Compounds with antitumor activity against cancer cells with EGFR or HER2 exon 20 insertions

Abstract

The present disclosure provides methods of treating cancer in patients determined to have an EGFR and / or HER2 exon 20 mutation, e.g., an insertion mutation, by administering a third-generation tyrosine kinase inhibitor, such as poziotinib or afatinib. TIFF2022527788000013.tif58128

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 826,843, filed March 29, 2019, which is incorporated by reference in its entirety.

[0002] Incorporating sequence tables The Sequence Listing, contained in a file entitled "UTFCP1383WO.txt", which is 3.59 KB (as calculated on Microsoft Windows) and was created on March 27, 2020, has been submitted electronically herewith and is incorporated by reference herein.

[0003] This invention was made with Government support under Grant No. CA190628 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0004] 1. Field The present invention relates generally to the fields of molecular biology and medicine. More specifically, the present invention relates to methods of treating patients with EGFR and / or HER2 exon 20 mutations, such as insertion mutations. [Background technology]

[0005] 2. Description of Related Technology Approximately 10-15% of NSCLCs harbor activating EGFR mutations. In the majority of these patients whose tumors harbor "classical" susceptibility mutations (L858R and exon 19 deletions), TKIs such as gefitinib and erlotinib have provided dramatic clinical benefit, with approximately 70% experiencing objective responses (ORs), improved progression-free survival (PFS) compared to chemotherapy alone, and improved quality of life (Maemondo et al., 2010). However, approximately 10-12% of EGFR mutant NSCLC tumors harbor in-frame insertions within exon 20 of EGFR (Arcila et al., 2012) and are generally resistant to EGFR TKIs. In addition, 90% of HER2 mutations in NSCLC are exon 20 mutations (Mazieres et al., 2013). Combined, EGFR and HER2 exon 20 mutations comprise approximately 4% of NSCLC patients. Data to date suggest that available HER2 TKIs (afatinib, lapatinib, neratinib, dacomitinib) have limited activity in patients with HER2 mutant tumors, with most studies reporting OR rates below 40% (Kosaka et al., 2017), although some preclinical activity has been observed in HER2 mouse models treated with afatinib (Perera et al., 2009).

[0006] Exon 20 of EGFR and HER2 contains two main regions, the c-helix (residues 762-766 in EGFR and 770-774 in HER2) and the loop following the c-helix (residues 767-774 in EGFR and 775-783 in HER2). Crystallography of the EGFR exon 20 insertion D770insNPG revealed a stabilized and robust active conformation that induces resistance to first generation TKIs upon insertion after residue 764. On the other hand, modeling of EGFR A763insFQEA showed that insertion before residue 764 does not have this effect and does not induce drug resistance (Yasuda et al., 2013). Furthermore, in a patient-derived xenograft (PDX) model of EGFR exon 20-driven NSCLC with an insertion in the loop following the c-helix (EGFR H773insNPH), the third-generation EGFR TKIs, osimertinib (AZD9291) and rociletinib (CO-1696), were found to have minimal activity (Yang et al., 2016). In a recent study of rare EGFR and HER2 exon 20 mutations, the authors found heterogeneous responses to covalent quinazoline-based second-generation inhibitors such as dacomitinib and afatinib; however, the concentrations required to target the more common exon 20 insertion mutations exceeded those achievable in the clinic (Kosaka et al., 2017). Thus, there is a significant clinical need to identify new therapies that overcome innate drug resistance in NSCLC tumors harboring exon 20 mutations, especially insertion mutations, in EGFR and HER2. Summary of the Invention

[0007] overview Embodiments of the present disclosure provide methods and compositions for treating cancer in patients with EGFR and / or HER2 exon 20 mutations, such as exon 20 insertion mutations. In one embodiment, a method of treating cancer in a subject is provided, comprising administering an effective amount of poziotinib to the subject, wherein the subject has been determined to have one or more EGFR exon 20 mutations, such as one or more EGFR exon 20 insertion mutations. In certain aspects, the subject is a human.

[0008] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as a tablet. In some embodiments, one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations.

[0009] In certain embodiments, the one or more EGFR exon 20 mutations comprise one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 763-778. In some embodiments, the subject is determined to have two, three, or four EGFR exon 20 mutations. In some embodiments, the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776.

[0010] In certain embodiments, the subject is determined to not have an EGFR mutation at residues C797 and / or T790, e.g., C797S and / or T790M. In some embodiments, the one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY In certain embodiments, the exon 20 mutation is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. In certain embodiments, the exon 20 mutation is A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, and / or N771dupNPH.

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

[0012] In certain embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 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 a 28-day cycle.

[0013] In certain embodiments, the subject is determined to have EGFR exon 20 mutation, for example, insertion mutation, by analyzing genomic sample from patient.In some embodiments, genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.In certain embodiments, the presence of EGFR exon 20 mutation is determined by nucleic acid sequencing (e.g., DNA sequencing of tumor tissue or plasma-derived circulating free DNA) or PCR analysis.

[0014] In certain embodiments, the method further comprises administering an additional 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 poziotinib administration and / or anti-cancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, in controlled release, in delayed release, as a suppository, or sublingually. In some embodiments, the administration of poziotinib and / or the administration of anti-cancer therapy includes local administration, regional administration, or systemic administration. In certain embodiments, the administration of poziotinib and / or the administration of anti-cancer therapy is administered more than once, for example, daily, every other day, or weekly.

[0015] In some embodiments, the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal 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 neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric 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 EGFR exon 20 mutations, such as one or more EGFR exon 20 insertion mutations. In certain aspects, the one or more EGFR exon 20 mutations include a point mutation, an insertion, and / or a deletion of 3-18 nucleotides at amino acids 763-778. In certain aspects, the subject has been determined to have two, three, or four EGFR20 mutations.

[0017] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as a tablet. In some embodiments, one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations.

[0018] In some embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 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 a 28-day cycle.

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

[0020] In some embodiments, the one or more EGFR exon 20 insertion mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, and H773. In certain embodiments, the subject has been determined to not have an EGFR mutation at residues C797 and / or T790, e.g., C797S and / or T790M. In certain embodiments, the one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. In some embodiments, the patient is being treated with an anti-cancer therapy.

[0021] In yet another embodiment, a method for predicting responsiveness to poziotinib alone or in combination with an anti-cancer therapy in a subject having cancer is provided, comprising detecting an EGFR exon 20 mutation (e.g., an EGFR exon 20 insertion mutation) in a genomic sample obtained from the patient, and if the sample is positive for the presence of an EGFR exon 20 mutation, the patient is predicted to have a favorable response to poziotinib alone or in combination with an anti-cancer therapy. In some aspects, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain aspects, the presence of an EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. In certain aspects, the EGFR exon 20 mutation comprises one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763-778. In some embodiments, the EGFR exon 20 mutation is present at residues A763, H773, A767, S768, V769, D770, N771, and / or D773. In some embodiments, the EGFR exon 20 mutation is selected from the group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, and N771dupNPH.

[0022] In certain embodiments, a favorable response to a poziotinib inhibitor alone or in combination with an anti-cancer therapy includes a reduction in tumor size or tumor burden, inhibition of tumor growth, relief of tumor-associated pain, relief of cancer-associated pathology, relief of cancer-associated symptoms, non-progression of cancer, extended disease-free interval, extended time to progression, induction of remission, reduced metastasis, or improved patient survival. In further embodiments, patients predicted to have a favorable response 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 a tablet. In some embodiments, one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations.

[0024] In some embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 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 a 28-day cycle.

[0025] In some embodiments, the subject has or has demonstrated resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or belatinib.

[0026] A further embodiment provides a method of treating cancer in a patient comprising administering an effective amount of poziotinib or afatinib to a subject, wherein the subject has been determined to have one or more HER2 exon 20 mutations selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V. In some embodiments, the one or more HER2 exon 20 mutations further comprise one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 770-785. In some embodiments, the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. In some embodiments, the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. In some embodiments, the HER2 exon 20 mutation is present at residues V773, A775, G776, S779, G778, and / or P780. In certain embodiments, the subject is a human.

[0027] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as a tablet. In some embodiments, one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations.

[0028] In some embodiments, poziotinib is administered orally. In some embodiments, poziotinib is administered at a dose of 5-25 mg, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 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 a 28-day cycle.

[0029] In some embodiments, the subject has or exhibits resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or belatinib.

[0030] In some embodiments, the method further comprises administering an mTOR inhibitor. In certain embodiments, the mTOR inhibitor is rapamycin, temsirolimus, everolimus, ridaforolimus, or MLN4924. In certain embodiments, the mTOR inhibitor is everolimus.

[0031] In certain embodiments, poziotinib or afatinib and / or mTOR inhibitor is administered intravenously, subcutaneously, intraosseously, orally, transdermally, sustained release, controlled release, delayed release, as a suppository, or sublingually.In some embodiments, the patient is determined to have HER2 exon 20 mutation by analyzing genomic samples from the patient.In certain embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.In some embodiments, the presence of HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis.

[0032] In additional aspects, the method further comprises administering an additional anti-cancer therapy, hi some aspects, the anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

[0033] In some embodiments, the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal 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 neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric 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.

[0034] In another embodiment, a pharmaceutical composition comprising poziotinib or afatinib is provided for a patient determined to have one or more HER2 exon 20 mutations selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V. In some embodiments, the one or more HER2 exon 20 mutations further comprise one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 770-785. In some embodiments, the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. In some embodiments, the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. In some embodiments, the HER2 exon 20 mutation is present at residues V773, A775, G776, S779, G778, and / or P780.In some embodiments, the patient is being treated with an anti-cancer therapy.

[0035] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as a tablet. In some embodiments, one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations.

[0036] In some embodiments, poziotinib is included in the composition at a dose of 5-25 mg, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 23, 24, or 25 mg. In some embodiments, poziotinib is at a dose of 8 mg, 12 mg, or 16 mg.

[0037] In some embodiments, the subject has or exhibits resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or belatinib.

[0038] In yet another embodiment, the present invention provides a method of predicting responsiveness to poziotinib alone or afatinib alone or poziotinib or afatinib in combination with an anti-cancer therapy in a patient having cancer, comprising the steps of: detecting A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del The method includes detecting a HER2 exon 20 mutation (e.g., a HER2 exon 20 insertion mutation) selected from the group consisting of insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V, and when the sample is positive for the presence of a HER2 exon 20 mutation, the patient is predicted to have a favorable response to poziotinib alone or afatinib alone or poziotinib or afatinib in combination with an anticancer therapy. In some embodiments, the one or more mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. In some embodiments, the HER2 exon 20 mutation further comprises one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 770-785. In certain embodiments, the HER2 exon 20 mutation is present at residues V773, A775, G776, V777, G778, S779, and / or P780. In other embodiments, the HER2 exon 20 mutation is present at residues A775, G776, S779, and / or P780.

[0039] In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain embodiments, the presence of HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. In certain embodiments, the anti-cancer therapy is an mTOR inhibitor. In some embodiments, favorable response to poziotinib inhibitor alone or afatinib inhibitor alone or in combination with anti-cancer therapy includes reduction in tumor size or tumor burden, inhibition of tumor growth, reduction in tumor-related pain, reduction in cancer-related pathology, reduction in cancer-related symptoms, non-progression of cancer, prolonged disease-free period, prolonged time to progression, induction of remission, reduction in metastasis, or improved patient survival. In further embodiments, patients predicted to have favorable response are administered poziotinib alone or in combination with a second anti-cancer therapy.

[0040] Also provided herein is a composition comprising a nucleic acid isolated from a human cancer cell; and a primer pair capable of amplifying at least a first portion of exon 20 of a human EGFR or HER2 coding sequence. In some embodiments, the composition further comprises a labeled probe molecule capable of specifically hybridizing to a first portion of exon 20 of a human EGFR or HER coding sequence when a mutation is present in the sequence. In certain embodiments, the composition further comprises a thermostable DNA polymerase. In some embodiments, the composition further comprises dNTPS. In some embodiments, the labeled probe is selected from the group consisting of A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY It hybridizes to the first portion of exon 20 of the human EGFR coding sequence when a mutation selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C is present.

[0041] In certain embodiments, the labeled probe hybridizes to a first portion of exon 20 of the human HER2 coding sequence when a mutation selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, and P780insGSP is present.

[0042] In another embodiment, an isolated nucleic acid is provided encoding a mutant EGFR protein, wherein the mutant protein differs from wild-type human EGFR by one or more EGFR exon 20 mutations comprising a point mutation, insertion, and / or deletion of 3-18 nucleotides at amino acids 763 to 778. In some aspects, the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776. In certain embodiments, the one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 8, 9, 10, 11, or 12.

[0043] In yet another embodiment, an isolated nucleic acid is provided encoding a mutant HER2 protein, the mutant protein differing from wild-type human HER2 by one or more HER2 exon 20 mutations comprising one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 770-785. In some aspects, the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780. In certain aspects, the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO:14, 15, 16, 17, or 18.

[0044] [The present invention 1001] 1. 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 EGFR exon 20 mutations. [The present invention 1002] The method of any one of claims 1 to 10, wherein said poziotinib is further defined as poziotinib hydrochloride. [The present invention 1003] The method of claim 1002, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1004] The method of any of claims 1001 to 1004, wherein said one or more EGFR exon 20 mutations are further defined as EGFR20 insertion mutations. [The present invention 1005] The method of any of claims 1001 to 1004, wherein said one or more EGFR exon 20 mutations are further defined as a de novo EGFR20 insertion mutation. [The present invention 1006] 6. The method of any of claims 1001 to 1005, wherein said one or more EGFR exon 20 mutations comprise a point mutation, an insertion, and / or a deletion of 3 to 18 nucleotides at amino acids 763 to 778. [The present invention 1007] The method of any one of claims 1001 to 1006, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S. [The present invention 1009] The method of any of claims 1001 to 1008, wherein the subject has previously been administered a tyrosine kinase inhibitor. [The present invention 1010] The method of claim 1009, wherein the subject is resistant to a previously administered tyrosine kinase inhibitor. [The present invention 1011] The method of the present invention 1010, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or beratinib. [The present invention 1012] 12. The method of any of claims 1001 to 1011, wherein the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, and V774. [The present invention 1013] 13. The method of any of claims 1001 to 1012, wherein the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776. [The present invention 1014] The method according to any one of claims 1001 to 1013, wherein the subject is determined to not have an EGFR mutation at residue C797. [The present invention 1015] Any of the methods of the present invention 1001 to 1014, wherein the one or more exon 20 mutations are selected from the group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV. [The present invention 1016] The one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY Any of the methods of 1001 to 1015, wherein the amino acid sequence is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1017] The method of any one of claims 1001 to 1016, wherein the exon 20 mutation is D770insNPG. [The present invention 1018] The method according to any one of claims 1001 to 1017, wherein the subject is determined to have an EGFR exon 20 mutation by analyzing a genomic sample derived from the patient. [The present invention 1019] The method of claim 1019, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1020] The method of any of claims 1001 to 1019, wherein the presence of an EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1021] The method of any of claims 1001 to 1020, wherein the poziotinib is administered orally. [The present invention 1022] The method of any of claims 1001 to 1021, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1023] The method of any of claims 1001 to 1022, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1024] The method of any of claims 1001 to 1023, wherein said poziotinib is administered daily. [The present invention 1025] The method of any of claims 1001 to 1024, wherein the poziotinib is administered continuously. [The present invention 1026] Any of the methods of claims 1001 to 1025, wherein the poziotinib is administered in a 28-day cycle. [The present invention 1027] The method of any one of claims 1001 to 1026, further comprising the step of administering an additional anticancer therapy. [The present invention 1028] The method of claim 1027, wherein said further anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy. [The present invention 1029] The method of claim 1027 or 1028, wherein said administration of poziotinib and / or anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, controlled release, delayed release, as a suppository, or sublingually. [The present invention 1030] The method of any of claims 1027 to 1030, wherein administering poziotinib and / or performing anticancer therapy includes local, regional, or systemic administration. [The present invention 1031] The method of any of claims 1027 to 1031, wherein the administration of poziotinib and / or the anticancer therapy is performed two or more times. [The present invention 1032] The method according to any one of claims 1001 to 1031, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or cancer of the hematopoietic system, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1033] The method according to any one of claims 1001 to 1032, wherein the cancer is non-small cell lung cancer. [The present invention 1034] The method of any one of claims 1001 to 1033, wherein the patient is a human. [The present invention 1035] A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more EGFR exon 20 mutations. [The present invention 1036] The composition of the present invention 1035 further defined as an oral composition. [The present invention 1037] The composition of any one of claims 1035 to 1036, comprising 5 to 25 mg of poziotinib. [The present invention 1038] Any of compositions 1035 to 1037 comprising 8 mg, 12 mg, or 16 mg of poziotinib. [The present invention 1039] The composition of any of claims 1035 to 1038, wherein said poziotinib is further defined as poziotinib hydrochloride. [The present invention 1040] The composition of any one of claims 1035 to 1039, which is formulated as a tablet. [The present invention 1041] The composition of any of claims 1035 to 1040, wherein said one or more EGFR exon 20 mutations are further defined as EGFR20 insertion mutations. [The present invention 1042] The composition of any of claims 1035 to 1041, wherein said one or more EGFR exon 20 mutations are further defined as a de novo EGFR20 insertion mutation. [The present invention 1043] 1043. Any of the compositions of claims 1035 to 1042, wherein said one or more EGFR exon 20 mutations comprise a point mutation, an insertion, and / or a deletion of 3 to 18 nucleotides at amino acids 763 to 778. [The present invention 1044] The composition of any of claims 1035 to 1043, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations. [The present invention 1045] The composition of any of claims 1035 to 1044, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S. [The present invention 1046] Any of the compositions of claims 1035 to 1045, wherein one or more EGFR exon 20 insertion mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, and V774. [The present invention 1047] Any of the compositions of claims 1035 to 1046, wherein the one or more EGFR exon 20 insertion mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776. [The present invention 1048] The composition of any of claims 1035 to 1047, wherein the subject is determined to not have an EGFR mutation at residue C797. [The present invention 1049] Any of the compositions of 1035 to 1048 of the present invention, wherein the one or more exon 20 mutations are selected from the group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV. [The present invention 1050] The one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY Any of the compositions of claims 1035 to 1049, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1051] The composition of any one of claims 1035 to 1050, wherein the patient is undergoing treatment with an anti-cancer therapy. [The present invention 1052] 1. A method for predicting responsiveness to poziotinib alone or in combination with a second anti-cancer therapy in a subject having cancer, comprising detecting an EGFR exon 20 mutation in a genomic sample obtained from the patient, wherein if the sample is positive for the presence of the EGFR exon 20 mutation, the patient is predicted to have a favorable response to poziotinib alone or in combination with a second anti-cancer therapy. [The present invention 1053] The method of claim 1052, wherein said EGFR exon 20 mutation is further defined as an exon 20 insertion mutation. [The present invention 1054] The method of any one of claims 1052 to 1053, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1055] The method of any of claims 1052 to 1054, wherein the presence of an EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1056] 6. The method of any of claims 1052 to 1055, wherein said EGFR exon 20 mutation comprises a point mutation, an insertion, and / or a deletion of 3 to 18 nucleotides at amino acids 763 to 778. [The present invention 1057] The method according to any one of claims 1052 to 1056, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S. [The present invention 1058] 1057. The method of any of claims 1052 to 1057, wherein the EGFR exon 20 mutation is present at residues A763, A767, S768, V769, D770, N771, P772, H773, and / or V774. [The present invention 1059] 9. The method of any of claims 1052 to 1058, wherein the EGFR exon 20 mutation is present at residues A763, A767, S768, V769, D770, N771, P772, H773, V774, and / or R776. [The present invention 1060] The method of any one of 1052 to 1059 of the present invention, wherein the EGFR exon 20 mutation is selected from the group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV. [The present invention 1061] The EGFR exon 20 mutation is A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY Any of the methods of claims 1052 to 1060, wherein the amino acid sequence is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1062] Any of the methods of 1052-1061, wherein the favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy comprises a reduction in tumor size or tumor burden, inhibition of tumor growth, reduction in tumor-associated pain, reduction in cancer-associated pathology, reduction in cancer-associated symptoms, non-progression of cancer, extended disease-free interval, extended time to progression, induction of remission, reduction in metastasis, or improved patient survival. [The present invention 1063] The method of any of claims 1052 to 1062, further comprising the step of administering poziotinib, alone or in combination with a second anti-cancer therapy, to said patient predicted to have a favorable response. [The present invention 1064] The method of any of claims 1052 to 1063, wherein the poziotinib is administered orally. [The present invention 1065] The method of any of claims 1052 to 1064, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1066] The method of any of claims 1062 to 1065, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1067] The method of any of claims 1062 to 1066, wherein said poziotinib is further defined as poziotinib hydrochloride. [The present invention 1068] The method of any one of claims 1062 to 1067, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1069] 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib or afatinib, wherein the subject has been determined to have one or more HER2 exon 20 mutations selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V. [The present invention 1070] 1069. The method of claim 1069, wherein the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. [The present invention 1071] The method of any one of claims 1069 to 1070, wherein said poziotinib is administered orally. [The present invention 1072] The method of any of claims 1069 to 1071, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1073] The method of any of claims 1069 to 1072, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1074] The method of any of claims 1069 to 1073, wherein said poziotinib is further defined as poziotinib hydrochloride. [The present invention 1075] The method of any one of claims 1069 to 1074, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1076] 6. The method of any of claims 1069 to 1075, wherein said one or more HER2 exon 20 mutations further comprise one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770 to 785. [The present invention 1077] 1077. The method of any of claims 1069 to 1076, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. [The present invention 1078] 1077. The method of any of claims 1069 to 1076, wherein said one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780. [The present invention 1079] The method of any of claims 1069 to 1078, wherein said HER exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [The present invention 1080] Any of the methods of claims 1069 to 1079, wherein the HER exon 20 insertion mutation is A775insYVMA. [The present invention 1081] The method of any one of claims 1069 to 1080, further comprising the step of administering an mTOR inhibitor. [The present invention 1082] The method of claim 1081, wherein the mTOR inhibitor is rapamycin, temsirolimus, everolimus, ridaforolimus, or MLN4924. [The present invention 1083] The method of claim 1081, wherein the mTOR inhibitor is everolimus. [The present invention 1084] The method of the present invention 1081, wherein said poziotinib or afatinib and / or said mTOR inhibitor is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, controlled release, delayed release, as a suppository, or sublingually. [The present invention 1085] The method of any one of claims 1069 to 1084, wherein the subject is determined to have a HER2 exon 20 mutation by analyzing a genomic sample derived from the patient. [The present invention 1086] The method of claim 1085, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1087] The method of any of claims 1069 to 1086, wherein the presence of a HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1088] The method of any one of claims 1069 to 1087, further comprising the step of administering an additional anticancer therapy. [The present invention 1089] The method according to any one of claims 1069 to 1088, wherein said further anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy. [The present invention 1090] The method according to any one of claims 1069 to 1089, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or cancer of the hematopoietic system, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1091] The method of any one of claims 1069 to 1090, wherein the cancer is non-small cell lung cancer. [The present invention 1092] The method of any one of claims 1069 to 1091, wherein the subject is a human. [The present invention 1093] A pharmaceutical composition comprising poziotinib or afatinib for use in a subject determined to have one or more HER2 exon 20 mutations selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V. [The present invention 1094] The composition of the present invention 1093, wherein the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, and V773M. [The present invention 1095] The composition of any one of claims 1093 to 1094, wherein said HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [The present invention 1096] 6. The composition of any of claims 1093 to 1095, wherein said HER2 exon 20 mutation further comprises one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770 to 785. [The present invention 1097] 1093. Any of the compositions of claims 1093 to 1096, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. [The present invention 1098] 1097. The composition of any of claims 1093 to 1096, wherein the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780. [This invention 1099] The pharmaceutical composition of any one of claims 1093 to 1098, wherein the patient is undergoing treatment with an anticancer therapy. [The present invention 1100] 1. A method for predicting responsiveness to poziotinib alone or afatinib alone, or poziotinib or afatinib in combination with an anti-cancer therapy, in a subject having cancer, comprising detecting in a genomic sample obtained from the subject A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del The method comprises detecting a HER2 exon 20 mutation selected from the group consisting of insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V, wherein if the sample is positive for the presence of a HER2 exon 20 mutation, the patient is predicted to have a favorable response to poziotinib alone or afatinib alone, or poziotinib or afatinib in combination with an anti-cancer therapy. [The present invention 1101] The method of claim 1100, wherein said HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [The present invention 1102] The method of any one of claims 1100 to 1101, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1103] The method of any of claims 1100 to 1102, wherein the presence of a HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1104] The method according to any one of claims 1100 to 1103, wherein said anticancer therapy is an mTOR inhibitor. [The present invention 1105] Any of the methods of the present invention 1100-1104, wherein the favorable responsiveness to the poziotinib inhibitor alone or afatinib inhibitor alone or in combination with an anti-cancer therapy comprises a reduction in tumor size or tumor burden, inhibition of tumor growth, reduction in tumor-associated pain, reduction in cancer-associated pathology, reduction in cancer-associated symptoms, non-progression of cancer, extended disease-free interval, extended time to progression, induction of remission, reduction in metastasis, or improved patient survival. [The present invention 1106] The method of any of claims 1100 to 1105, further comprising the step of administering poziotinib or afatinib, alone or in combination with a second anti-cancer therapy, to said patient predicted to have a favorable response. [The present invention 1107] (a) nucleic acid isolated from a human cancer cell; and (b) a primer pair capable of amplifying at least a first portion of exon 20 of the human EGFR or HER2 coding sequence; A composition comprising: [The present invention 1108] a labeled probe molecule capable of specifically hybridizing to said first portion of exon 20 of said human EGFR or HER coding sequence if a mutation is present in said sequence; The composition of claim 1107, further comprising: [The present invention 1109] The composition of any one of claims 1107 to 1108, further comprising a thermostable DNA polymerase. [The present invention 1110] The composition of any one of claims 1107 to 1109, further comprising dNTPS. [The present invention 1111] A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY Any of the compositions of claims 1108 to 1110, wherein the labeled probe hybridizes to the first portion of exon 20 of the human EGFR coding sequence when a mutation selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV is present. [The present invention 1112] A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY Any of the compositions of claims 1108 to 1111, wherein the labeled probe hybridizes to the first portion of exon 20 of the human EGFR coding sequence when a mutation selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C is present. [The present invention 1113] Any of the compositions of claims 1108 to 1111, wherein the labeled probe hybridizes to the first portion of exon 20 of the human HER2 coding sequence when a mutation selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V is present. [The present invention 1114] 1. An isolated nucleic acid encoding a mutant EGFR protein, wherein the mutant protein differs from wild-type human EGFR by one or more EGFR exon 20 mutations comprising a point mutation, an insertion, and / or a deletion of 3 to 18 nucleotides at amino acids 763-778. [The present invention 1115] 1114. The isolated nucleic acid of the present invention, wherein the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, and V774. [The present invention 1116] 1114. The isolated nucleic acid of claim 1115, wherein the one or more EGFR exon 20 mutations are present at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776. [The present invention 1117] The isolated nucleic acid of any one of claims 1114 to 1116, wherein the one or more exon 20 mutations are selected from the group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV. [The present invention 1118] The one or more exon 20 mutations are A763insFQEA, A763insLQEA, A767insASV, S768dupSVD, S768I, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY An isolated nucleic acid according to any one of claims 1114 to 1117, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1119] An isolated nucleic acid according to any one of claims 1114 to 1118, comprising the sequence of SEQ ID NO:8, 9, 10, 11, or 12. [The present invention 1120] 1. An isolated nucleic acid encoding a mutant HER2 protein, wherein the mutant protein differs from wild-type human HER2 by one or more HER2 exon 20 mutations comprising one or more point mutations, insertions, and / or deletions of 3-18 nucleotides at amino acids 770-785. [The present invention 1121] 1120. The isolated nucleic acid of the present invention, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. [The present invention 1122] 1120. The isolated nucleic acid of claim 1121, wherein the one or more HER2 exon 20 mutations are selected from the group consisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V. [The present invention 1123] An isolated nucleic acid of any of claims 1120 to 1122, comprising the sequence of SEQ ID NO:14, 15, 16, 17, or 18. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description herein. [Brief description of the drawings]

[0045] The accompanying drawings form part of the specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figures 1A-1J: Exon 20 insertion mutations induce de novo resistance to covalent and non-covalent TKIs. (Figure 1A) Progression-free survival (PFS) of patients with classical and exon 20 EGFR mutations resistant to first-line therapy. Patients with exon 20 insertions have reduced percent survival. (Figure 1B) Schematic of EGFR and HER2 exon 20 insertion mutations generated in stable Ba / F3 models. Dose-response curves of cell viability of Ba / F3 cell lines expressing EGFR (Figure 1C-E) and (Figure 1F-H) HER2 exon 20 insertion mutations treated with first-, second-, and third-generation TKIs for 72 hours. (Figure 1C-H) Mean values ​​± SEM of six cell lines are plotted for each concentration (n=3). (Figure 1I) 3-D modeling of EGFR D770insNPG and T790M. The shift of the P-loop and α-c-helix into the binding pocket results in steric hindrance that pushes AZD9291 out of the binding pocket. (FIG. 1J) 3-D modeling of HER2 A775insYVMA and WT. The overall shift of the P-loop and α-c-helix into the binding pocket results in an overall reduction in the size of the binding pocket. [Diagram 2]Figures 2A-2G: Poziotinib potently inhibits EGFR and HER2 exon 20 insertion mutations. Dose-response curves for cell viability of Ba / F3 cell lines expressing EGFR (Figure 2A) and HER2 (Figure 2B) exon 20 insertion mutations treated with poziotinib for 72 hours. Mean ± SEM for each individual cell line was plotted for each concentration (n=3). (Figure 2C) Western blotting confirms inhibition of p-EGFR and p-HER2 in Ba / F3 cell lines after 2 hours of poziotinib treatment (n=2). (Figure 2D) Correlation of Ba / F3 EGFR exon 20 insertion site with amino acid position (n=2). Pearson correlation coefficients and p-values ​​were determined using GraphPad Prism. (Figure 2E) Dose-response curves for cell viability of patient-derived cell lines CUTO14 expressing EGFR A767dupASV and (Figure 2F) YUL0019 expressing EGFR N771del insFH treated with poziotinib or afatinib for 72 h (n=3). (Figure 2F) IC50 values ​​of EGFR mutant Ba / F3 cells normalized to IC50 values ​​of Ba / F3 EGFR T790M cell lines after 72 h incubation with afatinib, osimertinib, rociletinib, or poziotinib (n=3). (Figure 2G) Bars represent mean ± SEM. Values ​​above 1 represent a lower strength of inhibition compared to T790M, and values ​​below 1 represent a stronger inhibition of the exon 20 insertion compared to T790M. [Diagram 3]Figures 3A-3H: Poziotinib reduces tumor burden in EGFR and HER2 exon 20 insertion mutant mouse models. EGFR D770insNPG (Figure 3A) or HER2 A775insYVMA (Figure 3B) mice were treated daily for 4 weeks with vehicle (EGFR n=5 and HER2 n=4), 20 mg / kg afatinib (EGFR n=4), or 10 mg / kg poziotinib (EGFR n=5 and HER2 n=6). Waterfall plots of tumor volume change measured by MRI show 85% and 60% tumor inhibition by poziotinib at 4 weeks in EGFR and HER2 GEMMs, respectively. (Figures 3A-B) p values ​​were calculated using a two-tailed Student's t-test. Representative MRI images of EGFR (Figure 3C) and HER2 (Figure 3D) GEMMs before and after 4 weeks of poziotinib treatment show robust tumor regression. Plots of tumor volume of EGFR D770insNPG (Figure 3E) (n=4) and HER2 A775insYVMA (Figure 3F) (n=6) treated with 10 mg / kg poziotinib 5 days / week for 12 weeks show that mice continue to respond to poziotinib treatment. (Figure 3G) YUL-0019 (EGFR N771delinsFH) cells treated with afatinib or poziotinib. Cells treated with 10 mg / kg poziotinib had the lowest tumor volume, and cells treated with 5 mg / kg had the second lowest tumor volume. (FIG. 3H) EGFR H773insNPH PDX mice were treated with vehicle control (n=6), 5 mg / kg (n=6), or 10 mg / kg (n=3) poziotinib. Mice treated with poziotinib had reduced tumor volume. Waterfall plots show that tumor burden was reduced by >85% in all poziotinib-treated mice, and 8 of 9 poziotinib-treated mice had complete shrinkage of xenografts to residual bolus. Statistical significance was determined using one-way ANOVA analysis combined with Tukey's test, ***, p<0.0001. [Figure 4]Figures 4A-4C: EGFR and HER2 exon 20 insertion mutations are activating mutations. (Figure 4A) Waterfall plots of individual patients with EGFR exon 20 insertions show de novo resistance to erlotinib, gefitinib, or afatinib. Patient mutations are listed under each representative bar. (Figure 4B) Stable Ba / F3 cell lines expressing EGFR exon 20 insertion mutations were viable in IL-3-independent conditions, unlike Ba / F3 cells expressing empty vector or EGFR WT, indicating that EGFR exon 20 insertions are activating mutations. (Figure 4C) IL-3-independent growth of 11 stable Ba / F3 cell lines expressing different HER2 mutations shows that the majority of HER2 activating mutations reside within exon 20 of HER2. With the exception of L755P, all activating mutations were HER2 exon 20 insertion mutations. (Figure 4B-C) Cell viability was measured by Cell Titer Glo assay. Mean values ​​± SEM were plotted for each cell line (n=3). [Diagram 5] Dose-response curves of cell viability for individual Ba / F3 cell lines expressing EGFR exon 20 insertion mutations treated with 1st, 2nd, and 3rd generation TKIs for 72 hours. Mean ± SEM is plotted for each concentration (n=3). [Figure 6] Dose-response curves for cell viability of individual Ba / F3 cell lines expressing HER2 exon 20 insertion mutations treated with 1st, 2nd, and 3rd generation TKIs for 72 hours. Mean ± SEM is plotted for each concentration (n=3). [Figure 7]Figures 7A-7D: EGFR and HER2 exon 20 insertion mutations after residue A763 are resistant to first and third generation TKIs. Ba / F3 cells harboring EGFR exon 20 insertions were serum starved for 1 h and then treated with the indicated doses of (Figure 7A) erlotinib or (Figure 7C) osimertinib for 2 h (N=2). p-EGFR and p-HER2 levels after (Figure 7B) erlotinib and (Figure 7D) osimertinib treatment were quantified using Photoshop. Values ​​were plotted in Graphpad Prism, bars represent mean ± SEM. (N=2) p<0.05 (*), p<0.01 (**), or p<0.001 (***). [Figure 8] Figures 8A-8E: EGFR and HER2 exon 20 insertion mutations are sensitive to poziotinib in vitro. (Figure 8A) Western blots of p-EGFR and p-HER2 after 2 hours of poziotinib treatment in the indicated Ba / F3 cell lines were quantified using Photoshop. Values ​​were plotted in Graphpad Prism, bars represent mean ± SEM. (N=2) (Figure 8B) Western blots of CUTO-14 patient-derived cell lines after 3 hours of the indicated doses of afatinib or poziotinib (N=3). (Figure 8C) Quantification of p-EGFR from Western blots after 3 hours of the indicated doses of afatinib or poziotinib in the CUTO-14 cell line. Poziotinib treatment resulted in decreased p-EGFR. (Figure 8D) Linear regression plot of IC50 values ​​against relative expression in Ba / F3 cell lines showed no correlation between expression and sensitivity to poziotinib (n=2). (Figure 8E) Linear regression plot of IC50 values ​​against location of mutations in the HER2 receptor showed no correlation between location and sensitivity to poziotinib in HER2 mutant Ba / F3 cell lines (n=2). Pearson correlation coefficients and p-values ​​were calculated using Graphpad prism. p<0.05 (*), p<0.01 (**), or p<0.001 (***). [Figure 9]C797S and EMT are two distinct mechanisms of poziotinib resistance in vitro. Dose-response curves of cell viability of EGFR mutant Ba / F3 cell lines treated with poziotinib for 72 hours. Mean values ​​± SEM are plotted for each concentration (n=3). [Figure 10] Dose-response curves for cell viability of MCF10A HER2 G776del insVC cell lines treated with the indicated TKIs. [Figure 11A] Dose-response curves for cell viability of EGFR mutant Ba / F3 cell lines treated with poziotinib or the indicated TKIs for 72 h. [Figure 11B] Dose-response curves for cell viability of EGFR mutant Ba / F3 cell lines treated with poziotinib or the indicated TKIs for 72 h. [Figure 11C] Dose-response curves for cell viability of EGFR mutant Ba / F3 cell lines containing resistance mutations treated with poziotinib or the indicated TKIs for 72 h. [Figure 11D] Dose-response curves for cell viability of EGFR mutant Ba / F3 cell lines containing resistance mutations treated with poziotinib or the indicated TKIs for 72 h. [Figure 12] Dose-response curves for cell viability of HER2 mutant Ba / F3 cell lines treated with poziotinib or the indicated TKIs for 72 h. [Figure 13] Figures 13A-13B: HER2 mutations occur in a wide variety of cancer types, with mutational hotspots occurring across the receptor. Bar plots of weighted average frequencies of HER2 mutations (A) and HER2 exon 20 mutations (B) by cancer. Bars represent weighted mean ± SEM. Dot size represents number of patients in each database. Frequencies of HER2 mutations detected by cfDNA reported by Guardant Health were corrected for clinical sensitivity as reported in Odegaard et al. 2018. [Figure 14]Figures 14A-14H: HER2 mutation hotspots vary by cancer type. Pie charts of frequency of HER2 mutation locations in (A) all cancers (N=2338), (B) lung cancer (N=177), (C) breast cancer (N=143), and (D) colorectal cancer (N=219) reported in the cBioportal and MD Anderson databases. (E) Lollipop plot of the 10 most frequent HER2 mutations in all cancers reported in cBioPortal and MD Anderson (N=2338 HER2 mutations). Bar length correlates with mutation frequency. (E-H) Lollipop plot of the 10 most frequent HER2 mutations in NSCLC (F, N=177), breast cancer (G, N=143), and colorectal cancer (H, N=219) in the cBioPortal and MD Anderson databases; bar length correlates with reported mutation frequency. [Figure 15] Figures 15A-15C: The most frequent HER2 variants in the tyrosine kinase domain are activating mutations. Cell viability of stable Ba / F3 cell lines expressing HER2 exon 19 (A), HER2 exon 20 (B), and HER2 exon 21 (C) mutations grown under IL-3-free conditions for 14 days. Cell viability was measured every 3 days by Cell Titer Glo assay. Mean values ​​± SEM were plotted for each cell (n=3 biologically independent experiments). [Figure 16]Figures 16A-16F: Poziotinib was the most potent inhibitor tested for HER2 mutations in Ba / F3 cells. (A) Heatmap of log IC50 values ​​calculated in GraphPad for Ba / F3 cells stably expressing the indicated mutations and drugs after 72 hours of drug treatment. Cell viability was measured by Cell Titer Glo assay (N>3). Mean IC50 values ​​for all Ba / F3 cell lines expressing HER2 mutations (B), HER2 exon 19 mutant cell lines (C), HER2 exon 20 mutant cell lines (D), or HER2 exon 21 mutant cell lines (E) after 72 hours of drug treatment with afatinib, neratinib, tarloxotinib-TKI, or poziotinib. Bars represent mean ± SEM (N>3). (C-E) Statistical significance between groups was determined using one-way ANOVA with Dunn's multiple comparison test. (F) Mean IC50 values ​​of Ba / F3 cells expressing L755S or L755P with the indicated inhibitors. Dots represent mean ± SEM (N > 3). Statistical significance was determined by paired t-test. [Figure 17] Figures 17A-17D: Molecular dynamics simulations of HER2 mutants show a putative mechanism of decreased drug sensitivity for Y772dupYVMA and L755P mutations. (A) α-C-helix positions for HER2 V777L and Y772dupYVMA exon 20 mutants during 150 ns accelerated molecular dynamics simulations. (B) Partial distribution of molecular dynamics snapshots for HER2 exon 20 mutants in α-C-helix "in" versus "out" conformations. (C) Molecular dynamics snapshots of V777L and Y772dupYVMA mutants. There was a significant shift in α-C-helix position while there was slight difference in P-loop and kinase hinge conformations. (D) Molecular dynamics snapshots of L755P and L755S HER2 mutants. The L755P mutant lacks the backbone hydrogen bond with V790, leading to destabilization of the kinase hinge and contraction of the P-loop towards the binding site. [Figure 18]Figures 18A-18F: Human cell lines expressing HER2 mutations are also most sensitive to poziotinib. Dose-response curves of MCF10A cells expressing exon 20 insertion mutations, HER2 G776delinsVC (A), HER2 Y772dupYVMA (B), HER2 G778dupGSP (C), treated for 72 hours with the indicated inhibitors. (D) Bar graphs of MCF10A HER2 selectivity index. The IC50 values ​​of the mutant cell lines were divided by the mean IC50 value of the HER2 WT expressing cell lines for each indicated drug. Dots represent the mean ± SEM for each cell line, and bars represent the mean ± min / max of all three cell lines (N > 3 for each cell line). (E) Dose-response curves of CW-2 colon cells harboring the HER2 exon 19 mutation L755S, treated for 72 hours with the indicated inhibitors. (A-C,E) Curves represent median ± SEM, N=3. (F) Bar graph of CW-2 tumor volume at day 21. Mice were treated 5 days / week with vehicle control (N=5), 30 mg / kg neratinib (N=5), 20 mg / kg afatinib (N=5), or 5 mg / kg poziotinib (N=5) and tumors were randomized at 350 mm3 and indicated by dotted lines. Dots represent individual tumors and bars represent mean ± SEM. Statistical significance was determined by one-way ANOVA. [Figure 19]Figures 19A-19D: NSCLC patients with HER2 mutations have a confirmed response rate of 42% to poziotinib. (A) Waterfall plot of response for the first 12 HER2 exon 20 patients in clinical trial NCT03066206. Objective partial response is shown (bars 7, 8, 10, 11, and 12 from the left), inconfirmed response (bar 9), stable disease (bars 3-6), and progression (bars 1-2). (B) Kaplan-Meier plot of progression-free survival for the first 12 HER2 exon 20 patients shows that mPFS was 5.6 months as of December 2018. (C) CT scan of a patient with HER2 Y772dupYVMA mutation 1 day before poziotinib treatment and after 8 weeks of treatment. (D) PET scan of a patient with HER2 L755P mutant NSCLC 1 day before and 4 weeks after poziotinib treatment. The patient had been previously treated with and progressed on platinum-based chemotherapy in combination with trastuzumab, nivolumab, and anti-TDM1, but had a -12% reduction in target lesions with poziotinib treatment. [Figure 20]Figures 20A-20G: Poziotinib treatment induces accumulation of HER2 on the cell surface, and the combination of poziotinib and T-DM1 treatment enhances antitumor activity. (A) FACS analysis of HER2 receptor expression on MC10A cell lines expressing HER2 Y772dupYVMA, HER2 G778dupGSP, and HER2 G776delinsVC after 24 hours of 10 nM poziotinib treatment. Bars represent mean ± SEM, and significant differences were determined by Student's t-test between DMSO-treated and poziotinib-treated groups. (B) Bar graphs of IC50 values ​​for MCF10A cell lines expressing HER2 Y772dupYVMA, HER2 G778dupGSP, and HER2 G776delinsVC treated with poziotinib, T-DM1, or poziotinib plus T-DM1 at the indicated doses. Bars represent mean ± SEM (n = 3 independent experiments); significance was determined by one-way ANOVA and Dunn's multiple comparison post-hoc analysis. (C) Tumor growth curves of HER2 Y772dupYVMA NSCLC PDXs treated with the indicated inhibitors. Poziotinib treatment was performed 5 days per week, and T-DM1 was administered once at the start of treatment. (D) Kaplan-Meier curves of progression-free survival (PFS), where PFS is defined as tumor doubling from best response. The Mantel-Cox log-rank test was used to determine significance between groups. Mice were censored at the time of euthanasia. (E) Dot plots of percent change in tumor volume in mice treated with the indicated inhibitors at day 15. (F) Diagram of the number of tumor-bearing mice in each group at days 15 and 45. (G) Spider plots of tumor volume in HER2 Y772dupYVMA mice treated with the indicated inhibitors. The red dotted line indicates the randomization point (300 mm3). [Figure 21] Figures 21A-21D: Exon 20 insertion mutation diversity varies by cancer type in the Guardant, cBioPortal, and MD Anderson databases. (A) Pie chart of HER2 exon 20 insertion mutation frequency in all cancer types, N=517. Exon 20 insertion mutation frequency was further analyzed by cancer type: (B) lung cancer, N=362, (C) breast cancer, N=30, and (D) other cancers, N=125. [Figure 22] Figures 22A-22B: Common HER2 mutations are constitutively phosphorylated, and p-HER2 expression does not correlate with drug sensitivity. (A) Relative p-HER2 expression was measured by using the ratio of p-HER2 to total HER2 determined by ELISA. Bars represent mean ± SEM, and n=3. ND=below detection limit. (B) Relative HER2 correlation was plotted against poziotinib IC50 values ​​for Ba / F3 HER2 mutant cell lines. Pearson correlation coefficients and p-values ​​were determined by GraphPad Prism (n=3). [Figure 23] Figures 23A-23B: Molecular modeling reveals that HER2 mutants have different binding pocket sizes. (A) HER2 kinase domain exon 19, 20, and 21 protein scaffolds colored blue, pink, and orange, respectively. Ligands from the template X-ray structure (PDB 3PP0) are depicted with green bars, and labels are given at mutated residues / insertion positions. (B) Binding pocket volume profiles for HER2 mutants obtained from accelerated molecular dynamics simulations. [Figure 24] Poziotinib inhibits p-HER2 in HER2 mutant cell lines. Western blot of MCF10A cells expressing G776delinsVC after 2 hours of treatment with the indicated drugs and doses. [Diagram 25]Poziotinib inhibits tumor growth in xenografts of exon 19 mutant colorectal cancer. CW-2 cells harboring the HER2 L755S mutation were injected into the flank of 6-week-old female nu / nu nude mice. When tumors reached 350 mm3, mice were randomized into 4 groups: 20 mg / kg afatinib, 5 mg / kg poziotinib, 30 mg / kg neratinib, or vehicle control. Tumor volumes were measured 3 times per week, and mice received drugs Monday-Friday (5 days per week). Symbols represent the mean ± SEM at each time point. Statistical significance was determined using two-way ANOVA with Tukey's multiple comparison test. Asterisks indicate significance between vehicle and poziotinib or neratinib. P values ​​for each comparison are listed below, starting from day 10, when significant differences were first detected. [Figure 26] Poziotinib is more effective than high-dose osimertinib in EGFR S768dupSVD PDX models. Female NSG mice 6-8 weeks of age were implanted with patient NSCLC tumor fragments harboring the EGFR S768dupSVD mutation, and when tumors reached 300 mm3, mice were randomized into four groups: vehicle control, poziotinib 2.5 mg / kg, osimertinib 5 mg / kg, or osimertinib 25 mg / kg. Drugs were administered to mice 5 days per week, and tumor volumes were measured three times per week. Symbols represent mean ± SEM tumor volume at each time point. Dot plots represent percent change in mean tumor volume at day 21, with each dot representing one mouse. [Figure 27]Poziotinib has higher antitumor activity than neratinib in PDX models of NSCLC harboring Y772dupYVMA. Female NSG mice 6-8 weeks of age were implanted with patient NSCLC tumor fragments harboring the HER2 Y772dupYVMA mutation, and when tumors reached 300 mm3, mice were randomized into three groups: vehicle control, poziotinib 2.5 mg / kg, or neratinib 30 mg / kg. Drugs were administered to mice 5 days per week, and tumor volumes were measured three times per week. Symbols represent mean ± SEM tumor volume at each time point. Dot plots represent percent change in mean tumor volume at day 21, with each dot representing one mouse. ANOVA was used to determine p values ​​for the indicated bars at the end of treatment time point. [Figure 28] Single agent poziotinib is more efficacious than neratinib in breast cancer PDXs harboring V777L. Female NSG mice 6-8 weeks of age were implanted with patient breast cancer tumor fragments harboring the HER2 V777L mutation, and when tumors reached 300 mm3, mice were randomized into three groups: vehicle control, poziotinib 2.5 mg / kg, or neratinib 30 mg / kg. Drugs were administered to mice 5 days per week, and tumor volumes were measured three times per week. Symbols represent mean ± SEM tumor volume at each time point. Dot plots represent percent change in mean tumor volume at day 30, with each dot representing one mouse. ANOVA was used to determine p values ​​for the indicated bars at the end of treatment time point. [Figure 29]Poziotinib has antitumor activity in various EGFR and HER2 exon 20 mutant in vivo models. In the PDX model, female NSG mice 6-8 weeks of age were implanted with the indicated tumor fragments carrying various EGFR or HER2 exon 20 mutations, and when tumors reached 300 mm3, mice were randomized into two groups: vehicle control or poziotinib 5 mg / kg. Drugs were administered to mice 5 days per week, and tumor volumes were measured three times per week. Bars represent the percent change in mean tumor volume at 4 weeks, and each dot represents one mouse. In the GEMM, tumors were induced by doxycycline diet, and tumor conformation by MRI was performed, and mice were treated daily for 4 weeks with vehicle or poziotinib 10 mg / kg. Bars represent the percent change in mean tumor volume at 4 weeks, and each dot represents one mouse, and tumor volumes were measured by MRI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Description of exemplary embodiments Although the majority of activating mutations in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are inherently resistant. This study used in silico, in vitro, and in vivo testing to model the structural changes induced by these exon 20 mutations and identify effective inhibitors. 3-D modeling revealed significant changes that limit the size of the drug binding pocket and impose constraints on the binding of large, rigid inhibitors. Poziotinib, due to its small size and flexibility, is able to circumvent these conformational changes and was found to be a potent and relatively selective inhibitor of EGFR or HER2 exon 20 mutant proteins. Poziotinib also has potent activity in mutant exon 20 EGFR or HER2 NSCLC patient-derived xenograft (PDX) models and genetically engineered mouse models. Thus, these data identify poziotinib as a potent, clinically active inhibitor of EGFR / HER2 exon 20 mutations and elucidate the molecular characteristics of kinase inhibitors that may circumvent the conformational changes induced by these insertions.

[0047] Thus, certain embodiments of the present disclosure provide a method for treating cancer patients with EGFR and / or HER2 exon 20 mutations, such as exon 20 insertions. Specifically, the method includes administering poziotinib (also known as HM781-36B) or afatinib to patients identified as having EGFR and / or HER exon 20 insertion mutations. The size and flexibility of poziotinib overcomes steric hindrance and inhibits EGFR and HER2 exon 20 mutants at low nanomolar concentrations. Thus, poziotinib or afatinib and structurally similar inhibitors are potent EGFR or HER2 inhibitors that can be used to target both EFGR and HER2 exon 20 insertions that are resistant to irreversible second and third generation TKIs.

[0048] I. Definition As used herein, "a" or "an" may include one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0049] Although the present disclosure supports definitions that refer to alternatives only and "and / or," use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive. As used herein, "another" can mean at least a second, or more.

[0050] The term "about" means ±5% of the indicated value.

[0051] "Treatment" or "treating" includes (1) inhibiting disease in a subject or patient experiencing or exhibiting disease pathology or symptomology (e.g., preventing further progression of the pathology and / or symptomology), (2) ameliorating disease in a subject or patient experiencing or exhibiting disease pathology or symptomology (e.g., reversing the pathology and / or symptomology), and / or (3) affecting any measurable reduction in disease in a subject or patient experiencing or exhibiting disease pathology or symptomology. For example, treatment can include administration of an effective amount of poziotinib.

[0052] "Prophylactically treating" includes (1) reducing or alleviating the risk of disease onset in a subject or patient who may be at risk and / or susceptible to the disease, but who has not yet experienced or displayed any or all of the disease's pathology or symptomatology, and / or (2) delaying the onset of disease pathology or symptomatology in a subject or patient who may be at risk and / or susceptible to the disease, but who has not yet experienced or displayed any or all of the disease's pathology or symptomatology.

[0053] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0054] The term "effective" as the term is used herein and / or in the 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 "pharmaceutical effective amount" means that the amount of the compound, when administered to a subject or patient for treating or preventing a disease, is sufficient to affect such treatment or prevention of the disease.

[0055] As used herein, the term "IC 50 " refers to an inhibitory dose, which is 50% of the maximal response obtained. This quantitative measurement indicates how much of a particular drug, or other substance (inhibitor) is needed to inhibit half of a particular biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0056] An "anti-cancer" agent can negatively affect cancer cells / tumors in a subject, for example, by promoting the killing of cancer cells, inducing apoptosis of cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing the size of a tumor, inhibiting the growth of a tumor, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the survival of a subject with cancer.

[0057] The term "insertion" or "insertion mutation" refers to the addition of one or more nucleotide base pairs to a DNA sequence. For example, an EGFR exon 20 insertion mutation can result in an insertion mutation of about 2-21 base pairs at amino acids 767-774. In another example, a HER2 exon 20 insertion mutation includes one or more insertions of 3-18 nucleotides at amino acids 770-785. Exemplary EGFR and HER exon 20 insertion mutations are illustrated in FIG. 1 of the present disclosure.

[0058] "Hybridize" or "hybridization" refers to the binding of nucleic acids. Hybridization conditions can vary depending on the sequence homology of the nucleic acids to be bound. Thus, when the sequence homology between the nucleic acids of interest is high, stringent conditions are used. When the sequence homology is low, moderate conditions are used. When hybridization conditions are stringent, hybridization specificity is increased, and this increase in hybridization specificity reduces the production of non-specific hybridization products. However, under moderate hybridization conditions, hybridization specificity is decreased, and this decrease in hybridization specificity increases the production of non-specific hybridization products.

[0059] "Probe" refers to a polynucleotide that has a length of at least 8 nucleotides and forms a hybrid structure with a target sequence due to the complementarity of at least one sequence in the probe with a sequence in the target region. The polynucleotide may be composed of DNA and / or RNA. The probe is detectably labeled in certain embodiments. The size of the probe may vary significantly. Generally, the length of the probe is, for example, at least 8-15 nucleotides long. Other probes are, for example, at least 20, 30, or 40 nucleotides long. Still other probes are somewhat longer, at least, for example, 50, 60, 70, 80, or 90 nucleotides long. The probe may also be of any particular length within the ranges. Preferably, the probe does not contain a sequence complementary to a sequence used to prime the target sequence during the polymerase chain reaction.

[0060] "Oligonucleotide" or "polynucleotide" refers to a polymer of single- or double-stranded deoxyribonucleotides or ribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.

[0061] "Modified ribonucleotides" or deoxyribonucleotides refer to molecules that can be used in place of natural bases in nucleic acids, 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 modifiers, terminal modifiers, spacer modifiers, and backbone-modified nucleotides, including, but not limited to, ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methylphosphonates, methylphosphoramidites, methylphosphonamidites, 5'-β-cyanoethylphosphoramidites, methylenephosphonates, phosphorodithioates, peptide nucleic acids, optically inactive and neutral internucleotide linkages.

[0062] "Variant" refers to a polynucleotide or polypeptide that differs from the wild type or form most prevalent in an individual population by the exchange, deletion, or insertion of one or more nucleotides or amino acids, respectively. The number of exchanged, deleted, or inserted nucleotides or amino acids 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.

[0063] "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. A primer can be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. A primer can contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of a primer are empirically determined. The lower limit of the length of a primer is the minimum length required to form a stable duplex upon hybridization with a target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3-4 nucleotides in length) do not form thermodynamically stable duplexes with a target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of having duplex formation in a region other than a predetermined nucleic acid sequence in the target nucleic acid. In general, suitable primer lengths are within the range of about 10 to about 40 nucleotides in length. In certain embodiments, for example, a primer can be 10 to 40, 15 to 30, or 10 to 20 nucleotides in length. A primer, when placed under appropriate conditions, is capable of acting as a point of initiation of synthesis on a polynucleotide sequence.

[0064] "Detection," "detectable," and their grammatical equivalents refer to a method of 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 a target nucleic acid can be characterized as "detecting" the target nucleic acid. The label attached to the probe can include any of a wide variety of labels 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.

[0065] "Amplify," "amplification," and their grammatical equivalents refer to any method that replicates at least a portion of a target nucleic acid sequence in a template-dependent manner, including, but not limited to, a wide variety of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for carrying out the amplification step include 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 strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, Cambridge, UK), and self-sustained sequence replication (3SR), as well as multiplex versions or combinations thereof, including, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as multiplex chain reaction-CCR), etc. Descriptions of such techniques are found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[0066] "EGFR" or "epidermal growth factor receptor" or "EGFR" refers to a tyrosine kinase cell surface receptor and is encoded by one of four alternative transcripts represented as GenBank Accession Nos. NM_005228.3, NM_201282.1, NM_201283.1, and NM_201284.1. A variant of EGFR contains an insertion in exon 20.

[0067] "HER2" or "ERBB2" is a member of the EGFR / ErbB family and is represented by GenBank Accession No. NM_004448.2. A variant of HER2 contains an insertion in exon 20.

[0068] Generally, as used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals, within the scope of sound medical judgment, with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic response, or other problem or complication.

[0069] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable as described above and has 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 with 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 acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanesulfonic acid, and the like. Included in the acid addition salts are those formed with organic acids such as pionic 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 acid, 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 may be formed when 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 particular anion or cation forming a part of any salt of this invention is not hazardous so long as the salt, as a whole, is pharmacologically acceptable.Further examples of pharma- ceutically acceptable salts and their methods of 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).

[0070] II. EGFR and HER2 exon 20 mutations Certain embodiments of the present disclosure relate to determining whether a subject has one or more EGFR and / or HER2 exon 20 mutations, such as insertion mutations, particularly one or more of the insertion mutations depicted in FIG. 1. A subject may have two, three, four, or more EGFR exon 20 mutations and / or HER2 exon 20 mutations. Mutation detection methods are known in the art and include PCR analysis and nucleic acid sequencing, as well as FISH and CGH. In certain aspects, exon 20 mutations are detected by DNA sequencing, such as from tumor or plasma-derived circulating free DNA.

[0071] The EGFR exon 20 mutations may include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 to 778. The one or more EGFR exon 20 mutations may be located at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, V774, and R776.

[0072] EGFR exon 20 insertions include H773_V774insH, A767_v769ASV, N771_P772insH, D770_N771insG, H779_V774insH, N771delinsHH, S768_D770dupDVD, A767_V769dupASV, A767_V769dupASV, and P772_H773d up, N771_H773dupNPH, S768_D770dupSVD, N771delinsGY, S768_D770delinsSVD, D770_D770delinsGY, A767_V769dupASV, H773dup, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and / or V774insHV. In certain embodiments, the exon 20 mutation is A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, and / or N771dupNPH.

[0073] In some embodiments, the subject may have or develop a mutation at EGFR residue C797, which may lead to resistance to TKIs 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, the subject with a T790 mutation, such as T790M, may be administered osimertinib, and the subject with a C797 mutation, such as C797S, may be administered chemotherapy and / or radiation therapy.

[0074] The HER2 exon 20 mutation may comprise one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770 to 785. The one or more HER2 exon 20 mutations may be present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. The one or more HER2 exon 20 mutations may be A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and / or L786V.

[0075] The patient sample can be any body tissue or fluid that contains nucleic acid from lung cancer in a 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 tissue, for example, lung tissue. The lung tissue can be derived from tumor tissue and can be fresh frozen or formalin-fixed, paraffin-embedded (FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

[0076] Suitable samples 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 mucosal scrapings of oral mucosa, but can also be extracted from other biological samples, including urine, tumors, or expectorants. The sample itself typically includes nucleated cells (e.g., blood or buccal cells) or tissues removed from a 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, for example, by drawing blood. In some embodiments, the sample is obtained without the cooperation of a medical institution, for example, the sample is obtained non-invasively, such as a sample including buccal cells obtained using a buccal swab or brush, or a mouthwash sample.

[0077] In some cases, the biological sample 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 collected from the biological sample using techniques well known in the art. For example, cells can be collected by centrifuging the 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, e.g., gDNA. See, e.g., Ausubel et al. (2003). The sample can be concentrated and / or purified to isolate DNA. All samples obtained from a subject, including those that are subjected to any kind of further processing, are considered to have been obtained from the subject. Genomic DNA can be extracted from the biological sample using routine methods, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted using kits such as QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.) and Wizard® Genomic DNA Purification Kit (Promega). Non-limiting examples of sample sources include urine, blood, and tissue.

[0078] The presence or absence of EGFR or HER2 exon 20 mutations, such as exon 20 insertion mutations, 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 insertion mutations. If desired, amplification of nucleic acid 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, such as a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe, of the gene in genomic DNA, RNA, or cDNA. The nucleic acid probe can be designed to specifically or preferentially hybridize with a particular mutation.

[0079] A set of probes typically refers to a set of primers (usually a primer pair) and / or detectably labeled probes used to detect target gene mutations (e.g., EGFR and / or HER2 exon 20 mutations) used in the present disclosure's possible recommended treatments. The primer pairs are used in an amplification reaction to define amplicons spanning the region for the target gene mutations for each of the genes. The set of amplicons is detected by a set of matched probes. In an exemplary embodiment, the method can use TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assays to detect a set of target gene mutations, e.g., EGFR and / or HER2 exon 20 mutations. In one embodiment, the set of probes is a set of primers used to generate amplicons that are detected by a nucleic acid sequencing reaction, such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) technology can be used.

[0080] Analysis of nucleic acid markers can be performed using techniques well known in the art, including but not limited to sequence analysis and electrophoretic analysis.Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., 1992), solid-phase sequencing (Zimmerman et al., 1992), sequencing with mass spectrometry, such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., 1998), and 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. Additionally, next generation sequencing can be performed using commercially available kits and equipment 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.

[0081] 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., 1996); and denaturing gel electrophoresis (DHPLC, Underhill et al., 1996). et al., 1997) infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry (WO 99 / 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 resolution (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, as well as combinations of such methods. See, e.g., U.S. Patent Application Publication No. 2004 / 0014095, which is incorporated herein by reference in its entirety.

[0082] In one example, a method for identifying EGFR and / or HER2 mutations in a sample comprises contacting a nucleic acid from such sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding a mutated EGFR or HER2 protein, or a fragment thereof that contains the mutation, and detecting said hybridization. In a particular embodiment, such a probe is a probe that is ... for example, a radioisotope ( 3 H, 32 P, or 33The probe is detectably labeled with an ion exchange polymerase (IP), a fluorescent agent (rhodamine, or fluorescein), or a coloring agent. In a particular embodiment, the probe is an antisense oligomer, such as PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be about 8 nucleotides to about 100 nucleotides, or about 10 to about 75, or about 15 to about 50, or about 20 to about 30. In another aspect, such a probe of the disclosure is provided in a kit for identifying EGFR or HER2 mutations in a sample, the kit comprising an oligonucleotide that specifically hybridizes to or is adjacent to a mutation site in the EGFR or HER2 gene. The kit further comprises instructions for treating a patient having a tumor containing an EGFR or HER2 insertion mutation with poziotinib or afatinib based on the results of a hybridization test using the kit.

[0083] In another embodiment, a method for detecting an exon 20 mutation in a sample comprises amplifying such a nucleic acid sample corresponding to exon 20 of such EGFR gene or HER2 or a fragment thereof suspected of containing a mutation, and comparing the electrophoretic mobility of the amplified nucleic acid with that of the corresponding wild-type EGFR or HER2 gene or a fragment thereof. A difference in mobility indicates the presence of a mutation in the amplified nucleic acid sequence. Electrophoretic mobility can be measured on a polyacrylamide gel.

[0084] Alternatively, nucleic acids can be analyzed for detection of mutations using enzymatic mutation detection (EMD) (Del Tito et al., 1998). EMD uses the bacteriophage resolvase T4 endonuclease VII, which scans along double-stranded DNA until it detects and resolves structural distortions caused by base pair mismatches resulting from point mutations, insertions, and deletions. Detection of two short fragments formed by resolvase degradation, for example by gel electrophoresis, indicates the presence of a mutation. The advantage of the EMD method is a single protocol for identifying point mutations, deletions, and insertions that are assayed directly from the PCR reaction, eliminating the need for sample purification, shortening hybridization times, and increasing the signal-to-noise ratio. Mixed samples containing up to 20-fold expression 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 steps are required to identify the mutation. As demonstrated in US Pat. No. 5,869,245, the CEL I enzyme can be used as well as resolvase T4 endonuclease VII.

[0085] III. Treatment method Further provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of poziotinib, afatinib, or a structurally similar inhibitor to a subject determined to have an EGFR and / or HER2 exon 20 mutation, e.g., an exon 20 insertion. The subject may have one or more EGFR and / or HER exon 20 mutations.

[0086] 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, preneoplastic lesions of the lung, colon cancer, melanoma, and bladder cancer. In certain aspects, the cancer is non-small cell lung cancer.

[0087] In some embodiments, the subject is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient with or at risk of having a disorder described herein). In one embodiment, the subject is in need of an enhanced immune response. In certain embodiments, the subject is or is at risk of being susceptible to infection. For example, the subject is undergoing or has undergone chemotherapeutic treatment and / or radiation therapy. Alternatively, or in combination, the subject is or is at risk of being susceptible to infection as a result of infection.

[0088] 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 subjects determined to have an EGFR or HER2 exon 20 mutation, such as an exon 20 insertion. Poziotinib is a quinazoline 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. Pat. 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 present invention.

[0089] 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 third day, or once a week. Administration may be on a continuous schedule, for example, a 28-day cycle.

[0090] In some embodiments, subjects with T790 mutations, such as T790M, can be administered osimertinib, and subjects with C797 mutations, such as C797S, can be administered chemotherapy and / or radiation therapy as described herein. Osimertinib administration, chemotherapy, and / or radiation administration can be performed alone or in combination with poziotinib. Osimertinib can be administered at a dose of 25 to 100 mg, for example, about 40 or 80 mg. Administration can be daily, every other day, every second day, every third day, or once a week. Osimertinib can be administered orally, for example, in tablets.

[0091] Afatinib may be administered at a dose of 10-50 mg, for example, 10, 20, 30, 40, or 50 mg.

[0092] B. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising poziotinib or afatinib and a pharma- ceutical acceptable carrier for a subject determined to have an EGFR or HER2 exon 20 mutation, e.g., an exon 20 mutation.

[0093] 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 pharma- ceutical acceptable carriers (see Remington's Pharmaceutical Sciences 22, 2003) in the form of a lyophilized formulation or an aqueous solution. ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium 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 dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharma- ceutically acceptable carriers herein further include insterstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0094] C. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment comprise poziotinib or afatinib in combination with at least one additional therapy.The additional therapy can be radiation therapy, 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 of the above.The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0095] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or a metastasis inhibitor. In some embodiments, the additional therapy is administration of an agent with reduced side effects (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy 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 known in the art.

[0096] Poziotinib or afatinib may be administered before, during, after, or in various combinations with additional cancer therapy, such as immune checkpoint therapy. Administration may range from simultaneous administration, to minutes apart, days apart, or weeks apart. In embodiments where poziotinib or afatinib is provided to the patient separately from the additional therapeutic agent, it is generally ensured that no significant period expires between each delivery time, so that the two compounds can still provide a beneficial combined effect to the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 hours of each other. In some circumstances, it may be desirable to provide an interval of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration, significantly extending the period for treatment.

[0097] Various combinations may be used. In the examples below, poziotinib or afatinib is "A" and the anti-cancer therapy is "B". TIFF0007650814000001.tif18128

[0098] Administration of any compound of the present embodiments or treatment regimen to a patient will follow standard protocols for administration of such compounds, taking into account the toxicity of the drug, if any. Thus, in some embodiments, there is a step of monitoring toxicity resulting from the combination therapy.

[0099] 1.Chemotherapy A variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to indicate a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within a cell, for example, whether they affect the cell cycle and which stage of the cell cycle they affect. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intervene in DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0100] 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; ethylenimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; catistatin; CC-1065 (including its azozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongiostatin; nitrogen mustards, such as chlorambucil, chlornaphazine, clophosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma lI and calicheamicin omega I1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin). sorbicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, protease inhibitors, and folate inhibitors. teropterin, and trimetrexate;purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifuridine, enocitabine, and floxuridine;androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;antiadrenal agents, such as mitotane and trilostane;folic acid supplements, such as For example, folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxatrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin;losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxine; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and and carboplatin; vinblastine; platinum; etopside (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluorodifluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0101] 2. Radiation therapy Other agents that cause DNA damage and are widely used include gamma radiation, known as X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, including microwave ovens, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV irradiation. All of these agents likely affect a wide range of damage to DNA, to the precursors of DNA, to DNA replication and repair, and to chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens for extended periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0102] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination or in conjunction with the method of this embodiment. 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 one example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can be used as the effector of the therapy, or the antibody can recruit other cells that actually act on the 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 with a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0103] Antibody-drug conjugates have emerged as a breakthrough approach in the development of cancer treatments. 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 MAbs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver payloads (drugs) to tumor cells with abundant levels of antigen. Targeted delivery of drugs also minimizes exposure of drugs in normal tissues, resulting in reduced toxicity and improved therapeutic index. 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, validates this approach. Currently, more than 30 ADC drug candidates are in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs depends heavily on the identification and validation of novel targets suitable for this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high level expression in tumor cells and robust internalization.

[0104] In one aspect of immunotherapy, tumor cells must have some markers that are suitable for targeting (i.e., not present in the majority of other cells). Many tumor markers exist, and any of these may be suitable for targeting in the context of this 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 anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines, e.g., IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, e.g., MIP-1, MCP-1, IL-8, and growth factors, e.g., FLT3 ligand.

[0105] Examples of immunotherapy include immune adjuvants, 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 interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1999). 8; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent 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. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0106] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. An immune checkpoint strengthens or weakens a signal (e.g., a costimulatory molecule). Inhibitory immune checkpoints that can be targeted by immune checkpoint interference 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 inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 system and / or CTLA-4.

[0107] The immune checkpoint inhibitor can be a drug, e.g., a small molecule, a recombinant form of a ligand or receptor, or is particularly 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 of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies. Those skilled in the art will recognize that alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this specification. For example, it is known that lambrolizumab is also known as MK-3475 and pembrolizumab, which are alternative and equivalent names.

[0108] 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 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.

[0109] 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 that includes an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an 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 WO 2006 / 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.

[0110] 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 costimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called 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 CTLA-4, an inhibitory receptor for the B7 molecule.

[0111] 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.

[0112] Suitable anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) for use in the present method can be made using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129; International Patent Publication Nos. WO01 / 14424, WO98 / 42752, and WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab); U.S. Patent No. 6,207,156; Hurwitz et al., 1998; Camacho et al., 2004; and Mokyr et al., 1998 can be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, and WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0113] An exemplary anti-CTLA-4 antibody is 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 binds to and / or competes for binding to the same epitope on CTLA-4 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence homology with the above antibodies (e.g., at least about 90%, 95%, or 99% variable region homology with ipilimumab).

[0114] Other molecules for regulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Nos. WO1995 / 001994 and WO1998 / 042752; all incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, incorporated herein by reference.

[0115] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, 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 may be combined with other therapies, such as the treatment 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 treatment includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0116] Removal of part or all of the cancer cells, tissue, or tumor may result in the formation of a cavity in the body. Treatment can be accomplished by perfusion, direct injection, or local application of the area with additional anticancer 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 at varying doses.

[0117] 5. Other drugs It is contemplated that other agents may be used in combination with certain 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, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intracellular signaling by increasing the number of GAP junctions may increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effect 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 antibody c225, may be used in combination with certain aspects of the present embodiment to improve the therapeutic effect.

[0118] IV. Kit Kits for detecting EGFR and / or HER2 exon 20 mutations, such as those disclosed herein, are also within the scope of this disclosure. One example of such a kit may include a set of primers specific for exon 20 mutations. The kit may further include instructions for using the primers to detect the presence or absence of a particular EGFR and / or HER2 exon 20 mutation described herein. The kit may further include instructions for diagnostic purposes indicating that positive identification of EGFR and / or HER2 exon 20 mutations described herein in a sample from a cancer patient is indicative of sensitivity to the tyrosine kinase inhibitors poziotinib or afatinib or structurally similar inhibitors. The kit may further include instructions indicating that positive identification of EGFR and / or HER2 exon 20 mutations described herein in a sample from a cancer patient indicates that the patient should be treated with poziotinib, afatinib, or structurally similar inhibitors. EXAMPLES

[0119] V. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow are techniques that the inventors have found to function well in the practice of the invention and therefore can be considered as constituting preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that numerous changes can be made in the specific embodiments disclosed herein and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0120] Example 1 - Identification of drugs against cancer cells with EGFR or HER exon 20 insertions Clinical response to TKIs was examined in patients whose tumors harbored EGFR exon 20 insertions in the clinical database; among 280 patients with EGFR mutant NSCLC treated with single-agent erlotinib, gefitinib, or afatinib, 129 patients were identified as harboring classical EGFR mutations (exon 19 deletions, L858R, and L861Q) and 9 patients were identified as harboring EGFR exon 20 insertions. NSCLC patients with classical EGFR mutations had a median PFS of 14 months, whereas patients with EGFR exon 20 insertions had a median PFS of only 2 months (p<0.0001, log-rank test; Figure 1A). Of the 9 EGFR exon 20 insertion patients, an OR was observed in only one patient harboring an S768del-insIL mutation who received afatinib (Figure 4A). This clinical data demonstrates that available EGFR TKIs have limited activity in EGFR exon 20 insertion-driven NSCLC and confirms that alternative treatment strategies are required in these specific tumors.

[0121] As a first step in drug screening, seven EGFR mutations and eleven HER2 mutations were expressed in Ba / F3 cells. The locations of EGFR and HER2 exon 20 mutations are summarized in Figure 1B. To evaluate which exon 20 mutations of EGFR and HER2 were activating, Ba / F3 cell lines were screened for IL-3-independent survival. All EGFR exon 20 insertions tested were found to be activating mutations (Figure 4B), and six HER2 exon 20 mutations and L755P located in exon 19 were found to be activating mutations (Figure 4C). Next, the sensitivity to clinically evaluated EGFR and HER2 TKIs, including reversible TKIs (first generation), irreversible TKIs (second generation), and irreversible mutant-specific TKIs (third generation), was tested for exon 20 insertions and then compared with the sensitivity to the classical susceptibility mutation EGFR L858R. With the exception of EGFR A763insFQEA, EGFR exon 20 insertions (n ​​= 6) were found in the first generation ( Fig. 1C , IC 50 =3.3 to >10 μM), second generation (Fig. 1d, IC 50 = 40–135 nM), and the third generation (Fig. le, IC 50 = 103-850 nM) (Figure 5, Table 1). In addition, HER2 exon 20 mutants (n = 6) were resistant to first-generation (Figure 1F, IC 50 = 1.2–13 μM) and the third generation (Figure 1H, IC 50 = 114-505 nM). Second-generation TKIs have some activity against Ba / F3 HER2 exon 20 mutant cell lines (Figure 1G, IC 50 =10-12 nM, Figure 6, Table 1). Consistent with the drug screening, Western blotting showed that, except for EGFR A763insFQEA, which showed partial inhibition at lower doses, erlotinib and osimertinib did not significantly inhibit p-EGFR2 in EGFR exon 20 insertion mutants, and significantly inhibited p-HER2 in HER2 exon 20 insertion mutants only at 500 nM (Figure 7A-D).

[0122] Table 1. IC50 values ​​of EGFR and HER2 exon 20 insertions by EGFR / HER2 TKIs TIFF0007650814000002.tif98155

[0123] To investigate why exon 20 insertions confer resistance to first- and third-generation EGFR TKIs, we performed 3-D modeling of the solved crystal structure of EGFR T790M with EGFR D770insNPG and EGFR WT to visualize changes within the drug-binding pocket. Modeling revealed that EGFR exon 20 insertions resemble the T790M mutation in the alignment of the gatekeeper residue T790, resulting in increased affinity for ATP and reduced binding of first-generation inhibitors, making these mutations resistant to noncovalent inhibitors. In addition, HER2 exon 20 insertions induced a constitutively active conformation, preventing the binding of the noncovalent HER2 inhibitor lapatinib, which binds to HER2 in an inactive conformation. Furthermore, EGFR and HER2 exon 20 insertions have dramatic effects on the drug-binding pocket. In silico modeling of EGFR (Figure 1I) and HER2 (Figure 1J) exon 20 insertions revealed a significant shift of the α-c-helix (arrow) into the drug binding pocket (Figure 1J) due to the insertion at the C-terminus of the α-c-helix, which forces the ridged configuration of the α-c-helix to an inward, activated position. In addition, 3-D modeling also showed a significant shift of the P-loop into the drug binding pocket (Figure 1I, 1J) of both receptors. These shifts together result in steric hindrance of the drug binding pocket from two directions in both EGFR and HER2 exon 20 mutant proteins. Consistent with the in vitro studies described above, 3-D modeling supports the observation that afatinib inhibits exon 20 insertions more effectively than osimertinib. Osimertinib has a large terminal 1-methylindole group directly linked to a rigid pyrimidine core. This large, inflexible group reduces osimertinib's ability to reach the C797 residue as efficiently as afatinib in EGFR exon 20 insertions (Figure 1I). Alternatively, afatinib has a smaller 1-chloro-2-fluorobenzene ring end group that is indirectly linked to the quinazoline core via a secondary amine group, allowing afatinib to fit within the sterically hindered binding pocket.Furthermore, steric hindrance prevents the binding of osimertinib to HER2 A775insYVMA. Collectively, in vitro data and in silico modeling indicate that small, flexible quinazoline derivatives may be able to target EGFR / HER2 exon 20 insertions.

[0124] We next sought to identify TKIs with enhanced activity against the exon 20 insertion. Poziotinib, like afatinib, contains a small terminal group and a flexible quinazoline core. On the other hand, poziotinib has a smaller substituent linking the Michael Acceptor group to the quinazoline core compared to afatinib, and increased halogenation of the terminal benzene ring compared to afatinib. This electron-rich moiety also interacts with basic residues of EGFR, such as K745, further stabilizing the binding. To that end, poziotinib was tested in the Ba / F3 system. In vitro, poziotinib potently inhibited the growth of EGFR exon 20 mutant Ba / F3 cell lines (Figure 2A) and HER2 exon 20 mutant Ba / F3 cells (Figure 2B). Poziotinib had an average IC of 1.0 nM in EGFR exon 20 mutant Ba / F3 cell lines. 50 poziotinib was approximately 100-fold more potent than osimertinib and 40-fold more potent than afatinib in vitro. Furthermore, poziotinib had a mean IC 50 poziotinib was >200-fold more potent than osimertinib and >6-fold more potent than afatinib in vitro. These results were verified by Western blotting, where poziotinib inhibited EGFR and HER2 phosphorylation at concentrations as low as 5 nM (Figures 2C, 8A). To further verify that poziotinib sensitivity was not due to the level of expression of EGFR or HER2 mutants, the expression of each mutant was measured by ELISA and then analyzed by IC 50 The IC values ​​were plotted against the IC values ​​(Figure 8D). 50No correlation was found between IC and expression (R = -0.056, p = 0.856), whereas in EGFR, a correlation was found between poziotinib sensitivity and the location of the EGFR mutation (R = 0.687, p = 0.044) (Figure 2D), with IC increasing as the insertion was further away from the α-c-helix. 50 The results suggest that poziotinib inhibits proliferation of patient-derived cell lines CUTO14 (EGFR A767dupASV) and YUL0019 (EGFR N771del insFH) with mean IC50 of 1.84 nM and 0.30 nM, respectively. Interestingly, this correlation was not observed for HER2 exon 20 mutations, where the size of the insertion rather than the location of the insertion was more variable (Figure 8E). This correlation suggests that the exact location of the mutation may have different effects on the drug binding pocket, contributing to the heterogeneity of drug response observed. In addition, poziotinib inhibited proliferation of patient-derived cell lines CUTO14 (EGFR A767dupASV) and YUL0019 (EGFR N771del insFH) with mean IC50 of 1.84 nM and 0.30 nM, respectively. 50 poziotinib effectively inhibited p-EGFR at 1000 nM levels, which was >15-fold more potent than afatinib in CUT014 and >100-fold more potent than afatinib in YUL0019 (Figure 2E,F). Western blotting of the CUT014 cell line determined that there was significant inhibition of p-EGFR with 10 nM poziotinib treatment, but p-EGFR was not significantly inhibited by afatinib up to 1000 nM (Figure 8B,C).

[0125] To determine the specificity of poziotinib in inhibiting exon 20 mutants compared with the T790M mutant, we compared the IC of afatinib, osimertinib, rociletinib, and poziotinib at exon 20 mutants. 50 Values ​​are the IC of afatinib, osimertinib, rociletinib, and poziotinib in EGFR T790M mutant Ba / F3 cell lines. 50 The IC 50Values ​​are displayed normalized to the single EGFR T790M mutation, with values ​​less than 1 indicating specificity for the exon 20 insertion compared to T790M (Figure 2G). Compared to EGFR T790M mutants, EGFR exon 20 insertions were >65-fold more sensitive to poziotinib. Furthermore, EGFR exon 20 insertion mutations were >1.4-fold more resistant to afatinib, >5.6-fold more resistant to osimertinib, and >24-fold more resistant to rociletinib than EGFR T790M mutants (Figure 2G).

[0126] To investigate why poziotinib selectively and potently inhibits the exon 20 mutant compared to the T790M mutation, but not third-generation TKIs such as osimertinib, 3-D modeling was performed to determine how changes in the drug-binding pocket affect drug binding. Osimertinib fits into the drug-binding pocket of the EGFR T790M mutant receptor (Figure 2H), whereas in the exon 20 mutant, the large changes in the binding pocket (Figure 2I) sterically hindered the binding of the third-generation inhibitor. On the other hand, poziotinib is smaller and has greater flexibility, allowing poziotinib to fit within the sterically hindered exon 20 binding pocket (Figure 2I). Furthermore, 3-D modeling of EGFR D770insNPG with poziotinib and afatinib suggests that the shifted P-loop within the drug-binding pocket allows poziotinib to bind more tightly within the binding pocket than afatinib. Structural modeling calculations show that the free energy of binding (London ΔG) for poziotinib is lower than that of afatinib, indicating a stronger binding affinity of poziotinib. 3-D modeling of WT HER2 with osimertinib shows that the binding pocket of WT HER2 is larger than that of HER2 A775insYVMA. Thus, poziotinib binds tightly deep within the sterically hindered drug binding pocket of HER2 A775insYVMA, overcoming the conformational change induced by the exon 20 insertion.

[0127] The efficacy of poziotinib was tested in vivo using GEM models of EGFR and HER2 exon 20 insertion-induced NSCLC. Lung tumors were induced in previously described EGFR D770insNPG (Cho et al., 2013) and HER2 A775insYVMA (Perera et al., 2009) mice and animals received poziotinib (10 mg / kg) or vehicle control daily for 4 weeks. As determined by MRI, poziotinib reduced tumor burden by 85% in EGFR exon 20 GEMMs (Figure 3A,C) and 60% in HER2 exon 20 GEMMs (Figure 3B,D), a higher level of inhibition than the 37% previously observed with afatinib in the same GEM models. Representative MRI images of tumors before and after poziotinib are shown for both EGFR and HER2 GEMMs (Figure 3C,D). In both EGFR and HER2 GEM models, mice treated with 10 mg / kg poziotinib showed sustained regression without signs of progression at week 12 (Figure 3E,F). In addition, poziotinib treatment (5 or 10 mg / kg) completely reduced tumors (>85% inhibition) by day 14 in the EGFR exon 20 insertion PDX model LU0387 (H773insNPH) (Figure 3G).

[0128] To determine whether poziotinib, like other irreversible inhibitors, binds covalently at C797, we engineered a Ba / F3 cell line that harbors the C797S mutation observed in approximately 30% of patients with osimertinib resistance (Thress et al., 2015). 50 In patients with poziotinib, induced resistance to poziotinib at baseline and baseline ≥ 100 mg / kg / day was observed. Together, these studies suggest that poziotinib may be susceptible to mechanisms of acquired resistance similar to other third-generation TKIs.

[0129] To verify the above findings, we performed a study using breast cancer cell line MCF10A with HER2 G776del insVC. The cells were treated with different inhibitors at various doses, and it was found that the breast cancer cell line was sensitive to poziotinib as was observed in other cell lines tested (Figure 10). Therefore, poziotinib can be used in the treatment of other cancers with exon 20 mutations.

[0130] Thus, exon 20 mutants were observed to confer de novo resistance to first-, second-, and third-generation TKIs. Using 3-D modeling of EGFR D770insNPG and HER2 A775insYVMA, poziotinib was identified as possessing structural features that may overcome the alterations in the drug-binding pocket induced by the insertion in exon 20. Furthermore, the predicted activity of poziotinib was confirmed using in vitro and in vivo models that demonstrated potent antitumor activity of poziotinib in cells harboring these mutations.

[0131] Poziotinib was found to be approximately 40-fold more potent than afatinib and 65-fold more potent than dacomitinib in EGFR exon 20 mutants. Additionally, poziotinib was 6-fold more potent than afatinib and dacomitinib in HER2 exon 20 mutants in vitro. Taken together, these data indicate that poziotinib shares a similar quinazoline scaffold with afatinib and dacomitinib, but additional features of the kinase inhibitor result in increased activity and relative specificity for EGFR exon 20 mutations compared to the more common T790M mutation.

[0132] 3-D modeling suggests that poziotinib's smaller size, increased halogenation, and flexibility confer a competitive advantage to the inhibitor in the sterically hindered drug-binding pocket of exon 20 mutant EGFR / HER2. A negative correlation was observed between the distance of the mutation from the α-c-helix and drug sensitivity. This association suggests that the exact location of the mutation affects the drug-binding pocket and / or binding affinity of the TKI. Furthermore, the data showed that the size of the insertion also affects drug sensitivity. Furthermore, a patient-derived cell line with a net gain of one amino acid, YUL0019 (N771del insFH), was more sensitive to quinazoline-based pan-HER inhibitors than cells with a larger EGFR exon 20 insertion.

[0133] Example 2 - Materials and Methods Patient population and statistical analysis: We identified patients with EGFR mutant NSCLC enrolled in the prospectively collected MD Anderson Lung Cancer Moon Shot GEMINI database. EGFR mutation status was determined using one of the PCR-based next-generation sequencing of a panel of 50, 134 or 409 genes used in routine clinical care. The Kaplan-Meier method was used to calculate PFS. PFS is defined as the time from initiation of EGFR TKI to radiological progression or death. Resting scans were obtained at 6-8 week intervals during treatment and retrospectively evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, to determine response rates in patients with EGFR exon 20 insertion NSCLC.

[0134] Cell line generation and IL-3 deprivation: Ba / F3 cell lines were cultured under sterile conditions in complete RPMI-1640 (R8758; Sigma Life Science) medium supplemented with L-glutamine, 10% heat-inactivated FBS (Gibco), 1% penicillin / streptomycin (Sigma Life Science), and 10 ng / ml mouse IL-3 (R&D systems). Stable cell lines were generated by retroviral transduction of Ba / F3 cell lines for 12 h. Retroviruses were generated by transfecting pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 2 into Phoenix 293T ampho-packing cell line (Orbigen) using Lipofectamine 2000 (Invitrogen). 72 h after transduction, 2 μg / ml puromycin (Invitrogen) was added to the medium. After 5 days of selection, cells were stained with FITC-HER2 (Biolegend) or PE-EGFR (Biolegend) and sorted by FACS. Cell lines were then grown for 15 days in the absence of IL-3 and cell viability was measured every 3 days using the Cell Titer Glo assay (Progema). The resulting stable cell lines were maintained in complete RPMI-1640 medium as described above without IL-3. HCC827 and HCC4006 lung cancer cell lines were obtained from ATCC and maintained in 10% RPMI medium under sterile conditions. Cell line identity was confirmed by DNA fingerprinting with short tandem repeats using the PowerPlex 1.2 kit (Promega). Fingerprinting results were compared to reference fingerprints maintained by the primary source of the cell lines. All cell lines were mycoplasma-free. To generate erlotinib-resistant cell lines, HCC827 and HCC4006 (both EGFR mutant) cells were cultured with increasing concentrations of erlotinib until resistant variants emerged.

[0135] Table 2. Vectors used to generate stable cell lines TIFF0007650814000003.tif253166

[0136] Cell viability assay and IC 50 Estimation: Cell viability was measured using the Cell Titer Glo assay (Promega). Cells were harvested from suspension medium, spun down at 300×g for 5 min, resuspended in fresh RPMI medium, and counted using a Countess automated cell counter and trypan blue (Invitrogen). 1500 cells were seeded per well in technical triplicates in 384-well plates (Greiner Bio-One). Cells were treated with seven different concentrations of inhibitors or vehicle alone in serial 3-fold dilutions of TKI in a final volume of 40 μL per well. After 72 hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 10 min and bioluminescence was measured using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). Bioluminescence values ​​were normalized to DMSO-treated cells and normalized values ​​were plotted in GraphPad Prism using a nonlinear regression fit to the normalized data with variable slope. IC 50 Values ​​were calculated by GraphPad Prism at 50% inhibition. Each experiment was repeated three times unless otherwise stated.

[0137] Tyrosine kinase inhibitors: lapatinib, afatinib, dacomitinib, AZD9291, CO-1686, EGF816, ibrutinib, and HM781-36B were purchased from Selleck Chemical. Erlotinib and gefitinib were obtained from the in-house pharmacy at The University of Texas MD Anderson Cancer Center. BI-694 was provided by Boehringer-Ingelheim. All inhibitors were dissolved in DMSO at a concentration of 10 mM and stored at -80°C.

[0138] 3-D modeling: The structure of EGFR D770insNPG protein was retrieved (Protein Data Bank entry code: 4LRM) and used as a template to build a molecular 3-D structural model of EGFR D770insNPG. HER2 A775insYVMA was built using the model previously published in Shen et al. A homology model was constructed using MODELLER 9v6 and further energetically minimized using the Molecular Operating Environment software package (Chemical Computing Group, Montreal, Canada). Molecular docking of TKIs to exon 20 mutant EGFR and HER2 was performed with default parameters using GOLD software, unless otherwise stated. Premature termination was prohibited in the docking process. Restraints were used to model the covalent bond formation between the receptor and the inhibitor. Flexibility of residues in the binding pocket was accommodated using GOLD software. Diagrams showing the correlation between EGFR / HER2 and inhibitors were visualized using PYMOL.

[0139] Western blotting of Ba / F3 mutants: For western blotting, cells were washed with phosphate-buffered saline and lysed with protein lysis buffer (ThermoFisher) and protease inhibitor cocktail tablets (Roche). Protein (30–40 μg) was loaded onto gels purchased from BioRad. BioRad semi-dry transfer was used and then probed with antibodies against pEGFR (#2234), EGFR (#4267), pHER2 (#2247), and HER2 (#4290) (1:1000; Cell Signaling). Blots were probed with antibodies against β-actin (Sigma-Aldrich, #A2228) or vinculin (Sigma-Aldrich, #V4505) as loading controls and exposed using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) and BioRad's ChemiDoc Touch Imaging System or radiographic film. Representative images of two different protein isolations are shown, with blots run in duplicate. Quantification of Western blots was completed in Photoshop and calculated as (background average intensity - sample average intensity) (number of pixels) = band intensity. Samples were first normalized to loading controls (β-actin or vinculin) and then normalized to DMSO and graphed in GraphPad Prism. Significance from DMSO was calculated in GraphPad Prism.

[0140] ELISA and correlation of Ba / F3 mutants: Protein was harvested from the parental Ba / F3 cell line and each of the Ba / F3 exon 20 mutants found to be activating mutations described above. ELISAs were performed as described in the manufacturer's instructions for total EGFR (Cell Signaling, #7250) and total HER2 (Cell Signaling, #7310). Relative expression measured by ELISA was calculated as IC50 calculated as described above. 50 The mean mean and mean p-values ​​were plotted against the mean mean and p-values, respectively. Pearson correlation coefficients and p-values ​​were determined by GraphPad Prism.

[0141] Patient-derived cell line testing: CUTO14 cells were generated from pleural effusion of a patient with lung adenocarcinoma after informed consent using culture methods previously described (Davies et al., 2013). Cell lines were treated with the indicated doses of afatinib or poziotinib for 72 h and cell viability was measured by MTS assay (Promega). IC50 was calculated as previously described (n=3). Western blotting with patient-derived cell lines was completed as previously described (Hong et al., 2007) (n=3). Cells were treated with the indicated doses of afatinib or poziotinib for 2 h. All antibodies were purchased from Cell Signaling Technology, except for total EGFR (BD Transduction Laboratories) and GAPDH (Calbiochem).

[0142] The YUL0019 cell line was established under an IRB approved protocol from malignant pericardial fluid obtained from a patient with advanced adenocarcinoma of the lung. The cell line was cultured in RPMI+L-glutamine (Corning) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals) and 1% penicillin / streptomycin (Corning). To confirm the presence of EGFR mutations, RNA was extracted from cell pellets using the RNeasy mini kit (Qiagen #74104) according to the manufacturer's instructions. cDNA was synthesized using the Superscript III First-Strand cDNA Synthesis kit (Invitrogen #18080-051) and used as a template to amplify EGFR. The PCR product was amplified using the following primers: The variants were sequenced by Sanger sequencing using TIFF0007650814000004.tif12163. Forward and reverse sequence traces were manually reviewed. The variant detected in the patient-derived cell line was a compound insertion in exon 20 of EGFR, resulting in the substitution of the amino acid asparagine at position 771 by two amino acids, phenylalanine and histidine (N771delinsFH). Cell viability and IC 50 The estimation was carried out as described above.

[0143] Patient-derived xenograft (PDX) testing: The LU0387 PDX study was completed by Crown BioSciences. Briefly, tumor fragments from EGFR H773insNPH-expressing tumors were inoculated into 5-6 week old female nu / nu nude mice. Tumors were 100-200 mm 3 Upon reaching 18 days, mice were randomized into three groups: 5 mg / kg poziotinib, 10 mg / kg poziotinib, or vehicle control (20% PEG-400, 3% Tween-80 in dH2O). Tumor volumes and body weights were measured twice weekly. Mice receiving 5 mg / kg poziotinib received the drug for 4-5 days, then had a 4-day break, then received an additional 4 days of treatment. Mice were then observed for an additional 2 days without treatment. Mice receiving 10 mg / kg poziotinib received the drug for 3-4 days, then were observed for 10 days without treatment. Mice that were humanely euthanized due to events unrelated to tumor burden were excluded from the final analysis.

[0144] Genetically Engineered Mouse Model (GEMM) testing: EGFR D770insNPG and HER2 A775insYVMA GEMMs were generated as previously described (Perera et al., 2009; Cho et al., 2013). Mice were handled in accordance with Good Animal Practices as defined by the Office of Laboratory Animal Welfare and performed under the approval of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (Boston, MA). Mice were fed a continuous doxycycline diet from 6 weeks of age. Tumor volumes were determined by MRI as previously described (Perera et al., 2009; Cho et al., 2013). Mice with equal initial tumor volumes were randomized in an open-label fashion to vehicle and 10 mg / kg poziotinib per day based on overt tumor formation as determined by MRI. Mice that were humanely euthanized due to events unrelated to tumor burden were excluded from the final analysis.

[0145] Example 3 - Identification of drugs for cancer cells with HER2 exon 21 mutations HER2 mutations occur most frequently in bladder, stomach, and bile duct cancers: To understand the diversity of HER2 mutations across cancer types, we queried multiple databases, including cohorts from cBioPortal, MD Anderson Cancer Center, and Foundation Medicine, as well as a cfDNA cohort from Guardant Health. Across all databases, all nonsynonymous HER2 mutations were analyzed within 25 different cancer types (Table 4). Weighted average frequencies for HER2 mutations were calculated. Similar to what was observed in the AACR GENIE database (Meric-Bernstam et al., 2018), HER2 mutations occurred most frequently in bladder (8.3%), bile duct (5.3%), and stomach (4.5%) cancers (Figure 13A); and HER2 exon 20 mutations occurred most frequently in small intestine (1.8%), lung (1.5%), and breast (0.9%) cancers (Figure 13B).

[0146] HER2 mutations occur most frequently in the tyrosine kinase domain of HER2, and mutation hotspots vary by malignancy: Next, mutation frequencies were analyzed within different regions of the HER2 receptor reported at cBioPortal and MD Anderson. Across all cancer types, HER2 mutations occurred most frequently in the tyrosine kinase domain (46%), including mutations in exon 20 (20%), exon 19 (11%), and exon 21 (9%) (Figure 14A). In addition, extracellular domain mutations comprised 37% of HER2 mutations. Across all queried cancers, the most common 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 14E). In lung cancer, the majority of HER2 mutations occurred within exon 20 (48%), with Y772_A775dupYVMA comprising 34% of all HER2 mutations (Figures 14B, 14F). In breast cancer, the majority of HER2 mutations occurred within exon 19 (37%), with the L755 mutation having the highest prevalence at 22% of HER2 mutations (Figure 14C). Meanwhile, unlike lung cancer, where one variant predominated, breast cancer had more mutational diversity within exon 19 mutations (Figure 14G). In colorectal cancer, HER2 mutations occurred most frequently in exon 21 (23%) and the extracellular domain (23%), with the V842I variant in exon 21 having the highest prevalence (19%) (Figures 14D, 14H).

[0147] Y772dupYVMA is the most common HER2 exon 20 insertion mutation across all cancer types: HER2 exon 20 mutations are the most common mutations occurring 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, MD Anderson, and Guardant Health databases. The Y772dupYVMA insertion was the most common HER2 exon 20 insertion, comprising 70% of all HER2 exon 20 insertions, with the p.G778dupGSP (14%) and p.G776del insVC (9%) insertions occurring at the second and third highest frequencies (Figure 21A). Exon 20 insertion mutations in NSCLC (N=362) showed the greatest diversity of exon 20 insertion mutations (Figure 21B), whereas exon 20 insertion mutations in breast cancer (N=30) showed little diversity of insertion sequences, with only three different variants reported (Figure 21C). In other cancer types, only a few additional insertion mutations were observed, with duplications at Y772 and G778 occurring most frequently in all cancer types analyzed (Figure 21D).

[0148] Frequently detected HER2 alterations are activating mutations: To evaluate the functional impact of common HER2 mutations, 16 of the most frequently detected HER2 mutations between exons 19, 20, and 21 were stably expressed in Ba / F3 cells. All 16 tested HER2 mutations were found to induce IL-3-independent survival of Ba / F3 cells (Figure 15A-C). Furthermore, expression of these 16 HER2 mutations led to the expression of phosphorylated HER2 (Figure 22A), indicating that these mutations lead to receptor activation.

[0149] Poziotinib was the most potent TKI tested and inhibited the most common HER2 mutations in vitro: Recent reports have highlighted the efficacy of covalent quinazolinamine-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, as well as cancer-specific and variant-specific differences in patient outcomes. To systematically evaluate drug sensitivity among the most commonly detected HER2 variants, a panel of HER2 mutant Ba / F3 cells was screened against 11 covalent and noncovalent EGFR and HER2 TKIs. HER2 mutants exhibited strong resistance to the noncovalent inhibitors, lapatinib and sapatinib (Figure 16A). The covalent TKIs osimertinib, ibrutinib, and nazartinib were not effective in suppressing cell viability in cells expressing exon 20 mutations; however, these TKIs showed activity against cells expressing the D769 variant (Figure 16A). In comparison, the covalent quinazolinamine-based TKIs afatinib, neratinib, dacomitinib, tarloxotinib-TKI, and poziotinib had inhibitory activity against HER2 mutants across all three exons (Figure 16A). Among all HER2 mutation variants and TKIs tested, poziotinib had the lowest mean IC 50 and was significantly more effective at reducing cell viability than afatinib, neratinib, or tarloxotinib-TKIs (Figure 16B). In addition, poziotinib was significantly more effective against HER2 exon 19 and 20 mutations than either afatinib, neratinib, or tarloxotinib-TKIs, but had a mean IC 50There was no significant difference in drug sensitivity across all TKIs tested (Figure S16C-E), suggesting that the mutation location affects drug binding. Furthermore, the L755S and L755P variants within exon 19 had significant differences in drug sensitivity across all TKIs tested (Figure S16F), indicating that specific amino acid changes at this site affected drug binding affinity.

[0150] HER2 Mutation Location and Amino Acid Changes Affect Drug Binding Affinity: To further understand how mutation location and amino acid changes can affect drug binding affinity and inhibitory effect, molecular dynamics simulations were used to investigate how these mutations affect the structure and dynamics of the HER2 kinase domain. Molecular models of the L755S, L755P, Y772dupYVMA, and V777L HER2 mutants (Figure 23A) 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 for the P-loop and α-C-helix positions, differed between these HER2 mutants. Even between exon 20 mutations, differences were evident, especially in the α-C-helix region, where the conformational persistence of the α-C-helix differed between "in" (active conformation with a smaller binding pocket) and "out" (inactive conformation with a larger binding pocket). The V777L mutant sampled the "out" conformation more frequently, while the Y772dupYVMA mutant sampled both the "in" and "out" conformations (Figure 17A). Overall, these differences in conformational states resulted in the Y772dupYVMA mutant existing in the "in" conformation (Figure 17B) more than 10-fold more frequently than the V777L mutant, and, on average, a smaller binding pocket in Y772dupYVMA compared to V777L (Figures 17C and 23B). In addition, because neratinib contains a pyridyl ring oriented toward the α-C-helix, the smaller binding pocket of Y772dupYVMA may contribute to the weakened potency of neratinib for Y772dupYVMA compared to V777L.

[0151] Further analysis of the HER2 mutant binding pocket volume (Figure 22B) demonstrated that mutations at the same residue can have dramatically different effects on protein conformation. Specifically, the proline residue of the L755P mutation lacks a hydrogen bond donor, disrupting the backbone hydrogen bond between the β3 and β5 strands between L755 and V790, respectively. The lack of stabilization between these two β strands led to destabilization of the β-sheet and structural rearrangements in the kinase hinge region (Figure 17D). Specifically, the L800 residue of L755P protruded into the active site, greatly reducing the pocket size. The change in β3 strand conformation also collapsed the P-loop inward, further reducing the pocket volume and making this mutant less sensitive to many TKIs. Furthermore, changes in hinge mobility may also play a role in kinase activation. These clear changes in the L755P mutant conformation contrasted with the behavior of the L755S mutant, which had a conformation and pocket volume profile that was more similar to wild-type HER2 (FIG. 23B).

[0152] HER2-mutant 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 quinazolinamine-based TKIs have activity in models of HER2-mutant disease, a panel of EGFR / HER2 TKIs was tested in human cancer cell lines. Preneoplastic MCF10A breast epithelial cells were transfected with HER2 exon 20 mutations and assessed in vitro for sensitivity to 12 EGFR / HER2 TKIs. MCF10A cells expressing the G776del insVC, Y772dupYVMA, or G778dupGSP HER2 mutations were most sensitive to poziotinib, with IC of 12 nM, 8.3 nM, and 4.5 nM, respectively. 50 In comparison, tarloxotinib-TKI and neratinib had mean IC values ​​of 21 nM and 150 nM, respectively. 50Values ​​were generated (Figure 18A-C), showing that poziotinib was 2.6-fold and 19-fold more potent than tarloxotinib-TKI and neratinib, respectively (p<0.001). Furthermore, western blotting of MCF10A HER2 G776delinsVC cells with poziotinib and neratinib showed that poziotinib completely inhibited p-HER2 at 10 nM, whereas neratinib did not (Figure 24A). Since wild-type (WT) HER2 does not transform Ba / F3 cells to grow IL-3-independently, MCF10A cells were used to determine the selectivity of TKIs for mutant HER2 compared to WT HER2. For this purpose, selectivity index (SI, IC 50 Value Mutant / IC 50 IC values ​​(WT) were calculated for each inhibitor and it was found that poziotinib was the most mutant-selective TKI (SI=0.028) tested in MCF10A cell lines, followed by pyrotinib (SI=0.063) and tarloxotinib-TKI (SI=0.111) (Figure 18D). In agreement with the data obtained with Ba / F3 cells (Figure 15C), in a model of HER2 exon 19 mutant colorectal cancer (CW-2), the difference in sensitivity between poziotinib, tarloxotinib-TKI, and neratinib was less dramatic but significant (p=0.02 and p=0.0004), with mean IC values ​​of 3.19 nM, 4.24 nM, and 68.8 nM, respectively. 50 (Figure 18E). Furthermore, in a CW-2 colorectal cell xenograft mouse model, at 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 increased tumor volume (28%) compared to vehicle controls (p=0.023), and afatinib (20 mg / kg) treatment did not significantly affect tumor growth compared to vehicle controls (Figures 18F, 25).

[0153] Poziotinib has antitumor activity in patients with NSCLC harboring HER2 mutations: Based on these preclinical data and previously published studies on exon 20 mutations (Robichaux et al., 2018), we initiated an investigator-initiated phase II clinical trial (NCT03066206) of poziotinib in EGFR and HER2 exon 20 mutant NSCLC. Patients were treated with poziotinib 16 mg orally daily until progression, death, or dropout. Objective response was assessed every 8 weeks according to RECIST v1.1. Of the first 12 evaluable patients harboring 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 2 months later (confirmed objective response rate, 42%) (Figure 19A). Of these 12 patients, 2 patients had progressive disease (PD) at first response evaluation, resulting in a disease control rate (DCR) of 83%. As of December 2018, 10 of the 12 patients were ongoing, and the median PFS of the first 12 patients was 5.6 months (Figure 19B). To date, all patients included in this study harbored one of the two most common HER2 exon 20 insertions, Y772dupYVMA and G778dupGSP (Figure 19A). Representative pre- and post-treatment (8 weeks) images of one NSCLC patient with the Y772dupYVMA mutation showed strong tumor shrinkage in the right lung (Figure 19C). Patient characteristics, including number of prior therapies, are seen in Table 3. In addition, one heavily pretreated NSCLC patient harboring a HER2 exon 19 point mutation, L755, was treated with a compassionate care protocol (C-IND18-0014). The patient was treated with poziotinib 16 mg daily and had tumor shrinkage at 4 weeks (Figure 19D, white box). The patient had stable disease (SD) (-12% reduction in target lesions) per RECIST v1.1. The patient continued poziotinib with disease control for more than 7 months until overt disease progression was imaged and poziotinib was discontinued. The patient was clinically well at the end of poziotinib treatment and continued to receive further systemic therapy.

[0154] Combining poziotinib with T-DM1 treatment enhances antitumor activity: Previous testing of the HER2 TKI lapatinib in HER2-positive breast cancer models and EGFR inhibitors in EGFR-mutant NSCLC models has shown that TKI treatment leads to increased receptor accumulation on the cell surface, and that increased cell surface HER2 / EGFR increases susceptibility to antibody-dependent cellular 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, it was observed that poziotinib treatment increases cell surface HER2 expression by 2-fold (Figure 20A, p<0.0001). We next tested whether the combination of poziotinib and T-DM1 could reduce cell viability in vitro. Although T-DM1 alone did not inhibit the viability of the MCF10A HER2 mutant cell line, the combination of T-DM1 and poziotinib had a significantly lower IC than either agent alone in a dose-dependent manner. 50It was found that the combination of low-dose poziotinib and a single dose of T-DM1 produced significant results (Figure 20B). To validate these findings in vivo, the combination of low-dose poziotinib with a single dose of T-DM1 was tested in a HER2 mutant NSCLC PDX model, HER2 Y772dupYVMA (Figure 20C). To evaluate the response to treatment, progression-free survival (PFS), defined as the time from best response to tumor doubling, was determined. Mice receiving vehicle control had a median PFS (mPFS) of 3 days, whereas mice receiving low-dose poziotinib or T-DM1 had mPFS of 15 and 27 days, respectively. However, mice receiving a single dose of T-DM1 in combination with low-dose poziotinib (14 / 20) remained tumor-free at day 45 (Figure 20D). Furthermore, at the time of best efficacy, day 15, the combination of low-dose poziotinib (2.5 mg / kg) with a single dose of T-DM1 (10 mg / kg) resulted in complete tumor regression in 20 / 20 mice (100%) compared to 2 / 9 mice receiving T-DM1 alone or 0 / 12 mice receiving low-dose poziotinib (Figure 20C-F). By day 30, tumor growth had begun in all mice receiving T-DM1 alone; however, there was no evidence of tumor regrowth in 14 / 20 mice receiving combination treatment (Figure 20F,G).

[0155] Further studies verified the efficacy of poziotinib compared to other TKIs. It was observed that poziotinib was more effective than high-dose osimertinib in EGFR S768dupSVD PDX models (Figure 26). It was also shown that poziotinib had higher antitumor activity than neratinib in NSCLC PDX models carrying Y772dupYVMA (Figure 27). Single-agent poziotinib was more effective than neratinib in breast cancer PDX models carrying V777L (Figure 28). A summary of the efficacy of poziotinib antitumor activity in various EGFR and HER2 exon 20 mutant in vivo models is shown in Figure 29.

[0156] Table 3: Table of average IC50 values ​​for Ba / F3 cells expressing the indicated EGFR exon 20 mutations. To determine IC50 values, Ba / F3 cells were generated. Cells were seeded in technical triplicates in 384-well plates at 2,000 cells per well. After 24 hours, cells were treated with seven different doses of poziotinib ranging from 150 nM to 0.01 nM. Percent viability was determined and normalized to DMSO-treated controls. IC50 values ​​for each biological replicate were calculated using nonlinear regression modeling in GraphPad Prism. Means and SEM are representative of three independent experiments. TIFF0007650814000005.tif19884

[0157] Herein, we report that HER2 mutations occur in various tumor types, but the specific mutation hotspots differ for each malignancy. Furthermore, sensitivity to HER2 TKIs is heterogeneous among mutation locations, and HER2 exon 20 insertions and L755P mutations are resistant to the majority of HER2 TKIs, likely due to a reduced volume of the drug-binding pocket. Furthermore, poziotinib was identified as a potent pan-HER2 mutant-selective inhibitor with clinical efficacy in NSCLC patients with HER2 exon 20 insertions and L755P mutations. Finally, we 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.

[0158] Pan-cancer analyses indicate that HER2 mutation hotspots vary across cancer types and have different sensitivities to HER2 TKIs in vitro, potentially affecting clinical efficacy. In the SUMMIT study, neratinib produced the highest efficacy in breast cancer patients, with the majority of responders positive for L755S, V777L, or L869R mutations. In vitro Ba / F3 drug screens showed that these mutations were associated with low IC 50In contrast, patients with colorectal cancer did not respond to neratinib. Consistent with this clinical observation, the V842I mutation was the most common 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 the differential TKI sensitivity among malignancies may be explained, in part, by cancer-specific mutation hotspots that directly affect drug sensitivity. However, significant questions remain as to why the distribution of HER2 mutations differs across tumor types and whether certain mutations produce similar drug responses in different tumor types. Data from the SUMMIT trial showed that although specific exon 20 insertions were associated with neratinib sensitivity in breast cancer patients, these same mutations were associated with resistance in all other cancer types, indicating that there may be potential mechanisms underlying these tumor type-specific differences in sensitivity that merit further investigation.

[0159] Exon 20 insertion mutations and exon 19 L755P mutations confer resistance to most HER2 TKIs. In vitro drug screening showed that exon 20 insertion mutations and L755P mutations had the highest IC 50 We showed that the HER2 exon 20 insertion mutations had a significant effect on the overall response to TKIs. Molecular dynamics simulations 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 clinical observations that patients with HER2 exon 20 insertion mutations have historically shown poor response to TKIs. In lung cancer, where exon 20 insertions occur frequently, patients carrying HER2 exon 20 insertion mutations had response rates of 0%, 11.5%, and 18.2%-18.8% to neratinib, dacomitinib, and afatinib, respectively. Furthermore, the L755S mutation has been shown to be responsive to neratinib, while the L755P mutation is highly resistant to both TKIs and antibody-drug conjugates.

[0160] Example 4 - Materials and Methods HER2 Mutation Prevalence and Variant Frequency Analysis: To determine the frequency of each HER2 mutation reported in databases from MD Anderson Cancer Center, cBioPortal, Foundation Medicine, or Guardant Health, each database was queried separately, and the frequency was then weighted by the total number of patients in each database and reported as a weighted average. To determine the frequency of HER2 mutations across cancer types in cBioPortal, all non-overlapping studies were selected and exported. For overlapping studies, only the largest data set 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 independent of cancer type. To determine the frequency of HER2 exon 20 mutations from Foundation Medicine, de-identified data for the number of patients with HER2 deletions, frameshifts, insertions, and point mutations were tabulated, and cancer types with fewer 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 (70-73 gene panel) 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 reported frequencies from Guardant Health were then normalized and corrected for clinical sensitivity as reported in Odegaard et al. 2018. Specifically, the frequency was divided by the percent clinical sensitivity, 85.9%.

[0161] Ba / F3 cell line generation and IL-3 deprivation: The Ba / F3 cell line was established as previously described. Briefly, stable Ba / F3 cell lines were generated by retroviral transduction of the Ba / F3 cell line for 12 hours. Retroviruses were generated 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, cells were stained with FITC-HER2 (Biolegend) and sorted by FACS. Cell lines were then grown in the absence of IL-3 for 2 weeks and cell viability was assessed 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.

[0162] 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, 384-well plates (Greiner Bio-One) were seeded with 2000–3000 cells per well in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone in a final volume of 40 μL per well. After 3 days, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 min and bioluminescence was measured using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). Bioluminescence values ​​were normalized to DMSO-treated cells and normalized values ​​were plotted in GraphPad Prism using a nonlinear regression fit to the normalized data with variable slope. IC 50 Values ​​were calculated at 50% inhibition using GraphPad Prism.

[0163] ELISA for phosphorylated and total HER2 and correlation with IC50 values: Protein was harvested from each of the parental Ba / F3 cell lines and the Ba / F3 cell lines expressing HER2 mutations described above. 5 μg / ml of protein was added to each ELISA plate and ESLISA was performed as described in the manufacturer's instructions for phosphorylated HER2 (Cell signaling, #7968) and total HER2 (Cell Signaling, #7310). Relative p-HER2 expression was measured by obtaining the ratio of p-HER2 to total HER2 determined by ELISA. Relative p-HER2 ratios were plotted against poziotinib IC50 values ​​calculated as above. Pearson correlation coefficients and p-values ​​were determined by GraphPad Prism.

[0164] Tyrosine Kinase Inhibitors and T-DM1: All inhibitors were purchased from Selleck Chemical, except for EGF816 and pyrotinib, which were purchased from MedChem Express. All inhibitors were dissolved in DMSO at a concentration of 10 mM and stored at -80°C. Inhibitors were restricted to disposal after two freeze-thaw cycles. T-DM1 was purchased and reconstituted from the in-house pharmacy at MD Anderson Cancer Center.

[0165] Molecular Dynamics Simulations: Protein structural models of HER2 mutants were constructed by introducing in silico mutations into the PDB 3PP0 X-ray structure using the MOE computer program (Chemical Computing Group). Classical and accelerated molecular dynamics simulations were performed using the NAMD simulation package. Further details are provided in the Supplemental Information section.

[0166] Human cell lines: MCF10A cells were purchased from ATCC and cultured in DMEM / F12 medium supplemented with 1% penicillin / streptomycin, 5% horse serum (sigma), 20ng / ml EGF, 0.5mg / ml hydrocortisone, and 10μg / ml insulin. Stable cell lines were generated by retroviral transduction, and retroviruses were generated by transfecting pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 1 into Phoenix 293T-ampho cells (Orbigen) using Lipofectamine 2000 (Invitrogen). Two days after transduction, 0.5μg / ml puromycin (Invitrogen) was added to the RPMI medium. After 14 days of selection, cells were tested in a cell viability assay as described above. CW-2 cells were provided by the Riken cell line database under MTA and maintained in RPMI containing 10% FBS and 1% penicillin / streptomycin.

[0167] In vivo xenograft testing: CW-2 cell line xenografts were grown at 1 × 10 6 Tumors were generated by injecting 100 cells into 6-week-old female nu / nu nude mice. 3 Once the tumor reached a median age of 10 days, mice were randomized into 4 groups: 20 mg / kg afatinib, 5 mg / kg poziotinib, 30 mg / kg neratinib, or vehicle control (in dH2O, 0.5% methylcellulose, 2% Tween-80). Tumor volumes were measured 3 times per week. Mice received drug Monday-Friday (5 days per week), with dosing starting on Wednesdays and a 2-day rest period following the first 3 days of dosing.

[0168] 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 tumors were measured when they reached 200-300 mm 3Once tumor volume reached 1000 mg / kg, mice were randomized into 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 volumes and body weights were measured three times per week. Mice treated with 2.5 mg / kg poziotinib received the drug orally Monday through Friday (5 days per week). Mice treated with 10 mg / kg T-DM1 received a single intravenous (IV) dose of T-DM1 on the day of randomization. Mice treated with the combination of poziotinib and T-DM1 received a single IV dose of T-DM1 and started 2.5 mg / kg poziotinib 5 days per week 3 days after the dose of T-DM1. Mice were given a break from dosing if their body weight dropped by more than 10% or fell below 20 grams. Progression-free survival was defined as the time from best response to tumor doubling on two consecutive measurements. Complete regression was defined as a >95% reduction in tumor burden, and in mice with complete regression, tumor doubling was defined as a tumor doubling of 75 mm on three or more consecutive measurements. 3 Experiments were completed in accordance with Good Animal Practices and with approval from the MD Anderson Cancer Center Institutional Animal Care and Use Committee (Houston, TX).

[0169] Table 3. Vectors used to generate stable cell lines TIFF0007650814000006.tif129169

[0170] Table 4. Total number of patients by cancer type in the entire database TIFF0007650814000007.tif225160

[0171] Table 5. Patient characteristics and number of prior treatments TIFF0007650814000008.tif82128

[0172] FACS: MCF10A cells overexpressing HER2 mutations were seeded overnight in 6-well plates and then treated with 10 nM poziotinib. After 24 hours, cells were washed twice with PBS and trypsinized. 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. Cells were washed twice with 0.5% FBS in PBS and analyzed by flow cytometry. IgG and unstained controls were used for gating.

[0173] Western blotting: For Western blotting, 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 BioRad semi-dry transfer and then probed with antibodies against pHER2, HER2, pPI3K, PI3K, p-AKT, AKT, p-ERK1 / 2, and ERK1 / 2 (1:1000; Cell Signaling). Blots were probed with antibodies against vinculin or β-actin (Sigma-Aldrich) as loading controls and exposed using ECL Western blotting substrate (Promega).

[0174] Correlation between HER2 expression levels and Ba / F3 mutant IC50: Protein was harvested from Ba / F cell line and ELISA was performed for total HER2 as described by the manufacturer's instructions (Cell Signaling, #7310). Relative expression measured by ELISA was plotted against IC50 values ​​calculated as above. Pearson correlation coefficients and p-values ​​were determined by GraphPad Prism.

[0175] Clinical Trials and CIND Identification Numbers: Patients provided written informed consent to treatment with poziotinib under either a compassionate use protocol (MD Anderson Cancer Center CIND-18-0014) or clinical trial NCT03066206. The protocols were approved by both the MD Anderson Cancer Center Institutional Review Board and the Food and Drug Administration.

[0176] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this 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 modifications may be applied to the methods and steps described herein, or to the sequence of steps of the methods, without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein, and still achieve the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0177] References The following references provide exemplary procedural or other details supplementary to the references set forth herein, and are specifically incorporated herein by reference. TIFF0007650814000009.tif194158TIFF0007650814000010.tif245112TIFF0007650814000011.tif216101

Claims

1. A pharmaceutical composition comprising poziotinib for treating cancer in a subject, wherein the subject has a tumor determined to have one or more EGFR exon 20 mutations selected from the group consisting of A767insTLA, V769insGVV, V769L, V769insGSV, V769insMASVD, H773insAH, H773insH, and V774insHV.

2. 2. The pharmaceutical composition of claim 1, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations.

3. The pharmaceutical composition of claim 1 or 2, wherein the subject does not have the EGFR mutation T790M and / or C797S.

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

5. The method of claim 4, wherein the subject has resistance to a previously administered tyrosine kinase inhibitor.

6. 6. The pharmaceutical composition of claim 5, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or neratinib.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the poziotinib is administered in a dose of 8 mg, 12 mg, or 16 mg.

8. The pharmaceutical composition according to any one of claims 1 to 7, for use in combination with a further anti-cancer therapy.

9. 9. The pharmaceutical composition of claim 8, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or cancer of the hematopoietic system, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the cancer is non-small cell lung cancer.

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