Compounds having antitumor activity against cancer cells having HER2 exon 21 insertion

Abstract

The present disclosure provides methods of treating cancer in patients determined to have a HER2 exon 21 mutation by administering a third generation tyrosine kinase inhibitor, such as poziotinib.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 826,758, filed Mar. 29, 2019, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grant number CA190628 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] 1. Field The present invention generally relates to the fields of molecular biology and medicine. More specifically, the present invention relates to methods of treating patients having a HER2 exon 21 mutation.

Background Art

[0004] 2. Description of Related Art Amplification of Erb-b2 receptor tyrosine kinase 2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2), occurs in many cancer types, and targeted agents such as trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1), lapatinib, and neratinib have been shown to improve clinical outcomes compared to chemotherapy alone (Vogel et al, 2002). Activating mutations of ERBB2 (HER2) have been reported in many cancer types (Kris et al, 2015). There are FDA-approved targeted therapies for cancers bearing HER2 amplification, but there are no approved targeted therapies specific for HER2 mutations. On the other hand, the National Comprehensive Cancer Network non-small cell lung cancer (NSCLC) guidelines recommend testing newly diagnosed patients by extensive molecular profiling to detect HER2 mutations (Ettinger et al, 2018).

[0005] Recent clinical trials of targeted agents for HER2 mutant cancers have focused on covalent second-generation tyrosine kinase inhibitors (TKIs) such as afatinib, neratinib, and dacomitinib. The SUMMIT pan-cancer trial reported that patients who received neratinib had an objective response rate (ORR) of less than 15% for all HER2 mutations (Hyman et al, 2018). However, across multiple trials, when patients were stratified by cancer type, patients with breast cancer had an ORR of 12.5% - 32% for single-agent neratinib (Hyman el al, 2018; Ma el al, 2017), whereas patients with lung cancer had an ORR of 0% - 4% for neratinib as a single agent (Hyman et al, 2018; Mazieres el al, 2015), indicating cancer-specific differences in the efficacy of HER2 inhibition. Interestingly, within a single cancer type, HER2-targeting agents appear to cause variant-specific differences. In the SUMMIT trial, patients with HER2 kinase domain point mutations had an ORR of 21.4%, whereas patients with exon 20 insertions had an ORR of 7.1% for neratinib (Hyman et al, 2018). Furthermore, dacomitinib had an ORR of 11.5% for HER2 mutant NSCLC, but no response occurred among patients with the HER2 exon 20 insertion mutation, p.Y772dupYVMA (Kris et al, 2015). In two separate trials of afatinib, patients with exon 20 insertion-positive NSCLC had ORRs of 18.2% and 18.8% for afatinib.

[0006] Trials of HER2 monoclonal antibodies and drug-antibody conjugates have shown similar results. The pan-cancer trial MyPathway tested the efficacy of the combination of the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab in 35 different tumor types and reported an ORR of 11% for all HER2 mutations and cancer types. In this trial, only 21% of NSCLC patients and 1 patient with cholangiocarcinoma out of the 35 tumor types included responded. In addition, in the pan-HER2 variant NSCLC trial testing the efficacy of T-DM1, patients with exon 20 insertion mutations had an ORR of 54.5%, while patients with exon 19 mutations had no partial response. These cancer-specific and variant-specific differences in patient outcomes indicate the unmet need for a detailed and systematic understanding of the HER2 mutation landscape across cancer types and the specific effective therapies for the various identified HER2 mutations.

[0007] Preclinical trials of HER2 activating mutations have also reported different sensitivities to various TKIs. Testing of mutations within the HER2 extracellular domain has shown that these mutations are associated with resistance to non-covalent inhibitors such as lapatinib, and furthermore show strong sensitivity to covalent TKIs including neratinib, afatinib, and osimertinib, while mutations within exon 19 have been shown to exhibit variable sensitivities to lapatinib and covalent inhibitors. Additionally, testing has demonstrated that HER2 exon 20 mutations have broad resistance to non-covalent and covalent TKIs such as osimertinib, nazartinib, rociletinib, and ormutinib, etc. Moreover, the covalent quinazoline amine-based TKIs neratinib, afatinib, and dacomitinib induce different responses to individual HER2 exon 20 mutations. However, only uncommon HER2 mutations have shown sensitivity to these TKIs at clinically significant concentrations. More recently, it has been reported that poziotinib effectively inhibits HER2 exon 20 insertion mutations at achievable concentrations in patients, and that poziotinib treatment induced a radiation response in one patient harboring a HER2 exon 20 mutation. Nevertheless, not even a single HER2 TKI targeting the most common variants of HER2 mutant cancers has been identified.

SUMMARY OF THE INVENTION

[0008] SUMMARY In embodiments of the present disclosure, the present disclosure provides methods and compositions for treating cancer in a patient having a HER2 exon 21 mutation. In one embodiment, a method of treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib, wherein the subject has been determined to have one or more HER exon 21 mutations, is provided. In certain aspects, the subject is human.

[0009] In some aspects, poziotinib is further defined as poziotinib hydrochloride. In certain aspects, poziotinib hydrochloride is formulated as a tablet.

[0010] In certain embodiments, one or more HER2 exon 21 mutations include one or more point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832-883. In some embodiments, a subject has been determined to have two, three, or four HER2 exon 21 mutations. In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842, R868, and L869. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842 and R868. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

[0011] In some embodiments, a subject is resistant to or exhibits resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or beratinib.

[0012] In certain embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In certain embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In some embodiments, poziotinib is administered daily. In certain embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

[0013] In certain embodiments, it is determined that the subject has a HER2 exon 21 mutation by analyzing a patient-derived genomic sample. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain embodiments, the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing (e.g., DNA sequencing of tumor tissue or plasma-derived circulating free DNA) or by PCR analysis.

[0014] In certain embodiments, the method further comprises the step of administering further anti-cancer therapy. In some embodiments, the anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In certain embodiments, the administration of poziotinib and / or the anti-cancer therapy is carried out intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, in controlled release, in delayed release, as a suppository, or sublingually. In some embodiments, administering poziotinib and / or carrying out the anti-cancer therapy includes local administration, topical administration, or systemic administration. In certain embodiments, the administration of poziotinib and / or the anti-cancer therapy is carried out more than once, for example, daily, every other day, or weekly.

[0015] In some embodiments, the cancer is oral cancer, hypopharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, cancer of the central nervous system or peripheral nervous system tissues, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, hypopharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0016] In another embodiment, a pharmaceutical composition comprising poziotinib is provided for a patient determined to have one or more HER2 exon 21 mutations. In certain embodiments, the one or more HER2 exon 21 mutations include point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832-883. In certain embodiments, the subject has been determined to have two, three, or four HER2 exon 21 mutations.

[0017] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as a tablet.

[0018] In some embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In certain embodiments, poziotinib is administered daily. In some embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

[0019] In some embodiments, the subject has resistance to or has shown resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or velatinib.

[0020] In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842, R868, and L869. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842 and R868. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I and R868W. In some embodiments, the patient is being treated by anti-cancer therapy.

[0021] In yet another embodiment, there is provided a method of predicting responsiveness to poziotinib alone or in combination with anti-cancer therapy in a subject having cancer, the method comprising detecting a HER2 exon 21 mutation in a genomic sample obtained from the patient, wherein when the sample is positive for the presence of the HER2 exon 21 mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone or in combination with anti-cancer therapy. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain embodiments, the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. In certain embodiments, the HER2 exon 21 mutation comprises one or more point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832-883. In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842, R868, and L869. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some embodiments, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842 and R868. In some embodiments, one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

[0022] In certain embodiments, a favorable responsiveness to a poziotinib inhibitor alone or in combination with an anti-cancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, alleviation of cancer-related conditions, alleviation of cancer-related symptoms, non-progression of cancer, extension of disease-free period, extension of the period to progression, induction of remission, reduction of metastasis, or improvement of patient survival. In further embodiments, patients predicted to have a favorable responsiveness are administered poziotinib alone or in combination with a second anti-cancer therapy.

[0023] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as tablets.

[0024] In some embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5 - 25 mg, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In certain embodiments, poziotinib is administered daily. In some embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

[0025] In some embodiments, the subject has had or shown resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or velatinib.

[0026] [The present invention 1001] A method for treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib, wherein the subject has been determined to have one or more HER2 exon 21 mutations, the method. [The present invention 1002] The method of the present invention 1001, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1003] The method of the present invention 1002, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1004] The method according to any one of the present inventions 1001 to 1003, wherein the one or more HER2 exon 21 mutations include point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832 to 883. [The present invention 1005] The method according to any one of the present inventions 1001 to 1004, wherein the subject has been determined to have two, three, or four HER exon 21 mutations. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, wherein the subject has been previously administered a tyrosine kinase inhibitor. [The present invention 1007] The method of the present invention 1006, wherein the subject is resistant to the previously administered tyrosine kinase inhibitor. [The present invention 1008] The method of the present invention 1007, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or beratinib. [The present invention 1009] The method of the present invention 1004, wherein the one or more HER2 exon 21 mutations are present in one or more residues selected from the group consisting of V842, R868, and L869. [The present invention 1010] The method of the present invention 1004, wherein the one or more HER2 exon 21 mutations are present in residues V842 and / or R868. [The present invention 1011] The method according to any one of the present inventions 1001 to 1010, wherein the subject has been determined not to have an EGFR mutation at residue C797. [The present invention 1012] The method according to any one of the present inventions 1001 to 1011, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. [The present invention 1013] The method according to any one of the present inventions 1001 to 1012, wherein the one or more HER2 exon 21 mutations are V842I and / or R868W. [The present invention 1014] Any of the methods of the present invention 1001 to 1013, wherein it is determined that the subject has a HER2 exon 21 mutation by analyzing a patient-derived genomic sample. [The present invention 1015] The method of the present invention 1015, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1016] Any of the methods of the present invention 1001 to 1015, wherein the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1017] Any of the methods of the present invention 1001 to 1016, wherein the poziotinib is administered orally. [The present invention 1018] Any of the methods of the present invention 1001 to 1017, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1019] Any of the methods of the present invention 1001 to 1018, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1020] Any of the methods of the present invention 1001 to 1019, wherein the poziotinib is administered daily. [The present invention 1021] Any of the methods of the present invention 1001 to 1020, wherein the poziotinib is administered continuously. [The present invention 1022] Any of the methods of the present invention 1001 to 1021, wherein the poziotinib is administered in a 28-day cycle. [The present invention 1023] Any of the methods of the present invention 1001 to 1022, further comprising the step of performing a further anti-cancer therapy. [The present invention 1024] The method of the present invention 1023, wherein the further anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1025] The method of the present invention 1023 or 1024, wherein the administration of the poziotinib and / or the anti-cancer therapy is performed intravenously, subcutaneously, intramedullary, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. [The present invention 1026] Any of the methods of the present invention 1023 to 1025, wherein the administration of the poziotinib and / or the anti-cancer therapy includes local administration, topical administration, or systemic administration. [The present invention 1027] Any of the methods of the present invention 1023 to 1026, wherein the administration of the poziotinib and / or the anti-cancer therapy is performed two or more times. [The present invention 1028] The method according to any one of 1001 to 1027 of the present invention, wherein the cancer is oral cancer, oropharyngeal cancer, hypopharyngeal cancer, respiratory cancer, urogenital cancer, digestive cancer, cancer of the central nervous system tissue or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, hypopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1029] The method according to any one of 1001 to 1028 of the present invention, wherein the cancer is non-small cell lung cancer. [The present invention 1030] The method according to any one of 1001 to 1029 of the present invention, wherein the patient is human. [The present invention 1031] A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more HER exon 21 mutations. [The present invention 1032] The composition of the present invention 1031, further defined as an oral composition. [The present invention 1033] The composition of the present invention 1031 or 1032, comprising 5 to 25 mg of poziotinib. [The present invention 1034] The composition according to any one of 1031 to 1033 of the present invention, comprising 8 mg, 12 mg, or 16 mg of poziotinib. [The present invention 1035] The composition according to any one of 1031 to 1034 of the present invention, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1036] The composition according to any one of 1031 to 1035 of the present invention, formulated as a tablet. [The present invention 1037] The composition according to any one of 1031 to 1036 of the present invention, wherein the one or more HER2 exon 21 mutations comprise point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832 to 883. [The present invention 1038] The composition according to any one of 1031 to 1037 of the present invention, wherein the subject is determined to have two, three, or four HER exon 21 mutations. [The present invention 1039] The composition of the present invention 1037, wherein the one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842, R868, and L869. [The present invention 1040] The method of the present invention 1037, wherein the one or more HER2 exon 21 mutations are present at residue V842 and / or R868. [The present invention 1041] The composition according to any one of the present inventions 1031 to 1040, wherein the subject is determined not to have an EGFR mutation at residue C797. [The present invention 1042] The composition according to any one of the present inventions 1031 to 1041, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. [The present invention 1043] The method according to any one of the present inventions 1031 to 1042, wherein the one or more exon 21 mutations are V842I and / or R868W. [The present invention 1044] The composition according to any one of the present inventions 1031 to 1043, wherein the subject is being treated by an anti-cancer therapy. [The present invention 1045] A method for predicting responsiveness to poziotinib alone or in combination with a second anti-cancer therapy in a subject having cancer, comprising the step of detecting a HER2 exon 21 mutation in a genomic sample obtained from the patient, wherein the patient is predicted to have a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy when the sample is positive for the presence of the HER2 exon 21 mutation. [The present invention 1046] The method of the present invention 1045, wherein the HER exon 21 mutation is further defined as an exon 20 insertion mutation. [The present invention 1047] The method according to the present invention 1045 or 1046, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1048] The method according to any one of the present inventions 1045 to 1047, wherein the presence of the HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1049] The method of the present invention 1048, wherein the HER2 exon 21 mutation comprises point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832 to 883. [The present invention 1050] The method of the present invention 1049, wherein the HER2 exon 21 mutation is present at one or more residues selected from the group consisting of V842, R868, and L869. [The present invention 1051] The method of the present invention 1049, wherein the one or more HER2 exon 21 mutations are present at residue V842 and / or R868. [The present invention 1052] Any of the methods of the present invention 1045-1051, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. [The present invention 1053] Any of the methods of the present invention 1045-1052, wherein a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related pathologies, reduction of cancer-related symptoms, non-progression of cancer, extension of disease-free period, extension of the period until progression, induction of remission, reduction of metastasis, or improvement of patient survival. [The present invention 1054] Any of the methods of the present invention 1045-1053, further comprising the step of administering poziotinib alone or in combination with a second anti-cancer therapy to the patient predicted to have a favorable responsiveness. [The present invention 1055] The method of the present invention 1054, wherein the poziotinib is administered orally. [The present invention 1056] The method of the present invention 1054 or 1055, wherein the poziotinib is administered at a dose of 5-25 mg. [The present invention 1057] Any of the methods of the present invention 1054-1056, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1058] Any of the methods of the present invention 1054-1057, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1059] Any of the methods of the present invention 1054-1058, wherein the poziotinib hydrochloride is formulated as a tablet. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, the detailed description and specific examples, while indicating preferred embodiments of the present invention, are intended for purposes of illustration only and it should be understood that they are not intended to limit the present invention.

Brief Description of the Drawings

[0027] The accompanying drawings, which form a part of this specification, are included to further demonstrate certain specific aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Mode for Carrying Out the Invention

[0028] Description of Exemplary Embodiments This study determined the frequency of genomic variants of HER2 mutations most commonly seen among various malignancies. Systematically, the activation potential was shown for 16 of the most frequent HER2 mutations, and their drug susceptibilities were evaluated with 11 commonly used EGFR and HER2 TKIs. It was found that exon 20 insertion mutations and the p.L755P (not p.L755S) mutation in exon 19 were resistant to many of the tested TKIs. Molecular dynamics modeling of the drug-resistant HER2 variants, L755P and exon 20 insertion, demonstrated that these mutations affected the receptor's conformational state and reduced the overall size of the drug-binding pocket. Furthermore, poziotinib was identified as a potent inhibitor of all evaluated HER2 mutations. Moreover, this study showed that poziotinib has clinical activity in NSCLC patients harboring the most resistant HER2 variants, exon 20 insertion, exon 21 mutations, and L755P. In conclusion, this study showed that poziotinib-mediated cell surface receptor accumulation enhances the activity of T-DM1 that can be utilized to increase antitumor activity in vivo and results in complete tumor regression in PDX models of HER2 mutant NSCLC.

[0029] Accordingly, certain embodiments of the present disclosure provide a method for treating cancer patients having a HER2 exon 21 mutation. Specifically, the method comprises administering poziotinib (also known as HM781-36B) or afatinib to patients identified as having a HER exon 19 point mutation. The size and flexibility of poziotinib overcome steric hindrance and inhibit the HER2 exon 21 variant at low nanomolar concentrations. Thus, poziotinib or afatinib and structurally similar inhibitors are potent HER2 inhibitors that can be used to target HER2 exon 21 insertions that are resistant to second- and third-generation irreversible TKIs.

[0030] I. Definitions As used herein, "a" or "an" can include one or more. When used in the claims of this specification, the terms "a" or "an" can mean one or more when used in conjunction with the term "comprising".

[0031] The present disclosure supports only alternatives as well as definitions that refer to "and / or". However, unless it is clearly indicated that only the alternatives are referred to or that the alternatives are mutually exclusive, the use of the term "or" in the claims is used to mean "and / or". As used herein, "another" can mean at least a second or more.

[0032] The term "about" means ±5% of the indicated value.

[0033] "Treat" or "treating" includes (1) inhibiting a disease in a subject or patient who is experiencing or showing the pathology or general symptoms of the disease (e.g., preventing further progression of the pathology and / or general symptoms), (2) improving a disease in a subject or patient who is experiencing or showing the pathology or general symptoms of the disease (e.g., reversing the pathology and / or general symptoms), and / or (3) acting on any measurable reduction of the disease in a subject or patient who is experiencing or showing the pathology or general symptoms of the disease. For example, treatment can include administration of an effective amount of poziotinib or afatinib.

[0034] "Preventively treat" includes (1) reducing or alleviating the risk of onset of a disease in a subject or patient who has a risk of the disease and / or is likely to be susceptible to the disease but has not yet experienced or shown any or all of the pathology or general symptoms of the disease, and / or (2) delaying the onset of the pathology or general symptoms of the disease in a subject or patient who has a risk of the disease and / or is likely to be susceptible to the disease but has not yet experienced or shown any or all of the pathology or general symptoms of the disease.

[0035] As used herein, the terms "patient" or "subject" refer to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a genetically engineered species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, minors, infants, and fetuses.

[0036] The term "effective," when used in this specification and / or claims, means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that is sufficient to act on the treatment or prevention of the disease when administered to the subject or patient for treating or preventing the disease.

[0037] As used herein, the term "IC 50 " refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measurement indicates how much of a particular drug, or other substance (inhibitor), is required to inhibit a particular biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism) by half.

[0038] An "anticancer" agent can, for example, promote the killing of cancer cells, induce apoptosis of cancer cells, reduce the growth rate of cancer cells, reduce the incidence or number of metastases, shrink the size of tumors, inhibit tumor growth, reduce the blood supply to tumors or cancer cells, promote the immune response against cancer cells or tumors, prevent or inhibit the progression of cancer, or extend the survival period of a subject having cancer, thereby having a negative impact on cancer cells / tumors in the subject.

[0039] The term "insertion" or "insertional mutation" refers to the addition of one or more nucleotide base pairs into a DNA sequence. For example, the HER2 exon 21 insertion mutation contains one or more insertions of 1 to 18 nucleotides at amino acids 832 and 883.

[0040] "Hybridize" or "hybridization" refers to the binding of nucleic acids to each other. The conditions for hybridization can vary depending on the sequence homology of the nucleic acids to be bound. Thus, when the sequence homology between the target nucleic acids is high, stringent conditions are used. When the sequence homology is low, intermediate conditions are used. When the hybridization conditions are stringent, the hybridization specificity increases, and this increase in hybridization specificity reduces the production of non-specific hybridization products. However, under intermediate hybridization conditions, the hybridization specificity decreases, and this decrease in hybridization specificity increases the production of non-specific hybridization products.

[0041] A "probe" refers to a polynucleotide having a length of at least 8 nucleotides and forming a hybrid structure with a target sequence due to the complementarity of at least one sequence in the probe and the sequence in the target region. The polynucleotide can be composed of DNA and / or RNA. The probe is detectably labeled in certain embodiments. The size of the probe can vary significantly. Generally, the length of the probe is, for example, at least 8 to 15 nucleotides in length. The length of other probes is, for example, at least 20, 30, or 40 nucleotides in length. The length of still other probes is somewhat longer and at least, for example, 50, 60, 70, 80, or 90 nucleotides in length. The probe can also be of any specific length within the above range. Preferably, the probe does not contain a sequence complementary to the sequence used to prime the target sequence during polymerase chain reaction.

[0042] "Oligonucleotide" or "polynucleotide" refers to a polymer of single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, which can be unmodified RNA or DNA or modified RNA or DNA.

[0043] "Modified ribonucleotide" or deoxyribonucleotide refers to a molecule that can be used in place of a natural base in a nucleic acid, including but not limited to modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized, and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifying factors, terminal modifying factors, spacer modifying factors, and backbone-modified nucleotides, including but not limited to ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methylphosphonates, methylphosphoramidites, methylphosphonamidites, 5'-β-cyanoethyl phosphoramidites, methylene phosphonates, phosphorodithioates, peptide nucleic acids, optically inactive and neutral internucleotide linkages.

[0044] "Variant" refers to a polynucleotide or polypeptide that is different from the wild-type or the most frequently occurring form in an individual population by one or more nucleotide or amino acid exchanges, deletions, or insertions. The number of nucleotides or amino acids exchanged, deleted, or inserted can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, for example, 25, 30, 35, 40, 45, or 50.

[0045] "Primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer can be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. The primer can contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the primer length are determined empirically. The lower limit of the primer length is the minimum length required to form a stable double-strand upon hybridization to the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3-4 nucleotide lengths) do not form a thermodynamically stable double-strand with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of duplex formation in regions other than the predetermined nucleic acid sequence in the target nucleic acid. Generally, the appropriate primer length is in the range of about 10 to about 40 nucleotide lengths. In certain embodiments, for example, the primer can be 10-40, 15-30, or 10-20 nucleotide lengths. The primer can act as a starting point for synthesis on a polynucleotide sequence when placed under appropriate conditions.

[0046] "Detect", "detectable", and their grammatical equivalents refer to methods for determining the presence, and / or amount, and / or homology of a target nucleic acid sequence. In some embodiments, detection results in amplification of the target nucleic acid sequence. In other embodiments, sequencing of the target nucleic acid can be characterized as "detecting" the target nucleic acid. The label bound to the probe can include any of a variety of labels well known in the art that are detectable, for example, by chemical or physical means. Labels that can be attached to the probe include, for example, fluorescent and luminescent materials.

[0047] "Amplify", "amplification", and their grammatical equivalents refer to any method of replicating at least a portion of a target nucleic acid sequence in a template-dependent manner, including but not limited to a wide range of techniques for amplifying nucleic acid sequences either linearly or exponentially. Exemplary means for performing the amplification step include, but are not limited to, ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR), as well as their multiplex versions or combinations, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as composite chain reaction - CCR), etc. Descriptions of such techniques can be found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[0048] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, organs, and / or body fluids within the scope of sound medical judgment, with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic reaction, or other problems or complications.

[0049] "Pharmaceutically acceptable salts" means salts of the compounds of the present invention that are pharmaceutically acceptable as described above and have the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphor-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that the specific anions or cations forming part of any salt of the present invention are not dangerous as long as the salt as a whole is pharmaceutically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0050] II. HER2 Exon 21 Mutations Certain embodiments of the present disclosure relate to determining whether a subject has one or more HER2 exon 21 mutations, particularly one or more insertion mutations shown in FIG. 2. The subject may have two, three, four, or more HER2 exon 21 mutations. Methods for detecting mutations are known in the art and include PCR analysis and nucleic acid sequencing, as well as FISH and CGH. In certain aspects, exon 21 mutations are detected, for example, by DNA sequencing from, e.g., tumors or plasma-derived circulating free DNA.

[0051] HER2 exon 21 mutations may include one or more point mutations, insertions, and / or deletions of 1 to 18 nucleotides at amino acids 832 - 883. In some aspects, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842, R868, and L869. In some aspects, one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some aspects, one or more HER2 exon 21 mutations are present at one or more residues selected from the group consisting of V842 and R868. In some aspects, one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

[0052] In some embodiments, the subject may have or develop a mutation at EGFR residue C797 that confers resistance to a TKI, such as poziotinib. Thus, in certain embodiments, the subject is determined not to have a mutation at EGFR C797 and / or T790, such as C797S and / or T790M. In some embodiments, a subject having a T790 mutation, such as T790M, may be administered osimertinib, and a subject having a C797 mutation, such as C797S, may be administered chemotherapy and / or radiation therapy.

[0053] The patient sample can be any bodily tissue or fluid that contains nucleic acids derived from lung cancer in the subject. In certain embodiments, the sample is a blood sample that contains circulating tumor cells or cell-free DNA. In other embodiments, the sample can be a tissue, such as lung tissue. The lung tissue can be derived from tumor tissue and can be fresh frozen or formalin-fixed and paraffin-embedded (FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

[0054] Samples suitable for use in the methods described herein include genetic material, such as genomic DNA (gDNA). Genomic DNA is typically extracted from biological samples such as blood or buccal mucosal scrapings, but can also be extracted from other biological samples including urine, tumors, or sputum. The sample itself typically comprises nucleated cells (e.g., blood or buccal cells) or tissue removed from the subject, including normal or tumor tissue. Methods and reagents for obtaining, processing, and analyzing samples are well known in the art. In some embodiments, the sample is obtained with the cooperation of a medical institution, such as by collecting blood. In some embodiments, the sample is obtained without the cooperation of a medical institution, such as the sample is obtained non-invasively, such as a sample containing buccal cells obtained using a buccal swab or brush, or a mouthwash sample.

[0055] In some cases, biological samples can be processed for DNA isolation. For example, DNA in a cell or tissue sample can be separated from other components of the sample. Cells can be recovered from biological samples using techniques well known in the art. For example, cells can be recovered by centrifuging a cell sample and resuspending the pelleted cells. The cells can be resuspended in a buffer such as phosphate buffered saline (PBS). After centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, such as gDNA. See, for example, Ausubel et al. (2003). Samples can be concentrated and / or purified to isolate DNA. All samples obtained from a subject, including those subjected to any kind of further processing, are considered to be those obtained from the subject. For example, genomic DNA can be extracted from biological samples using conventional methods, including phenol extraction. Alternatively, genomic DNA can be extracted using kits such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.), and the Wizard® Genomic DNA Purification Kit (Promega). Non-limiting examples of sample sources include urine, blood, and tissue.

[0056] The presence or absence of a HER2 exon 21 mutation as described herein can be determined using methods well known in the art. For example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and / or arrays can be used to detect the presence or absence of an insertion mutation. If desired, amplification of nucleic acids can be accomplished using methods well known in the art, such as PCR. In one example, a sample (e.g., a sample containing genomic DNA) is obtained from a subject. The DNA in the sample is then tested to determine the characteristics of the insertion mutation as described herein. The insertion mutation can be detected by any method described herein, such as by sequencing or by hybridization to a nucleic acid probe of a gene in genomic DNA, RNA, or cDNA, e.g., a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to hybridize specifically or preferentially to a particular variant.

[0057] A set of probes typically refers to a set of primers (usually primer pairs) and / or detectably labeled probes used to detect target gene mutations (e.g., HER2 exon 21 mutations) used in the recommended treatments feasible with the present disclosure. The primer pairs are used in an amplification reaction to define amplicons spanning regions for the target gene mutations for each of the said genes. The set of amplicons is detected by a set of matching probes. In an exemplary embodiment, the method can use a TaqMan (trademark) (Roche Molecular Systems, Pleasanton, Calif.) assay to detect a set of target gene mutations, e.g., HER2 exon 21 mutations. In one embodiment, the set of probes is a set of primers used to produce amplicons detected by a nucleic acid sequencing reaction such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ (trademark) (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ (trademark) (Illumina, San Diego, Calif.) technologies can be used.

[0058] Analysis of nucleic acid markers can be carried out using techniques well known in the art, including but not limited to sequence analysis and electrophoretic analysis. Non-limiting examples of sequence analysis include sequencing with mass spectrometry such as Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., 1992), solid-phase sequencing (Zimmerman et al., 1992), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., 1998), as well as sequencing by hybridization (Chee et al., 1996; Drmanac et al., 1993; Drmanac et al., 1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Furthermore, next-generation sequencing methods can be carried out using commercially available kits and apparatuses from companies such as the Life Technologies / Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche / 454 next-generation sequencing system.

[0059] Other methods of nucleic acid analysis include direct manual sequencing (Church and Gilbert, 1988; Sanger et al., 1977; U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-strand conformation polymorphism assay (SSCP) (Schafer et al., 1995); clamped denaturing gel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformation-sensitive gel electrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield et al., 1989); denaturing high performance liquid chromatography (DHPLC, Underhill et al., 1997) infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry (WO99 / 57318); mobility shift analysis (Orita et al., 1989); restriction enzyme analysis (Flavell et al., 1978; Geever et al., 1981); quantitative real-time PCR (Raca et al., 2004); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., 1985); RNase protection assay (Myers et al., 1985); use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR, and may include combinations of such methods. See, for example, U.S. Patent Application Publication No. 2004 / 0014095. This patent is hereby incorporated by reference in its entirety.

[0060] In one example, a method of identifying HER2 mutations in a sample comprises contacting nucleic acid derived from such sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding a mutated HER2 protein, or a fragment thereof that contains the mutation, and detecting such hybridization. In a particular embodiment, such probe is, for example, a radioisotope ( 3 H, 32 P, or 33P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent, and is detectably labeled. In certain embodiments, the probe is an antisense oligomer such as PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be from about 8 nucleotides to about 100 nucleotides, or from about 10 to about 75, or from about 15 to about 50, or from about 20 to about 30. In another aspect, such a probe of the present disclosure is provided in a kit for identifying HER2 mutations in a sample, and such a kit contains oligonucleotides that specifically hybridize to or are adjacent to the mutation sites in the HER2 gene. The kit further contains instructions for treating a patient having a tumor containing a HER2 insertion mutation with poziotinib or afatinib based on the results of a hybridization assay using the kit.

[0061] In another aspect, a method for detecting an exon 21 mutation in a sample includes amplifying such a nucleic acid sample corresponding to exon 21 of the HER2 gene or a fragment thereof that is considered to contain the mutation, and comparing the electrophoretic mobility of the amplified nucleic acid with the electrophoretic mobility of the corresponding wild-type HER2 gene or a fragment thereof. A difference in mobility indicates the presence of a mutation in the amplified nucleic acid sequence. The electrophoretic mobility can be measured on a polyacrylamide gel.

[0062] Alternatively, nucleic acids can be analyzed for the detection of mutations using Enzymatic Mutation Detection (EMD) (Del Tito et al., 1998). EMD uses bacteriophage resolvase T4 endonuclease VII, which scans along double-stranded DNA until it detects and resolves the structural distortions caused by base pair mismatches due to point mutations, insertions, and deletions. Detection of two short fragments formed, for example, by gel electrophoresis upon resolvase cleavage indicates the presence of a mutation. The advantage of the EMD method is that it is assayed directly from the PCR reaction, eliminating the need for sample purification, shortening hybridization times, increasing the signal-to-noise ratio, and is a single protocol for identifying point mutations, deletions, and insertions. Mixed samples containing up to 20-fold overexpression of normal DNA and fragments up to 4 kb in size can be assayed. However, EMD scanning does not identify the specific base changes that occur in mutation-positive samples and, if necessary, further sequencing procedures are required to identify the mutations. As demonstrated in U.S. Patent No. 5,869,245, CEL I enzyme can be used in a similar manner to resolvase T4 endonuclease VII.

[0063] III. Treatment Methods Also provided herein is a method for treating or delaying the progression of cancer in an individual, the method comprising administering to the individual an effective amount of poziotinib, afatinib, or a structurally similar inhibitor to a subject determined to have a HER2 exon 21 mutation, such as an exon 21 insertion. The subject may have one or more HER exon 21 mutations.

[0064] Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-neoplastic lesions of the lung, colon cancer, melanoma, and bladder cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0065] In some embodiments, the subject is a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient having or at risk of having the disorders described herein). In one embodiment, the subject is in need of enhancing an immune response. In certain embodiments, the subject is susceptible or at risk of being susceptible. For example, the subject has received or is receiving chemotherapy and / or radiation therapy. Alternatively, or in combination, the subject is susceptible or at risk of being susceptible as a result of an infection.

[0066] Certain embodiments relate to the administration of poziotinib (also known as HM781-36B, HM781-36, and 1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]prop-2-en-1-one) to a subject determined to have a HER2 exon 21 mutation. Poziotinib is a quinazoline-based pan-HER inhibitor that irreversibly blocks signaling through the HER family of tyrosine kinase receptors, including HER1, HER2, and HER4. Poziotinib or structurally similar compounds (e.g., U.S. Patent No. 8,188,102 and U.S. Patent Application Publication No. 2013 / 0071452; incorporated herein by reference) may be used in the methods of the invention.

[0067] Poziotinib, poziotinib hydrochloride, may be administered orally, for example, in tablets. Poziotinib may be administered at a dose of 4-25 mg, for example, at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg. Administration may be daily, every other day, every three days, or once a week. Administration may be on a continuous schedule, for example, in a 28-day cycle.

[0068] In some embodiments, a subject having a T790 mutation, such as T790M, may be administered osimertinib, and a subject having a C797 mutation, such as C797S, may be administered chemotherapy and / or radiotherapy as described herein. Osimertinib administration, chemotherapy, and / or radiotherapy may be performed alone or in combination with poziotinib. Osimertinib may be administered at a dose of 25 to 100 mg, such as about 40 or 80 mg. The administration may be daily, every other day, every two days, every three days, or once a week. Osimertinib may be administered orally, for example, in tablet form.

[0069] Afatinib may be administered at a dose of 10 to 50 mg, such as 10, 20, 30, 40, or 50 mg. Afatinib may be administered daily, every other day, every two days, every three days, or weekly. Afatinib may be administered orally, for example, in tablet form.

[0070] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising poziotinib or afatinib and one or more pharmaceutically acceptable carriers for a subject determined to have a HER2 exon 21 mutation, such as an exon 21 mutation.

[0071] The pharmaceutical compositions and formulations described herein comprise an active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity in one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington’s Pharmaceutical Sciences 22 ndIt can be prepared by mixing with (edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto. In the present specification, an exemplary pharmaceutically acceptable carrier further includes an interstitial drug dispersant such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use including rHuPH20 are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glucosaminoglycanases such as chondroitinase.

[0072] B. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment include poziotinib or afatinib in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0073] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or a metastasis suppressor. In some embodiments, the additional therapy is the administration of an agent that reduces side effects (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an antiemetic). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents well known in the art.

[0074] Ponatinib or afatinib can be administered before, during, after, or in various combinations with additional cancer therapies such as immune checkpoint therapies. Administration can range from simultaneous administration to intervals of minutes, days, or weeks. In embodiments where ponatinib or afatinib is provided to the patient separately from the additional therapeutic agent, generally, a significant period does not elapse between each delivery time to ensure that the two compounds can still provide an advantageous combined effect to the patient. In such cases, it is contemplated that antibody therapy and anti-cancer therapy may be provided to the patient within about 12 - 24 or 72 hours of each other, and more specifically, within about 6 - 12 hours of each other. In some situations, it may be desirable to space the intervals between each administration by several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) to significantly extend the treatment period.

[0075] Various combinations can be used. In the following examples, ponatinib or afatinib is "A" and anti-cancer therapy is "B". TIFF0007700043000001.tif18128

[0076] Administration of any compound or implementation of a therapy for the patient in this embodiment follows the general protocol for administration of such a compound, taking into account the toxicity of the drug if present. Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy.

[0077] 1. Chemotherapy According to this embodiment, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to denote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at which stage of the cell cycle they act. Alternatively, the agents may be characterized based on their ability to directly crosslink to DNA, to intervene in DNA, or to induce chromosomal and mitotic abnormalities by acting on nucleic acid synthesis.

[0078] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially, bullatacin and bullatacinone); camptothecin (including synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its azozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin gammaII and calicheamicin omegaI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotic chromophores, actinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, pteropterin, and trimethoprim; purine analogs, for example, fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, and floxuridine; androgens, for example, calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal, for example, mitotane and trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;Roxanthrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lysoxine; sizofiran; spirigermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecene (especially, T-2 toxin, verracurin A, lolitrem A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoid, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complex, for example, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS2000; difluorodifluoromethylornithine (DMFO); retinoid, for example, retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabiene, navelbine, farnesyl-protein transferase inhibitor, transplatin, and any pharmaceutically acceptable salt, acid, or derivative of the above are included.;

[0079] 2. Radiation therapy Other factors that cause DNA damage and are widely used include those known as gamma rays, X-rays, and / or the targeted delivery of radioisotopes to tumor cells. Other forms of DNA-damaging agents are contemplated, such as electron ranges, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents are likely to affect a wide range of damage to DNA, to precursors of DNA, to DNA replication and repair, and to the assembly and maintenance of chromosomes. The dose range for X-rays spans from a daily dose of 50-200 roentgens for a long period (3-4 weeks) to a single dose of 2000-6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by neoplastic cells.

[0080] 3. Immunotherapy One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the present embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone can be used as an effector of the therapy, or the antibody may recruit other cells that actually act in cell killing. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a molecular targeting agent. Alternatively, the effector can be a lymphocyte having surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0081] Antibody-drug conjugates have emerged as an epoch-making approach in cancer therapy development. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to cell-killing drugs. This approach combines the high specificity of the Mab for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells along with abundant levels of antigen. The targeted delivery of the drug also minimizes drug exposure in normal tissues, leading to reduced toxicity and improved therapeutic indices. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, has validated this approach. Currently, more than 30 ADC drug candidates are at various stages of clinical trials for cancer therapy (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the drug discovery and development of new ADCs rely heavily on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high-level expression in tumor cells and robust internalization.

[0082] In one aspect of immunotherapy, tumor cells need to have several markers that are suitable for targeting (i.e., not present in most other cells). There are many tumor markers, and any of these may be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect and an immune-stimulating effect. There are also immune-stimulating molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0083] Examples of immunotherapy include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in combination with the antibody therapy described herein.

[0084] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance or attenuate signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target PD-1 family and / or CTLA-4.

[0085] The immune checkpoint inhibitor can be a drug, e.g., a small molecule, a recombinant form of a ligand or receptor, or in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both documents are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, in particular, chimeric, humanized, or human forms of antibodies may be used. Those skilled in the art will appreciate that alternative and / or equivalent names may be used for the specific antibodies referred to in the present disclosure. Such alternative and / or equivalent names are interchangeable in the context of this specification. For example, pembrolizumab is known to be also known as MK-3475 and lambrolizumab as alternative and equivalent names.

[0086] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 system antagonists for use in the methods provided herein are well known in the art and are described, for example, in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0087] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0088] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of inhibitory receptors for CTLA-4 and B7 molecules.

[0089] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0090] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, U.S. Patent No. 8,119,129; International Publication Nos. WO01 / 14424, WO98 / 42752, and WO00 / 37504 (CP675,206, tremelimumab; also known as previous ticilimumab); U.S. Patent No. 6,207,156; Hurwitz et al., 1998; Camacho et al., 2004; and anti-CTLA-4 antibodies disclosed in Mokyr et al., 1998 can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with these antibodies recognized in the art for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. 2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0091] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence homology with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region homology with ipilimumab).

[0092] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995 / 001994 and WO1998 / 042752; all incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867, incorporated herein by reference.

[0093] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery, including prophylactic, diagnostic or staging, therapeutic, and palliative surgery. Therapeutic surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can also be combined with other therapies, such as the therapy of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrocautery, and microsurgery (Mohs surgery).

[0094] When all or part of a cancer cell, tissue, or tumor is resected, a cavity may be formed in the body. Treatment can be accomplished by perfusion, direct injection, or topical application of the area for further anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages.

[0095] 5. Other Agents It is contemplated that other agents may be used in combination with specific aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that act on the upregulation of cell surface receptors and GAP junctions, cell growth inhibitors and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intracellular signaling by increasing the number of GAP junctions can increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell growth inhibitors or differentiating agents can be used in combination with specific aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effects of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with specific aspects of the present embodiment to improve the therapeutic effect.

[0096] IV. Kit Kits for detecting HER2 exon 21 mutations, such as those disclosed herein, are also within the scope of the present disclosure. An example of such a kit may include a set of primers specific for the exon 21 mutation. The kit may further include instructions for using the primers to detect the presence or absence of specific HER2 exon 21 mutations described herein. The kit may further include instructions for diagnosis indicating that identification of a positive for the HER2 exon 21 mutations described herein in a sample from a cancer patient is an indicator of sensitivity to the tyrosine kinase inhibitor poziotinib or afatinib or structurally similar inhibitors. The kit may further include instructions indicating that identification of a positive for the HER2 exon 21 mutations described herein in a sample from a cancer patient indicates that the patient should be treated with poziotinib, afatinib, or a structurally similar inhibitor.

Examples

[0097] V. Examples The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples are those that the inventors have found to function well in the practice of the present invention and can, therefore, be considered to constitute a preferred mode for its implementation. However, those skilled in the art should understand that, in light of the present disclosure, numerous changes can be made to the specific embodiments disclosed herein and that the same or similar results can still be obtained without departing from the spirit and scope of the present invention.

[0098] Example 1 - Identification of Drugs against Cancer Cells with HER2 Exon 21 Mutations HER2 mutations occur most frequently in cancers of the bladder, stomach, and bile duct: To understand the diversity of HER2 mutations among cancer types, multiple databases were queried, including cohorts from cBioPortal, MD Anderson Cancer Center, and Foundation Medicine, as well as the cfDNA cohort from Guardant Health. Across all databases, all non-synonymous HER2 mutations were analyzed within the range of 25 different cancer types (Table 2). The weighted average frequency for HER2 mutations was calculated. Similar to what was observed in the AACR GENIE database (Meric-Bernstam et al, 2018), HER2 mutations occurred most frequently in cancers of the bladder (8.3%), bile duct (5.3%), and stomach (4.5%) (Figure 1A); and HER2 exon 20 mutations occurred most frequently in cancers of the small intestine (1.8%), lung (1.5%), and breast (0.9%) (Figure 1B).

[0099] HER2 mutations occur most frequently in the tyrosine kinase domain of HER2, and the mutation hotspots vary by malignancy: Next, the frequencies of mutations were analyzed within the ranges of different regions of the HER2 receptor reported in cBioPortal and MD Anderson. Across all cancer types, HER2 mutations occurred most frequently in the tyrosine kinase domain (46%), which included mutations in exon 20 (20%), exon 19 (11%), and exon 21 (9%) (Figure 2A). In addition, extracellular domain mutations consisted of 37% of HER2 mutations. Across all cancers queried, the most frequently observed HER2 mutations were p.S310F / Y (11.0%), p.Y772_A775dupYVMA (5.7%), p.L755P / S (4.6%), p.V842I (4.4%), and p.V777L / M (4.0%) (Figure 2E). In lung cancer, most HER2 mutations occurred within exon 20 (48%), and Y772_A775dupYVMA accounted for 34% of all HER2 mutations (Figures 2B, 2F). In breast cancer, most HER2 mutations occurred within exon 19 (37%), and the L755 mutation had the highest prevalence at 22% of HER2 mutations (Figure 2C). On the other hand, unlike lung cancer where one variant was dominant, there was more mutational diversity within exon 19 mutations in breast cancer (Figure 2G). In colorectal cancer, HER2 mutations occurred most frequently in exon 21 (23%) and the extracellular domain (23%), and the V842I variant in exon 21 had the highest prevalence (19%) (Figures 2D, 2H).

[0100] Y772dupYVMA is the most frequently observed HER2 exon 20 insertion mutation across all cancer types: HER2 exon 20 mutations are the most frequent mutations within the tyrosine kinase domain of HER2 (16% of all HER2 mutations and 43% of tyrosine kinase domain mutations), and HER2 exon 20 insertion mutations remain a clinical challenge. To understand the diversity and prevalence of exon 20 insertions, the frequency of HER2 exon 20 insertion sequences was analyzed by cancer type in the cBioportal database, MD Anderson database, and Guardant Health database. The Y772dupYVMA insertion is the most frequently observed HER2 exon 20 insertion, accounting for 70% of all HER2 exon 20 insertions. The p.G778dupGSP (14%) and p.G776del insVC (9%) insertions occurred with the second and third highest frequencies (Figure 9A). Exon 20 insertion mutations in NSCLC (N = 362) showed the greatest diversity of exon 20 insertion mutations (Figure 9B), while exon 20 insertion mutations in breast cancer (N = 30) showed little diversity in the insertion sequences, with only three different variants reported (Figure 9C). In other cancer types, additional insertion mutations were rarely observed, and duplications at Y772 and G778 occurred with the highest frequencies in all cancer types analyzed (Figure 9D).

[0101] Frequently detected HER2 changes are activating mutations: To evaluate the functional impact of frequently observed HER2 mutations, 16 HER2 mutations most frequently detected between exons 19, 20, and 21 were stably expressed in Ba / F3 cells. All 16 HER2 mutations tested were found to induce IL-3-independent survival of Ba / F3 cells (Figures 3A - C). Furthermore, the expression of these 16 HER2 mutations resulted in the expression of phosphorylated HER2 (Figure 10A), indicating that these mutations lead to receptor activation.

[0102] Poziotinib is the most potent TKI tested and inhibited the most frequently seen HER2 mutations in vitro: Recent reports have highlighted the efficacy of covalent quinazoline amine-based TKIs (i.e., afatinib, dacomitinib, poziotinib, neratinib) in preclinical models of HER2 mutant disease, but clinical trials of afatinib, dacomitinib, and neratinib have low ORRs and cancer-specific and variant-specific differences in patient outcomes. To systematically evaluate drug sensitivity among the most frequently detected HER2 variants, a panel of HER2 variant Ba / F3 cells was screened against 11 covalent and noncovalent EGFR and HER2 TKIs. HER2 variants showed strong resistance to the noncovalent inhibitors, lapatinib and sapatinib (Figure 4A). The covalent TKIs osimertinib, ibrutinib, and nazartinib were not effective in suppressing cell viability in cells expressing exon 20 mutations; however, these TKIs were active against cells expressing the D769 variant (Figure 4A). In comparison, the covalent quinazoline amine-based TKIs, afatinib, neratinib, dacomitinib, talroxitinib-TKI, and poziotinib had inhibitory activity against HER2 variants across all three exons (Figure 4A). Among all HER2 mutant variants and TKIs tested, poziotinib had the lowest average IC 50 and was significantly more effective than afatinib, neratinib, or talroxitinib-TKI in reducing cell viability (Figure 4B). In addition, poziotinib was significantly more effective than any of afatinib, neratinib, or talroxitinib-TKI against HER2 exon 19 and 20 mutations, but the average IC against exon 21 variants 50There was no significant difference (Figs. 4C - E), suggesting that the mutation positions affected drug binding. Furthermore, the L755S and L755P variants within exon 19 had significant differences in drug sensitivity across all the TKIs tested (Fig. 4F), indicating that specific amino acid changes at this site affected drug binding affinity.

[0103] Positions and amino acid changes of HER2 mutations affect drug binding affinity: To further understand how the positions and amino acid changes of mutations can affect drug binding affinity and inhibitory effects, molecular dynamics simulations were used to examine how these mutations affect the structure and dynamics of the HER2 kinase domain. Molecular models of the L755S, L755P, Y772dupYVMA, and V777L HER2 variants (Fig. 11A) were constructed using the publicly available X-ray structure (PDB 3PP0) as a template and subjected to accelerated molecular dynamics to increase protein conformational sampling. The range of sampled protein conformations, especially regarding the P-loop and α-C-helix positions, differed among these HER2 variants. Even among exon 20 mutations, differences were evident, especially in the α-C-helix region, where the persistence of the α-C-helix conformation differed between "in" (active conformation with a smaller binding pocket) and "out" (inactive conformation with a larger binding pocket). The V777L variant sampled a large number of "out" conformations, while the Y772dupYVMA variant sampled both "in" and "out" conformations (Fig. 5A). Overall, these conformational state differences resulted in the Y772dupYVMA variant being present in the "in" conformation (Fig. 5B) at a frequency more than 10-fold higher than the V777L variant and, on average, a smaller binding pocket in Y772dupYVMA compared to V777L (Figs. 5C and 11B). In addition, since neratinib contains a pyridyl ring directed towards the α-C-helix, the smaller binding pocket of Y772dupYVMA may be responsible for weakening the efficacy of neratinib against Y772dupYVMA compared to V777L.

[0104] Further analysis of the HER2 variant binding pocket volume (Figure 10B) demonstrated that mutations at the same residues can have dramatically different effects on the protein's three-dimensional structure. Specifically, the proline residue of the L755P mutation lacks a hydrogen bond donor and disrupts the backbone hydrogen bonds between the β3 and β5 strands between L755 and V790, respectively. The lack of stabilization between these two β strands led to the destabilization of the β-sheet and structural rearrangement in the kinase hinge region (Figure 5D). Specifically, the L800 residue of L755P protruded into the active site, significantly reducing the pocket size. The change in the β3 strand structure also caused the P-loop to collapse inward, further reducing the pocket volume and the sensitivity of this variant to many TKIs. Additionally, the change in hinge mobility may also play a role in kinase activation. These obvious changes in the L755P variant structure are in contrast to the behavior of the L755S variant and have a three-dimensional structure and pocket volume profile more similar to wild-type HER2 (Figure 11B).

[0105] HER2 variant human cancer cell lines showed enhanced sensitivity to poziotinib: Clinical studies testing HER2 inhibitors have revealed cancer type-specific differences in drug sensitivity (Hyman et al, 2018). To determine whether covalent quinazoline amine-based TKIs have activity in models of HER2 variant diseases, a panel of EGFR / HER2 TKIs was tested in human cancer cell lines. HER2 exon 20 mutations were transfected into pre-tumorigenic MCF10A mammary epithelial cells, and the sensitivity to 12 EGFR / HER2 TKIs was evaluated in vitro. MCF10A cells expressing the G776del insVC, Y772dupYVMA, or G778dupGSP HER2 mutations were the most sensitive to poziotinib, with IC 50 values of 12 nM, 8.3 nM, and 4.5 nM, respectively (Figures 6A - C). In comparison, talroxitinib-TKI and neratinib had average IC 50Values were generated (Figures 6A - C), and poziotinib was shown to be more than 2.6 - fold and 19 - fold more potent than tarlotrectinib - TKI and neratinib, respectively (p < 0.001). Further, Western blotting of MCF10A HER2 G776delinsVC cells with poziotinib and neratinib showed that poziotinib completely suppressed p - HER2 at 10 nM, while neratinib did not (Figure 12A). Since wild - type (WT) HER2 does not transform Ba / F3 cells to grow independently of IL - 3, MCF10A cells were used to determine the selectivity of TKIs for mutant HER2 compared to WT HER2. For this purpose, the selectivity index (SI, IC 50 value mutant / IC 50 value WT) was calculated for each inhibitor, and it was found that poziotinib was the most mutant - selective TKI tested in the MCF10A cell line (SI = 0.028), followed by pyrotinib (SI = 0.063) and tarlotrectinib - TKI (SI = 0.111) (Figure 6D). Consistent with the data obtained using Ba / F3 cells (Figure 3C), in a model of HER2 exon 19 mutant colorectal cancer (CW - 2), the differences in sensitivity among poziotinib, tarlotrectinib - TKI, and neratinib were not as dramatic but were significant (p = 0.02 and p = 0.0004), with mean IC 50 values of 3.19 nM, 4.24 nM, and 68.8 nM, respectively (Figure 6E). Further, in a xenograft mouse model of CW - 2 colorectal cells, on day 21, animals treated with poziotinib (5 mg / kg) showed a 58% reduction in tumor volume compared to the vehicle - treated group (p = 0.011). In comparison, animals treated with neratinib (30 mg / kg) showed an increased tumor volume (28%) compared to the vehicle control (p = 0.023), and afatinib (20 mg / kg) treatment had no significant effect on tumor growth compared to the vehicle control (Figures 6F, 13).

[0106] Poziotinib has antitumor activity in NSCLC patients with HER2 mutations: Based on these preclinical data and previously published studies on exon 20 mutations (Robichaux et al, 2018), a physician-led phase II clinical trial of poziotinib (NCT03066206) in EGFR and HER2 exon 20 mutant NSCLC was initiated. Patients were treated with 16 mg of poziotinib orally daily until progression, death, or withdrawal. Objective responses were evaluated every 8 weeks based on RECIST v1.1. Of the first 12 evaluable patients with HER2 exon 20 insertion mutations, 6 / 12 (50%) patients had a best response of partial response (PR). This response was confirmed in 5 / 12 by serial scans after 2 months (confirmed objective response rate, 42%) (Figure 7A). Of these 12 patients, 2 patients had progression (PD) at the time of the first efficacy determination, and an 83% disease control rate (DCR) was obtained. As of December 2018, 10 of the 12 patients were continuing, and the median PFS of the first 12 patients was 5.6 months (Figure 7B). To date, all patients included in this trial harbored one of the two most common HER2 exon 20 insertions, Y772dupYVMA and G778dupGSP (Figure 7A). Representative images before and after (8 weeks) treatment of one NSCLC patient with the Y772dupYVMA mutation showed a marked reduction in tumor in the right lung (Figure 7C). Patient characteristics, including the number of prior treatment histories, are shown in Table 3. In addition, one heavily pretreated NSCLC patient with the HER2 exon 19 point mutation, L755, was treated under a compassionate use protocol (C-IND18-0014). The patient was treated with 16 mg of poziotinib daily and had tumor shrinkage at 4 weeks (Figure 7D, white frame). The patient had stable disease (SD) (-12% reduction in target lesions) according to RECIST v1.1. The patient had disease control and continued on poziotinib for over 7 months until obvious disease progression was imaged and poziotinib was discontinued. The patient was clinically well at the end of poziotinib treatment and subsequently received further systemic treatment.

[0107] The combination of poziotinib and T-DM1 treatment enhances antitumor activity: Previous studies of the HER2 TKI lapatinib in HER2-positive breast cancer models and EGFR inhibitors in EGFR mutant NSCLC models have shown that TKI treatment results in increased receptor accumulation on the cell surface and that increased cell surface HER2 / EGFR increases sensitivity to antibody-dependent cell cytotoxicity (ADCC). To determine whether poziotinib treatment increases total HER2 receptor expression on the cell surface, cell surface HER2 expression was analyzed by FACS 24 hours after low-dose poziotinib treatment. On average, poziotinib treatment was found to increase cell surface HER2 expression two-fold (Figure 8A, p<0.0001). Next, it was tested whether the combination of poziotinib and T-DM1 could reduce cell viability in vitro. T-DM1 alone did not suppress the viability of MCF10A HER2 mutant cell lines, but the combination of T-DM1 and poziotinib was significantly lower than either agent alone in a dose-dependent manner for the IC 50It was found to have provided a value (Figure 8B). To verify these findings in vivo, the combination of low-dose poziotinib and a single dose of T-DM1 was tested in a HER2 mutant NSCLC PDX model, HER2 Y772dupYVMA (Figure 8C). To evaluate the response to treatment, the progression-free survival (PFS), defined as the time from the best effect to tumor doubling, was determined. Mice receiving the vehicle control had a median PFS (mPFS) of 3 days, whereas mice receiving low-dose poziotinib or T-DM1 had mPFSs of 15 days and 27 days, respectively. However, mice (14 / 20) receiving a single dose of T-DM1 in combination with low-dose poziotinib remained tumor-free on day 45 (Figure 8D). Furthermore, at the time of the best effect, day 15, the combination of low-dose poziotinib (2.5 mg / kg) and a single dose of T-DM1 (10 mg / kg) resulted in complete tumor regression in 20 / 20 mice (100%), compared with 2 / 9 mice receiving T-DM1 alone or 0 / 12 mice receiving low-dose poziotinib (Figures 8C–F). By day 30, tumor growth had started in all mice receiving T-DM1 alone; however, in 14 / 20 mice receiving the combination treatment, there was no evidence of tumor recurrence (Figures 8F, G).

[0108] It is reported herein that HER2 mutations occur in various tumor types, but the specific mutation hotspots differ for each malignancy. Furthermore, the sensitivity to HER2 TKIs is heterogeneous among mutation positions, and HER2 exon 20 insertions and L755P mutations are resistant to most HER2 TKIs, probably due to a reduction in the volume of the drug-binding pocket. Furthermore, poziotinib has been identified as a potent pan-HER2 variant-selective inhibitor with clinical efficacy in NSCLC patients with HER2 exon 20 insertions and L755P mutations. Finally, it was established that poziotinib treatment induces the accumulation of HER2 on the cell surface and that the combination of poziotinib and T-DM1 treatment enhances antitumor activity in vitro and in vivo.

[0109] Pan-cancer analysis indicates that HER2 mutation hotspots vary across cancer types and have different sensitivities to HER2 TKIs in vitro, which may affect clinical efficacy. In the SUMMIT trial, neratinib produced the highest efficacy in breast cancer patients, and the majority of responders were positive for the L755S, V777L, or L869R mutations. In in vitro Ba / F3 drug screening, these mutations correlated with low IC 50 values. In contrast, patients with colorectal cancer did not respond to neratinib. Consistent with this clinical observation, the V842I mutation is the most frequently observed HER2 mutation in colorectal cancer cases, and this specific mutation was found to be insensitive to neratinib in drug screening assays. These data suggest that differential TKI sensitivity among malignancies is, in part, explained by cancer-specific mutation hotspots and may directly affect drug sensitivity. However, significant questions remain regarding the reasons for the different distributions of HER2 mutations across tumor types and whether certain mutations produce similar drug responses in different tumor types. Data from the SUMMIT trial indicate that specific exon 20 insertions were associated with neratinib sensitivity in breast cancer patients, but these same mutations were associated with resistance in all other cancer types, indicating that potential mechanisms underlying these tumor type-specific differences in sensitivity are worthy of further investigation.

[0110] Exon 20 insertion mutations and exon 19 L755P mutations are resistant to most HER2 TKIs. In vitro drug screening shows that exon 20 insertion mutations and L755P mutations have the highest IC 50It has been shown to have a value. Molecular dynamics simulations have revealed that these mutations induce conformational changes that affect the overall size and mobility of the drug-binding pocket. Collectively, these in vitro and in silico findings are consistent with the clinical observation that patients with HER2 exon 20 insertion mutations have historically had poor responses to TKIs. In lung cancer where exon 20 insertions occur at a high frequency, patients harboring the HER2 exon 20 insertion mutation had response rates of 0%, 11.5%, and 18.2% - 18.8% to neratinib, dacomitinib, and afatinib, respectively. Furthermore, while the L755S mutation has been shown to respond to neratinib, the L755P mutation has strong resistance to both TKIs and antibody-drug conjugates.

[0111] Example 2 - Materials and Methods Analysis of HER2 Mutation Retention Rate and Variant Frequency: To determine the frequency of each HER2 mutation reported in the databases from MD Anderson Cancer Center, cBioPortal, Foundation Medicine, or Guardant Health, each database was queried individually, and then the frequencies were weighted by the total number of patients in each database and reported as a weighted average. To determine the frequency of HER2 mutations among cancer types in cBioPortal, all non-overlapping tests were selected and exported. For overlapping tests, only the largest dataset was used. To determine the HER2 mutation frequency at MD Anderson Cancer Center, the Institute for Personalized Cancer Therapy database was queried for all HER2 mutations regardless of cancer type. To determine the frequency of HER2 exon 20 mutations from Foundation Medicine, unspecified data on the number of patients with HER2 deletions, frameshifts, insertions, and point mutations were tabulated, and cancer types with less than five mutations were excluded. Finally, to determine the frequency of HER2 exon 20 mutations at Guardant Health, the Guardant360 clinical database was queried for samples (70- to 73-gene panels) tested between October 2015 and May 2018 with ERBB2 exon 20 mutations. Guardant360® is a CLIA-certified CAP / NYSDOH-approved comprehensive cfDNA NGS test that reports SNVs, indels, fusions, and SNVs in up to 73 genes. The frequencies reported from Guardant Health were then normalized and clinically sensitive corrected as reported by Odegaard et al 2018. Specifically, the frequencies were divided by the percent clinical sensitivity, 85.9%.

[0112] Generation of Ba / F3 cell line and IL-3 deficiency: The Ba / F3 cell line was established as previously described (Robichaux et al, 2018). Briefly, stable Ba / F3 cell lines were generated by retroviral transduction of the Ba / F3 cell line over 12 hours. Retroviruses were produced by transfecting Phoenix 293T-ampho cells (Orbigen) with pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 1 using Lipofectamine 2000 (Invitrogen). Three days after transduction, 2 μg / ml puromycin (Invitrogen) was added to the RPMI medium. After 5 days of selection, the cells were stained with FITC-HER2 (Biolegend) and sorted by FACS. Subsequently, the cell line was grown for 2 weeks in the absence of IL-3, and cell viability was evaluated every 3 days using the Cell Titer Glo assay (Progema). The resulting stable cell lines were maintained in RPMI-1640 medium containing 10% FBS without IL-3.

[0113] Cell viability assay and IC 50 Estimation: Cell viability was measured using the Cell Titer Glo assay (Promega) as previously described (Robichaux et al, 2018). Briefly, 2000 - 3000 cells per well were seeded technically in triplicate in 384-well plates (Greiner Bio-One). The cells were treated with 7 different concentrations of tyrosine kinase inhibitor or vehicle alone at a final volume of 40 μL per well. Three days later, 11 μL of Cell Titer Glo was added to each well. The plates were shaken for 15 minutes, and bioluminescence was measured using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). The bioluminescence values were normalized to DMSO-treated cells, and the normalized values were plotted using GraphPad Prism with a non-linear regression fit to the normalized data using a variable slope. IC 50 values were calculated by GraphPad Prism at 50% inhibition.

[0114] Correlation between ELISA for phosphorylated HER2 and total HER2 and IC50 values: Proteins were recovered from each of the parental Ba / F3 cell line and the Ba / F3 cell line expressing the HER2 mutation described above. 5 μg / ml of protein was added to each ELISA plate, and ELISA was performed for phosphorylated HER2 (Cell signaling, #7968) and total HER2 (Cell Signaling, #7310) as described in the manufacturer's instructions. Relative p-HER2 expression was measured by obtaining the ratio of p-HER2 to total HER2 determined by ELISA. The relative p-HER2 ratio was plotted against the poziotinib IC50 value calculated as described above. Pearson correlation coefficient and p-value were determined by GraphPad Prism.

[0115] Tyrosine kinase inhibitors and T-DM1: Except for EGF816 and pyrotinib purchased from MedChem Express, all inhibitors were purchased from Selleck Chemical. All inhibitors were dissolved in DMSO at a concentration of 10 mM and stored at -80 °C. Inhibitors were restricted to be discarded after two freeze-thaw cycles. T-DM1 was purchased and reconstituted from the institutional pharmacy of M.D. Anderson Cancer Center.

[0116] Molecular dynamics simulations: Protein structure models of HER2 variants were constructed using the MOE computer program (Chemical Computing Group) by introducing in-silico mutations into the PDB 3PP0 X-ray structure. Classical and accelerated molecular dynamics simulations were performed using the NAMD simulation package. Further details are provided in the Supplemental Information section.

[0117] Human cell line: MCF10A cells were purchased from ATCC and cultured in DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 5% fetal bovine serum (sigma), 20 ng / ml EGF, 0.5 mg / ml hydrocortisone, and 10 μg / ml insulin. Stable expressing cell lines were generated by retroviral transduction. Retroviruses were produced by transfecting Phoenix 293T-ampho cells (Orbigen) with the pBabe-Puro based vectors (Addgene and Bioinnovatise) summarized in Table 1 using Lipofectamine 2000 (Invitrogen). Two days after transduction, 0.5 μg / ml puromycin (Invitrogen) was added to the RPMI medium. After 14 days of selection, the cells were tested in a cell viability assay as described above. CW-2 cells were provided by the Riken cell line database under an MTA and maintained in RPMI containing 10% FBS and 1% penicillin / streptomycin.

[0118] In vivo xenograft studies: CW-2 cell line xenografts were generated by injecting 1 × 10 6 cells in 50% Matrigel into 6-week-old female nu / nu nude mice. When the tumors reached 350 mm 3 in size, the mice were randomized into 4 groups: 20 mg / kg afatinib, 5 mg / kg poziotinib, 30 mg / kg neratinib, or vehicle control (0.5% methylcellulose, 2% Tween-80 in dH2O). Tumor volumes were measured three times a week. Mice received drugs on Monday to Friday (5 days a week), starting on Wednesday and given two drug-free days after the first three days of dosing.

[0119] Y772dupYVMA PDX mice were purchased from Jax Labs (Model#TM01446). Tumor-derived fragments expressing HER2 Y772dupYVMA were inoculated into 5 - 6-week-old female NSG mice (Jax Labs #005557). Mice were measured three times a week and when the tumors reached 200 - 300 mm 3Once the tumor volume reached, the mice were randomized into the following four treatment groups: vehicle control (0.5% methylcellulose, 0.05% Tween-80 in dH2O), 2.5 mg / kg poziotinib, 10 mg / kg T-DM1, or a combination of 2.5 mg / kg poziotinib and 10 mg / kg T-DM1. Tumor volume and body weight were measured three times a week. Mice treated with 2.5 mg / kg poziotinib received the drug orally from Monday to Friday (5 days a week). Mice treated with 10 mg / kg T-DM1 received a single intravenous (IV) administration of T-DM1 on the day of randomization. Mice treated with the combination of poziotinib and T-DM1 received a single IV administration of T-DM1 and started 2.5 mg / kg poziotinib 5 days a week 3 days after the administration of T-DM1. If the body weight of the mice decreased by more than 10% or dropped below 20 grams, the mice were given a break from dosing. Progression-free survival was defined as from the best response in two consecutive measurements to tumor doubling. Complete regression was defined as a decrease of more than 95% of the tumor mass, and in mice with complete regression, tumor doubling was defined as greater than 75 mm in three or more consecutive measurements. The experiment was conducted in accordance with Good Animal Practices and completed with the approval of the MD Anderson Cancer Center Institutional Animal Care and Use Committee (Houston, TX). 3 was defined as greater than. The experiment was conducted in accordance with Good Animal Practices and completed with the approval of the MD Anderson Cancer Center Institutional Animal Care and Use Committee (Houston, TX).

[0120] (Table 1) Vectors used to generate stable cell lines TIFF0007700043000002.tif129170

[0121] (Table 2) Total number of patients by cancer type across the entire database TIFF0007700043000003.tif220134

[0122] (Table 3) Number of patient characteristics and prior treatment histories TIFF0007700043000004.tif82128

[0123] FACS: MCF10A cells overexpressing HER2 mutations were seeded in 6-well plates overnight and then treated with 10 nM poziotinib. After 24 hours, the cells were washed twice with PBS and trypsinized. The cells were then resuspended in 0.5% FBS in PBS and stained with anti-HER2-FITC antibody from Biolegend (#324404) for 45 minutes on ice. The cells were washed twice with 0.5% FBS in PBS and analyzed by flow cytometry. IgG and unstained controls were used for gating.

[0124] Western blotting: For western blotting, the cells were washed with PBS and lysed with RIPPA lysis buffer (ThermoFisher) and protease inhibitor cocktail tablets (Roche). Protein (30 - 40 μg) was loaded onto gels purchased from BioRad. Using a BioRad semidry transfer, the blot was then probed with antibodies against pHER2, HER2, pPI3K, PI3K, p-AKT, AKT, p-ERK1 / 2, and ERK1 / 2 (1:1000; Cell Signaling). The blot was probed with an antibody against vinculin or β-actin (Sigma-Aldrich) as a loading control and exposed using ECL western blotting substrate (Promega).

[0125] Correlation of HER2 expression levels with Ba / F3 variant IC50: Protein was recovered from Ba / F cell lines and ELISA was performed as described by the manufacturer's instructions for total HER2 (Cell Signaling, #7310). The relative expression measured by ELISA was plotted against the IC50 values calculated as described above. Pearson correlation coefficients and p-values were determined by GraphPad Prism.

[0126] Clinical Trials and CIND Identification Number: Patients provided written informed consent for treatment with poziotinib in either the compassionate use protocol (MD Anderson Cancer Center CIND-18-0014) or clinical trial NCT03066206. The protocol has been approved by both the MD Anderson Cancer Center Institutional Review Board and the US Food and Drug Administration.

[0127] All of the methods disclosed and claimed herein can be constructed and implemented without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and processes described herein, or to the order of the steps of such methods, without departing from the concept, spirit, and scope of the present invention. More specifically, certain chemically and physiologically related agents may be used in place of the agents described herein, and it will be apparent that they may achieve the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0128] References The following references provide exemplary procedural or other details that supplement the references shown herein and are specifically incorporated herein by reference. TIFF0007700043000005.tif201158TIFF0007700043000006.tif252112TIFF0007700043000007.tif238101

Claims

1. A medicament comprising an effective amount of poziotinib for use in a method of treating cancer in a subject, which is not used in combination with trastuzumab emtansine (T-DM1), the subject has been determined to have one or more HER2 exon 21 mutations, and the one or more HER2 exon 21 mutations include V842I.

2. The medicament according to claim 1, wherein the poziotinib is further defined as poziotinib hydrochloride.

3. The medicament according to claim 1 or 2, wherein the subject has been determined to have two, three, or four HER2 exon 21 mutations.

4. The medicament according to any one of claims 1 to 3, wherein the subject has been previously administered a tyrosine kinase inhibitor.

5. The medicament according to claim 4, wherein the subject is resistant to the previously administered tyrosine kinase inhibitor.

6. The medicament according to claim 5, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or beratinib.

7. The medicament according to any one of claims 1 to 6, wherein the subject has been determined not to have a mutation at residue C797 of EGFR.

8. The medicament according to any one of claims 1 to 7, wherein the one or more HER2 exon 21 mutations further include R868W.

9. The medicament according to any one of claims 1 to 8, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg.

10. The medicament according to any one of claims 1 to 9, wherein the method further comprises the step of performing a further anti-cancer therapy.

11. The medicament according to claim 10, wherein the further anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy.

12. The cancer is oral cancer, pharyngeal cancer, hypopharyngeal cancer, respiratory cancer, urogenital cancer, digestive cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, pharyngeal cancer, hypopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer, and the pharmaceutical according to any one of claims 1 to 11.

13. The cancer is non-small cell lung cancer, and the pharmaceutical according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more HER2 exon 21 mutations, not used in combination with T-DM1, and the one or more HER2 exon 21 mutations including V842I, the pharmaceutical composition.

15. The composition according to claim 14, comprising 8 mg, 12 mg, or 16 mg of poziotinib.

Images