21 of a cycle.

[0318] The investigator may initiate a dose reduction or treatment discontinuation for reasons other than those listed below if deemed in the best interest of patient safety.

[0319] Doses used to maintain toxicity will not be replaced.

[0320] Study treatment may be continued for up to 6 weeks prior to local CNS therapy. Maintain oral study medication (tucatinib / placebo and capecitabine) for 1 week prior to planned CNS-directed therapy. Tucatinib may be maintained prior to CNS-directed radiotherapy if necessary. Capecitabine is a known radiosensitizer and therefore needs to be maintained prior to CNS-directed radiotherapy. Trastuzumab has been shown not to enhance radiation and therefore may be continued during radiotherapy as planned. Oral study medication may be restarted 7 or more days after completion of SRS / SRT, 21 or more days after WBRT, or 28 or more days after surgical resection. Plans to maintain and restart study medication prior to and following local therapy need to be discussed with the medical supervisor and documented in writing.

[0321] Tucatinib or placebo dose reduction

[0322] Tables 2-7 provide dose adjustment requirements for Tucatinib or placebo. Dose reductions greater than those required by these tables may be performed at the discretion of the investigator. A maximum of 3 dose reductions of Tucatinib or placebo are permitted, but dose reductions below 150 mg BID are not permitted. In the opinion of the investigator, study treatment is discontinued for patients who will require a dose reduction to <150 mg BID or who may require a fourth dose reduction of Tucatinib.

[0323] After a dose reduction, the tocatinib or placebo dose was not re-escalated.

[0324] Table 2: Recommended dose reduction schedule for tucatinib or placebo

[0325]

[0326] a. Dose reductions in more steps than those listed in this table (i.e., individual dose reductions greater than 50 mg) may be used if the investigator deems it clinically appropriate. However, tucatinib or placebo may not be reduced to less than 150 mg BID.

[0327] Trastuzumab dose adjustments

[0328] There are no dose reductions for trastuzumab. Trastuzumab may also be given on a weekly basis, 2 mg / kg IV every 7 days, but only if the trastuzumab infusion has been delayed, and the weekly infusion requires resynchronization of the cycle length to 21 days, after discussion with the medical supervisor. The subcutaneous dose of trastuzumab (600 mg) cannot be adjusted because it is only given once every 3 weeks. If trastuzumab cannot be restarted at the same dose after being maintained for an AE, then dosing must be discontinued. Because trastuzumab is given as an IV infusion, infusion-related reactions (IARs) may occur.

[0329] If significant IAR occurs, the infusion will be interrupted and appropriate medical treatment will be administered (see below). Permanent discontinuation may be considered in patients with severe IAR. The clinical evaluation is based on the severity of the aforementioned reactions and the response to treatment given for the adverse reaction.

[0330] If a patient developed an IAR, the investigator could treat the patient according to the following instructions or according to customary guidelines, as appropriate: stop the infusion and notify the physician; assess vital signs; administer acetaminophen 650 mg PO; consider administering pethidine 50 mg IM, diphenhydramine 50 mg IV, ranitidine 50 mg IV or cimetidine 300 mg IV, dexamethasone 10 mg IV, or famotidine 20 mg IV; if vital signs are stable, resume the trastuzumab infusion.

[0331] If a patient has developed infusion syndrome, standard premedication is not required for subsequent treatment. Patients may receive acetaminophen prior to treatment. Severe reactions have been treated with supportive care, such as oxygen, beta-agonists, corticosteroids, and withdrawal of study agents as indicated.

[0332] Table 3: Dose modifications of tucatinib or placebo and trastuzumab for clinical adverse events other than left ventricular dysfunction or hepatotoxicity related to tucatinib or placebo and / or trastuzumab*

[0333]

[0334] a. Hair loss does not require dose adjustment.

[0335] *Note that if the AE in question does not recover to the grade required to restart study drug as listed in the table, then the patient may need to discontinue the drug completely. Patients who require tocatinib treatment for >6 weeks must discontinue study treatment unless a longer extension is approved by the medical supervisor.

[0336] Capecitabine dose adjustment

[0337] Capecitabine doses were adjusted as described in Table 4 below.

[0338] Capecitabine was withheld (as determined by the investigator) for any patient experiencing a grade 2 or higher AE considered related to capecitabine or to the combination of tucatinib or placebo and capecitabine and / or trastuzumab.

[0339] After a dose reduction, the capecitabine dose was not re-escalated.

[0340] Table 4: Dose Modifications of Capecitabine for Clinical Adverse Events Considered to be Related to Capecitabine

[0341]

[0342] Abbreviations: Common Terminology Criteria for Adverse Events (CTCAE); not applicable (NA).

[0343] a. Dosage adjustment table is based on Package insert; round doses according to customary guidance.

[0344] b. In certain cases of asymptomatic or mildly symptomatic Grade 2 laboratory abnormalities (e.g., anemia), the investigator may elect to maintain the capecitabine dose level and / or resume capecitabine prior to recovery to Grade 1. This should be done only when the risk to the patient of a capecitabine dose interruption and / or reduction outweighs the risk to the patient of the adverse event and when this is consistent with routine and customary clinical practice. If the investigator wishes to follow an alternative dose adjustment schedule for capecitabine in this situation, approval of the medical supervisor is required.

[0345] Dose Modifications for Hepatotoxicity

[0346] Dose adjustments may be required in the presence of abnormal liver function. For dose adjustments of trastuzumab or placebo and capecitabine, see Table 5 below. Dose adjustments of trastuzumab are not required, but the dose may be determined at the investigator's discretion.

[0347] Table 5: Dose adjustments of tratotinib or placebo and capecitabine for abnormal liver function

[0348]

[0349] Abbreviations: alanine aminotransferase (ALT); aspartate aminotransferase (AST); upper limit of normal (ULN).

[0350] Dose Adjustment for Left Ventricular Dysfunction

[0351] Guidance for dose modifications of tucatinib or placebo and trastuzumab for left ventricular dysfunction is provided in Table 6 .

[0352] Table 6: Dosage adjustments for left ventricular dysfunction

[0353]

[0354] Abbreviations: Congestive heart failure (CHF); left ventricular ejection fraction (LVEF).

[0355] Permanently discontinue tucatinib or placebo and trastuzumab for persistent (ie, >4 weeks) LVEF decline or >3 dose interruptions due to LVEF decline.

[0356] Dose Adjustment for Prolonged QTc Interval

[0357] Guidance for dose adjustments of tucatinib or placebo for prolonged QTc interval is provided in Table 7.

[0358] Table 7: Dose adjustments of tucatinib or placebo for prolonged QTc interval, regardless of relationship to medication

[0359]

[0360] Security Assessment

[0361] Safety assessments include: monitoring and recording of AEs and SAEs; physical examinations and vital signs; and measurement of protocol-specified clinical laboratory tests, ECGs, and ECHO or MUGA scans that are critical to the safety assessment of the study drug. Clinically significant changes in these parameters may be captured as AEs.

[0362] The Investigator is responsible for the appropriate medical care and safety of patients entering this study. The Investigator must record all AEs and inform the Sponsor of any SAEs experienced by patients entering this study.

[0363] Data Monitoring Committee

[0364] The independent DMC is responsible for regularly reviewing the safety of patients in the study. The DMC will regularly review blinded and unblinded data, including deaths, discontinuations, dose reductions, AEs, and SAEs. The DMC makes recommendations to the sponsor regarding the conduct of the study, including continuing the study as planned or with protocol modifications, or stopping the study early due to excessive toxicity. A separate DMC charter outlines the composition of the committee, the roles and responsibilities of the members, and describes DMC procedures. The sponsor provides a copy of each DMC recommendation to the investigator.

[0365] Clinical laboratory evaluation

[0366] All safety labs were analyzed by a local laboratory on site. During prescreening and screening, a central laboratory was used to perform HER2 confirmatory testing.

[0367] The chemistry panel included tests for: calcium, magnesium, inorganic phosphorus, uric acid, total protein, lactate dehydrogenase (LDH), albumin, blood urea nitrogen (BUN), creatinine, bicarbonate, glucose, potassium, chloride, and sodium.

[0368] Liver function tests (LFTs) included the following: AST / SGOT, ALT / SGPT, total bilirubin, and alkaline phosphatase.

[0369] The hematology panel included the following tests: complete blood count (CBC) differential, hemoglobin, hematocrit (Hct), and platelets.

[0370] The coagulation panel included the following tests: INR, prothrombin time (PT), and aPTT.

[0371] Urinalysis includes, but is not limited to, the following tests: color, appearance, pH, protein, glucose, ketones, and blood.

[0372] Cardiotoxicity Safety Program

[0373] Trastuzumab and other HER2-targeted therapies are known to increase the risk of developing asymptomatic and symptomatic declines in LVEF. Asymptomatic heart failure has been reported rarely in patients receiving tucatinib in combination with trastuzumab alone or in combination with capecitabine. Therefore, cardiac function is closely monitored.

[0374] Patients were closely monitored throughout the study for the occurrence of any other expected and / or unexpected toxicities. Cardiac ejection fraction was assessed by MUGA or ECHO at screening and every 12 weeks thereafter until study discontinuation and 30 days after the last dose (unless completed within 12 weeks prior to the 30-day follow-up visit).

[0375] The risk of prolonged QTc with tratocinib is not fully understood. For patients with conditions that may prolong QTc, tratocinib must be used with caution. These conditions include patients with uncorrected hypokalemia or hypomagnesemia and the presence of drugs with an accepted or possible association with QTc interval prolongation or induction of torsades de pointes (see, Table 13 at the end of this example). Patients excluded from this study had congenital or acquired long QT syndrome, a family history of sudden death, a history of previous drug-induced QT prolongation, and current use of drugs with a known and acceptable association with QT prolongation (see, Table 13 at the end of this example).

[0376] Safety Plan for Hepatotoxicity

[0377] Although not one of the most common adverse reactions reported in patients receiving tucatinib, grade 3 and 4 elevations in LFTs have been observed in some patients receiving tucatinib in the study. Liver function tests should be monitored in any patient taking tucatinib.

[0378] Because of the known risk of elevated liver enzymes with tucatinib, patients' LFTs (ALT, AST, total bilirubin, alkaline phosphatase) were closely monitored. If liver function tests were elevated, tucatinib was maintained per protocol and normalization to appropriate levels was monitored per protocol before restarting study drug.

[0379] The identification of liver enzyme abnormalities as a potential adverse reaction to tucatinib does not impact the expected favorable benefit-risk profile of tucatinib and is therefore consistent with the type and severity of AEs seen with other cancer therapies for patients with metastatic breast cancer.

[0380] Safety Planning for Patients with Brain Metastases

[0381] Patients with brain metastases are at risk for AEs due to the presence of CNS lesions, disease progression, and toxicity potentially related to study treatment. Sometimes, the use of systemic or radiotherapy to treat brain metastases is associated with local edema, which is believed to be due to the effect of treatment rather than tumor progression. Shortly after starting treatment with tucatinib, capecitabine, and trastuzumab, patients with known brain metastases in study ONT-380-005 were found to have cerebral edema in the area surrounding the known metastases in the thalamus. The patient's symptoms responded rapidly and completely to systemic corticosteroids. It is unclear whether the patient's symptoms were due to local progression or treatment-related toxicity. Similarly, patients treated with tucatinib and trastuzumab alone experienced expansion of previously irradiated CNS lesions during study treatment. Patients underwent surgical resection, and no tumor survival was found. The resected lesions are believed to represent treatment-related necrosis.

[0382] In order to minimize the risk of symptomatic cerebral edema in patients with brain metastasis in this study, patients with high-risk metastasis are excluded from the trial, including those who need immediate local treatment, those with rapidly progressive lesions, those who need corticosteroids (> 2mg dexamethasone / day or equivalent) to control CNS symptoms at the beginning of the study, and those with larger untreated lesions. However, if these patients can accept direct CNS-directed therapy using surgery or radiation, they can receive local therapy and are then eligible for the trial. In the case of selection, as long as contrast brain MRI does not show clear evidence of CNS progression, patients can receive corticosteroid therapy for short-term management and control of symptomatic local edema. All such cases require the approval of the research medical guardian.

[0383] Safety plan to prevent pregnancy

[0384] Because of the potential for effects on embryo-fetal development, all study patients must use effective contraception as described above from the time informed consent is signed until 7 months after the last dose of study drug or investigational medicinal product. Females of childbearing potential (i.e., females who have not undergone surgical sterilization by hysterectomy, bilateral salpingectomy, and / or bilateral oophorectomy; and females who are not postmenopausal, defined as ≥12 months of amenorrhoea at the time of decision) must have a negative pregnancy test prior to starting the trial and must use an effective method of contraception during the trial. Effective methods of contraception include: combined (estrogen and progestin) hormonal contraception associated with ovulation inhibition (oral, intravaginal, or transdermal); progestin-only hormonal contraception associated with ovulation inhibition (oral, injectable, or implantable); intrauterine device; intrauterine hormone-releasing system; bilateral tubal occlusion / ligation; vasectomized partner or sexual abstinence. Male patients with partners of childbearing potential must use barrier contraception.

[0385] Patients of reproductive potential should undergo a urine pregnancy test on Day 1 of each treatment cycle.

[0386] Adverse Events

[0387] definition

[0388] An "adverse event (AE)" is defined as any untoward medical occurrence in a patient or clinical study patient administered a pharmaceutical product and which does not necessarily have a causal relationship to the treatment methods described herein.

[0389] An AE can therefore be any unfavorable and unexpected sign (e.g., abnormal laboratory finding), symptom or illness transiently associated with the use of a medicinal product, whether or not considered related to the medicinal product (International Conference on Harmonisation (ICH) E2A Guideline; Definitions and Standards for Expedited Reporting; 21 CFR 312.32 IND Safety Reporting).

[0390] When determining whether to record a test result or medical condition as an AE, consider the following factors.

[0391] Any new adverse medical event or unfavorable or unexpected change in a pre-existing condition occurring during or after treatment with study drug was recorded as an AE.

[0392] Complications occurring as a result of protocol-authorized interventions (eg, invasive procedures such as biopsy) were recorded as AEs.

[0393] Neither elective procedures nor routinely scheduled treatments were considered AEs. However, adverse medical events occurring during a previously planned elective procedure were recorded as AEs.

[0394] Baseline conditions were not considered AEs unless they worsened after study drug administration. Any change assessed as a clinically significant worsening of baseline disease must be recorded as an AE. Baseline conditions existing before consent were recorded as medical history.

[0395] Clinically significant laboratory abnormalities or vital signs (e.g., requiring intervention, meeting criteria for severity, leading to termination or discontinuation of study treatment, or related to dose or symptoms) were recorded as AEs. When possible, abnormal laboratory results that met the AE definition were recorded as clinical diagnoses rather than the abnormality itself (e.g., "anemia" rather than "decreased blood count").

[0396] A “serious adverse event (SAE)” is defined as an adverse event that meets one of the following criteria:

[0397] Table 8: Classification of serious adverse events

[0398]

[0399] "Overdose" is defined as the administration of an investigational medicinal product in an amount greater than the maximum dose per dose or cumulatively according to the protocol.

[0400] A "medication error" is an unexpected error in dispensing or administering an investigational medicinal product that is not in accordance with the regimen described in this example.

[0401] "Misuse" is defined as any intentional and inappropriate use of an investigational medicinal product not in accordance with the protocol.

[0402] "Abuse" is defined as persistent or occasional intentional excessive use of an investigational medicinal product with adverse physical or psychological effects.

[0403] Information on overdose, medication errors, abuse, and misuse is collected as needed as part of the investigational medicinal product dosing information and / or as a protocol violation.

[0404] Any AEs related to study drug overdose, medication errors, abuse, and misuse were recorded on the AE eCRF and the diagnosis of the AE was documented.

[0405] An “adverse event (AE) of special interest” may be any serious or non-serious AE that the sponsor determines is of scientific or medical concern and is specific to the project, for which continued monitoring and rapid communication to the sponsor may be appropriate.

[0406] Report the following AEs of special interest to the Sponsor within 24 hours regardless of regulatory severity criteria or causality.

[0407] Potential drug-induced liver injury

[0408] Any potential for drug-induced liver injury assessed by laboratory criteria for Hy's Law was considered a program-defined event of interest. The following laboratory abnormalities defined potential Hy's Law conditions: AST or ALT elevation >3X ULN and concurrent elevation of total bilirubin >2X ULN (within 21 days of AST and / or ALT elevation), except in patients with documented Gilbert's syndrome.

[0409] Asymptomatic left ventricular systolic dysfunction

[0410] Generally, an asymptomatic decrease in LVEF should not be reported as an AE because LVEF data are collected separately in the eCRF. However, an asymptomatic decrease in LVEF that leads to a change in study treatment or discontinuation of study treatment is considered an event of special interest and a serious adverse event and must be reported to the sponsor.

[0411] Brain edema

[0412] Any event of cerebral edema not clearly attributed to disease progression was reported as an event of special interest.

[0413] AE severity was graded using the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI CTCAE) (version 4.03). These criteria are provided in Table 12 at the end of this example.

[0414] AE severity and severity are assessed independently. Severity characterizes the intensity of the AE. Severity serves as a guide for sponsors in determining regulatory reporting requirements (see definition of SAE above).

[0415] The relationship of AEs to all study drugs (tucatinib / placebo, capecitabine, and trastuzumab) was assessed using the guidance shown in Table 9 below. AEs not reported to have a causal relationship required follow-up to determine causality.

[0416] Table 9: Guidance on AE causality

[0417]

[0418] Procedures for initiating and recording adverse events

[0419] Cause adverse events

[0420] Investigators assessed patients for the occurrence of AEs at all scheduled and unscheduled visits. The occurrence of AEs was sought through non-byte questioning of patients at each visit. AEs may also be detected when patients voluntarily provide AEs during or between visits or through physical examinations or other assessments.

[0421] All AEs reported by patients were reviewed by the investigator and had to be documented in the original documentation and in the provided AEeCRF.

[0422] Recording adverse events

[0423] All serious and non-serious AEs occurring during the protocol-defined reporting period were recorded in the eCRF, regardless of their relationship to study drug. SAEs occurring between the prescreening consent and primary consent do not need to be recorded unless they result from a study procedure (e.g., biopsy).

[0424] For each AE, the following information was assessed and recorded in the eCRF: description of the AE (including onset and resolution date), severity (see above definitions), relationship to each study drug (see above definitions), outcome of each event, severity (see above definitions), and actions taken for each study drug.

[0425] Diagnosis and signs or symptoms

[0426] Whenever possible, researchers grouped signs or symptoms that constituted a single diagnosis under a single event term. For example, cough, rhinitis, and sneezing might be grouped together as "upper respiratory tract infection." Symptoms were grouped into diagnoses only when the component signs or symptoms were part of a medically confirmed diagnosis (as evidenced by standard medical textbooks). If any aspect of a sign or symptom did not fit the classic pattern of a diagnosis, then the individual symptoms were reported as separate events.

[0427] Progression of underlying malignancy

[0428] Progression of negative underlying malignancies was assessed as an efficacy variable and was not reported as an AE or SAE. Symptomatic clinical worsening due to disease progression as determined by the investigator was also not reported as an AE or SAE.

[0429] However, if the symptom cannot be attributed solely to progression of the underlying malignancy or does not fit the expected pattern of progression of the disease under study, clinical symptoms of progression may be reported as an AE or SAE. In addition, complications from progression of the underlying malignancy are reported as AEs or SAEs.

[0430] Reporting timing and follow-up of adverse events and serious adverse events

[0431] All AEs identified during the clinical studies (tucatinib / placebo, capecitabine, or trastuzumab) were reported from the time the patient signed the informed consent form to the 30-day follow-up.

[0432] Any SAE that the Investigator believed was related to any study drug and occurred after the patient discontinued study treatment was reported to the Sponsor.

[0433] All SAEs and AEs of interest were followed until the acute event had recovered or stabilized, even if the patient discontinued study treatment prior to recovery of the SAE. Non-serious AEs were followed during each reporting period, as described above.

[0434] If a non-serious AE continued at the 30-day follow-up, the AE was recorded as ongoing.

[0435] Serious Adverse Events and Events of Special Interest Reporting Procedure

[0436] All SAEs / EOIs must be reported to the Sponsor as an SAE / EOI within 24 hours of discovery of the event, regardless of their relationship to the study drug, that occur after the first dose of study drug. SAEs that occur after informed consent but before the administration of study drug and that may be related to a protocol procedure must also be reported to the Sponsor within 24 hours of discovery of the event. Any new or subsequent information regarding a previously reported SAE / EOI must be reported to the Sponsor within 24 hours of becoming aware of the new or subsequent information.

[0437] For initial SAE / EOI reports, the available details of the situation should be recorded in the SAE / EOI form, including at least the following: patient changes, AE items (including severity criteria and onset date), study treatment, and causality assessment.

[0438] The process for reporting and documenting SAEs and EOIs is provided in the Research Binder. It is the responsibility of the investigator to report these events to their IRB and / or IEC in accordance with federal and local agency laws and regulations.

[0439] New or follow-up information should be faxed to the Sponsor's Clinical Safety Department. Medical concerns or questions regarding safety will be sent directly to the Medical Guardian.

[0440] When recording a SAE, consider the following factors.

[0441] Death is the result of an event. Events leading to death are recorded and reported on the SAE / EOI form and eCRF.

[0442] For hospitalizations, surgical procedures, or diagnostic procedures, record the illness leading up to the surgery or diagnostic procedure as the SAE, not the procedure itself.

[0443] Sponsor safety reporting to regulatory authorities

[0444] Investigators were required to report all SAEs to the Sponsor. The Sponsor performed safety reporting to regulatory authorities, IRBs, and IECs as required by local regulatory reporting requirements. SAEs assessed as related to tucatinib / placebo and unexpected (as per IB) were blinded by the Sponsor to identify study treatment and reported in accordance with local regulatory reporting requirements. Investigators received all expedited reports in a blinded manner.

[0445] Pregnancy Report

[0446] Pregnancy was reported until 6 months after the last dose of study drug (tucatinib, capecitabine, or trastuzumab, whichever was most recent). The Sponsor was notified if a patient or a female partner of a male patient became pregnant while participating in the study. If a study participant became pregnant during dosing, treatment was discontinued.

[0447] All pregnancies were reported to the sponsor within 24 hours by the investigators, including the male patients' partners. The sponsor requested follow-up evaluation of the pregnancy, fetus, and child.

[0448] Abortions, whether accidental, therapeutic, or spontaneous, were reported as SAEs. Congenital anomalies or birth defects were also reported as SAEs, as described above. All pregnancies were monitored throughout the duration; all perinatal and neonatal outcomes were reported. Infants were followed for at least 8 weeks. Pregnancies were reported to the Sponsor's Clinical Safety Department using the Pregnancy Report Form.

[0449] Table 10: Selected strong inhibitors and inducers of CYP2C8 and their elimination half-lives

[0450]

[0451] a. FDA. “Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers”

[0452] (www.fda.gov / Drugs / DevelopmentApprovalProcess / DevelopmentResources / DrugInteractionsLabeling / ucm093664.htm#potency).

[0453] b.EMA. “Guideline on the investigation of drug interactions”

[0454] www.ema.europa.eu / docs / en_GB / document_library / Scientific_guideline / 2012 / 07 / WC500129606.pdf

[0455] Table 11: Selected strong and moderate inhibitors or inducers of CYP3A4 and their elimination half-lives

[0456]

[0457] Table 12: Adverse Event Severity Grading Scale (CTCAE Version 4.03)

[0458]

[0459]

[0460] Table 13: Drugs Acceptable or Possibly Associated with Risk of QT Prolongation or Torsades de pointes

[0461]

[0462] Guidance for Industry, E14 Clinical Evaluation of QT / QTc Interval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs. USD Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER). October 2005. ICH. Geoffrey K Isbister and Colin B Page. Drug induced QT prolongation: the measurement and assessment of the QT interval in clinical practice. Br J Clin Pharmacol. 2013 Jul;76(1):48–57.

[0463] Glossary and terminology

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] References

[0470] 1. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 [Internet]. International Agency for Research on Cancer. 2010 [cited June 6, 2013]. Available from: globocan.iarc.fr.

[0471] 2. USCSW Group. United States Cancer Statistics: 1999–2009 Incidence and Mortality Web-based Report. In: Health and Human Services CfDCaP, and National Cancer Institute, eds. Atlanta, Georgia, 2013.

[0472] 3. Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clinical breast cancer. 2004; 5(1): 63-9.

[0473] 4. Giordano SH, Temin S, Kirshner JJ, Chandarlapaty S, Crews JR, Davidson NE, et al. Systemic therapy for patients with advanced human epidermal growth factor receptor 2-positive breast cancer: American Society of Clinical Oncology clinical practice guideline. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2014; 32(19): 2078-99.

[0474] 5. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2 / neu oncogene. Science. 1987; 235(4785): 177-82.

[0475] 6. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. The New England journal of medicine. 2001; 344(11): 783-92.

[0476] 7. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. The New England journal of medicine. 2012; 367(19): 1783-91.

[0477] 8. Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, et al. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. The New England journal of medicine. 2012; 366(2): 109-19.

[0478] 9. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. The New England journal of medicine. 2006; 355(26): 2733-43.

[0479] 10. (Lapatinib) T. [Packaging insert]: Novartis; 2015 [cited 2015]. Available from: www.gsksource.com / pharma / content / dam / GlaxoSmithKline / US / en / Prescribing_Information / Tykerb / pdf / TYKERB-PI-PIL.PDF.

[0480] 11. Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2002; 20(3): 719-26.

[0481] 12. Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). Journal of the National Cancer Institute. 2001; 93(24): 1852-7.

[0482] 13. Scaltriti M, Rojo F, Ocana A, Anido J, Guzman M, Cortes J, et al. Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. Journal of the National Cancer Institute. 2007; 99(8): 628-38.

[0483] 14. Pohlmann PR, Mayer IA, Mernaugh R. Resistance to Trastuzumab in Breast Cancer. Clinical cancer research: an official journal of the American Association for Cancer Research. 2009; 15(24): 7479-91.

[0484] 15. Nahta R, Esteva FJ. HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast cancer research: BCR. 2006; 8(6): 215.

[0485] 16. Baselga J, Bradbury I, Eidtmann H, Di Cosimo S, de Azambuja E, Aura C, et al. Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lancet. 2012; 379(9816): 633-40.

[0486] 17. Blackwell KL, Burstein HJ, Storniolo AM, Rugo H, Sledge G, Koehler M, et al. Randomized study of Lapatinib alone or in combination with trastuzumab in women with ErbB2-positive, trastuzumab-refractory metastatic breast cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010; 28(7): 1124-30.

[0487] 18. Blackwell KL, Burstein HJ, Storniolo AM, Rugo HS, Sledge G, Aktan G, et al. Overall survival benefit with lapatinib incombination with trastuzumab for patients with human epidermal growth factor receptor 2-positive metastatic breast cancer: final results from the EGF104900 Study. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2012; 30(21): 2585-92.

[0488] 19. Network NCC. NCCN & Clinical Resources 2013 (NCCN & Clinical Resources 2013) [cited 15 August 2013]. Available from: www.nccn.org.

[0489] 20. Clayton AJ, Danson S, Jolly S, Ryder WD, Burt PA, Stewart AL, et al. Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer. British journal of cancer. 2004; 91(4): 639-43.

[0490] 21. Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, de Azambuja E, Procter M, Suter TM, et al. 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): an open-label, randomised controlled trial. Lancet. 2013;382(9897):1021-8.

[0491] 22. Pestalozzi BC, Holmes E, de Azambuja E, Metzger-Filho O, Hogge L, Scullion M, et al. CNS relapses in patients with HER2-positive early breast cancer who have and have not received adjuvant trastuzumab: a retrospective substudy of the HERA trial (BIG 1-01). The Lancet Oncology. 2013; 14(3): 244-8.

[0492] 23. Lin NU, Bellon JR, Winer EP. CNS metastases in breast cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2004; 22(17): 3608-17.

[0493] 24. Ekenel M, Hormigo AM, Peak S, Deangelis LM, Abrey LE. Capecitabine therapy of central nervous system metastases from breast cancer. Journal of neuro-oncology. 2007; 85(2): 223-7.

[0494] 25. Ramakrishna N, Temin S, Chandarlapaty S, Crews JR, Davidson NE, Esteva FJ, et al. Recommendations on disease management for patients with advanced human epidermal growth factor receptor 2-positive breast cancer and brain metastases: American Society of Clinical Oncology clinical practice guideline. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2014; 32(19): 2100-8.

[0495] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes made therefrom should be understood by those skilled in the art, and they are included in the subject matter and interests of this application and the scope of the appended claims. All publications, patents, patent applications, and serial numbering cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. Use of tucatinib in the preparation of a medicament for treating or alleviating the effects of a combination therapy for HER2-positive cancer in a subject, wherein the medicament is to be administered in combination with trastuzumab to treat HER2-positive cancer, wherein the HER2-positive cancer is selected from the group consisting of colorectal cancer, gastric cancer, non-small cell lung cancer, gallbladder cancer, and esophageal cancer.

2. The use according to claim 1, wherein The combination therapy further comprises a chemotherapeutic agent.

3. The use according to claim 2, wherein The chemotherapeutic agent is an antimetabolite.

4. The use according to claim 3, wherein The antimetabolite is selected from the group consisting of capecitabine, carmofur, doxifluridine, 5-fluorouracil, pyrimidine and combinations thereof.

5. The use according to claim 4, wherein The antimetabolite is capecitabine.

6. The use according to any one of claims 1 to 5, wherein The cancer is colorectal cancer.

7. The use according to any one of claims 1 to 5, wherein The cancer is esophageal cancer.

8. The use according to any one of claims 1 to 5, wherein The cancer is gastric cancer.

9. The use according to any one of claims 1 to 5, wherein The cancer is gallbladder cancer.

10. The use according to claim 9, wherein The bile cancer is cholangiocarcinoma.

11. The use according to claim 1, wherein The cancer is non-small cell lung cancer.

12. The use according to claim 1, wherein The cancer is an unresectable locally advanced cancer or a metastatic cancer.

13. The use according to claim 1, wherein The subject had received prior treatment with trastuzumab, pertuzumab, and T-DM1.

14. The use according to claim 1, wherein The administration of trastuzumab is performed before, during or after the administration of tucatinib.

15. The use according to claim 1, wherein The cancer includes wild-type KRAS Cells with exon 2 genotype.

16. The use according to claim 1, wherein The cancer includes wild-type NRAS Genotype of cells.

17. The use according to claim 1, wherein The cancer includes wild-type BRAF Genotype of cells.

18. The use according to claim 1, wherein The cancer is relapsed or refractory to standard of care.

19. The use according to claim 1, wherein Treating the subject results in a tumor growth inhibition (TGI) index of at least 85%.

20. The use according to claim 1, wherein Treatment of the subject resulted in a TGI index of 100%.

21. The use according to claim 1, wherein The combination of trastuzumab and tucatinib is synergistic.

22. The use according to claim 1, wherein Treatment of the subject results in a TGI index that is greater than that observed with trastuzumab or tucatinib alone.

23. The use according to claim 1, wherein The dose of tucatinib was 3-7 mg / kg subject body weight twice daily.

24. The use according to claim 1, wherein The dose of tucatinib was 300 mg twice daily.

25. The use according to claim 1, wherein The trastuzumab dose was 6 mg-8 mg / kg subject body weight once every three weeks.

26. The use according to claim 1, wherein The dose of trastuzumab was 600 mg once every three weeks.

27. The use according to claim 1, wherein The tucatinib or the trastuzumab is administered orally.

28. The use according to claim 3, wherein The antimetabolite is administered orally.

29. The use according to claim 28, wherein The dose of the antimetabolite was 1,000 mg / m 2 Subjects' body surface area, twice daily.

30. The use according to claim 28 or 29, wherein The trastuzumab is administered intravenously or subcutaneously.

31. The use according to claim 1, wherein One or more therapeutic effects in a subject are improved relative to baseline following administration of the combination therapy.

32. The use according to claim 31, wherein The one or more therapeutic effects are selected from the group consisting of cancer-derived tumor size, objective response rate, duration of response, time to response, progression-free survival, and overall survival.

33. The use according to claim 32, wherein The size of the cancer-derived tumor is reduced by at least 10% relative to the size of the cancer-derived tumor prior to administration of the combination therapy.

34. The use according to claim 32, wherein The objective response rate is at least 20%.

35. The use according to claim 32, wherein The subject exhibits progression-free survival of at least 1 month following administration of the combination therapy.

36. The use according to claim 32, wherein The subject exhibits overall survival of at least 1 month following administration of the combination therapy.

37. The use according to claim 1, wherein The duration of response to the combination therapy is at least 1 month after administration of the combination therapy.

38. The use according to claim 1, wherein The subject has one or more adverse events and further receives an additional therapeutic agent to eliminate or reduce the severity of the one or more adverse events.

39. The use according to claim 1, wherein The subject is at risk of developing one or more adverse events and is further receiving an additional therapeutic agent to prevent or reduce the severity of the one or more adverse events.

40. The use according to claim 38 or 39, wherein The one or more adverse events are grade 2 or higher adverse events.

41. The use according to claim 40, wherein The one or more adverse events are grade 3 or higher adverse events.

42. The use according to claim 38, wherein The one or more adverse events are serious adverse events.

43. The use according to claim 1, wherein The subject is a human being.

44. Use of tucatinib in the preparation of a medicament for a combination therapy for treating a HER2-positive cancer in a subject who has exhibited an adverse event after initiating treatment with a combination therapy comprising an initial dose level of trastuzumab and tucatinib, wherein the medicament is to be administered to the subject in combination with the trastuzumab at a reduced dose level, wherein the HER2-positive cancer is selected from the group consisting of colorectal cancer, gastric cancer, non-small cell lung cancer, gallbladder cancer, and esophageal cancer.

45. The use according to claim 44, wherein The combination therapy further comprises a chemotherapeutic agent.

46. ​​The use according to claim 45, wherein The chemotherapeutic agent is an antimetabolite.

47. The use according to claim 46, wherein The antimetabolite is selected from the group consisting of capecitabine, carmofur, doxifluridine, 5-fluorouracil, pyrimidine and combinations thereof.

48. The use according to claim 47, wherein The antimetabolite is capecitabine.

49. The use according to claim 44, wherein The cancer is colorectal cancer.

50. The use according to claim 44, wherein The cancer is esophageal cancer.

51. The use according to claim 44, wherein The cancer is gastric cancer.

52. The use according to claim 44, wherein The cancer is gallbladder cancer.

53. The use according to claim 52, wherein The bile cancer is cholangiocarcinoma.

54. The use according to claim 44, wherein The cancer is non-small cell lung cancer.

55. The use according to claim 44, wherein The adverse events were grade 2 or higher.

56. The use according to claim 55, wherein The adverse events were grade 3 or higher.

57. The use according to claim 44, wherein The adverse event was hepatotoxicity.

58. The use according to claim 44, wherein The adverse event was left ventricular dysfunction.

59. The use according to claim 44, wherein The adverse event was prolongation of the QTc interval.

60. The use according to claim 44, wherein The cancer is an unresectable locally advanced cancer or a metastatic cancer.

61. The use according to claim 44, wherein The subject had received prior treatment with trastuzumab, pertuzumab, and T-DM1.

62. The use according to claim 44, wherein The initial dose level of tucatinib was 300 mg twice daily.

63. The use according to claim 44, wherein The reduced dose level of tucatinib was 250 mg twice daily.

64. The use according to claim 44, wherein The reduced dose level of tucatinib was 200 mg twice daily.

65. The use according to claim 44, wherein The reduced dose level of tucatinib was 150 mg twice daily.

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