Compounds with Anti-tumor activity against cancer cells bearing EGFR or her2 exon 20 insertions

Abstract

To provide methods of treating cancer in a patient determined to have an EGFR and / or HER2 exon 20 mutation, such as an insertion mutation.SOLUTION: The present invention provides a method for treating cancer in a subject determined to have one or more EGFR exon 20 mutations, the method comprising a step of administering to the subject a third-generation tyrosine kinase inhibitor such as poziotinib or afatinib in an effective amount.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Incorporation of Sequence Listing A sequence listing contained in a file named "UTFCP1383WO.txt", which is 3.59 KB (as calculated on Microsoft Windows) and was created on Mar. 27, 2020, is electronically submitted herewith and is hereby incorporated by reference.

[0003] This invention was made with government support under grant number 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 having EGFR and / or HER2 exon 20 mutations, such as insertion mutations.

Background Art

[0005] 2. Description of Related Art Approximately 10-15% of NSCLC patients harbor activating EGFR mutations. In the majority of these patients whose tumors have "classical" sensitive mutations (L858R and exon 19 deletions), TKIs such as gefitinib and erlotinib have shown dramatic clinical utility, with approximately 70% experiencing objective response (OR), progression-free survival (PFS) compared to chemotherapy alone, and improvement in quality of life (Maemondo et al., 2010). However, approximately 10-12% of EGFR mutant NSCLC tumors have an in-frame insertion 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). Together, EGFR and HER2 exon 20 mutations constitute approximately 4% of NSCLC patients. Previous data have shown that available HER2 TKIs (afatinib, lapatinib, neratinib, dacomitinib) have limited activity in patients with HER2 mutant tumors, with many trials 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] Exons 20 of EGFR and HER2 contain two main regions, the c-helix (residues 762-766 in EGFR and residues 770-774 in HER2) and the loop following the c-helix (residues 767-774 in EGFR and residues 775-783 in HER2). Crystallography of the EGFR exon 20 insertion D770insNPG has 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 insertions before residue 764 do not exhibit such an effect and do not induce drug resistance (Yasuda et al., 2013). Furthermore, in a patient-derived xenograft (PDX) model of EGFR exon 20-induced NSCLC with an insertion in the loop after 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 recent studies of rare EGFR and HER2 exon 20 mutations, the authors found heterogeneous responses to covalent kinase inhibitor-based second-generation inhibitors such as dacomitinib and afatinib; however, the concentrations required to target more common exon 20 insertion mutations exceeded clinically achievable concentrations (Kosaka et al, 2017). Therefore, there is a significant clinical need to identify new treatments to overcome the innate drug resistance of NSCLC tumors harboring exon 20 mutations, particularly insertion mutations, in EGFR and HER2. Summary of the Invention

[0007] Summary Embodiments of the present disclosure provide methods and compositions for treating cancer in patients having EGFR and / or HER2 exon 20 mutations, such as exon 20 insertion mutations. In one embodiment, a method of treating cancer in a subject, the method 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, such as one or more EGFR exon 20 insertion mutations, is provided. In certain aspects, the subject is human.

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

[0009] In certain aspects, the one or more EGFR exon 20 mutations include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763-778. In some aspects, the subject has been determined to have two, three, or four EGFR exon 20 mutations. 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.

[0010] In certain embodiments, the subject is determined to not have an EGFR mutation, such as C797S and / or T790M, at residue C797 and / or T790. In some embodiments, one or more exon 20 mutations are 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 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 resistance to, or shows 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, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In certain embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In some embodiments, poziotinib is administered daily. In certain embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

[0013] In certain embodiments, it is determined that the subject has an EGFR exon 20 mutation, such as an insertion mutation, by analyzing a patient-derived genomic sample. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain embodiments, the presence of the EGFR exon 20 mutation is determined by nucleic acid sequencing (e.g., DNA sequencing of tumor tissue or plasma-derived cell-free DNA) or PCR analysis.

[0014] In certain embodiments, the method further comprises the step of performing 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 administration of poziotinib and / or the anti-cancer therapy is performed intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. In some embodiments, administering poziotinib and / or performing the anti-cancer therapy includes local administration, topical administration, or systemic administration. In certain embodiments, the administration of poziotinib and / or the anti-cancer therapy is performed more than once, such as daily, every other day, or weekly.

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

[0016] In another embodiment, there is provided a pharmaceutical composition comprising poziotinib 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 embodiments, the one or more EGFR exon 20 mutations include point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763-778. In certain embodiments, the subject is 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, the 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, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In certain embodiments, poziotinib is administered daily. In some embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in a 28-day cycle.

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

[0020] In some embodiments, one or more 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 is determined to not have an EGFR mutation, such as C797S and / or T790M, at residues C797 and / or T790. In specific embodiments, one or more exon 20 mutations are 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. In some embodiments, the patient is being treated by 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, the method comprising detecting an EGFR exon 20 mutation (e.g., an EGFR exon 20 insertion mutation) in a genomic sample obtained from the patient, wherein when the sample is positive for the presence of the EGFR exon 20 mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain embodiments, the presence of the EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. In certain embodiments, 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, favorable responsiveness to a poziotinib inhibitor alone or in combination with an anti-cancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related morbidity, reduction of cancer-related symptoms, non-progression of cancer, prolongation of disease-free period, prolongation of time to progression, induction of remission, reduction of metastasis, or improvement in patient survival. In further embodiments, patients predicted to have favorable responsiveness are administered poziotinib alone or in combination with a second anti-cancer therapy.

[0023] In some embodiments, poziotinib is further defined as poziotinib hydrochloride. In certain embodiments, poziotinib hydrochloride is formulated as tablets. 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, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In certain embodiments, poziotinib is administered daily. In some embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

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

[0026] A further embodiment is a method of treating cancer in a patient, 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. In some embodiments, the 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 770-785. 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 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, the 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, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In certain embodiments, poziotinib is administered daily. In some embodiments, poziotinib is administered continuously. In some embodiments, poziotinib is administered in 28-day cycles.

[0029] In some embodiments, the subject is resistant to or shows resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or velatinib.

[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 a particular embodiment, the mTOR inhibitor is everolimus.

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

[0032] In an additional aspect, the method further includes the step of administering an additional anti-cancer therapy. In some aspects, the anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy.

[0033] In some aspects, the cancer is oral cancer, hypopharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital 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, hypopharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain aspects, the cancer is non-small cell lung cancer.

[0034] In another embodiment, provided is a pharmaceutical composition comprising poziotinib or afatinib 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 to 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 by 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, the 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 dosage of 5 to 25 mg, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg. In some embodiments, poziotinib is at a dosage of 8 mg, 12 mg, or 16 mg.

[0037] In some embodiments, the subject is resistant to or shows resistance to a previously administered tyrosine kinase inhibitor. In certain embodiments, the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, neratinib, or velatinib.

[0038] In yet another embodiment, there is provided a method of predicting responsiveness to poziotinib alone, afatinib alone, or poziotinib or afatinib in combination with an anti-cancer therapy in a patient having cancer, the method comprising detecting in a genomic sample obtained from the patient a HER2 exon 20 mutation (e.g., a HER2 exon 20 insertion 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, wherein when the sample is positive for the presence of the HER2 exon 20 mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone, afatinib alone, or poziotinib or afatinib in combination with an anti-cancer therapy. In some aspects, 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 aspects, the HER2 exon 20 mutation further comprises one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770-785. In certain aspects, the HER2 exon 20 mutation is present at residues V773, A775, G776, V777, G778, S779, and / or P780. In other aspects, 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 the HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. In particular embodiments, the anti-cancer therapy is an mTOR inhibitor. In some embodiments, a favorable responsiveness to a poziotinib inhibitor alone or an afatinib inhibitor alone or a poziotinib inhibitor or an afatinib inhibitor in combination with an anti-cancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, alleviation of cancer-related conditions, alleviation of cancer-related symptoms, non-progression of cancer, prolongation of disease-free period, prolongation of time to progression, induction of remission, reduction of metastasis, or improvement of patient survival. In further embodiments, patients predicted to have a favorable responsiveness are administered poziotinib alone or in combination with a second anti-cancer therapy.

[0040] This specification also provides 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 the human EGFR or HER2 coding sequence. In some embodiments, the composition further comprises a labeled probe molecule capable of specifically hybridizing to the first portion of exon 20 of the sequence when a mutation is present in the human EGFR or HER coding 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 hybridizes to the first portion of exon 20 of the human EGFR coding sequence when a mutation 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C is present.

[0041] In certain embodiments, 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, and P780insGSP is present.

[0042] In another embodiment, there is provided an isolated nucleic acid encoding a mutant EGFR protein, wherein the mutant protein differs from wild-type human EGFR only by one or more EGFR exon 20 mutations that include point mutations, insertions, and / or deletions of 3 to 18 nucleotides in amino acids 763-778. In some embodiments, the one or more EGFR exon 20 mutations are present in 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 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. In a particular embodiment, 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 encoding a mutant HER2 protein, wherein the mutant protein differs from wild-type human HER2 only by one or more HER2 exon 20 mutations comprising one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770-785, is provided. In some embodiments, the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780. In certain 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 a particular embodiment, the nucleic acid comprises the sequence of SEQ ID NO:14, 15, 16, 17, or 18.

[0044] [Inventive Concept 1001] 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. [Inventive Concept 1002] The method of Inventive Concept 1001, wherein the poziotinib is further defined as poziotinib hydrochloride. [Inventive Concept 1003] The method of Inventive Concept 1002, wherein the poziotinib hydrochloride is formulated as a tablet. [Inventive Concept 1004] The method according to any one of Inventive Concepts 1001 to 1004, wherein the one or more EGFR exon 20 mutations are further defined as EGFR20 insertion mutations. [Inventive Concept 1005] The method according to any one of Inventive Concepts 1001 to 1004, wherein the one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations. [Inventive Concept 1006] Any of the methods of the present invention 1001-1005, wherein the one or more EGFR exon 20 mutations comprise point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763-778. [The present invention 1007] Any of the methods of the present invention 1001-1006, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations. [The present invention 1008] Any of the methods of the present invention 1001-1007, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S. [The present invention 1009] Any of the methods of the present invention 1001-1008, wherein the subject has been previously administered a tyrosine kinase inhibitor. [The present invention 1010] The method of the present invention 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] Any of the methods of the present invention 1001-1011, wherein the one or more EGFR exon 20 mutations are present in one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, and V774. [The present invention 1013] Any of the methods of the present invention 1001-1012, wherein the one or more EGFR exon 20 mutations are present in 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] Any of the methods of the present invention 1001-1013, wherein the subject is determined to not have an EGFR mutation at residue C797. [The present invention 1015] Any one 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] Any one of the methods of the present invention 1001 to 1015, wherein the one or more exon 20 mutations are 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1017] Any one of the methods of the present invention 1001 to 1016, wherein the exon 20 mutation is D770insNPG. [The present invention 1018] Any one of the methods of the present invention 1001 to 1017, wherein it is determined that the subject has an EGFR exon 20 mutation by analyzing a genomic sample derived from the patient. [The present invention 1019] The method of the present invention 1019, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1020] The method according to any one of the present inventions 1001 to 1019, wherein the presence of the EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1021] The method according to any one of the present inventions 1001 to 1020, wherein the poziotinib is administered orally. [The present invention 1022] The method according to any one of the present inventions 1001 to 1021, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1023] The method according to any one of the present inventions 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 according to any one of the present inventions 1001 to 1023, wherein the poziotinib is administered daily. [The present invention 1025] The method according to any one of the present inventions 1001 to 1024, wherein the poziotinib is administered continuously. [The present invention 1026] The method according to any one of the present inventions 1001 to 1025, wherein the poziotinib is administered in a 28-day cycle. [The present invention 1027] The method according to any one of the present inventions 1001 to 1026, further comprising the step of performing a further anti-cancer therapy. [The present invention 1028] The method of the present invention 1027, wherein the further anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1029] The method of the present invention 1027 or 1028, wherein the poziotinib administration and / or the anti-cancer therapy is performed intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. [The present invention 1030] The method according to any one of aspects 1027 to 1030 of the present invention, wherein administering the said poziotinib and / or performing anti-cancer therapy includes local administration, topical administration, or systemic administration. [Aspect 1031 of the present invention] The method according to any one of aspects 1027 to 1031 of the present invention, wherein the said poziotinib administration and / or anti-cancer therapy is performed two or more times. [Aspect 1032 of the present invention] The method according to any one of aspects 1001 to 1031 of the present invention, wherein the said cancer is oral cancer, hypopharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, digestive cancer, cancer of the central nervous system tissue or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, hypopharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [Aspect 1033 of the present invention] The method according to any one of aspects 1001 to 1032 of the present invention, wherein the said cancer is non-small cell lung cancer. [Aspect 1034 of the present invention] The method according to any one of aspects 1001 to 1033 of the present invention, wherein the said patient is a human. [Aspect 1035 of the present invention] A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more EGFR exon 20 mutations. [Aspect 1036 of the present invention] The composition according to aspect 1035 of the present invention, further defined as an oral composition. [Aspect 1037 of the present invention] The composition according to aspect 1035 or 1036 of the present invention, comprising 5 to 25 mg of poziotinib. [Aspect 1038 of the present invention] The composition according to any one of aspects 1035 to 1037 of the present invention, comprising 8 mg, 12 mg, or 16 mg of poziotinib. [The present invention 1039] A composition according to any one of the present inventions 1035 to 1038, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1040] A composition according to any one of the present inventions 1035 to 1039, formulated as a tablet. [The present invention 1041] A composition according to any one of the present inventions 1035 to 1040, wherein the one or more EGFR exon 20 mutations are further defined as EGFR20 insertion mutations. [The present invention 1042] A composition according to any one of the present inventions 1035 to 1041, wherein the one or more EGFR exon 20 mutations are further defined as de novo EGFR20 insertion mutations. [The present invention 1043] A composition according to any one of the present inventions 1035 to 1042, wherein the one or more EGFR exon 20 mutations include point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 to 778. [The present invention 1044] A composition according to any one of the present inventions 1035 to 1043, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations. [The present invention 1045] A composition according to any one of the present inventions 1035 to 1044, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S. [The present invention 1046] A composition according to any one of the present inventions 1035 to 1045, wherein one or more EGFR exon 20 insertion mutations are present in one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, H773, and V774. [The present invention 1047] A composition according to any one of the present inventions 1035 to 1046, wherein one or more EGFR exon 20 insertion mutations are present in 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] Any composition according to any of aspects 1035 - 1047 of the present invention, wherein the subject is determined to not have an EGFR mutation at residue C797. [Aspect 1049] Any composition according to any of aspects 1035 - 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. [Aspect 1050] Any composition according to any of aspects 1035 - 1049 of the present invention, wherein the one or more exon 20 mutations are 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [Aspect 1051] Any composition according to any of aspects 1035 - 1050 of the present invention, wherein the patient is being treated by anti - cancer therapy. [Aspect 1052] A method for predicting responsiveness to poziotinib alone or in combination with a second anti-cancer therapy in a subject having cancer, comprising the step of detecting an EGFR exon 20 mutation in a genomic sample obtained from the patient, wherein when the sample is positive for the presence of the EGFR exon 20 mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy. [Inventive Concept 1053] The method of Inventive Concept 1052, wherein the EGFR exon 20 mutation is further defined as an exon 20 insertion mutation. [Inventive Concept 1054] The method of Inventive Concept 1052 or 1053, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [Inventive Concept 1055] The method according to any one of Inventive Concepts 1052 to 1054, wherein the presence of the EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [Inventive Concept 1056] The method according to any one of Inventive Concepts 1052 to 1055, wherein the EGFR exon 20 mutation comprises point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 to 778. [Inventive Concept 1057] The method according to any one of Inventive Concepts 1052 to 1056, wherein one or more of the EGFR exon 20 mutations are not T790M and / or C797S. [Inventive Concept 1058] The method according to any one of Inventive Concepts 1052 to 1057, wherein the EGFR exon 20 mutation is present at residues A763, A767, S768, V769, D770, N771, P772, H773, and / or V774. [Inventive Concept 1059] The method according to any one of Inventive Concepts 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. [Inventive Concept 1060] The method according to 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 method according to any one of 1052 to 1060 of the present invention, wherein the EGFR exon 20 mutation 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [The present invention 1062] The method according to any one of 1052 to 1061 of the present invention, wherein a favorable responsiveness to poziotinib alone or poziotinib in combination with an anticancer therapy includes a decrease in tumor size or tumor volume, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related conditions, reduction of cancer-related symptoms, non-progression of cancer, extension of the disease-free period, extension of the period until progression, induction of remission, reduction of metastasis, or improvement of patient survival. [The present invention 1063] The method according to any one of 1052 to 1062 of the present invention, further comprising administering poziotinib alone or in combination with a second anti-cancer therapy to the patient predicted to have favorable responsiveness. [1064 of the present invention] The method according to any one of 1052 to 1063 of the present invention, wherein the poziotinib is administered orally. [1065 of the present invention] The method according to any one of 1052 to 1064 of the present invention, wherein the poziotinib is administered at a dose of 5 to 25 mg. [1066 of the present invention] The method according to any one of 1062 to 1065 of the present invention, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [1067 of the present invention] The method according to any one of 1062 to 1066 of the present invention, wherein the poziotinib is further defined as poziotinib hydrochloride. [1068 of the present invention] The method according to any one of 1062 to 1067 of the present invention, wherein the poziotinib hydrochloride is formulated as a tablet. [1069 of the present invention] A method of treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib or afatinib, wherein the subject is 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. [1070 of the present invention] The method of the present invention 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 the present invention 1069 or 1070, wherein the poziotinib is administered orally. [The present invention 1072] The method of any one of the present inventions 1069 to 1071, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1073] The method of any one of the present inventions 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 one of the present inventions 1069 to 1073, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1075] The method of any one of the present inventions 1069 to 1074, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1076] The method of any one of the present inventions 1069 to 1075, wherein the 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] The method of any one of the present inventions 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] The method of any one of the present inventions 1069 to 1076, wherein the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780. [The present invention 1079] Any of the methods of the invention from 1069 to 1078, wherein the HER exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [The invention 1080] Any of the methods of the invention from 1069 to 1079, wherein the HER exon 20 insertion mutation is A775insYVMA. [The invention 1081] Any of the methods of the invention from 1069 to 1080, further comprising the step of administering an mTOR inhibitor. [The invention 1082] The method of the invention 1081, wherein the mTOR inhibitor is rapamycin, temsirolimus, everolimus, ridaforolimus, or MLN4924. [The invention 1083] The method of the invention 1081, wherein the mTOR inhibitor is everolimus. [The invention 1084] The method of the invention 1081, wherein the poziotinib or afatinib and / or the mTOR inhibitor is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, in controlled release, in delayed release, as a suppository, or sublingually. [The invention 1085] Any of the methods of the invention from 1069 to 1084, wherein it is determined that the subject has a HER2 exon 20 mutation by analyzing a genomic sample derived from the patient. [The invention 1086] The method of the invention 1085, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The invention 1087] Any of the methods of the invention from 1069 to 1086, wherein the presence of the HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [The invention 1088] Any of the methods of the invention from 1069 to 1087, further comprising the step of performing an additional anti-cancer therapy. [The invention 1089] The method according to any one of the present invention 1069 to 1088, wherein the further anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1090] The method according to any one of the present invention 1069 to 1089, wherein the cancer is oral cancer, pharyngeal cancer, hypopharyngeal cancer, respiratory cancer, urogenital cancer, digestive cancer, cancer of the central nervous system tissue or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, pharyngeal cancer, hypopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1091] The method according to any one of the present invention 1069 to 1090, wherein the cancer is non-small cell lung cancer. [The present invention 1092] The method according to any one of the present invention 1069 to 1091, wherein the subject is 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 the present invention 1093 or 1094, wherein the HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [The present invention 1096] The composition of any one of the present inventions 1093 to 1095, wherein the 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] The composition of any one of the present inventions 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] The composition of any one of the present inventions 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. [The present invention 1099] The pharmaceutical composition of any one of the present inventions 1093 to 1098, wherein the patient is being treated by anti-cancer therapy. [The present invention 1100] A method for predicting responsiveness to poziotinib alone, afatinib alone, or poziotinib or afatinib in combination with an anticancer therapy in a subject having cancer, the method comprising detecting, in a genomic sample obtained from the subject, a HER2 exon 20 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, wherein when the sample is positive for the presence of the HER2 exon 20 mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone, afatinib alone, or poziotinib or afatinib in combination with an anticancer therapy. [Inventive concept 1101] The method of inventive concept 1100, wherein the HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation. [Inventive concept 1102] The method of inventive concept 1100 or 1101, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [Inventive concept 1103] The method according to any one of inventive concepts 1100 to 1102, wherein the presence of the HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis. [Inventive concept 1104] The method according to any one of inventive concepts 1100 to 1103, wherein the anticancer therapy is an mTOR inhibitor. [Inventive concept 1105] A preferred responsiveness to a poziotinib inhibitor alone, an afatinib inhibitor alone, or a poziotinib inhibitor or afatinib inhibitor in combination with an anti-cancer therapy, including a reduction in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related pathologies, reduction of cancer-related symptoms, non-progression of cancer, prolongation of disease-free period, prolongation of time to progression, induction of remission, reduction of metastasis, or improvement of patient survival, according to any of the methods 1100 - 1104 of the present invention. [The present invention 1106] The method according to any of 1100 - 1105 of the present invention, further comprising the step of administering poziotinib or afatinib alone or in combination with a second anti-cancer therapy to the patient predicted to have a preferred responsiveness. [The present invention 1107] (a) 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 the first portion of exon 20 of the said sequence when a mutation is present in the human EGFR or HER coding sequence The composition of the present invention 1107, further comprising. [The present invention 1109] The composition of the present invention 1107 or 1108, further comprising a thermostable DNA polymerase. [The present invention 1110] The composition according to any of 1107 - 1109 of the present invention, further comprising dNTPS. [The present invention 1111] When there is a mutation 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 labeled probe hybridizes to the first part of exon 20 of the human EGFR coding sequence, any composition of the present invention from 1108 to 1110. [Invention 1112] When there is a mutation 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C, the labeled probe hybridizes to the first part of exon 20 of the human EGFR coding sequence, any composition of the present invention from 1108 to 1111. [Invention 1113] When there is 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, the labeled probe hybridizes to the first part of exon 20 of the human HER2 coding sequence, and the composition according to any one of inventions 1108 to 1111 of the present invention. [Invention 1114] An isolated nucleic acid encoding a mutant EGFR protein, wherein the mutant protein differs from wild-type human EGFR only by one or more EGFR exon 20 mutations including point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 to 778, and the isolated nucleic acid. [Invention 1115] The isolated nucleic acid according to invention 1114, 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. [Invention 1116] The isolated nucleic acid according to invention 1114 or 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. [Invention 1117] The isolated nucleic acid of any one of aspects 1114 - 1116 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. [Aspect 1118] The isolated nucleic acid of any one of aspects 1114 - 1117 of the present invention, wherein the one or more exon 20 mutations are 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 H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C. [Aspect 1119] The isolated nucleic acid of any one of aspects 1114 - 1118 of the present invention, comprising the sequence of SEQ ID NO:8, 9, 10, 11, or 12. [Aspect 1120] An isolated nucleic acid encoding a mutant HER2 protein, wherein the mutant protein differs from wild - type human HER2 only by one or more HER2 exon 20 mutations, the 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] The isolated nucleic acid of the present invention 1120, 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] The isolated nucleic acid of the present invention 1120 or 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] The isolated nucleic acid of any one of the present inventions 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. However, it should be understood that, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, the detailed description and specific examples are presented for the purpose of illustration only and are not intended to limit the present invention.

Brief Description of the Drawings

[0045] The accompanying drawings, which form a part of this specification, are included to further demonstrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

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Figure 27

Figure 28

Figure 29

Mode for Carrying Out the Invention

[0046] Description of Exemplary Embodiments The majority of activating mutations in epidermal growth factor receptor (EGFR) variant non-small cell lung cancer (NSCLC) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), but a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant. This study used in silico, in vitro, and in vivo assays 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 constrain the binding of large, rigid inhibitors. Poziotinib, due to its small size and flexibility, was found to be able to avoid these steric changes and is a potent and relatively selective inhibitor of EGFR or HER2 exon 20 mutant proteins. Poziotinib also has potent activity in xenograft (PDX) models and genetically engineered mouse models derived from mutant exon 20 EGFR or HER2 NSCLC patients. Thus, these data identify poziotinib as a potent and clinically active inhibitor of EGFR / HER2 exon 20 mutations and elucidate the molecular characteristics of kinase inhibitors that can avoid the steric changes induced by these insertions.

[0047] Accordingly, certain embodiments of the present disclosure provide a method for treating cancer patients having an EGFR and / or HER2 exon 20 mutation, such as an exon 20 insertion. Specifically, the method comprises administering poziotinib (also known as HM781-36B) or afatinib to a patient identified as having an EGFR and / or HER exon 20 insertion mutation. The size and flexibility of poziotinib overcome steric hindrance and inhibit 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 EGFR and HER2 exon 20 insertions that are resistant to irreversible second- and third-generation TKIs.

[0048] I. Definitions As used herein, "a" or "an" can include one or more. When used in the claims of this specification, the terms "a" or "an" can mean one or more when used in conjunction with the word "comprising".

[0049] The present disclosure supports only alternatives and definitions that refer to "and / or", but the use of the term "or" in the claims is used to mean "and / or" unless it is clearly indicated that it refers only to alternatives or that 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] "Treat" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or showing the pathology or general symptoms of the disease (e.g., preventing further progression of the pathology and / or general symptoms), (2) improving the disease in a subject or patient experiencing or showing the pathology or general symptoms of the disease (e.g., reversing the pathology and / or general symptoms), and / or (3) acting on any measurable reduction of the disease in a subject or patient experiencing or showing the pathology or general symptoms of the disease. For example, treatment can include administration of an effective amount of poziotinib.

[0052] "Preventively treat" includes (1) reducing or alleviating the risk of onset of a disease in a subject or patient having a risk of the disease and / or being likely to contract the disease, but who has not yet experienced or shown any or all of the pathology or general symptoms of the disease, and / or (2) delaying the onset of the pathology or general symptoms of the disease in a subject or patient having a risk of the disease and / or being likely to contract the disease, but who has not yet experienced or shown any or all of the pathology or general symptoms of the disease.

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

[0054] The term "effective", when used in this specification and / or claims, means sufficient to achieve the desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" means an amount of the compound that is sufficient to act on the treatment or prevention of a disease when administered to the subject or patient for treating or preventing the disease.

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

[0056] An "anticancer" agent can, for example, promote the killing of cancer cells, induce apoptosis of cancer cells, reduce the growth rate of cancer cells, reduce the incidence or number of metastases, shrink the size of tumors, inhibit tumor growth, reduce the blood supply to tumors or cancer cells, promote the immune response against cancer cells or tumors, prevent or inhibit the progression of cancer, or extend the survival period of a subject having cancer, thereby having a negative impact on cancer cells / tumors in the subject.

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

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

[0059] A "probe" refers to a polynucleotide having a length of at least 8 nucleotides and forming a hybrid structure with a target sequence due to the complementarity between at least one sequence in the probe and a sequence in the target region. The polynucleotide can be composed of DNA and / or RNA. In certain embodiments, the probe is detectably labeled. The size of the probe can vary significantly. Generally, the length of the probe is, for example, at least 8 to 15 nucleotides in length. The length of other probes is, for example, at least 20, 30, or 40 nucleotides in length. The length of still other probes is somewhat longer, at least, for example, 50, 60, 70, 80, or 90 nucleotides in length. The probe can also be of any specific length within the said range. Preferably, the probe does not contain a sequence complementary to the sequence used for priming the target sequence during polymerase chain reaction.

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

[0061] A "modified ribonucleotide" or deoxyribonucleotide refers to a molecule that can be used in place of a natural base in a nucleic acid, including, but not limited to, modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized, and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifying factors, terminal modifying factors, spacer modifying factors, and backbone-modified nucleotides, including, but not limited to, ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methylphosphonates, methylphosphoramidites, methylphosphonamidites, 5′-β-cyanoethyl phosphoramidites, methylene phosphonates, phosphorodithioates, peptide nucleic acids, optically inactive and neutral internucleotide linkages.

[0062] A "variant" refers to a polynucleotide or polypeptide that differs from the wild-type or most frequently occurring form in an individual population by one or more nucleotide or amino acid exchanges, deletions, or insertions. The number of nucleotides or amino acids exchanged, deleted, or inserted can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, for example, 25, 30, 35, 40, 45, or 50.

[0063] A "primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer can be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. The primer can contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the primer length are determined empirically. The lower limit of the primer length is the minimum length required to form a stable double-strand upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 - 4 nucleotides in length) do not form a thermodynamically stable double-strand with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of duplex formation in regions other than a predetermined nucleic acid sequence in the target nucleic acid. Generally, the appropriate primer length is in the range of about 10 to about 40 nucleotides in length. In certain embodiments, for example, the primer can be 10 - 40, 15 - 30, or 10 - 20 nucleotides in length. The primer can act as a starting point for synthesis on a polynucleotide sequence when placed under appropriate conditions.

[0064] "Detect," "detectable," and their grammatical equivalents refer to methods for determining the presence, and / or amount, and / or homology of a target nucleic acid sequence. In some embodiments, detection results in amplification of the target nucleic acid sequence. In other embodiments, sequencing of the target nucleic acid can be characterized as "detecting" the target nucleic acid. The label attached to the probe can include any of a wide variety of labels well known in the art that are detectable, for example, by chemical or physical means. Labels that can be attached to the probe include, for example, fluorescent and luminescent materials.

[0065] "Amplify," "amplification," and their grammatical equivalents refer to any method of replicating at least a portion of a target nucleic acid sequence in a template-dependent manner, including but not limited to a wide range of techniques for amplifying nucleic acid sequences either linearly or exponentially. Exemplary means for performing the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR), as well as their multiplex versions or combinations, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as composite chain reaction - CCR), etc. Descriptions of such techniques can be found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[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 numbers NM_005228.3, NM_201282.1, NM_201283.1, and NM_201284.1. Variants of EGFR include insertions in exon 20.

[0067] "HER2" or "ERBB2" is a member of the EGFR / ErbB family and is represented as GenBank accession number NM_004448.2. Variants of HER2 include insertions in exon 20.

[0068] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues, organs, and / or body fluids within the scope of sound medical judgment, with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic response, or other problems or complications.

[0069] "Pharmaceutically acceptable salts" means salts of the compounds of the present invention that are pharmaceutically acceptable as described above and have the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphor-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that a particular anion or cation forming part of any salt of the present invention is not dangerous as long as the salt as a whole is pharmaceutically acceptable.Further examples of pharmaceutically acceptable salts and methods for their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[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 insertion mutations illustrated in FIG. 1. The 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 embodiments, exon 20 mutations are detected by DNA sequencing, for example, from a tumor or plasma-derived circulating free DNA.

[0071] EGFR exon 20 mutations can include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763-778. One or more EGFR exon 20 mutations can 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 can include H773_V774insH, A767_v769ASV, N771_P772insH, D770_N771insG, H779_V774insH, N771delinsHH, S768_D770dupDVD, A767_V769dupASV, A767_V769dupASV, P772_H773dup, 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 that confers 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, a subject having a T790 mutation, such as T790M, may be administered osimertinib, and a subject having a C797 mutation, such as C797S, may be administered chemotherapy and / or radiation therapy.

[0074] HER2 exon 20 mutations may include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 770 to 785. One or more HER2 exon 20 mutations may be present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786. 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] A patient sample can be any bodily tissue or fluid that contains nucleic acids derived 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 a tissue, e.g., lung tissue. The lung tissue can be derived from tumor tissue and may be fresh frozen or formalin-fixed and paraffin-embedded (FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

[0076] Samples suitable for use in the methods described herein include genetic material, e.g., genomic DNA (gDNA). Genomic DNA is typically extracted from biological samples such as blood or buccal mucosal scrapings, but can also be extracted from other biological samples including urine, tumors, or sputum. The sample itself typically comprises nucleated cells (e.g., blood or buccal cells) or tissue removed from 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, e.g., by collecting blood. In some embodiments, the sample is obtained without the cooperation of a medical institution, e.g., the sample is obtained non-invasively, such as a sample comprising buccal cells obtained using a buccal swab or brush, or an oral wash sample.

[0077] In some cases, biological samples can be processed for DNA isolation. For example, DNA in a cell or tissue sample can be separated from other components of the sample. Cells can be recovered from biological samples using techniques well known in the art. For example, cells can be recovered by centrifuging a cell sample and resuspending the pelleted cells. The cells can be resuspended in a buffer such as phosphate buffered saline (PBS). After centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, e.g., gDNA. See, e.g., Ausubel et al. (2003). Samples can be concentrated and / or purified to isolate DNA. All samples obtained from a subject, including those subjected to any kind of further processing, are considered to be those obtained from the subject. For example, genomic DNA can be extracted from biological samples using conventional methods including phenol extraction. Alternatively, genomic DNA can be extracted using kits such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.) and the Wizard® Genomic DNA Purification Kit (Promega). Non-limiting examples of sample sources include urine, blood, and tissue.

[0078] The presence or absence of an EGFR or HER2 exon 20 mutation, such as an exon 20 insertion mutation, as described herein can be determined using methods well known in the art. For example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and / or arrays can be used to detect the presence or absence of the insertion mutation. If desired, amplification of the 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 of the gene in genomic DNA, RNA, or cDNA, such as a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to hybridize specifically or preferentially to a particular variant.

[0079] A set of probes typically refers to a set of primers (usually primer pairs) and / or detectably labeled probes used to detect target gene mutations (e.g., EGFR and / or HER2 exon 20 mutations) used in the recommended treatments feasible with the present disclosure. The primer pairs are used in an amplification reaction to define amplicons spanning regions for the target gene mutations for each of the said genes. The set of amplicons is detected by a set of matching probes. In an exemplary embodiment, the method can use a TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assay 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 produce 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] The analysis of nucleic acid markers can be carried out using techniques well-known in the art, including but not limited to sequence analysis and electrophoretic analysis. Non-limiting examples of sequence analysis include sequencing with mass spectrometry such as Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., 1992), solid-phase sequencing (Zimmerman et al., 1992), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., 1998), 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. Further, next-generation sequencing methods can be carried out using kits and apparatuses commercially available 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., 1997) infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry (WO99 / 57318); mobility shift analysis (Orita et al., 1989); restriction enzyme analysis (Flavell et al., 1978; Geever et al., 1981); quantitative real-time PCR (Raca et al., 2004); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., 1985); RNase protection assay (Myers et al., 1985); use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR, and may include combinations of such methods. See, for example, U.S. Patent Application Publication No. 2004 / 0014095. This patent is hereby incorporated by reference in its entirety.

[0082] In one example, a method for identifying EGFR and / or HER2 mutations in a sample comprises contacting a nucleic acid derived from such sample with a nucleic acid probe that can specifically hybridize to a nucleic acid encoding a mutated EGFR or HER2 protein, or a fragment thereof that contains the mutation, and detecting the hybridization. In a particular embodiment, such probe may be, for example, a radioisotope ( 3 H, 32 P, or 33P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent, and is detectably labeled. In certain embodiments, the probe is an antisense oligomer such as PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be from about 8 nucleotides to about 100 nucleotides, or from about 10 to about 75, or from about 15 to about 50, or from about 20 to about 30. In another aspect, such a probe of the present disclosure is provided in a kit for identifying EGFR or HER2 mutations in a sample, and such a kit contains oligonucleotides that specifically hybridize to or are adjacent to the mutation sites in the EGFR or HER2 gene. The kit further includes 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 assay using the kit.

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

[0084] Alternatively, nucleic acids can be analyzed for the detection of mutations using Enzymatic Mutation Detection (EMD) (Del Tito et al., 1998). EMD uses bacteriophage resolvase T4 endonuclease VII, which scans along double-stranded DNA until it detects and resolves the structural distortions caused by base pair mismatches due to point mutations, insertions, and deletions. Detection of two short fragments formed by, for example, gel electrophoresis upon resolvase cleavage indicates the presence of a mutation. The advantages of the EMD method are that it is assayed directly from the PCR reaction, eliminating the need for sample purification, shortening hybridization times, increasing the signal-to-noise ratio, and is a single protocol for identifying point mutations, deletions, and insertions. Mixed samples containing up to 20-fold overexpression of normal DNA and fragments up to 4 kb in size can be assayed. However, EMD scanning does not identify the specific base changes that occur in mutation-positive samples and, if necessary, further sequencing procedures are required to identify the mutations. As demonstrated in U.S. Patent No. 5,869,245, CEL I enzyme can be used in a similar manner to resolvase T4 endonuclease VII.

[0085] III. Treatment Methods Also provided herein is a method for treating or delaying the progression of cancer in an individual, the method comprising administering to the individual an effective amount of poziotinib, afatinib, or a structurally similar inhibitor to a subject determined to have an EGFR and / or HER2 exon 20 mutation, such as 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, pre-neoplastic lesions of the lung, colon cancer, melanoma, and bladder cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0087] In some embodiments, the subject is a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient having or at risk of having the disorders described herein). In one embodiment, the subject is in need of enhancing an immune response. In certain embodiments, the subject is susceptible or at risk of being susceptible. For example, the subject has received or is receiving chemotherapy and / or radiation therapy. Alternatively, or in combination, the subject is susceptible or at risk of being susceptible as a result of an infection.

[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 a subject determined to have an EGFR or HER2 exon 20 mutation, such as an exon 20 insertion. Poziotinib is a quinazoline-based pan-HER inhibitor that irreversibly blocks signaling through the HER family of tyrosine kinase receptors, including HER1, HER2, and HER4. Poziotinib or structurally similar compounds (e.g., U.S. Patent No. 8,188,102 and U.S. Patent Application Publication No. 2013 / 0071452; incorporated herein by reference) may be used in the methods of the invention.

[0089] Poziotinib, poziotinib hydrochloride, may be administered orally, for example, in tablets. Poziotinib may be administered at a dose of 4 to 25 mg, for example, at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg. Administration may be daily, every other day, every three days, or once a week. Administration may be on a continuous schedule, for example, in a 28-day cycle.

[0090] In some embodiments, a subject having a T790 mutation, such as T790M, may be administered osimertinib, and a subject having a C797 mutation, such as C797S, may be administered chemotherapy and / or radiation therapy as described herein. Osimertinib administration, chemotherapy, and / or radiation administration may be performed alone or in combination with poziotinib. Osimertinib may be administered at a dose of 25 to 100 mg, such as about 40 or 80 mg. The administration may be daily, every other day, every two days, every three days, or once a week. Osimertinib may be administered orally, for example, in tablet form.

[0091] Afatinib may be administered at a dose of 10 to 50 mg, such as 10, 20, 30, 40, or 50 mg. Afatinib may be administered.

[0092] B. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising poziotinib or afatinib and one or more pharmaceutically acceptable carriers for a subject determined to have an EGFR or HER2 exon 20 mutation, such as 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 pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington’s Pharmaceutical Sciences 22 ndIt can be prepared by mixing with (edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited to these. In this specification, an exemplary pharmaceutically acceptable carrier further includes an interstitial drug dispersant such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use including rHuPH20 are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glucosaminoglycanases such as chondroitinase.

[0094] C. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment include poziotinib or afatinib in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0095] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or a metastasis inhibitor. In some embodiments, the additional therapy is the administration of an agent that reduces side effects (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an antiemetic). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents well known in the art.

[0096] Ponatinib or afatinib can be administered before, during, after, or in various combinations with additional cancer therapies such as immune checkpoint therapies. Administration can range from co - administration to intervals of minutes, days, or weeks. In embodiments where ponatinib or afatinib is provided to the patient separately from an additional therapeutic agent, generally, a significant period does not elapse between each delivery time to ensure that the two compounds can still provide an advantageous combined effect to the patient. In such cases, it is contemplated that antibody therapy and anti - cancer therapy may be provided to the patient within about 12 - 24 or 72 hours of each other, more specifically, within about 6 - 12 hours of each other. In some situations, it may be desirable to space the intervals between each administration by several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) to significantly extend the period of treatment.

[0097] Various combinations can be used. In the following examples, ponatinib or afatinib is "A" and anti - cancer therapy is "B". TIFF2025090740000001.tif18128

[0098] The administration of any compound or the implementation of a therapy for a patient in this embodiment follows the general protocol for the administration of such a compound, taking into account the toxicity of the drug if present. Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy.

[0099] 1. Chemotherapy According to this embodiment, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to denote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, e.g., whether they affect the cell cycle and at which stage of the cell cycle they act. Alternatively, the agents may be characterized based on their ability to directly crosslink DNA, to intervene in DNA, or to induce chromosomal and mitotic abnormalities by acting on 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; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially, bratasin and bratasinone); camptothecin (including synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its azozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobemycin, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin gammaII and calicheamicin omegaI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotic chromophores, actinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, pteropterin, and trimetrexate; purine analogs, for example, fludarabine, 6-mercaptopurine, thiampurine, and thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, and floxuridine; androgens, for example, calusterone, drostanolone propionate, epithioestanol, mepitiostane, and testolactone; antiadrenal, for example, mitotane and trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;Roxanthrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lysoxine; schizophyllan; spirigermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecene (especially, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoid, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complex, for example, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS2000; difluorodifluoromethylornithine (DMFO); retinoid, for example, retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabiene, navelbine, farnesyl-protein transferase inhibitor, transplatin, and any pharmaceutically acceptable salt, acid, or derivative of the foregoing are included.;

[0101] 2. Radiation therapy Other factors that cause DNA damage and are widely used include those known as gamma rays, X-rays, and / or the targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are contemplated, such as electron beams, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are likely to affect a wide range of damage to DNA, to precursors of DNA, to DNA replication and repair, and to the assembly and maintenance of chromosomes. The dose range for X-rays spans from a daily dose of 50 - 200 roentgens for a long period (3 - 4 weeks) to a single dose of 2,000 - 6,000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by neoplastic cells.

[0102] 3. Immunotherapy One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the present 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 an example. Immune effectors can be, for example, antibodies specific for some markers on the surface of tumor cells. The antibody alone can be used as an effector of the therapy, or the antibody may recruit other cells that actually act on cell killing. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a molecular targeting agent. Alternatively, the effector can be a lymphocyte having surface molecules that directly or indirectly interact with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0103] Antibody-drug conjugates have emerged as an epoch-making approach in the development of cancer therapy. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to cell-killing drugs. This approach combines the high specificity of the Mab for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells along with abundant levels of antigen. Targeted delivery of the drug also minimizes drug exposure in normal tissues, resulting in reduced toxicity and improved therapeutic indices. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, has validated this approach. Currently, more than 30 ADC drug candidates are at various stages of clinical trials for cancer therapy (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs rely heavily on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high-level expression in tumor cells and robust internalization.

[0104] In one aspect of immunotherapy, tumor cells need to have several markers that are suitable for targeting (i.e., not present in most other cells). There are many tumor markers, and any of these may be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect and an immune-stimulating effect. There are also immune-stimulating molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0105] Examples of immunotherapy include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in combination with the antibody therapy described herein.

[0106] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance or weaken signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target PD-1 family and / or CTLA-4.

[0107] The immune checkpoint inhibitor can be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or in particular, an antibody, such as a human antibody (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both documents are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs can be used, and in particular, chimeric, humanized, or human forms of antibodies can be used. Those skilled in the art will appreciate that alternative and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this specification. For example, pembrolizumab is known to be also known as MK-3475 and lambrolizumab as alternative and equivalent names.

[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 well known in the art and are described, for example, in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

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

[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 co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of inhibitory receptors for CTLA-4 and B7 molecules.

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

[0113] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above 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 modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Patent Nos. WO1995 / 001994 and WO1998 / 042752; all incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867, incorporated herein by reference.

[0115] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery, including prophylactic, diagnostic or staging, therapeutic, and palliative surgery. Therapeutic surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can also be combined with other treatment methods, such as the treatment of the present embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative treatment methods. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electro-surgery, and microscope-controlled surgery (Mohs surgery).

[0116] When part or all of the cancer cells, tissue, or tumor is resected, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or topical application of the area using additional anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages.

[0117] 5. Other Agents It is contemplated that other agents may be used in combination with specific aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that act on the upregulation of cell surface receptors and GAP junctions, cell growth inhibitors and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intracellular signaling by increasing the number of GAP junctions can increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell growth inhibitors or differentiating agents can be used in combination with specific aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effects of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with specific aspects of the present embodiment to improve the therapeutic effect.

[0118] IV. Kit Kits for detecting EGFR and / or HER2 exon 20 mutations, such as those disclosed herein, are also within the scope of the present disclosure. An example of such a kit may include a set of primers specific for the exon 20 mutation. The kit may further include instructions for using the primers to detect the presence or absence of specific EGFR and / or HER2 exon 20 mutations described herein. The kit may further include instructions for diagnosis indicating that identification of a positive for the EGFR and / or HER2 exon 20 mutations described herein in a sample from a cancer patient is an indicator of sensitivity to poziotinib or afatinib or a structurally similar inhibitor that is a tyrosine kinase inhibitor. The kit may further include instructions indicating that identification of a positive for the 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 a structurally similar inhibitor.

Example

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

[0120] Example 1 - Identification of Drugs Against Cancer Cells with EGFR or HER Exon 20 Insertion The clinical response to TKI was examined in patients with tumors bearing EGFR exon 20 insertion in a clinical database; among 280 patients with EGFR mutant NSCLC treated with single-agent erlotinib, gefitinib, or afatinib, 129 patients were identified as having classical EGFR mutations (exon 19 deletion, L858R, and L861Q), and 9 patients were identified as having EGFR exon 20 insertion. NSCLC patients with classical EGFR mutations had a median PFS of 14 months, while patients with EGFR exon 20 insertion had a median PFS of only 2 months (p < 0.0001, log-rank test; Figure 1A). Among the 9 EGFR exon 20 insertion patients, an OR was observed only in 1 patient bearing the S768del-insIL mutation who received afatinib (Figure 4A). This clinical data shows that the activity of available EGFR TKIs is limited in EGFR exon 20 insertion-induced NSCLC and confirms that alternative treatment strategies are needed in these specific tumors.

[0121] As the first step of drug screening, seven types of EGFR mutations and eleven types of HER2 mutations were expressed in Ba / F3 cells. The positions of EGFR and HER2 exon 20 mutations are summarized in Figure 1B. To evaluate which exon 20 mutations of EGFR and HER2 are activated, the Ba / F3 cell line was screened for IL-3 independent survival. All EGFR exon 20 insertions tested were activating mutations (Figure 4B), and it was found that six HER2 exon 20 mutations and L755P located in exon 19 were 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 sensitive mutation EGFR L858R. Except for EGFR A763insFQEA, EGFR exon 20 insertions (n = 6) were resistant to first generation (Figure 1C, IC 50 = 3.3 to >10 μM), second generation (Figure 1d, IC 50 = 40 to 135 nM), and third generation (Figure 1e, IC 50 = 103 to 850 nM) EGFR TKIs (Figure 5, Table 1). In addition, HER2 exon 20 mutants (n = 6) were resistant to first generation (Figure 1F, IC 50 = 1.2 to 13 μM) and third generation (Figure 1H, IC 50 = 114 to 505 nM) TKIs. Second generation TKIs have some activity against the Ba / F3 HER2 exon 20 mutant cell line (Figure 1G, IC 50 = 10 to 12 nM, Figure 6, Table 1). Except for EGFR A763insFQEA, which showed partial inhibition at lower doses, consistent with the drug screening, Western blotting showed that erlotinib and osimertinib did not significantly inhibit p-EGFR2 in EGFR exon 20 insertion mutations and significantly inhibited p-HER2 only in HER2 exon 20 insertion mutants at 500 nM (Figure 7A - D).

[0122] (Table 1) IC50 values of EGFR and HER2 exon 20 insertions by EGFR / HER2 TKIs TIFF2025090740000002.tif98155

[0123] To investigate the reasons why the exon 20 insertions confer resistance to first- and third-generation EGFR TKIs, 3-D modeling was performed on the resolved crystal structure of EGFR T790M against EGFR D770insNPG and EGFR WT to visualize the changes within the drug-binding pocket. Modeling revealed that the EGFR exon 20 insertions are similar to the T790M mutation in the alignment of the gatekeeper residue T790, resulting in an increased affinity for ATP and a decreased binding of first-generation inhibitors, and these mutations confer resistance to non-covalent inhibitors. In addition, the HER2 exon 20 insertion induced a constitutively active conformation and interfered with the binding of the non-covalent HER2 inhibitor lapatinib, which binds to the inactive conformation of HER2. Furthermore, the EGFR and HER2 exon 20 insertions have dramatic effects on the drug-binding pocket. In silico modeling of the EGFR (Figure 1I) and HER2 (Figure 1J) exon 20 insertions revealed a significant shift (arrow) of the α-c-helix into the drug-binding pocket at the C-terminus of the α-c-helix, which forced the ridged arrangement of the α-c-helix to move to the inner activation position (Figure 1J). In addition, 3-D modeling also showed a significant shift of the P-loop into the drug-binding pocket of both receptors (Figure 1I, 1J). 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 above in vitro tests, 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 the ability of osimertinib to reach the C797 residue as efficiently as afatinib in the EGFR exon 20 insertion (Figure 1I). Alternatively, afatinib has a smaller 1-chloro-2-fluorobenzene ring terminal group indirectly linked to the quinazoline core via a secondary amine group, allowing afatinib to fit into the sterically hindered binding pocket.Furthermore, steric hindrance prevents the binding of osimertinib to HER2 A775insYVMA. In summary, in vitro data and in silico modeling indicate that small and flexible quinazoline derivatives may be able to target EGFR / HER2 exon 20 insertions.

[0124] Next, a search was conducted to identify TKIs with enhanced activity against exon 20 insertions. Poziotinib contains a small terminal group and a flexible quinazoline core, similar to afatinib. 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 the basic residues of EGFR, such as K745, further stabilizing its binding. Therefore, 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 50 value of 1.0 nM in the EGFR exon 20 mutant Ba / F3 cell line, and poziotinib was approximately 100-fold more potent than osimertinib and more than 40-fold more potent than afatinib in vitro. Furthermore, poziotinib had an average IC 50 value of 1.9 nM in the HER2 exon 20 mutant Ba / F3 cell line, and poziotinib was more than 200-fold more potent than osimertinib and more than 6-fold more potent than afatinib in vitro. These results were verified by Western blotting, where poziotinib inhibited the phosphorylation of EGFR and HER2 at a low concentration of 5 nM (Figure 2C, 8A). Furthermore, to verify that poziotinib sensitivity was not due to the level of expression of the EGFR or HER2 mutants, the expression of each mutant was measured by ELISA and then plotted against the IC 50 value (Figure 8D). The IC 50No correlation was observed between expression and responsiveness (R = -0.056, p = 0.856), but for EGFR, a correlation was observed between poziotinib sensitivity and the position of EGFR mutations (R = 0.687, p = 0.044) (Figure 2D). As the insertion was further away from the α-c-helix, higher IC 50 values were suggested. Interestingly, this correlation was not observed for HER2 exon 20 mutations, where the size of the insertion changed more significantly than the insertion position (Figure 8E). This correlation suggests that the exact position of the mutation has various effects on the drug-binding pocket and contributes to the observed heterogeneity of drug responses. Additionally, poziotinib effectively inhibited the growth of patient-derived cell lines CUTO14 (EGFR A767dupASV) and YUL0019 (EGFR N771del insFH) at mean IC 50 values of 1.84 nM and 0.30 nM, respectively, which were more than 15-fold more potent than afatinib in CUT014 and more than 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 to the T790M mutant, the IC 50 values of afatinib, osimertinib, rociletinib, and poziotinib in exon 20 mutants were compared with the IC 50 values of afatinib, osimertinib, rociletinib, and poziotinib in the EGFR T790M mutant Ba / F3 cell line. The IC 50Values normalized for a single EGFR T790M mutation and shown to be less than 1 indicate specificity for exon 20 insertions compared to T790M (Figure 2G). The EGFR exon 20 insertion had more than 65-fold greater sensitivity to poziotinib compared to the EGFR T790M mutant. Furthermore, the EGFR exon 20 insertion mutation had more than 1.4-fold greater resistance to afatinib, more than 5.6-fold greater resistance to osimertinib, and more than 24-fold greater resistance to rociletinib compared to the EGFR T790M mutant (Figure 2G).

[0126] To investigate the reason why poziotinib selectively and potently inhibits exon 20 mutants compared to the T790M mutation, while third-generation TKIs such as osimertinib do not, 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), and in the exon 20 mutant, 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 it to fit into the exon 20-binding pocket with steric hindrance (Figure 2I). Furthermore, 3-D modeling of EGFR D770insNPG with poziotinib and afatinib suggests that a shifted P-loop within the drug-binding pocket binds poziotinib more firmly in the binding pocket than afatinib. Calculations of structural modeling show that the free energy of binding (London ΔG) for poziotinib is lower than that for afatinib, indicating a stronger binding affinity for 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 drug-binding pocket with steric hindrance of HER2 A775insYVMA, overcoming the structural changes induced by the exon 20 insertion.

[0127] Using GEM models of EGFR and HER2 exon 20 insertion-induced NSCLC, the efficacy of poziotinib was tested in vivo. Lung tumors were induced in previously described EGFR D770insNPG (Cho et al, 2013) and HER2 A775insYVMA (Perera et al, 2009) mice, and the animals were given poziotinib (10 mg / kg) or vehicle control daily for 4 weeks. As determined by MRI, poziotinib reduced tumor volume at inhibition levels higher than 37% previously observed with afatinib in the same GEM models, at 85% in EGFR exon 20 GEMMs (Figure 3A, C) and 60% in HER2 exon 20 GEMMs (Figure 3B, D). 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 12 weeks (Figure 3E, F). Additionally, poziotinib treatment (5 or 10 mg / kg) completely reduced tumors by 14 days in the EGFR exon 20 insertion PDX model LU0387 (H773insNPH) (>85% inhibition) (Figure 3G).

[0128] To determine whether poziotinib covalently binds at C797, similar to other irreversible inhibitors, Ba / F3 cell lines with the C797S mutation observed in approximately 30% of patients with osimertinib resistance (Thress et al, 2015) were generated. The C797S mutation was found to confer resistance to poziotinib at IC 50 values >10 μM. Taken together, these studies suggested that poziotinib may be susceptible to the effects of similar acquired resistance mechanisms as other third-generation TKIs.

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

[0130] Thus, exon 20 variants were found to exhibit de novo resistance to first-generation, second-generation, and third-generation TKIs. Using 3-D modeling of EGFR D770insNPG and HER2 A775insYVMA, poziotinib was identified as having structural features that can overcome the changes 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 the potent antitumor activity of poziotinib in cells with these mutations.

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

[0132] 3D modeling suggests that the smaller size, increased halogenation, and flexibility of poziotinib 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 correlation suggests that the exact position of the mutation affects the drug-binding pocket and / or the binding affinity of the TKI. Furthermore, the data showed that the size of the insertion also affects drug sensitivity. Additionally, the patient-derived cell line YUL0019 (N771del insFH), which had a net increase of only 1 amino acid, was more sensitive to a quinazoline-based pan-HER inhibitor than cells with larger EGFR exon 20 insertions.

[0133] Example 2 - Materials and Methods Patient Population and Statistical Analysis: Patients with EGFR mutant NSCLC registered in the prospectively collected MD Anderson Lung Cancer Moon Shot GEMINI database were identified. EGFR mutation status was determined using one of the 50-, 134-, or 409-gene panel PCR-based next-generation sequencing used in routine clinical care. Progression-free survival (PFS) was calculated using the Kaplan-Meier method. PFS was defined as the time from the start of EGFR TKI to radiographic progression or death. Restaging scans were obtained at 6- to 8-week intervals during treatment and retrospectively evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, to determine the response rate in patients with EGFR exon 20 insertion NSCLC.

[0134] Cell line preparation and IL-3 depletion: The Ba / F3 cell line was cultured 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 of mouse IL-3 (R&D systems) under sterile conditions. Stable cell lines were generated by retroviral transduction of the Ba / F3 cell line for 12 hours. Retroviruses were produced by transfecting the pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 2 into Phoenix 293T ampho packaging cell line (Orbigen) using Lipofectamine 2000 (Invitrogen). 72 hours 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. Subsequently, the cell lines were 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 the above complete RPMI-1640 medium without IL-3. The HCC827 and HCC4006 lung cancer cell lines were obtained from ATCC and maintained in 10% RPMI medium under sterile conditions. The identity of the cell lines was confirmed by DNA fingerprinting using short tandem repeats with the PowerPlex 1.2 kit (Promega). The fingerprinting results were compared with the reference fingerprint maintained by the original source of the cell line. All cell lines were mycoplasma-free. To generate erlotinib-resistant cell lines, HCC827 and HCC4006 (both EGFR mutants) cells were cultured with increasing concentrations of erlotinib until resistant variants emerged.

[0135] (Table 2) Vectors used to generate stable cell lines TIFF2025090740000003.tif253166

[0136] Cell viability assay and IC 50 Estimation: Cell viability was measured using the Cell Titer Glo assay (Promega). Cells were collected from suspension medium, centrifuged at 300×g for 5 minutes, resuspended in fresh RPMI medium, and counted using a Countess automated cell counter and trypan blue (Invitrogen). 1500 cells per well were seeded in triplicate technically in a 384-well plate (Greiner Bio-One). Cells were treated with inhibitors or vehicle alone at seven different concentrations of the TKI serially diluted three-fold with a final volume of 40 μL per well. After 72 hours, 11 μL of Cell Titer Glo was added to each well. The plate was shaken for 10 minutes and bioluminescence was measured using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). The bioluminescence values were normalized to DMSO-treated cells and the normalized values were plotted in GraphPad Prism using a non-linear regression fit to the normalized data with variable slope. IC 50 values were calculated by GraphPad Prism at 50% inhibition. Unless otherwise specified, each experiment was repeated three times.

[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 institutional pharmacy of 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] 3D Modeling: The structure of the EGFR D770insNPG protein was retrieved (Protein Data Bank entry code: 4LRM) and used as a template to construct a molecular 3D structure model of EGFR D770insNPG. HER2 A775insYVMA was constructed using a previously published model by Shen et al. Homology models were constructed using MODELLER 9v6 and further energy minimized using the Molecular Operating Environment software package (Chemical Computing Group, Montreal, Canada). Molecular docking of TKI exon 20 mutants to EGFR and HER2 was performed using GOLD software with default parameters unless otherwise stated. Early termination was prohibited in the docking process. Covalent bond formation between the receptor and the inhibitor was modeled using restraints. The flexibility of the residues within the binding pocket was accommodated using GOLD software. Figures showing the correlation between EGFR / HER2 and the inhibitor were visualized using PYMOL.

[0139] Western Blotting of Ba / F3 Variants: 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. Using a BioRad semidry transfer, the blot was then probed with antibodies against pEGFR (#2234), EGFR (#4267), pHER2 (#2247), HER2 (#4290) (1:1000; Cell Signaling). The blot was probed with an antibody against β-actin (Sigma-Aldrich, #A2228) or vinculin (Sigma-Aldrich, #V4505) as a loading control and exposed using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) and the BioRad ChemiDoc Touch Imaging System or X-ray film. Representative images of two different protein isolations are shown and the blot was run in duplicate. Quantification of Western blotting was completed in Photoshop and calculated as (background average intensity - sample average intensity) (number of pixels) = band intensity. Samples were first normalized to the loading control (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 Variants: Protein was recovered from each of the parental Ba / F3 cell line and the Ba / F3 exon 20 variant found to be the activating mutation described above. ELISA was performed for total EGFR (Cell signaling, #7250) and total HER2 (Cell Signaling, #7310) as per the manufacturer's instructions. The relative expression measured by ELISA was plotted against the IC 50 values calculated as described above. Pearson correlation coefficient and p-value were determined by GraphPad Prism.

[0141] Testing of patient-derived cell lines: CUTO14 cells were generated from the pleural effusion of a patient with lung adenocarcinoma after informed consent using the previously described culture method (Davies et al, 2013). The cell lines were treated with the indicated doses of afatinib or poziotinib for 72 hours, and cell viability was measured by MTS assay (Promega). The 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). The cells were treated with the indicated doses of afatinib or poziotinib for 2 hours. 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 lung adenocarcinoma. 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 products were amplified using the following primers: Sequencing was performed by Sanger sequencing using TIFF2025090740000004.tif12163. Forward and reverse sequence traces were reviewed manually. The variant detected in the patient-derived cell line was a complex insertion in exon 20 of EGFR, resulting in substitution of the amino acid asparagine at position 771 by two amino acids, phenylalanine and histidine (N771delinsFH). Cell viability and IC 50 Estimation was performed as described above.

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

[0144] Testing of Genetically Engineered Mouse Model (GEMM): EGFR D770insNPG and HER2 A775insYVMA GEMMs were generated as previously described (Perera et al, 2009; Cho el al, 2013). Mice were handled in accordance with Good Animal Practices as defined by the Office of Laboratory Animal Welfare and were conducted under the approval of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (Boston, MA). Mice were given continuous doxycycline diet starting at 6 weeks of age. Tumor volume was determined by MRI as previously described (Perera et al, 2009; Cho et al, 2013). Mice with equal initial tumor volumes were non-blinded and randomly assigned to vehicle and 10 mg / kg per day poziotinib based on palpable tumor formation determined by MRI. Mice that were humanely euthanized for events unrelated to tumor burden were excluded from the final analysis.

[0145] Example 3 - Identification of Drugs Against 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 among cancer types, we queried multiple databases, including cohorts from cBioPortal, MD Anderson Cancer Center, and Foundation Medicine, as well as the cfDNA cohort from Guardant Health. Across all databases, all non-synonymous HER2 mutations were analyzed within a range of 25 different cancer types (Table 4). The weighted average frequency for HER2 mutations was calculated. Similar to what was observed in the AACR GENIE database (Meric-Bernstam et al, 2018), HER2 mutations occurred most frequently in 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 the mutation hotspots vary by malignancy: Next, the frequencies of mutations were analyzed within the ranges of the various regions of the HER2 receptor reported in cBioPortal and MD Anderson. Across all cancer types, HER2 mutations occurred most frequently in the tyrosine kinase domain (46%), which included mutations in exon 20 (20%), exon 19 (11%), and exon 21 (9%) (Figure 14A). In addition, extracellular domain mutations consisted of 37% of HER2 mutations. Across all cancers queried, the most commonly seen 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, most HER2 mutations occurred within exon 20 (48%), and Y772_A775dupYVMA accounted for 34% of all HER2 mutations (Figures 14B, 14F). In breast cancer, most HER2 mutations occurred within exon 19 (37%), and the L755 mutation had the highest prevalence at 22% of HER2 mutations (Figure 14C). On the other hand, unlike lung cancer where one variant was dominant, there was more mutational diversity within exon 19 mutations in breast cancer (Figure 14G). In colorectal cancer, HER2 mutations occurred most frequently in exon 21 (23%) and the extracellular domain (23%), and the V842I variant in exon 21 had the highest prevalence (19%) (Figures 14D, 14H).

[0147] Y772dupYVMA is the most frequently observed HER2 exon 20 insertion mutation across all cancer types: HER2 exon 20 mutations are the most frequent mutations within the tyrosine kinase domain of HER2 (16% of all HER2 mutations and 43% of tyrosine kinase domain mutations), and HER2 exon 20 insertion mutations remain a clinical challenge. To understand the diversity and prevalence of exon 20 insertions, the frequency of HER2 exon 20 insertion sequences was analyzed by cancer type in the cBioportal database, MD Anderson database, and Guardant Health database. The Y772dupYVMA insertion is the most frequently observed HER2 exon 20 insertion, accounting for 70% of all HER2 exon 20 insertions. The p.G778dupGSP (14%) and p.G776del insVC (9%) insertions occurred with the second and third highest frequencies (Figure 21A). Exon 20 insertion mutations in NSCLC (N = 362) showed the greatest diversity of exon 20 insertion mutations (Figure 21B), while exon 20 insertion mutations in breast cancer (N = 30) showed little diversity in the insertion sequences, with only three different variants reported (Figure 21C). In other cancer types, additional insertion mutations were rarely observed, and duplications at Y772 and G778 occurred with the highest frequencies in all cancer types analyzed (Figure 21D).

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

[0149] Ponatinib is the most potent TKI tested and inhibited the most frequently seen HER2 mutations in vitro: Recent reports have highlighted the efficacy of covalent quinazoline amine-based TKIs (i.e., afatinib, dacomitinib, ponatinib, neratinib) in preclinical models of HER2 variant disease, but clinical trials of afatinib, dacomitinib, and neratinib have low ORRs and cancer-specific and variant-specific differences in patient outcomes. To systematically evaluate drug sensitivity among the most frequently detected HER2 variants, a panel of HER2 variant Ba / F3 cells was screened against 11 covalent and non-covalent EGFR and HER2 TKIs. HER2 variants showed strong resistance to the non-covalent 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 quinazoline amine-based TKIs, afatinib, neratinib, dacomitinib, taloxotininib-TKI, and ponatinib had inhibitory activity against HER2 variants across all three exons (Figure 16A). Among all HER2 variant and TKI tested, ponatinib had the lowest average IC 50 and was significantly more effective in reducing cell viability than afatinib, neratinib, or taloxotininib-TKI (Figure 16B). In addition, ponatinib was significantly more effective than any of afatinib, neratinib, or taloxotininib-TKI against HER2 exon 19 and 20 mutations, but the average IC against exon 21 variants 50There was no significant difference (Figs. 16C-E), suggesting that the mutation sites affect drug binding. Furthermore, the L755S and L755P variants within exon 19 had significant differences in drug sensitivity across all TKIs tested (Fig. 16F), indicating that specific amino acid changes at this site affected drug binding affinity.

[0150] The position and amino acid changes of HER2 mutations affect drug binding affinity: To further understand how the position and amino acid changes of mutations can affect drug binding affinity and inhibitory effects, molecular dynamics simulations were used to examine how these mutations affect the structure and dynamics of the HER2 kinase domain. Molecular models of the L755S, L755P, Y772dupYVMA, and V777L HER2 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. In particular, the range of sampled protein conformations with respect to the P-loop and α-C-helix positions differed among these HER2 mutants. Even among exon 20 mutations, the differences were evident, especially in the α-C-helix region, where the persistence of the α-C-helix conformation differed between "in" (active conformation with a smaller binding pocket) and "out" (inactive conformation with a larger binding pocket). The V777L mutant sampled a large number of "out" conformations, while the Y772dupYVMA mutant sampled both "in" and "out" conformations (Figure 17A). Overall, these conformational state differences resulted in the Y772dupYVMA mutant being present in the "in" conformation (Figure 17B) with a frequency more than 10-fold higher than that of the V777L mutant, and on average, a smaller binding pocket in Y772dupYVMA compared to V777L (Figures 17C and 23B). In addition, since neratinib contains a pyridyl ring directed towards the α-C-helix, the smaller binding pocket of Y772dupYVMA may be responsible for weakening the efficacy of neratinib against Y772dupYVMA compared to V777L.

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

[0152] HER2 variant human cancer cell lines showed enhanced sensitivity to poziotinib: Clinical studies testing HER2 inhibitors have revealed cancer type-specific differences in drug sensitivity (Hyman et al, 2018). To determine whether covalent quinazoline amine-based TKIs are active in models of HER2 variant disease, a panel of EGFR / HER2 TKIs was tested in human cancer cell lines. Premalignant MCF10A breast epithelial cells were transfected with a HER2 exon 20 mutation and their sensitivity to 12 EGFR / HER2 TKIs was evaluated in vitro. MCF10A cells expressing the G776del insVC, Y772dupYVMA, or G778dupGSP HER2 mutations were the most sensitive to poziotinib, with IC 50 values of 12 nM, 8.3 nM, and 4.5 nM, respectively (186A–C). In comparison, talotrectinib-TKI and neratinib had mean IC 50Values were generated (Figures 18A - C), and poziotinib was shown to be more than 2.6 - fold and 19 - fold more potent than tarlotrectinib - TKI and neratinib, respectively (p < 0.001). Further, Western blotting of MCF10A HER2 G776delinsVC cells with poziotinib and neratinib showed that poziotinib completely suppressed p - HER2 at 10 nM, while neratinib did not (Figure 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, the selectivity index (SI, IC 50 value mutant / IC 50 value WT) was calculated for each inhibitor, and poziotinib was found to be the most mutant - selective TKI tested in the MCF10A cell line (SI = 0.028), followed by pyrotinib (SI = 0.063) and tarlotrectinib - TKI (SI = 0.111) (Figure 18D). Consistent with the data obtained using Ba / F3 cells (Figure 15C), in a model of HER2 exon 19 mutant colorectal cancer (CW - 2), the differences in sensitivity among poziotinib, tarlotrectinib - TKI, and neratinib were not as dramatic but were significant (p = 0.02 and p = 0.0004), with mean IC 50 values of 3.19 nM, 4.24 nM, and 68.8 nM, respectively (Figure 18E). Further, in a xenograft mouse model of CW - 2 colorectal cells, on day 21, animals treated with poziotinib (5 mg / kg) showed a 58% reduction in tumor volume compared to the vehicle - treated group (p = 0.011). In comparison, animals treated with neratinib (30 mg / kg) showed an increased tumor volume (28%) compared to the vehicle control (p = 0.023), and afatinib (20 mg / kg) treatment had no significant effect on tumor growth compared to the vehicle control (Figures 18F, 25).

[0153] Poziotinib has antitumor activity in NSCLC patients with HER2 mutations: Based on these preclinical data and previously published studies on exon 20 mutations (Robichaux et al, 2018), a physician-led phase II clinical trial of poziotinib (NCT03066206) in EGFR and HER2 exon 20 mutant NSCLC was initiated. Patients were treated with 16 mg of poziotinib orally daily until progression, death, or withdrawal. Objective responses were evaluated every 8 weeks based on RECIST v1.1. Among the first 12 evaluable patients with HER2 exon 20 insertion mutations, 6 / 12 (50%) patients had a best response of partial response (PR). This response was confirmed in 5 / 12 by serial scans after 2 months (confirmed objective response rate, 42%) (Figure 19A). Among these 12 patients, 2 patients had progression (PD) at the time of the first efficacy determination, and a disease control rate (DCR) of 83% was obtained. As of December 2018, 10 out of 12 patients were continuing, and the median PFS of the first 12 patients was 5.6 months (Figure 19B). To date, all patients included in this trial harbored one of the two most common HER2 exon 20 insertions, Y772dupYVMA and G778dupGSP (Figure 19A). Representative images before and after (8 weeks) treatment of one NSCLC patient with the Y772dupYVMA mutation showed significant tumor shrinkage in the right lung (Figure 19C). Patient characteristics, including the number of prior treatment histories, are shown in Table 3. In addition, one heavily pretreated NSCLC patient with the HER2 exon 19 point mutation, L755, was treated under a compassionate use protocol (C-IND18-0014). The patient was treated with 16 mg of poziotinib daily and had tumor shrinkage at 4 weeks (Figure 19D, white frame). The patient had stable disease (SD) (-12% decrease in target lesions) according to RECIST v1.1. The patient had disease control and continued with poziotinib for over 7 months until obvious disease progression was imaged and poziotinib was discontinued. The patient was clinically well at the end of poziotinib treatment and subsequently received further systemic treatment.

[0154] The combination of poziotinib and T-DM1 treatment enhances antitumor activity: Previous studies of the HER2 TKI lapatinib in HER2-positive breast cancer models and EGFR inhibitors in EGFR mutant NSCLC models have shown that TKI treatment results in increased receptor accumulation on the cell surface and that increased cell surface HER2 / EGFR increases sensitivity to antibody-dependent cell cytotoxicity (ADCC). To determine whether poziotinib treatment increases total HER2 receptor expression on the cell surface, cell surface HER2 expression was analyzed by FACS 24 hours after low-dose poziotinib treatment. On average, poziotinib treatment was found to increase cell surface HER2 expression two-fold (Figure 20A, p<0.0001). Next, it was tested whether the combination of poziotinib and T-DM1 could reduce cell viability in vitro. T-DM1 alone did not suppress the viability of the MCF10A HER2 mutant cell line, but the combination of T-DM1 and poziotinib was significantly lower than either agent alone in a dose-dependent manner for the IC 50It was found to have brought about a value (Figure 20B). To verify these findings in vivo, a combination of low-dose poziotinib and a single dose of T-DM1 was tested in a HER2 variant NSCLC PDX model, HER2 Y772dupYVMA (Figure 20C). To evaluate the response to treatment, the progression-free survival (PFS), defined as the time from the best effect to tumor doubling, was determined. Mice receiving the vehicle control had a median PFS (mPFS) of 3 days, while mice receiving low-dose poziotinib or T-DM1 had mPFSs of 15 days and 27 days, respectively. However, mice (14 / 20) receiving a single dose of T-DM1 in combination with low-dose poziotinib remained tumor-free on day 45 (Figure 20D). Furthermore, on day 15, the time point of the best effect, the combination of low-dose poziotinib (2.5 mg / kg) and a single dose of T-DM1 (10 mg / kg) resulted in complete tumor regression in 20 / 20 mice (100%), compared to 2 / 9 mice receiving T-DM1 alone or 0 / 12 mice receiving low-dose poziotinib (Figures 20C - F). By day 30, tumor growth had started in all mice receiving T-DM1 alone; however, in 14 / 20 mice receiving the combination treatment, there was no evidence of tumor recurrence (Figures 20F, G).

[0155] Further tests verified the effectiveness of poziotinib compared to other TKIs. It was found that poziotinib was more effective than high-dose osimertinib in an EGFR S768dupSVD PDX model (Figure 26). It was also shown that poziotinib had higher antitumor activity than neratinib in a PDX model of NSCLC harboring Y772dupYVMA (Figure 27). Single-agent poziotinib was more effective than neratinib in a breast cancer PDX model harboring V777L (Figure 28). An overview of the effectiveness of poziotinib antitumor activity in various EGFR and HER2 exon 20 variant in vivo models is shown in Figure 29.

[0156] Table showing the mean IC50 values for Ba / F3 cells expressing the EGFR exon 20 mutations shown in (Table 3). Ba / F3 cells were generated to determine the IC50 values. Cells were seeded technically in triplicate in 384-well plates at 2,000 cells per well. After 24 hours, the cells were treated with 7 different doses of poziotinib ranging from 150 nM to 0.01 nM. Percent survival was determined and normalized to DMSO-treated controls. The IC50 values for each biological replicate were calculated using non-linear regression modeling in GraphPad Prism. The mean values and SEM are representative of 3 independent experiments. TIFF2025090740000005.tif19884

[0157] In this specification, it is reported that HER2 mutations occur in various tumor types, but the specific mutation hotspots vary for each malignancy. Furthermore, sensitivity to HER2 TKIs is heterogeneous among mutation positions, and HER2 exon 20 insertions and the L755P mutation are resistant to most HER2 TKIs, presumably due to a reduction in the volume of the drug-binding pocket. Furthermore, poziotinib has been identified as a potent pan-HER2 variant-selective inhibitor with clinical efficacy in NSCLC patients with HER2 exon 20 insertions and the L755P mutation. Finally, it was established that poziotinib treatment induces the accumulation of HER2 on the cell surface and that the combination of poziotinib and T-DM1 treatment enhances antitumor activity in vitro and in vivo.

[0158] Pan-cancer analysis indicates that HER2 mutation hotspots vary by cancer type and have different sensitivities to HER2 TKIs in vitro, which may affect clinical efficacy. In the SUMMIT trial, neratinib produced the highest efficacy in breast cancer patients, and most responders were positive for the L755S, V777L, or L869R mutations. In in vitro Ba / F3 drug screening, these mutations had low IC 50correlated with the values. In contrast, patients with colorectal cancer did not respond to neratinib. Consistent with this clinical observation, the V842I mutation is the most frequently observed HER2 mutation in colorectal cancer cases, and this specific mutation was found to be insensitive to neratinib in drug screening assays. These data suggest that different TKI sensitivities among malignancies are, in part, explained by cancer-specific mutation hotspots and may directly affect drug sensitivity. However, significant questions remain regarding the reasons for the different distributions of HER2 mutations among tumor types and whether certain mutations produce similar drug responses in different tumor types. Data from the SUMMIT trial showed that specific exon 20 insertions were associated with neratinib sensitivity in breast cancer patients, but these same mutations were associated with resistance in all other cancer types, indicating that potential mechanisms underlying these tumor type-specific differences in sensitivity may exist and warrant further investigation.

[0159] Exon 20 insertion mutations and exon 19 L755P mutations are resistant to most HER2 TKIs. In vitro drug screening showed that exon 20 insertion mutations and L755P mutations had the highest IC 50 values for each TKI tested. 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 the clinical observation that patients with HER2 exon 20 insertion mutations have historically had poor responses to TKIs. In lung cancer, where exon 20 insertions occur with high frequency, patients harboring the HER2 exon 20 insertion mutation had response rates of 0%, 11.5%, and 18.2% - 18.8% to neratinib, dacomitinib, and afatinib, respectively. Furthermore, while the L755S mutation has been shown to respond to neratinib, the L755P mutation has strong resistance to both TKIs and antibody-drug conjugates.

[0160] Example 4 - Materials and Methods Analysis of HER2 Mutation Prevalence and Variant Frequency: 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 individually, and then the frequencies were weighted by the total number of patients in each database and reported as a weighted average. To determine the frequency of HER2 mutations between cancer types in cBioPortal, all non-overlapping tests were selected and exported. For overlapping tests, only the largest dataset was used. To determine the HER2 mutation frequency at MD Anderson Cancer Center, the Institute for Personalized Cancer Therapy database was queried for all HER2 mutations regardless of cancer type. To determine the frequency of HER2 exon 20 mutations from Foundation Medicine, unspecified data on the number of patients with HER2 deletions, frameshifts, insertions, and point mutations were tabulated, and cancer types with 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 - to 73-gene panels) tested between October 2015 and May 2018 with ERBB2 exon 20 mutations. Guardant360® is a CLIA-certified CAP / NYSDOH-approved comprehensive cfDNA NGS test that reports SNVs, indels, fusions, and SNVs in up to 73 genes. The frequencies reported from Guardant Health were then normalized and clinically sensitive corrected as reported by Odegaard et al 2018. Specifically, the frequencies were divided by the percent clinical sensitivity, 85.9%.

[0161] Generation of Ba / F3 cell line and IL-3 deficiency: The Ba / F3 cell line was established as previously described. Briefly, stable Ba / F3 cell lines were generated by retroviral transduction of Ba / F3 cell lines over 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 RPMI medium. After 5 days of selection, cells were stained with FITC-HER2 (Biolegend) and sorted by FACS. Then, the cell line was grown for 2 weeks in the absence of IL-3, and cell viability was evaluated every 3 days using the Cell Titer Glo assay (Progema). The resulting stable cell lines were maintained in RPMI-1640 medium containing 10% FBS without IL-3.

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

[0163] ELISA for phosphorylated HER2 and total HER2 and correlation with IC50 values: Proteins were recovered from each of the parental Ba / F3 cell line and Ba / F3 cell lines expressing HER2 mutations as described above. 5 μg / ml of protein was added to each ELISA plate, and ELISA 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. The relative p-HER2 ratio was plotted against the poziotinib IC50 value calculated as described above. Pearson correlation coefficient and p-value were determined by GraphPad Prism.

[0164] Tyrosine kinase inhibitors and T-DM1: All inhibitors were purchased from Selleck Chemical, except for EGF816 and pyrotinib 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 be discarded after two freeze-thaw cycles. T-DM1 was purchased from the institutional pharmacy at M.D. Anderson Cancer Center and reconstituted.

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

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

[0167] In vivo xenograft studies: CW-2 cell line xenografts were generated by injecting 1 × 10 6 cells in 50% Matrigel into 6-week-old female nu / nu nude mice. When the tumors reached 350 mm 3 in size, the mice were randomized into 4 groups: 20 mg / kg afatinib, 5 mg / kg poziotinib, 30 mg / kg neratinib, or vehicle control (0.5% methylcellulose, 2% Tween-80 in dH2O). Tumor volumes were measured three times a week. Mice received drugs on Monday to Friday (5 days a week), starting on Wednesday and given 2 days off after 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- to 6-week-old female NSG mice (Jax Labs #005557). Mice were measured three times a week, and when the tumors reached 200 - 300 mm 3Once the tumor volume reached [the specified volume], the mice were randomized into the following four treatment groups: vehicle control (0.5% methylcellulose, 0.05% Tween-80 in dH2O), 2.5 mg / kg poziotinib, 10 mg / kg T-DM1, or a combination of 2.5 mg / kg poziotinib and 10 mg / kg T-DM1. Tumor volume and body weight were measured three times a week. Mice treated with 2.5 mg / kg poziotinib received the drug orally from Monday to Friday (5 days a week). Mice treated with 10 mg / kg T-DM1 received a single intravenous (IV) administration of T-DM1 on the day of randomization. Mice treated with the combination of poziotinib and T-DM1 received a single IV administration of T-DM1 and started 2.5 mg / kg poziotinib orally 5 days a week, 3 days after the administration of T-DM1. If the body weight of the mice decreased by more than 10% or dropped below 20 grams, the mice were given a break from dosing. Progression-free survival was defined as the time from the best response in two consecutive measurements to tumor doubling. Complete regression was defined as a decrease of more than 95% in tumor volume, and in mice with complete regression, tumor doubling was defined as exceeding 75 mm 3 in three or more consecutive measurements. The experiment was conducted in accordance with Good Animal Practices and was completed with the approval of the MD Anderson Cancer Center Institutional Animal Care and Use Committee (Houston, TX).

[0169] (Table 3) Vectors used to generate stable cell lines TIFF2025090740000006.tif129169

[0170] (Table 4) Total number of patients by cancer type across the entire database TIFF2025090740000007.tif225160

[0171] (Table 5) Number of patient characteristics and prior treatment histories TIFF2025090740000008.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, the cells were washed twice with PBS and trypsinized. The cells were then resuspended in 0.5% FBS in PBS and stained with anti-HER2-FITC antibody from Biolegend (#324404) for 45 minutes on ice. The cells were washed twice with 0.5% FBS in PBS and analyzed by flow cytometry. IgG and unstained controls were used for gating.

[0173] Western blotting: For western blotting, the cells were washed with PBS and lysed with RIPPA lysis buffer (ThermoFisher) and protease inhibitor cocktail tablets (Roche). Protein (30 - 40 μg) was loaded onto gels purchased from BioRad. Using a BioRad semi-dry transfer, the blot was then probed with antibodies against pHER2, HER2, pPI3K, PI3K, p-AKT, AKT, p-ERK1 / 2, and ERK1 / 2 (1:1000; Cell Signaling). The blot was probed with an antibody against vinculin or β-actin (Sigma-Aldrich) as a loading control and exposed using ECL western blotting substrate (Promega).

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

[0175] Clinical Trials and CIND Identification Number: Patients provided written informed consent for treatment with poziotinib either under the compassionate use protocol (MD Anderson Cancer Center CIND-18-0014) or in clinical trial NCT03066206. The protocol has been approved by both the MD Anderson Cancer Center Institutional Review Board and the U.S. Food and Drug Administration.

[0176] All of the methods disclosed and claimed herein can be constructed and implemented without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and procedures described herein, or to the order of the method steps, without departing from the concept, spirit, and scope of the invention. More specifically, certain chemically and physiologically related agents may be used in place of the agents described herein, and it will be apparent that they may achieve the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0177] References The following references provide exemplary procedural or other details that supplement the references set forth herein and are specifically incorporated herein by reference. TIFF2025090740000009.tif194158TIFF2025090740000010.tif245112TIFF2025090740000011.tif216101

[0178] Sequence Information SEQUENCE LISTING <110> BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM <120> COMPOUNDS WITH ANTI-TUMOR ACTIVITY AGAINST CANCER CELLS BEARING EGFR OR HER2 EXON 20 INSERTIONS <150> US 62 / 826,843 <151> 2019-03-29 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> EGFR mutation <400> 1 Phe Gln Glu Ala 1 <210> 2 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> EGFR mutation <400> 2 tccaggaagc ct 12 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> HER2 mutation <400> 3 Tyr Val Met Ala 1 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> HER2 mutation <400> 4 tatgtcatgg ct 12 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cttacaccca gtggagaagc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 accaagcgac ggtcctccaa 20 <210> 7 <211> 45 <212> DNA <213> Homo sapiens <400> 7 gaagcctacg tgatggccag cgtggacaac ccccacgtgt gccgc 45 <210> 8 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20 A763insFQEA <400> 8 gaagcctcca ggaagcctta cgtgatggcc agcagcgtgg acgtggacaa cccccacgtg 60 tgccgc 66 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20 S768dupSVD <400> 9 gaagcctacg tgatggccag cgtggacaac ccccacgtgt gccgc 45 <210> 10 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20 V769insASV <400> 10 gaagcctacg tgatggccag cgtgccagcg tgggacaacc cccacgtgtg ccgc 54 <210> 11 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20 D770insSVD <400> 11 gaagcctacg tgatggccag cgtggacgcg tggacaaacc cccacgtgtg ccgc 54 <210> 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20 H773insNPH <400> 12 gaagcctacg tgatggccag cgtggacaac ccccacaacc cccacgtgtg ccgc 54 <210> 13 <211> 48 <212> DNA <213> Homo sapiens <400> 13 gaagcatacg tgatggctgg tgtgggctcc ccatatgtct cccgcctt 48 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> HER2 exon 20 G776V <400> 14 gaagcatacg tgatggctgt tgtgggctcc ccatatgtct cccgcctt 48 <210> 15 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HER2 exon 20 G776V V777insV <400> 15 gaagcatacg tgatggcttg tgtgttgggc tccccatatg tctcccgcct t 51 <210> 16 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HER2 exon 20 G776del insVV <400> 16 gaagcatacg tgatggctgt tgttgtgggc tccccatatg tctcccgcct t 51 <210> 17 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> HER2 exon 20 G776del ins V <400> 17 cgaagcatac gtgatggctg gtgtgtctgg ctccccatat gtctcccgcc tt 52 <210> 18 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> HER2 exon P780insGSP <400> 18 gaagcatacg tgatggctgg tgtgggctcc ccatggctcc cctatgtctc ccgcctt 57

Claims

1. 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.

2. The method of claim 1, wherein said poziotinib is further defined as poziotinib hydrochloride.

3. The method of claim 2, wherein the poziotinib hydrochloride is formulated as a tablet.

4. The method of any one of claims 1 to 4, wherein the one or more EGFR exon 20 mutations are further defined as EGFR20 insertion mutations.

5. The method of any one of claims 1 to 4, wherein the one or more EGFR exon 20 mutations are further defined as a de novo EGFR20 insertion mutation.

6. The method of any one of claims 1 to 5, wherein the 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.

7. The method of any one of claims 1 to 6, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations.

8. The method of any one of claims 1 to 7, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S.

9. The method of any one of claims 1 to 8, wherein the subject has previously been administered a tyrosine kinase inhibitor.

10. The method of claim 9 , wherein the subject is resistant to a previously administered tyrosine kinase inhibitor.

11. 11. The method of claim 10, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, or beratinib.

12. 12. The method of any one of claims 1 to 11, 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.

13. 13. The method of any one of claims 1 to 12, 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.

14. The method of any one of claims 1 to 13, wherein the subject is determined to not have an EGFR mutation at residue C797.

15. 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 15. The method of any one of claims 1 to 14, wherein the nucleotide sequence is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV.

16. The one or more exon 20 mutations are 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 16. The method of any one of claims 1 to 15, wherein the nucleotide sequence is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C.

17. The method of any one of claims 1 to 16, wherein the exon 20 mutation is D770insNPG.

18. The method of any one of claims 1 to 17, wherein the subject is determined to have an EGFR exon 20 mutation by analyzing a genomic sample from the patient.

19. 20. The method of claim 19, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

20. The method of any one of claims 1 to 19, wherein the presence of an EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis.

21. The method of any one of claims 1 to 20, wherein the poziotinib is administered orally.

22. The method of any one of claims 1 to 21, wherein the poziotinib is administered at a dose of 5 to 25 mg.

23. The method of any one of claims 1-22, wherein the poziotinib is administered in a dose of 8 mg, 12 mg, or 16 mg.

24. The method of any one of claims 1 to 23, wherein the poziotinib is administered daily.

25. The method of any one of claims 1 to 24, wherein the poziotinib is administered continuously.

26. The method of any one of claims 1 to 25, wherein the poziotinib is administered in 28 day cycles.

27. The method of any one of claims 1 to 26, further comprising administering a further anti-cancer therapy.

28. 28. The method of claim 27, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

29. 29. The method of claim 27 or 28, wherein the 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.

30. The method of any one of claims 27 to 30, wherein administering poziotinib and / or administering anti-cancer therapy comprises administering locally, regionally, or systemically.

31. The method of any one of claims 27 to 31, wherein the administration of poziotinib and / or anti-cancer therapy is administered two or more times.

32. 32. The method of any one of claims 1 to 31, 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.

33. The method of any one of claims 1 to 32, wherein the cancer is non-small cell lung cancer.

34. The method of any one of claims 1 to 33, wherein the patient is a human.

35. A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more EGFR exon 20 mutations.

36. 36. The composition of claim 35, further defined as an oral composition.

37. The composition of claim 35 or 36, comprising 5 to 25 mg of poziotinib.

38. 38. The composition of any one of claims 35-37, comprising 8 mg, 12 mg, or 16 mg of poziotinib.

39. The composition of any one of claims 35-38, wherein said poziotinib is further defined as poziotinib hydrochloride.

40. The composition of any one of claims 35 to 39, formulated as a tablet.

41. The composition of any one of claims 35 to 40, wherein the one or more EGFR exon 20 mutations are further defined as an EGFR20 insertion mutation.

42. The composition of any one of claims 35 to 41, wherein the one or more EGFR exon 20 mutations are further defined as a de novo EGFR20 insertion mutation.

43. The composition of any one of claims 35 to 42, wherein the 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.

44. The composition of any one of claims 35 to 43, wherein the subject is determined to have two, three, or four EGFR exon 20 mutations.

45. The composition of any one of claims 35 to 44, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S.

46. 46. ​​The composition of any one of claims 35-45, 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, and V774.

47. 47. The composition of any one of claims 35 to 46, 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.

48. The composition of any one of claims 35 to 47, wherein the subject is determined to not have an EGFR mutation at residue C797.

49. 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 49. The composition of any one of claims 35 to 48, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV.

50. The one or more exon 20 mutations are 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 50. The composition of any one of claims 35 to 49, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C.

51. The composition of any one of claims 35 to 50, wherein the patient is being treated with an anti-cancer therapy.

52. 1. A method of 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 an anti-cancer therapy.

53. 53. The method of claim 52, wherein the EGFR exon 20 mutation is further defined as an exon 20 insertion mutation.

54. 54. The method of claim 52 or 53, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

55. The method of any one of claims 52 to 54, wherein the presence of an EGFR exon 20 mutation is determined by nucleic acid sequencing or PCR analysis.

56. 56. The method of any one of claims 52 to 55, wherein the 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.

57. The method of any one of claims 52 to 56, wherein the one or more EGFR exon 20 mutations are not T790M and / or C797S.

58. 58. The method of any one of claims 52 to 57, wherein the EGFR exon 20 mutation is present at residues A763, A767, S768, V769, D770, N771, P772, H773, and / or V774.

59. 59. The method of any one of claims 52 to 58, wherein the EGFR exon 20 mutation is present at residues A763, A767, S768, V769, D770, N771, P772, H773, V774, and / or R776.

60. The EGFR exon 20 mutation is A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY 60. The method of any one of claims 52 to 59, wherein the variant is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV.

61. 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 61. The method of any one of claims 52-60, wherein the amino acid sequence is selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C.

62. 62. The method of any one of claims 52-61, 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.

63. The method of any one of claims 52-62, further comprising administering poziotinib, alone or in combination with a second anti-cancer therapy, to said patients predicted to have a favorable response.

64. The method of any one of claims 52-63, wherein the poziotinib is administered orally.

65. The method of any one of claims 52-64, wherein the poziotinib is administered at a dose of 5-25 mg.

66. The method of any one of claims 62-65, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg.

67. The method of any one of claims 62-66, wherein said poziotinib is further defined as poziotinib hydrochloride.

68. The method of any one of claims 62-67, wherein the poziotinib hydrochloride is formulated as a tablet.

69. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib or afatinib, wherein the subject is a patient having any of the following cancer types: A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del The method of the present invention relates to a patient having a HER2 exon 20 mutation selected from the group consisting of insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V.

70. 70. The method of claim 69, 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.

71. 71. The method of claim 69 or 70, wherein the poziotinib is administered orally.

72. The method of any one of claims 69-71, wherein the poziotinib is administered at a dose of 5-25 mg.

73. 73. The method of any one of claims 69-72, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg.

74. The method of any one of claims 69-73, wherein said poziotinib is further defined as poziotinib hydrochloride.

75. The method of any one of claims 69 to 74, wherein the poziotinib hydrochloride is formulated as a tablet.

76. 76. The method of any one of claims 69-75, wherein 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.

77. 77. The method of any one of claims 69-76, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786.

78. 77. The method of any one of claims 69 to 76, wherein the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780.

79. The method of any one of claims 69 to 78, wherein the HER exon 20 mutation is further defined as a HER2 exon 20 insertion mutation.

80. The method of any one of claims 69 to 79, wherein the HER exon 20 insertion mutation is A775insYVMA.

81. 81. The method of any one of claims 69 to 80, further comprising administering an mTOR inhibitor.

82. 82. The method of claim 81, wherein the mTOR inhibitor is rapamycin, temsirolimus, everolimus, ridaforolimus, or MLN4924.

83. 82. The method of claim 81, wherein the mTOR inhibitor is everolimus.

84. 82. The method of claim 81, wherein the poziotinib or afatinib and / or the mTOR inhibitor is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, controlled release, delayed release, as a suppository, or sublingually.

85. The method of any one of claims 69 to 84, wherein the subject is determined to have a HER2 exon 20 mutation by analyzing a genomic sample from the patient.

86. 86. The method of claim 85, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

87. The method of any one of claims 69 to 86, wherein the presence of a HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis.

88. 88. The method of any one of claims 69 to 87, further comprising administering an additional anti-cancer therapy.

89. 89. The method of any one of claims 69 to 88, wherein said further anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

90. 90. The method of any one of claims 69 to 89, wherein 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 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.

91. 91. The method of any one of claims 69 to 90, wherein the cancer is non-small cell lung cancer.

92. The method of any one of claims 69 to 91, wherein the subject is a human.

93. 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.

94. 94. The composition of claim 93, 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.

95. The composition of claim 93 or 94, wherein the HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation.

96. The composition of any one of claims 93 to 95, wherein the 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.

97. 97. The composition of any one of claims 93 to 96, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786.

98. 97. The composition of any one of claims 93 to 96, wherein the one or more HER2 exon 20 mutations are present at residues V773, A775, G776, V777, G778, S779, and / or P780.

99. The pharmaceutical composition of any one of claims 93 to 98, wherein the patient is being treated with an anti-cancer therapy.

100. 1. A method of 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.

101. 101. The method of claim 100, wherein the HER2 exon 20 mutation is further defined as a HER2 exon 20 insertion mutation.

102. 102. The method of claim 100 or 101, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

103. The method of any one of claims 100 to 102, wherein the presence of a HER2 exon 20 mutation is determined by nucleic acid sequencing or PCR analysis.

104. 104. The method of any one of claims 100-103, wherein the anti-cancer therapy is an mTOR inhibitor.

105. The method of any one of claims 100-104, wherein the favorable responsiveness to the poziotinib inhibitor alone or the 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, increased disease-free interval, increased time to progression, induction of remission, reduction in metastasis, or improved patient survival.

106. The method of any one of claims 100-105, further comprising administering to said patient predicted to have a favorable response poziotinib or afatinib, alone or in combination with a second anti-cancer therapy.

107. (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:

108. 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 107, further comprising:

109. 109. The composition of claim 107 or 108, further comprising a thermostable DNA polymerase.

110. The composition of any one of claims 107 to 109, further comprising a dNTPS.

111. A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, N771dupNPH, A767insTLA, V769insGVV, V769L, V769insGSV, V769ins MASVD, D770del ins GY, D770insG, D770insY 111. The composition of any one of claims 108-110, wherein the labeled probe hybridizes to the first portion of exon 20 of the human EGFR coding sequence if a mutation selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV is present.

112. 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 112. The composition of any one of claims 108-111, wherein the labeled probe hybridizes to the first portion of exon 20 of the human EGFR coding sequence if a mutation selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C is present.

113. A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, P780insGSP, V777L, G778insLPS, V773M, Y772dupYVMA, G776del 112. The composition of any one of claims 108-111, wherein the labeled probe hybridizes to the first portion of exon 20 of the human HER2 coding sequence if a mutation selected from the group consisting of: insLC, G778dupGSP, V777insCG, G776V / S, V777M, M774dupM, A775insSVMA, A775insVA, and L786V is present.

114. 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, including a point mutation, an insertion, and / or a deletion of 3-18 nucleotides at amino acids 763-778.

115. 115. The isolated nucleic acid of claim 114, 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.

116. 116. The isolated nucleic acid of claim 114 or 115, 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.

117. 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 117. The isolated nucleic acid of any one of claims 114 to 116, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, P772insDNP, H773insAH, H773insH, and V774insHV.

118. The one or more exon 20 mutations are 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 118. The isolated nucleic acid of any one of claims 114 to 117, selected from the group consisting of H773Y, N771insSVDNR, N771insHH, N771dupN, P772insDNP, H773insAH, H773insH, V774M, V774insHV, R776H, and R776C.

119. 119. The isolated nucleic acid of any one of claims 114 to 118, comprising the sequence of SEQ ID NO: 8, 9, 10, 11, or 12.

120. 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.

121. 121. The isolated nucleic acid of claim 120, wherein the one or more HER2 exon 20 mutations are present at residues Y772, A775, M774, G776, G778, V777, S779, P780, and / or L786.

122. 122. The isolated nucleic acid of claim 120 or 121, 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.

123. 123. The isolated nucleic acid of any one of claims 120 to 122, comprising the sequence of SEQ ID NO: 14, 15, 16, 17, or 18.

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