Compound for cancer having tyrosine kinase inhibitor-resistant EGFR mutation

Abstract

The present disclosure provides methods of treating cancer in patients determined to have an osimertinib-resistant EGFR mutation by administering a third-generation tyrosine kinase inhibitor, such as poziotinib. TIFF2022529945000014.tif59128

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 835,343, filed Apr. 17, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] 1. Field The present invention generally relates to the fields of molecular biology and medicine. More specifically, the present invention relates to methods for treating patients having tyrosine kinase inhibitor-resistant EGFR mutations.

Background Art

[0003] 2. Description of the Related Art Approximately 10% of non-small cell lung cancers (NSCLCs) have mutations in the epidermal growth factor receptor (EGFR), which mutations increase sensitivity to tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and osimertinib. Recently, osimertinib has been approved as first-line therapy for EGFR-mutated NSCLC4, but de novo and acquired resistance remain a treatment obstacle for many patients. A series of acquired non-canonical EGFR mutations can have the potential to confer osimertinib resistance. It has been shown by testing that these acquired non-canonical resistance mutations change the conformation of the drug-binding pocket near the solvent in front of osimertinib, causing a change in the binding affinity between the drug and the receptor. Thus, there is an unmet need for new therapies for treating resistant EGFR-mutated cancers.

Summary of the Invention

[0004] Summary Aspects of the present disclosure provide methods and compositions for treating cancer in patients having resistant EGFR mutations. In a first aspect, 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 epithelial growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistant mutations. In certain aspects, the patient is human.

[0005] In some aspects, poziotinib is further defined as poziotinib hydrochloride. In certain aspects, poziotinib hydrochloride is formulated as a tablet.

[0006] In certain aspects, one or more EGFR TKI-resistant mutations include point mutations, insertions, and / or deletions (1 to 18 nucleotides) in exon 18, 19, 20, or 21. In some aspects, one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 688 - 728 of exon 18. In certain aspects, one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, G724, and T725. In certain aspects, one or more EGFR exon 18 mutations include E709A, E709K, L718Q, L718V, G719A, G719S, S720P, G724S, and / or T725M. In some aspects, one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 729 - 761 of exon 19. In certain aspects, one or more EGFR exon 19 mutations are located at one or more residues selected from the group consisting of I744, L747, L747, K754, A755, K757, and / or D761. In certain aspects, one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 763 - 823 of exon 20. In certain aspects, one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, A767, S768, V769, N771, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776.In some scenarios, one or more EGFR exon 20 mutations include A767ASV, D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, N771dupN, R776H, L792H, G796D, S784F, C775Y, and / or S811F. In certain scenarios, one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 824 - 875 of exon 21. In certain scenarios, one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L861, L862, L844, and L858. In some scenarios, one or more EGFR exon 21 mutations may include L833F, L833V, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some scenarios, the subject is determined to have two, three, or four EGFR TKI-resistant mutations. In certain scenarios, one or more EGFR TKI-resistant mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. In some scenarios, the subject is determined not to have an EGFR mutation at residue C797 or T790. In certain scenarios, the subject is determined not to have an EGFR mutation at residue T790. In other scenarios, the subject is determined to have the T790 mutation alone or in combination with another mutation (e.g., a G719 mutation, e.g., G719A or G719S). In certain scenarios, the subject is determined to have a mutation at the residue at C797. In some scenarios, one or more EGFR TKI-resistant mutations are selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid.In certain aspects, one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S.

[0007] In some aspects, the subject has previously received administration of a TKI. In certain aspects, the subject is resistant to the previously administered TKI. In some aspects, the TKI is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, naquotinib, or neratinib. In certain aspects, the TKI is osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, or naquotinib. In certain aspects, the TKI is osimertinib.

[0008] In certain aspects, the subject is determined to have an EGFR TKI resistance mutation by analyzing a patient-derived genomic sample. In some aspects, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain aspects, the presence of the EGFR TKI resistance mutation is determined by nucleic acid sequencing or PCR analysis.

[0009] In certain aspects, poziotinib is administered orally. In some aspects, poziotinib is administered at a dose of 5 - 25 mg. In certain aspects, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In some aspects, poziotinib is administered daily. In certain aspects, poziotinib is administered continuously. In some aspects, poziotinib is administered in a 28-day cycle.

[0010] In a further aspect, the method further includes a step of administering a further anti-cancer therapy. In some aspects, the further anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In certain aspects, poziotinib and / or the anti-cancer therapy is administered intravenously, subcutaneously, intrathecally, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. In some aspects, the administration of poziotinib and / or the anti-cancer therapy includes local, topical, or systemic administration. In certain aspects, poziotinib and / or the anti-cancer therapy is administered more than once.

[0011] 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.

[0012] In another aspect, there is provided a pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more EGFR TKI-resistant mutations. In some aspects, the composition is further defined as an oral composition. In certain aspects, the composition comprises 5 to 25 mg of poziotinib. In certain aspects, the composition comprises 8 mg, 12 mg, or 16 mg of poziotinib. In some aspects, poziotinib is further defined as poziotinib hydrochloride. In some aspects, the composition is formulated as a tablet. In some aspects, the subject is undergoing treatment with anti-cancer therapy.

[0013] In certain aspects, one or more EGFR TKI resistance mutations include point mutations, insertions, and / or deletions (1 to 18 nucleotides) in exon 18, 19, 20, or 21. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 688 - 728 of exon 18. In certain aspects, one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. In certain aspects, one or more EGFR exon 18 mutations include E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 729 - 761 of exon 19. In certain aspects, one or more EGFR exon 19 mutations are located at one or more residues selected from the group consisting of I744, L747, L747, A755, K757, and / or D761. In certain aspects, one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 763 - 823 of exon 20. In certain aspects, one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776.In some situations, one or more EGFR exon 20 mutations include D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, S784F, C775Y, and / or S811F. In certain situations, one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 824 to 875 of exon 21. In certain situations, one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L861, L862, L844, and L858. In some situations, one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some situations, the subject is determined to have two, three, or four EGFR TKI-resistant mutations. In certain situations, one or more EGFR TKI-resistant mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. In some situations, the subject is determined not to have an EGFR mutation at residue C797 or T790. In certain situations, the subject is determined not to have an EGFR mutation at residue T790. In other situations, the subject is determined to have the T790 mutation alone or in combination with another mutation. In certain situations, the subject is determined to have a mutation at the residue at C797. In some situations, one or more EGFR TKI-resistant mutations are selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid.In certain aspects, one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S.

[0014] In another aspect, a method for predicting responsiveness to poziotinib alone or in combination with a second anti-cancer therapy in a subject having cancer, the method comprising detecting an EGFR TKI resistance mutation in a genomic sample obtained from the patient, wherein when the sample is positive for the presence of an EGFR TKI resistance mutation, the patient is predicted to have a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy.

[0015] In certain aspects, one or more EGFR TKI resistance mutations include point mutations, insertions, and / or deletions (1 to 18 nucleotides) in exons 18, 19, 20, or 21. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 688 to 728 of exon 18. In certain aspects, one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. In certain aspects, one or more EGFR exon 18 mutations include E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 729 to 761 of exon 19. In certain aspects, one or more EGFR exon 19 mutations are located at one or more residues selected from the group consisting of I744, L747, L747, A755, K757, and / or D761. In certain aspects, one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 763 to 823 of exon 20. In certain aspects, one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776.In some scenarios, one or more EGFR exon 20 mutations include D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, S784F, C775Y, and / or S811F. In certain scenarios, one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 824 - 875 of exon 21. In certain scenarios, one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L861, L862, L844, and L858. In some scenarios, one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some scenarios, the subject is determined to have two, three, or four EGFR TKI resistance mutations. In certain scenarios, one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. In some scenarios, the subject is determined not to have an EGFR mutation at residue C797 or T790. In certain scenarios, the subject is determined not to have an EGFR mutation at residue T790. In other scenarios, the subject is determined to have the T790 mutation alone or in combination with another mutation. In certain scenarios, the subject is determined to have a mutation at the residue at C797. In some scenarios, one or more EGFR TKI resistance mutations are selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid.In certain aspects, one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S.

[0016] In some aspects, a favorable responsiveness to poziotinib alone or in combination with an anticancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related conditions, 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.

[0017] In a further aspect, the method further comprises administering poziotinib alone or in combination with a second anticancer therapy to a patient predicted to have a favorable responsiveness. In some aspects, poziotinib is administered orally. In certain aspects, poziotinib is administered at a dose of 5 - 25 mg. In certain aspects, poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. In some aspects, poziotinib is further defined as poziotinib hydrochloride. In certain aspects, poziotinib hydrochloride is formulated as tablets.

[0018] [The present invention 1001] A method for treating cancer in a subject, comprising administering to the subject an effective amount of poziotinib, wherein the subject has been determined to have one or more epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mutations. [The present invention 1002] The method of the present invention 1001, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1003] The method of the present invention 1001 or 1002, wherein the poziotinib hydrochloride is formulated as a tablet. [The present invention 1004] The method according to any one of the present inventions 1001 to 1003, wherein the one or more EGFR TKI resistance mutations include point mutations, insertions, and / or deletions of 1 to 18 nucleotides in exon 18, 19, 20, or 21. [The present invention 1005] The method according to any one of the present inventions 1001 to 1004, wherein the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides in amino acids 688 to 728 of exon 18. [The present invention 1006] The method of the present invention 1005, wherein the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. [The present invention 1007] The method of the present invention 1005 or 1006, wherein the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, G724, and T725. [The present invention 1008] The method according to any one of the present inventions 1005 to 1007, wherein the one or more EGFR exon 18 mutations include E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. [The present invention 1009] The method according to any one of the present inventions 1005 to 1008, wherein the one or more EGFR exon 18 mutations include E709A, E709K, L718Q, L718V, G719A, G719S, S720P, G724S, and / or T725M. [The present invention 1010] The method according to any one of the present inventions 1001 to 1009, wherein the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides in amino acids 729 to 761 of exon 19. [The present invention 1011] The method of the present invention 1010, wherein the one or more EGFR exon 19 mutations are located at one or more residues selected from the group consisting of I744, L747, L747, A755, K757, and / or D761. [The present invention 1012] The method of the present invention 1010 or 1011, wherein the one or more EGFR exon 19 mutations are located at one or more residues selected from the group consisting of I744, L747, L747, K754, A755, K757, and / or D761. [The present invention 1013] The method according to any one of the present inventions 1010 to 1012, wherein the one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. [The present invention 1014] The method according to any one of the present inventions 1010 to 1013, wherein the one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747P, L747FS, K754E, A755T, K757R, and / or D761N. [The present invention 1015] The method according to any one of the present inventions 1001 to 1014, wherein the one or more EGFR TKI-resistant mutations include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 to 823 of exon 20. [The present invention 1016] The method of the present invention 1015, wherein the one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. [The present invention 1017] The method of the present invention 1015 or 1016, wherein the one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, A767, S768, V769, N771, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. [The present invention 1018] The method according to any one of 1015 to 1018 of the present invention, wherein the one or more EGFR exon 20 mutations include D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, S784F, C775Y, and / or S811F. [The present invention 1019] The method according to any one of 1015 to 1018 of the present invention, wherein the one or more EGFR exon 20 mutations include A767ASV, D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, N771dupN, R776H, L792H, G796D, S784F, C775Y, and / or S811F. [The present invention 1020] The method according to any one of 1001 to 1019 of the present invention, wherein the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 824 to 875 of exon 21. [The present invention 1021] The method according to 1020 of the present invention, wherein the one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L861, L862, L844, and L858. [The present invention 1022] The method according to 1020 or 1021 of the present invention, wherein the one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. [The present invention 1023] The method according to any one of 1020 to 1022 of the present invention, wherein the one or more EGFR exon 21 mutations may include L833F, L833V, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. [The present invention 1024] The method according to any one of 1001 to 1023 of the present invention, wherein the subject is determined to have two, three, or four EGFR TKI resistance mutations. [The present invention 1025] The method according to any one of the present invention 1001 to 1024, wherein the subject has previously received an administration of a TKI. [The present invention 1026] The method according to the present invention 1025, wherein the subject is resistant to the TKI previously administered. [The present invention 1027] The method according to the present invention 1025 or 1026, wherein the TKI is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, naquotinib, or neratinib. [The present invention 1028] The method according to any one of the present invention 1025 to 1027, wherein the TKI is osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, or naquotinib. [The present invention 1029] The method according to any one of the present invention 1025 to 1028, wherein the TKI is osimertinib. [The present invention 1030] The method according to any one of the present invention 1001 to 1029, wherein the one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [The present invention 1031] The method according to any one of the present invention 1001 to 1030, wherein the subject is determined not to have an EGFR mutation at residue C797 or T790. [The present invention 1032] The method according to any one of the present invention 1001 to 1031, wherein the subject is determined not to have an EGFR mutation at residue T790. [The present invention 1033] The method according to any one of the present invention 1001 to 1030, wherein the subject has a T790 mutation. [The present invention 1034] The method according to the present invention 1033, wherein the subject has the T790 mutation in combination with at least one additional mutation. [The present invention 1035] The method according to the present invention 1034, wherein the subject has the T790M and G719A mutations. [The present invention 1036] The method according to the present invention 1034, wherein the subject has the T790M and G719S mutations. [The present invention 1037] The method according to any one of the present invention 1032 to 1036, wherein the subject is determined to have a mutation at the residue at C797. [The present invention 1038] The method according to any one of the present invention 1001 to 1037, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid. [The present invention 1039] Any of the methods of the present invention 1001 to 1038, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. [The present invention 1040] Any of the methods of the present invention 1001 to 1039, wherein the subject is determined to have an EGFR TKI resistance mutation by analyzing a patient-derived genomic sample. [The present invention 1041] The method of the present invention 1041, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1042] Any of the methods of the present invention 1001 to 1041, wherein the presence of the EGFR TKI resistance mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1043] Any of the methods of the present invention 1001 to 1042, wherein the poziotinib is administered orally. [The present invention 1044] Any of the methods of the present invention 1001 to 1043, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1045] Any of the methods of the present invention 1001 to 1044, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1046] Any of the methods of the present invention 1001 to 1045, wherein the poziotinib is administered daily. [The present invention 1047] Any of the methods of the present invention 1001 to 1046, wherein the poziotinib is administered continuously. [The present invention 1048] Any of the methods of the present invention 1001 to 1047, wherein the poziotinib is administered in a 28-day cycle. [The present invention 1049] Any of the methods of the present invention 1001 to 1048, further comprising the step of administering a further anti-cancer therapy. [The present invention 1050] The method of the present invention 1049, wherein the further anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1051] The method of the present invention 1049 or 1050, wherein the poziotinib and / or the anti-cancer therapy is applied intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. [The present invention 1052] Any of the methods of the present invention 1049 to 1051, wherein the application of the poziotinib and / or the anti-cancer therapy includes topical, local, or systemic application. [The present invention 1053] Any method according to any of claims 1049 to 1052 of the present invention, wherein the poziotinib and / or the anti-cancer therapy is applied two or more times. [invention 1054] Any method according to any of claims 1001 to 1053 of the present invention, wherein 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, 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. [invention 1055] Any method according to any of claims 1001 to 1054 of the present invention, wherein the cancer is non-small cell lung cancer. [invention 1056] Any method according to any of claims 1001 to 1055 of the present invention, wherein the patient is human. [invention 1057] A pharmaceutical composition comprising poziotinib for use in a subject determined to have one or more EGFR TKI resistance mutations. [invention 1058] The composition of claim 1057, further defined as an oral composition. [invention 1059] The composition of claim 1057 or 1058, comprising 5 to 25 mg of poziotinib. [invention 1060] The composition of claim 1057 or 1058, comprising 8 mg, 12 mg, or 16 mg of poziotinib. [invention 1061] The composition of claim 1057, wherein the poziotinib is further defined as poziotinib hydrochloride. [invention 1062] The composition of claim 1057 or 1058, formulated as a tablet. [invention 1063] Any composition according to any of claims 1057 to 1062, wherein the one or more EGFR TKI resistance mutations comprise point mutations, insertions, and / or deletions of 1 to 18 nucleotides in exon 18, 19, 20, or 21. [invention 1064] Any composition according to any of claims 1057 to 1063, wherein the subject is determined to have two, three, or four EGFR TKI resistance mutations. [invention 1065] The composition according to any one of aspects 1057 to 1064 of the present invention, wherein the one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [Aspect 1066] The composition according to any one of aspects 1057 to 1065 of the present invention, wherein the subject is determined to not have an EGFR mutation at residue C797 or T790. [Aspect 1067] The composition according to any one of aspects 1057 to 1066 of the present invention, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid. [Aspect 1068] The composition according to any one of aspects 1057 to 1067 of the present invention, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. [Aspect 1069] The composition according to any one of aspects 1057 to 1068 of the present invention, wherein the subject is undergoing treatment with an anti-cancer therapy. [Aspect 1070] A method for predicting responsiveness to poziotinib alone or in combination with a second anti-cancer therapy in a subject having cancer, the method comprising detecting an EGFR TKI resistance mutation in a genomic sample obtained from the patient, and predicting that the patient has a favorable responsiveness to poziotinib alone or in combination with an anti-cancer therapy when the sample is positive for the presence of the EGFR TKI resistance mutation. [Aspect 1071] The method according to aspect 1070 of the present invention, wherein the EGFR TKI resistance mutation is at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [Aspect 1072] The method according to aspect 1070 or 1071 of the present invention, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [Aspect 1073] The method according to any one of aspects 1070 to 1072 of the present invention, wherein the presence of a HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. [Aspect 1074] The method according to any one of 1070 to 1073 of the present invention, wherein the EGFR TKI-resistant mutation is selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, and X is any amino acid. [The present invention 1075] The method according to any one of 1070 to 1074 of the present invention, wherein the EGFR TKI-resistant mutation is selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. [The present invention 1076] A preferred responsiveness to poziotinib alone or 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 method according to any one of 1070 to 1075 of the present invention. [The present invention 1077] The method according to any one of 1070 to 1076 of the present invention, further comprising administering poziotinib alone or in combination with a second anticancer therapy to the patient predicted to have a preferred responsiveness. [The present invention 1078] The method according to any one of 1070 to 1077 of the present invention, wherein the poziotinib is administered orally. [The present invention 1079] The method according to any one of 1070 to 1078 of the present invention, wherein the poziotinib is administered at a dose of 5 to 25 mg. [The present invention 1080] The method according to any one of 1070 to 1079 of the present invention, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg. [The present invention 1081] The method according to any one of 1070 to 1080 of the present invention, wherein the poziotinib is further defined as poziotinib hydrochloride. [The present invention 1082] The method of the present invention 1081, wherein the poziotinib hydrochloride is formulated as a tablet. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, the detailed description and specific examples are provided by way of illustration only and not by way of limitation. It should be understood that while the preferred embodiments of the invention have been shown and described, various modifications thereof may be made by those skilled in the art without departing from the spirit and scope of the invention.

Brief Description of the Drawings

[0019] The following drawings form a part of this specification and are included to further demonstrate specific aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020]

Figure 1

Figure 2-1

Figure 2-2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0021] Description of Exemplary Embodiments In this study, osimertinib-resistant EGFR mutations across various malignancies such as NSCLC were identified. The drug sensitivity of resistant mutations was systematically evaluated across multiple TKIs. Resistant EGFR mutations were found to be sensitive to poziotinib.

[0022] Accordingly, certain aspects of the present disclosure provide methods for treating cancer patients having osimertinib-resistant EGFR mutations. In particular, the methods include administration of poziotinib (also known as HM781-36B) to patients identified as having one or more osimertinib-resistant EGFR mutations, such as exon 18, 19, 20, or 21 mutations. The size and flexibility of poziotinib overcome steric hindrance to inhibit EGFR variants at low nanomolar concentrations. Accordingly, poziotinib, and structurally similar inhibitors, are potent EGFR inhibitors that can be used to target osimertinib-resistant EGFR mutations.

[0023] I. Definitions As used herein, "a" or "an" can mean one or more. As used in the claims of this specification, when used in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one.

[0024] The use of the term "or" in the claims is used to mean "and / or" unless it is explicitly specified to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports definitions that refer only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0025] The term "essentially" is understood as a method or composition containing only steps or materials that do not significantly affect the particular steps or materials, as well as the basic and novel features of those methods and compositions.

[0026] The term "substantially free of" is used for a composition or particle that is substantially free of 98% of the listed components and less than 2% of the components that it is substantially free of.

[0027] As used herein, the terms "substantially" or "about" may be applied to modify any quantitative comparison, value, measurement, or other expression that can vary to an extent that is acceptable without changing the basic function associated therewith.

[0028] The term "about" generally means within one standard deviation of a specified value as determined using standard analytical techniques for measuring the specified value. The term can also be used to refer to the specified value ±5%.

[0029] "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 a disease in a subject or patient experiencing or showing the pathology or general symptoms of the disease (e.g., causing regression of 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.

[0030] "Preventively treating" includes (1) reducing or mitigating the risk of developing a disease in a subject or patient having a risk of the disease and / or being likely to be susceptible to the disease but not experiencing or showing 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 be susceptible to the disease but not yet experiencing or showing any or all of the pathology or general symptoms of the disease.

[0031] As used herein, the term "patient" or "subject" refers to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a 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.

[0032] As used herein, the term "effective" when used in this specification and / or claims means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient for treating or preventing a disease, is sufficient to act on the treatment or prevention of such disease.

[0033] As used herein, the term "IC 50 " refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measurement indicates how much of a particular drug or other substance (inhibitor) is required to half-inhibit a particular biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0034] 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 a subject.

[0035] The term "insertion" or "insertion mutation" refers to the addition of one or more nucleotide base pairs into a DNA sequence.

[0036] "Hybridize" or "hybridization" refers to the binding between nucleic acids. 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, mild conditions are used. When the hybridization conditions are stringent, the hybridization specificity increases, and this increase in hybridization specificity results in a decrease in the amount of non-specific hybridization products. However, under mild hybridization conditions, the hybridization specificity decreases, and this decrease in hybridization specificity results in an increase in the amount of non-specific hybridization products.

[0037] "Probe" refers to a polynucleotide that is at least 8 nucleotides in length and forms 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. The probe is detectably labeled in certain embodiments. The probe can vary greatly in its size. Generally, the probe is, for example, at least 8 - 15 nucleotides in length. Other probes are, for example, at least 20, 30, or 40 nucleotides in length. Still other probes are somewhat longer, at least, for example, 50, 60, 70, 80, or 90 nucleotides in length. Also, the probe can be of any specific length within the above ranges. Preferably, the probe does not contain a sequence complementary to the sequence used to prime the target sequence during polymerase chain reaction.

[0038] "Oligonucleotide" or "polynucleotide" refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotides, which can be unmodified RNA or DNA, or modified RNA or DNA.

[0039] A "modified ribonucleotide" or deoxyribonucleotide refers to a molecule that can be used in place of a natural base in a nucleic acid and includes, but is not limited to: modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized, and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifiers, terminal modifiers, spacer modifiers, and nucleotides with backbone modifications including, but not limited to: ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methylphosphonates, methylphosphoramidites, methylphosphonamidites, 5'-β-cyanoethylphosphoramidites, methylenephosphonates, phosphorodithioates, peptide nucleic acids, achiral, and neutral internucleotide linkages.

[0040] A "variant" is a polynucleotide or polypeptide that differs from the wild type or from the most widely observed form in a population of individuals due to one or more substitutions, deletions, or insertions of nucleotides or amino acids, respectively. The number of nucleotides or amino acids that are substituted, 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.

[0041] "Primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., an amplified DNA template) to prime a nucleic acid synthesis reaction. The primer may be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of the primer are determined experimentally. The lower limit of the length of the primer is the minimum length required to form a stable double strand upon hybridization to the target nucleic acid under the conditions of the nucleic acid amplification reaction. 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 forming a double strand in a region other than the predetermined nucleic acid sequence in the target nucleic acid. Generally, the length of a suitable primer ranges from about 10 to about 40 nucleotides in length. In certain embodiments, for example, the primer may 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.

[0042] "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 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.

[0043] "Amplifying," "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 multiplex versions or combinations thereof, 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), and the like. Descriptions of such techniques can be found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[0044] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, organs, and / or body fluids within the scope of sound medical judgment, with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic response, or other problems or complications.

[0045] "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).

[0046] II. Resistance EGFR Mutations Certain aspects of the present disclosure relate to determining whether a subject has one or more osimertinib-resistant EGFR mutations (e.g., mutations in exon 18, 19, 20, or 21). The subject may have two, three, four, or more EGFR exon 20 mutations. One or more EGFR mutations may be located at one or more residues selected from the group consisting of E709, L718, G719, G724, C797, V843, T854, L861, and L792 in exon 18 or 20. One or more EGFR mutations can be G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid. Methods for detecting mutations are known in the art and include PCR analysis and nucleic acid sequencing, as well as FISH and CGH. In certain aspects, EGFR mutations are detected by DNA sequencing, for example, from tumor or plasma-derived circulating free DNA.

[0047] EGFR exon 18 mutations can include one or more point mutations, insertions, and / or deletions (3 - 18 nucleotides) at amino acids 688 - 728, and in-frame deletions between amino acids of exon 18. One or more EGFR exon 18 mutations may be located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. One or more EGFR exon 18 mutations can include E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S.

[0048] EGFR exon 19 mutations can include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 729-761, and in-frame deletions between amino acids of exon 19. One or more EGFR exon 19 mutations can be located at one or more residues selected from the group consisting of I744, L747, L747, A755, K757, and / or D761. One or more EGFR exon 19 mutations can include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N.

[0049] EGFR exon 20 mutations can include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 763-823. In certain aspects, one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of A763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. In some aspects, one or more EGFR exon 20 mutations include D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, S784F, C775Y, and / or S811F.

[0050] EGFR exon 21 mutations can include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides), and in-frame deletions between amino acids of exon 21, in amino acids 824 to 875. One or more EGFR exon 21 mutations can be located in one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, L844, and L858. One or more EGFR exon 21 mutations can include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I.

[0051] In some scenarios, the subject may have or may develop mutations at residues C797 and T790 of EGFR, where such mutations can confer resistance to TKIs such as poziotinib. Thus, in certain scenarios, the subject is determined to not have mutations (e.g., C797S and / or T790M) at C797 and / or T790 of EGFR. In some scenarios, subjects having a T790 mutation (e.g., T790M) may receive administration of osimertinib, and subjects having a C797 mutation (e.g., C797S) may be administered chemotherapy and / or radiation therapy. For example, if C797S is acquired in the context of a classical EGFR mutation (e.g., L858R or exon 19 deletion) and T790M is absent, these mutations may be sensitive to poziotinib. However, if the C797S mutation is acquired along with a T790M mutation or an exon 20 insertion mutation, these mutations may be resistant to poziotinib. Further, if the T790M mutation is acquired along with a classical mutation (e.g., L858R or exon 19 deletion), these mutations may be resistant to poziotinib but may be sensitive to osimertinib. Also, in vitro, when the T790M mutation is acquired along with a point mutation in exon 18 (G719X / T790M), these mutations appear to remain sensitive to poziotinib. In some scenarios, the L858R / C797S, Ex19del / C797S, or G719X / T790M variants are sensitive to both poziotinib and other quinazoline amine TKIs. However, in certain scenarios, the L858R / T790M / C797S, exon 19 deletion / T790M / C797S, and exon 20 insertion + C797S or T790M variants are resistant to EGFR TKIs.

[0052] 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.

[0053] Samples suitable for use in the methods described herein contain 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 contains 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 containing buccal cells obtained using a buccal swab or brush, or a mouthwash sample.

[0054] In some cases, a biological sample can be processed for DNA isolation. For example, DNA in a cell or tissue sample can be separated from other components of the sample. Cells can be recovered from a biological sample 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, for example, Ausubel et al. (2003). The sample can be concentrated and / or purified to isolate the 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 a biological sample 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 sources of samples include urine, blood, and tissue.

[0055] The presence or absence of the resistant EGFR mutations described herein can be determined using methods well known in the art. For example, the presence or absence of an insertion mutation can be detected using gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and / or arrays. If desired, amplification of nucleic acids can be accomplished using methods well known in the art, such as PCR. In one example, a sample (e.g., a sample containing genomic DNA) is obtained from a subject. The DNA in the sample is then tested to determine the characteristics of the insertion mutations described herein. The insertion mutations can be detected by any method described herein, such as by sequencing or by hybridization to a nucleic acid probe of a gene in genomic DNA, RNA, or cDNA, 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.

[0056] 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 mutations) used in the recommended treatments feasible in 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 TaqMan (trademark) (Roche Molecular Systems, Pleasanton, Calif.) assays to detect a set of target gene mutations (e.g., EGFR mutations). In one embodiment, the set of probes is a set of primers used to produce amplicons detected by a nucleic acid sequencing reaction such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ (trademark) (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ (trademark) (Illumina, San Diego, Calif.) technologies can be utilized.

[0057] 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. Furthermore, next-generation sequencing methods can be carried out using kits and devices 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.

[0058] 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, e.g., the E. coli mutS protein; allele-specific PCR, and combinations of such methods may be included. See, for example, U.S. Patent Publication No. 2004 / 0014095. This patent is hereby incorporated by reference in its entirety.

[0059] In one example, a method of identifying an EGFR mutation in a sample involves 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 protein or a fragment thereof that contains the mutation, and detecting the hybridization. In a particular embodiment, such probe is, for example, a radioisotope ( 3 H, 32 P, or 33P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent, and is detectably labeled. In certain embodiments, the probe is an antisense oligomer, such as a 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 probes of the present disclosure are provided in a kit for identifying EGFR mutations in a sample, and such kit contains oligonucleotides that specifically hybridize to or adjacent to the mutation sites in the EGFR gene. The kit further includes instructions for treating a patient having a tumor containing an EGFR mutation with poziotinib based on the results of a hybridization assay using the kit.

[0060] In another aspect, a method for detecting an EGFR mutation in a sample includes amplifying such a nucleic acid sample corresponding to such an EGFR gene or a fragment thereof suspected of containing the mutation, and comparing the electrophoretic mobility of the amplified nucleic acid to the electrophoretic mobility of the corresponding wild-type EGFR 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.

[0061] Alternatively, nucleic acids can be analyzed for mutations using Enzymatic Mutation Detection (EMD) (Del Tito et al., 1998). EMD uses bacteriophage resolvase T4 endonuclease VII, which scans along double-stranded DNA until it detects and resolves the structural distortions caused by base pair mismatches due to point mutations, insertions, and deletions. Detection of two short fragments formed, for example, by gel electrophoresis upon resolvase digestion indicates the presence of a mutation. The advantage of the EMD method is that it is assayed directly from the PCR reaction, eliminating the need for sample purification, shortening hybridization times, and 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 the expression of normal DNA and fragments up to 4 kb in size can be assayed. However, EMD scanning does not identify the specific base changes that occur in a mutation-positive sample, and additional sequencing procedures are required to identify the mutation if necessary. As demonstrated in U.S. Patent No. 5,869,245, the CEL I enzyme can be used in a similar manner to bacteriophage resolvase T4 endonuclease VII.

[0062] III. Treatment Methods Also provided herein are methods for treating or delaying the progression of cancer in an individual, comprising administering to the individual, a subject determined to have a resistant EGFR mutation, an effective amount of poziotinib, or a structurally similar inhibitor. The subject can have one or more EGFR mutations.

[0063] 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 aspects, the cancer is non-small cell lung cancer.

[0064] 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 treatment and / or radiation therapy. Alternatively, or in combination, the subject is susceptible or at risk of being susceptible as a result of an infection.

[0065] 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 osimertinib-resistant EGFR mutation. Poziotinib is a quinazoline-based pan-HER inhibitor that irreversibly inhibits signaling through the HER family of receptor tyrosine kinases, including HER1, HER2, and HER4. Poziotinib, or a structurally similar compound (e.g., U.S. Patent No. 8,188,102 and U.S. Patent Application Publication No. 20130071452; incorporated herein by reference) may be used in the methods of the invention.

[0066] Poziotinib (e.g., poziotinib hydrochloride) can be administered orally, for example, as tablets. Poziotinib can be administered at a dose of 4-25 mg, for example, at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg. The dosing can be daily, every other day, every three days, or once a week. The dosing can be on a continuous schedule (e.g., a 28-day cycle).

[0067] In some scenarios, as described herein, a subject having a T790 mutation (e.g., T790M) may receive osimertinib, and a subject having a C797 mutation (e.g., C797S) may be administered chemotherapy and / or radiotherapy. Osimertinib, chemotherapy, and / or radiotherapy may be applied alone or in combination with poziotinib. Osimertinib may be administered at a dose of 25-100 mg, for example, at a dose of about 40 or 80 mg. The dosing may be daily, every other day, every two days, every three days, or once a week. Osimertinib may be administered orally, for example, as a tablet.

[0068] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising poziotinib and a pharmaceutically acceptable carrier for a subject determined to have a resistant EGFR mutation.

[0069] The pharmaceutical compositions and formulations described herein comprise an active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity, in the form of a lyophilized formulation or an aqueous solution, in one or more optional pharmaceutically acceptable carriers (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 polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto. In the present specification, an exemplary pharmaceutically acceptable carrier further includes an interstitial drug dispersant such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use including rHuPH20 are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glucosaminoglycanases such as chondroitinase.

[0070] B. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment include poziotinib in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., breast tumor excision 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.

[0071] 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 a 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.

[0072] Ponatinib can be administered before, during, after, or in various combinations with additional cancer therapies such as immune checkpoint therapies. Administration can range from concurrent administration to intervals of minutes, days, or weeks. In embodiments where ponatinib 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 provide an advantageous combined effect on 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 duration of treatment.

[0073] Various combinations can be utilized. In the following examples, ponatinib is "A" and the anti-cancer therapy is "B". TIFF0007709384000001.tif18128

[0074] Administration of any compound or implementation of a treatment method of this aspect to a patient follows the general protocol for administration of such a compound, taking into account the toxicity of the drug if present. Thus, in some aspects, there is a step of monitoring the toxicity resulting from the combination therapy.

[0075] 1. Chemotherapy According to this aspect, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to denote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at which stage of the cell cycle they act. Alternatively, the agents may be characterized based on their ability to directly crosslink DNA, to intervene in DNA, or to induce chromosomal and mitotic abnormalities by acting on nucleic acid synthesis.

[0076] 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 methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially, bullatacin and bullatacinone); camptothecin (including synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its azozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin gammaII and calicheamicin omegaI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromophore of pigment protein engine antibiotics, 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, querramycin, 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 trimethoprim; purine analogs, for example, fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, and floxuridine; androgens, for example, calusterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal, for example, mitotane and trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine 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, lolitrem A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoid, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complex, for example, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS2000; difluorodifluoromethylornithine (DMFO); retinoid, for example, retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitor, transplatin, and any pharmaceutically acceptable salt, acid, or derivative of the above is included.;

[0077] 2. Radiation therapy Other factors that cause DNA damage and are widely used include those known as gamma rays, X-rays, and / or the targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are contemplated, such as electron ranges, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are likely to affect DNA, its precursors, DNA replication and repair, and chromosomal assembly and maintenance in a wide range of damage. The dose range for X-rays spans from a daily dose of 50 - 200 roentgens for a long period (3 - 4 weeks) to a single dose of 2000 - 6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0078] 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 aspect. 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. Antibodies alone can be used as effectors of the therapy, or the antibody may recruit other cells that actually act in cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as molecular targeting agents. Alternatively, the effector can be lymphocytes having surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells

[0079] 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 Mabs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells along with abundant levels of antigen. The targeted delivery of the drug also minimizes drug exposure in normal tissues, leading to reduced toxicity and improved therapeutic indices. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, has validated this approach. Currently, more than 30 ADC drug candidates are at various stages of clinical trials for cancer therapy (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the drug discovery and development of new ADCs rely heavily on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high-level expression in tumor cells and robust internalization.

[0080] 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 aspect. Common tumor markers include CD20, carcinoembryonic antigen, tyrosine kinase (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 with 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.

[0081] Examples of immunotherapy include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as 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 therapies described herein.

[0082] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance or attenuate signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target PD-1 family and / or CTLA-4.

[0083] The immune checkpoint inhibitor can be a drug, e.g., a small molecule, a recombinant form of a ligand or receptor, or in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both documents are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, in particular, chimeric, humanized, or human forms of antibodies may be used. Those skilled in the art will appreciate that alternative and / or equivalent names may be used for the specific antibodies mentioned 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.

[0084] In some embodiments, a 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, a 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, a 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 Publications Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0085] 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 an 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.

[0086] 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.

[0087] 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.

[0088] 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 Publications WO01 / 14424, WO98 / 42752, and WO00 / 37504 (CP675,206, tremelimumab; also known as prior 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 Applications WO2001 / 014424, and WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0089] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence homology with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region homology with ipilimumab).

[0090] 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 Applications 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.

[0091] 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 this aspect, chemotherapy, radiation therapy, 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 microscopically controlled surgery (Mohs surgery).

[0092] In the resection of part or all of cancer cells, tissue, or tumor, a cavity may be formed in the body. The treatment can be achieved by perfusion, direct injection, or topical application of the area using further anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may have varying dosages.

[0093] 5. Other Agents It is contemplated that other agents may be used in combination with specific aspects of the present aspect 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 antiproliferative effect on adjacent hyperproliferative cell populations. In other aspects, cell growth inhibitors or differentiating agents can be used in combination with specific aspects of the present aspect to improve the antiproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effects of the present aspect. 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 aspect to improve the therapeutic effect.

[0094] IV. Kit Kits for detecting osimertinib-resistant EGFR mutations (e.g., 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 osimertinib-resistant EGFR mutations. The kit may further include instructions for using the primers to detect the presence or absence of specific osimertinib-resistant EGFR mutations described herein. The kit may further include instructions for diagnostic purposes indicating that identifying a sample from a cancer patient as positive for an osimertinib-resistant EGFR mutation described herein can serve as an indicator of sensitivity to the tyrosine kinase inhibitor poziotinib or a structurally similar inhibitor. The kit may further include instructions indicating that identifying a sample from a cancer patient as positive for an osimertinib-resistant EGFR mutation described herein indicates that the patient should be treated with poziotinib or a structurally similar inhibitor.

Examples

[0095] V. Examples The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples are those found by the inventors to function well in the practice of the present invention and can, therefore, be considered to constitute 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 similar or analogous results can still be obtained without departing from the spirit and scope of the present invention.

[0096] Example 1 - Identification of Drugs against Cancer Cells with Osimertinib-Resistant EGFR Mutations A panel of Ba / F3 cell lines expressing osimertinib-resistant mutations or erlotinib-resistant mutations, including non-canonical EGFR mutations spanning exons 18-21 and classical EGFR mutations, was generated. The transforming ability of the mutations was then evaluated by the persistence of cell viability after exclusion of IL-3. The activated EGFR mutant Ba / F3 cells were then screened against poziotinib. Cell viability was determined by the Cell Titer Glo assay.

[0097] Ponatinib inhibited the growth of Ba / F3 cell lines expressing non-canonical mutations (e.g., L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S) with IC50 values of less than 3 nM. In silico modeling of additional de novo resistance mutations (e.g., I740duplPVAIK) revealed that changes in the receptor's kinase hinge could prevent the binding of osimertinib, but deeper residues in the drug-binding pocket to which ponatinib binds were unaffected. In silico modeling of mutant EGFR demonstrated that the P-loop of exon 18 is important for the binding of osimertinib but not for the binding of ponatinib (Figure 1). In silico modeling of osimertinib bound to EGFR exon 19 del (E746_A7450del) revealed unique π-stacking interactions between the indole ring of osimertinib and the P-loop of EGFR exon 18 containing amino acids V726 and F723. Ponatinib extended further into the drug-binding pocket and interacted with the hydrophobic pocket containing T790. In silico modeling of EGFR G719S with ponatinib showed no predicted changes in either the binding of ponatinib or the TKI-protein interaction (Figure 1C). Molecular modeling of the L719Q mutation demonstrated that Q719 disrupted the interaction between osimertinib and M793 and shifted the Michael acceptor (reactive group) away from the alignment with C797. In contrast, ponatinib was less affected by Q719 and remained in a position to react with C797 even under the context of the L719Q mutation (Figure 1D).

[0098] Figure 2A shows that ponatinib is more potent and more selective than osimertinib in vitro in non-canonical EGFR mutations. Furthermore, in vivo, non-canonical P-loop exon 18 mutations were shown to confer primary resistance to osimertinib but not to ponatinib (Figure 3A).

[0099] Further tests showed that the acquired non-canonical mutations confer resistance to osimertinib but sensitivity to quinazoline TKIs, and that the drug sensitivity / resistance profiles of co-occurring mutations can be brought about by the primary mutation (Figure 4).

[0100] Therefore, poziotinib is an effective inhibitor against both de novo and acquired non-canonical osimertinib-resistant EGFR mutant NSCLC, including L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. This study demonstrated that second-generation TKIs, particularly poziotinib, overcome osimertinib resistance in non-canonical EGFR mutant NSCLC.

[0101] IC50 values of Ba / F3 cells expressing the primary non-canonical mutations described in (Table 1) after 72 hours of treatment with poziotinib or osimertinib. TIFF0007709384000002.tif135128TIFF0007709384000003.tif240111TIFF0007709384000004.tif39128

[0102] IC50 values of Ba / F3 cells expressing the acquired non-canonical mutations described in (Table 2) after 72 hours of treatment with poziotinib or osimertinib. TIFF0007709384000005.tif173128

[0103] (Table 3) List of mutations and sequences used to generate Ba / F3 cell lines. TIFF0007709384000006.tif221160TIFF0007709384000007.tif240160TIFF0007709384000008.tif244160TIFF0007709384000009.tif93160

[0104] Example 2 - Materials and Methods Generation of Ba / F3 cell line and exclusion of IL-3: The Ba / F3 cell line was established as previously described (Robichaux et al., 2018). Briefly, the stable Ba / F3 cell line was generated by retroviral transduction of the Ba / F3 cell line over 12 hours. The retrovirus was generated by transfecting the pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 1 into Phoenix 293T-ampho cells (Orbigen) using Lipofectamine 2000 (Invitrogen). Three days after transduction, 2 μg / ml puromycin (Invitrogen) was added to the RPMI medium. The cell line was then 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 line was maintained in RPMI-1640 medium containing 10% FBS and no IL-3.

[0105] Cell viability assay and IC 50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described (Robichaux et al., 2018). Briefly, 2000 - 3000 cells per well were plated in 384-well plates (Greiner Bio-One) with technical replicates = 3. 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 determined using a FLUOstar OPTIMA multimode microplate reader (BMG LABTECH). Bioluminescence values were normalized to cells treated with DMSO, and normalized values were plotted in GraphPad Prism using non-linear regression fitting to the normalized data with variable slope. IC 50The value was calculated by GraphPad Prism as the 50% inhibition.

[0106] All of the methods disclosed and claimed herein can be constructed and implemented without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and to the steps or the order of the steps described herein without departing from the concept, spirit, and scope of the present invention. More specifically, certain agents that are both chemically and physiologically relevant may be used in place of the agents described herein, and it will be apparent that they may achieve the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0107] References The following references are specifically incorporated herein by reference to the extent that they supplement the exemplary procedures shown herein or provide other details. TIFF0007709384000010.tif202158TIFF0007709384000011.tif238113TIFF0007709384000012.tif252107

Claims

**Claim 1** A pharmaceutical composition for treating cancer in a subject, comprising an effective amount of poziotinib, wherein the subject has been determined to have one or more epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mutations, and the one or more EGFR TKI resistance mutations include the following, pharmaceutical composition: (a) one or more EGFR exon 18 mutations located at one or more residues selected from the group consisting of E709, L718, G719, S720, and T725, (b) one or more EGFR exon 19 mutations located at one or more residues selected from the group consisting of I744, L747, K754, A755, K757, and D761, (c) one or more EGFR exon 20 mutations located at one or more residues selected from the group consisting of C775, S784, L792, G796, S811, and R776, or (d) one or more EGFR exon 21 mutations located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, and L844. **Claim 2** The pharmaceutical composition according to claim 1, wherein the one or more EGFR exon 18 mutations include E709A, E709K, L718Q, L718V, G719A, G719S, S720P, and / or T725M. **Claim 3** The pharmaceutical composition according to claim 1, wherein the one or more EGFR exon 19 mutations include I744V, I744T, L747S, L747P, L747FS, K754E, A755T, K757R, and / or D761N. **Claim 4** The pharmaceutical composition according to claim 1, wherein the one or more EGFR exon 20 mutations include S784F, R776C, S768I, V774M, S768I, H773insAH, V774A, V769L, V769M, A763insLQEA, R776H, L792H, G796D, S784F, C775Y, and / or S811F. **Claim 5** The pharmaceutical composition according to claim 1, wherein the one or more EGFR exon 21 mutations include L833F, L833V, V834L, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. **Claim 6** The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is determined to have two, three, or four EGFR TKI resistance mutations.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject has previously received administration of a TKI.

8. The pharmaceutical composition according to claim 7, wherein the subject is resistant to the TKI previously administered.

9. The pharmaceutical composition according to claim 7 or 8, wherein the TKI is lapatinib, afatinib, dacomitinib, osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, naquotinib, or neratinib.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject is determined not to have an EGFR mutation at residue C797 or T790.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject further has a T790 mutation.

12. The pharmaceutical composition according to claim 11, wherein the subject has T790M and G719A mutations.

13. The pharmaceutical composition according to claim 11, wherein the subject has T790M and G719S mutations.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the subject further has a C797 mutation.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the poziotinib is administered at a dose of 8 mg, 12 mg, or 16 mg.

16. The pharmaceutical composition according to any one of claims 1 to 15, which is administered in combination with a further anti-cancer therapy.

17. 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, the pharmaceutical composition according to any one of claims 1 to 16.

18. The cancer is non-small cell lung cancer, the pharmaceutical composition according to any one of claims 1 to 17.

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