Compounds with antitumor activity against cancer cells with tyrosine kinase inhibitor-resistant EGFR mutations

Abstract

The present disclosure provides methods of treating cancer in patients determined to have an osimertinib-resistant EGFR mutation by administering a second-generation quinazolinamine derivative tyrosine kinase inhibitor.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 835,354, filed April 17, 2019, the entirety of which is incorporated herein by reference.

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

[0003] 2. Description of Related Art Approximately 10% of non-small cell lung cancer (NSCLC) patients have epidermal growth factor receptor (EGFR) mutations, which increase sensitivity to tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and osimertinib. Recently, osimertinib has been approved as a first-line therapy for EGFR-mutant NSCLC4, but de novo resistance and acquired resistance remain treatment obstacles for many patients. A series of acquired atypical EGFR mutations may have the potential to confer osimertinib resistance. Studies have shown that these acquired atypical resistance mutations change the conformation of the solvent-proximal drug-binding pocket in front of osimertinib, causing changes in the binding affinity between the drug and the receptor. Therefore, there is an unmet need for novel therapies to treat cancers with resistant EGFR mutations. Summary of the Invention

[0004] overview The present disclosure provides a method and composition for treating cancer in patients with resistant EGFR mutations.In a first aspect, a method for treating cancer in a subject is provided, comprising administering an effective amount of a quinazolinamine derivative tyrosine kinase inhibitor (TKI) to the subject, wherein the subject is determined to have one or more epidermal growth factor receptor (EGFR) TKI resistance mutations.In some aspects, the patient is human.

[0005] In certain aspects, the quinazolinamine derivative TKI is a covalently bonded quinazolinamine TKI. In some aspects, the covalently bonded quinazolinamine TKI is afatinib, tarloxotinib-TKI, dacomitinib, pelitinib, or allitinib. In certain aspects, the quinazolinamine derivative TKI is a non-covalently bonded quinazolinamine TKI. In some aspects, the non-covalently bonded quinazolinamine TKI is sapatinib, AZD3759, vallitinib, TAK-285, or gefitinib. In certain aspects, the quinazolinamine derivative TKI is formulated as a tablet.

[0006] In certain aspects, the one or more EGFR TKI resistance mutations comprise point mutations, insertions, and / or deletions (1-18 nucleotides) in exons 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 688-728 of exon 18. In certain aspects, 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. In certain aspects, the one or more EGFR exon 18 mutations comprise E709A, E790K, L718Q, L718V, G719A, G719S, S720P, and / or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 729-761 of exon 19. In certain aspects, 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. In certain aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 763-823 of exon 20. In certain aspects, 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.In some aspects, the one or more EGFR exon 20 mutations include D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, G796S, S784F, C775Y, and / or S811F. In certain aspects, the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 824-875 of exon 21. In certain aspects, 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. In some aspects, the one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some aspects, the subject has been determined to have two, three, or four EGFR TKI resistance mutations. In certain aspects, one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792.In some aspects, the subject is determined not to have the EGFR mutation at residue C797 or T790.In certain aspects, the subject is determined not to have the EGFR mutation at residue T790.In other aspects, the subject is determined to have the T790 mutation alone or in combination with another mutation.In certain aspects, the subject is determined to have the mutation at residue C797. In some aspects, 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.In certain aspects, the 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 been administered with 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, nacotinib, or neratinib. In certain aspects, the TKI is osimertinib, ibrutinib, nazartinib, olmutinib, rociletinib, or nacotinib. In certain aspects, the TKI is osimeritinib.

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

[0009] In some aspects, the quinazolinamine derivative TKI is administered orally. In certain aspects, the quinazolinamine derivative TKI is administered daily. In certain aspects, the quinazolinamine derivative TKI is administered continuously. In some aspects, the quinazolinamine derivative TKI is administered every 28 days.

[0010] In a further aspect, the method further comprises administering an additional anticancer therapy. In some aspects, the additional anticancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, antiangiogenic therapy, or immunotherapy. In certain aspects, poziotinib and / or the anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, controlled release, delayed release, as a suppository, or sublingually. In some aspects, the administration of poziotinib and / or the anticancer therapy includes local, regional, or systemic administration. In certain aspects, poziotinib and / or the anticancer therapy is administered more than once.

[0011] In some aspects, the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or cancer of the hematopoietic system, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, 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] Further provided herein is a pharmaceutical composition comprising a quinazolinamine derivative TKI for use in a subject determined to have one or more EGFR TKI resistance mutations. In some aspects, the composition is further defined as an oral composition. In certain aspects, the composition comprises 5-25 mg of a quinazolinamine derivative TKI. In some aspects, the composition is formulated as a tablet. In some aspects, the subject is undergoing treatment with an anti-cancer therapy.

[0013] In certain aspects, the one or more EGFR TKI resistance mutations comprise point mutations, insertions, and / or deletions (1-18 nucleotides) in exons 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 688-728 of exon 18. In certain aspects, 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. In certain aspects, the one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 729-761 of exon 19. In certain aspects, 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. In certain aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 763-823 of exon 20. In certain aspects, 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.In some aspects, 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. In certain aspects, the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 824-875 of exon 21. In certain aspects, 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. In some aspects, the one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some aspects, the subject has been determined to have two, three, or four EGFR TKI resistance mutations. In certain aspects, one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792.In some aspects, the subject is determined not to have the EGFR mutation at residue C797 or T790.In certain aspects, the subject is determined not to have the EGFR mutation at residue T790.In other aspects, the subject is determined to have the T790 mutation alone or in combination with another mutation.In certain aspects, the subject is determined to have the mutation at residue C797. In some aspects, 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.In certain aspects, the 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 embodiment, a method for predicting the response of a cancer subject to quinazolinamine derivative TKI alone or in combination with a second anticancer therapy is provided, the method comprising detecting EGFR TKI resistance mutation in a genome sample obtained from the patient, wherein if the sample is positive for the presence of EGFR TKI resistance mutation, the patient is predicted to have a favorable response to quinazolinamine derivative TKI alone or in combination with a second anticancer therapy.In some embodiments, the genome sample is isolated from saliva, blood, urine, normal tissue or tumor tissue.In certain aspects, the presence of HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis.

[0015] In certain aspects, the one or more EGFR TKI resistance mutations comprise point mutations, insertions, and / or deletions (1-18 nucleotides) in exons 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 688-728 of exon 18. In certain aspects, 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. In certain aspects, the one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 729-761 of exon 19. In certain aspects, 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. In certain aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 763-823 of exon 20. In certain aspects, 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.In some aspects, 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. In certain aspects, the one or more EGFR TKI resistance mutations include one or more point mutations, insertions, and / or deletions (3-18 nucleotides) in amino acids 824-875 of exon 21. In certain aspects, 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. In some aspects, the one or more EGFR exon 21 mutations may include L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. In some aspects, the subject has been determined to have two, three, or four EGFR TKI resistance mutations. In certain aspects, one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792.In some aspects, the subject is determined not to have the EGFR mutation at residue C797 or T790.In certain aspects, the subject is determined not to have the EGFR mutation at residue T790.In other aspects, the subject is determined to have the T790 mutation alone or in combination with another mutation.In certain aspects, the subject is determined to have the mutation at residue C797. In some aspects, 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.In certain aspects, the 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 a further aspect, a favorable response to a quinazolinamine derivative TKI alone or in combination with an anti-cancer therapy is provided, including a reduction in tumor size or tumor burden, inhibition of tumor growth, relief of tumor-associated pain, relief of cancer-associated pathology, relief of cancer-associated symptoms, non-progression of cancer, extended disease-free interval, extended time to progression, induction of remission, reduced metastasis, or improved patient survival.

[0017] In a further aspect, the method further comprises administering the quinazolinamine derivative TKI alone or in combination with a second anticancer therapy to the patient predicted to have a favorable response. In some aspects, the quinazolinamine derivative TKI is orally administered. In certain aspects, the quinazolinamine derivative TKI is administered at a dose of 5 to 25 mg. In some aspects, the quinazolinamine derivative TKI is formulated as a tablet.

[0018] [The present invention 1001] A method of treating cancer in a subject, comprising administering to the subject an effective amount of a quinazolinamine derivative tyrosine kinase inhibitor (TKI), wherein the subject has been determined to have one or more epidermal growth factor receptor (EGFR) TKI resistance mutations. [The present invention 1002] 1001. The method of claim 1001, wherein said EGFR TKI resistance mutation is an acquired atypical EGFR mutation. [The present invention 1003] The method of the present invention 1001 or 1002, wherein the quinazolinamine derivative TKI is a covalent quinazolinamine TKI. [The present invention 1004] The method of claim 1003, wherein the covalent quinazolinamine TKI is afatinib, tarloxotinib-TKI, dacomitinib, pelitinib, or allitinib. [The present invention 1005] The method of any one of claims 1001 to 1002, wherein the quinazolinamine derivative TKI is a non-covalent quinazolinamine TKI. [The present invention 1006] 1005. The method of claim 10, wherein said non-covalent quinazolinamine TKI is sapatinib, AZD3759, varlitinib, TAK-285, or gefitinib. [The present invention 1007] The method of any one of claims 1001 to 1006, wherein the quinazolinamine derivative TKI is formulated as a tablet. [The present invention 1008] 8. The method of any of claims 1001 to 1007, wherein said one or more EGFR TKI resistance mutations comprise a point mutation, an insertion, and / or a deletion of 1 to 18 nucleotides in exon 18, 19, 20, or 21. [The present invention 1009] 9. The method of any of claims 1001 to 1008, wherein said one or more EGFR TKI resistance mutations comprise 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 1010] 1009. The method of claim 1009, wherein said 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 1011] 1010. The method of claim 1009 or 1010, wherein said one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and / or G724S. [The present invention 1012] 1012. The method of any of claims 1008 to 1011, wherein said one or more EGFR exon 18 mutations comprise E709A, E790K, L718Q, L718V, G719A, G719S, S720P, and / or G724S. [The present invention 1013] 1013. The method of any of claims 1001 to 1012, wherein said one or more EGFR TKI resistance mutations comprise 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 1014] 1013. The method of claim 1013, wherein said 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 1015] 1015. The method of claim 1013 or 1014, wherein said one or more EGFR exon 19 mutations comprise I744V, I744T L747S, L747FS, A755T, K757R, and / or D761N. [The present invention 1016] 1016. The method of any of claims 1001 to 1015, wherein said one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides in amino acids 763 to 823 of exon 20. [The present invention 1017] 1016. The method of claim 1016, wherein said 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 1018] 1017. The method of claim 1016, wherein the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, S784F, C775Y, and / or S811F. [The present invention 1019] 1016. Any of the methods of claims 1016 to 1018, wherein the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, A763insLQEA, L792H, G796D, G796S, S784F, C775Y, and / or S811F. [The present invention 1020] 1018. The method of any of claims 1001 to 1018, wherein said one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides in amino acids 824 to 875 of exon 21. [The present invention 1021] 1020. The method of claim 1020, wherein said 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] 1022. The method of claim 1020 or 1021, wherein said one or more EGFR exon 21 mutations may comprise L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I. [The present invention 1023] The method of any of claims 1001 to 1022, wherein the subject is determined to have two, three, or four EGFR TKI resistance mutations. [The present invention 1024] The method of any one of claims 1001 to 1023, wherein the subject has previously received a TKI. [The present invention 1025] The method of claim 1024, wherein the subject is resistant to a previously administered TKI. [The present invention 1026] The method of any one of claims 1024 to 1025, wherein the TKI is lapatinib, erlotinib, gefitinib, rociletinib, olmutinib, nacotinib, osimertinib, ibrutinib, or nazartinib. [The present invention 1027] The method of any of claims 1024 to 1026, wherein the TKI is gefitinib, erlotinib, or osimertinib. [The present invention 1028] The method of any of claims 1024 to 1027, wherein the TKI is osimertinib. [The present invention 1029] 1029. The method of any of claims 1001 to 1028, wherein said one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [The present invention 1030] The method of any of claims 1001 to 1029, wherein said subject has been determined to not have an EGFR mutation at residue C797 or T790. [The present invention 1031] The method of any of claims 1001 to 1030, wherein said subject is determined to not have an EGFR mutation at residue T790. [The present invention 1032] The method of any one of claims 1001 to 1029, wherein the subject has a T790 mutation. [The present invention 1033] The method of claim 1032, wherein said subject has a T790 mutation in combination with at least one additional mutation. [The present invention 1034] The method of claim 1031, wherein said subject is determined to have a mutation at residue C797. [This invention 1035] 1035. The method of any of claims 1001 to 1034, 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, wherein X is any amino acid. [The present invention 1036] 1036. The method of any of claims 1001 to 1035, wherein said one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. [This invention 1037] The method of any of claims 1001 to 1036, wherein the subject is determined to have an EGFR TKI resistance mutation by analyzing a genomic sample derived from the patient. [The present invention 1038] 1037. The method of claim 1037, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [This invention 1039] The method of any of claims 1001 to 1038, wherein the presence of said EGFR TKI resistance mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1040] The method of any one of claims 1001 to 1039, wherein the quinazolinamine derivative TKI is administered orally. [The present invention 1041] The method of any one of claims 1001 to 1040, wherein the quinazolinamine derivative TKI is administered daily. [The present invention 1042] 1041. The method of claim 1041, wherein said quinazolinamine derivative TKI is administered continuously. [This invention 1043] The method of claim 1042, wherein the quinazolinamine derivative TKI is administered in a 28-day cycle. [This invention 1044] The method of any one of claims 1001 to 1043, further comprising the step of administering an additional anticancer therapy. [This invention 1045] 104. The method of claim 1044, wherein said further anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy. [The present invention 1046] The method of claim 1044, wherein said quinazolinamine derivative TKI and / or said anti-cancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in sustained release, controlled release, delayed release, as a suppository, or sublingually. [This invention 1047] 104. The method of claim 1044, wherein administering said quinazolinamine derivative TKI and / or said anti-cancer therapy comprises topical, local, or systemic administration. [This invention 1048] 104. The method of claim 1044, wherein said quinazolinamine derivative TKI and / or said anti-cancer therapy is administered more than once. [This invention 1049] Any of the methods of inventions 1001 to 1048, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or cancer of the hematopoietic system, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1050] The method of any one of claims 1001 to 1049, wherein the cancer is non-small cell lung cancer. [This invention 1051] The method of any one of claims 1001 to 1050, wherein the patient is a human. [This invention 1052] A pharmaceutical composition comprising a quinazolinamine derivative TKI for use in a subject determined to have one or more EGFR TKI resistance mutations. [This invention 1053] The composition of the present invention 1052 further defined as an oral composition. [This invention 1054] The composition of any one of claims 1052 to 1053, comprising 5 to 25 mg of a quinazolinamine derivative TKI. [This invention 1055] 1052. The composition of any one of claims 1052 to 1054, which is formulated as a tablet. [This invention 1056] 6. The composition of any of claims 1052 to 1055, wherein said one or more EGFR TKI resistance mutations comprise a point mutation, an insertion, and / or a deletion of 1 to 18 nucleotides in exon 18, 19, 20, or 21. [This invention 1057] The composition of any of claims 1052 to 1056, wherein the subject is determined to have two, three, or four EGFR TKI resistance mutations. [This invention 1058] 1058. The composition of any of claims 1052 to 1057, wherein said one or more EGFR TKI resistance mutations are at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [This invention 1059] The composition of any of claims 1052 to 1058, wherein the subject is determined to not have an EGFR mutation at residue C797 or T790. [The present invention 1060] 1059. The composition of any of claims 1052 to 1059, 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, wherein X is any amino acid. [This invention 1061] 1059. The composition of any of claims 1052 to 1059, 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 1062] The composition of any one of claims 1052 to 1061, wherein the subject is undergoing treatment with an anti-cancer therapy. [The present invention 1063] 1. A method for predicting responsiveness to a quinazolinamine derivative TKI alone or in combination with a second anticancer therapy in a subject with cancer, the method comprising the step of detecting an EGFR TKI resistance mutation in a genomic sample obtained from the patient, wherein if the sample is positive for the presence of the EGFR TKI resistance mutation, the patient is predicted to have a favorable response to the quinazolinamine derivative TKI alone or in combination with a second anticancer therapy. [The present invention 1064] 1063. The method of claim 1063, wherein said EGFR TKI resistance mutation is at residues E709, L718, G719, G724, C797, V843, T854, L861, and / or L792. [This invention 1065] 1065. The method of any one of claims 1063 to 1064, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1066] 1066. The method of any of claims 1063 to 1065, wherein the presence of a HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. [This invention 1067] 1067. The method of any one of claims 1063 to 1066, wherein the EGFR TKI resistance 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 1068] The method of any of claims 1063 to 1067, wherein the EGFR TKI resistance mutation is selected from the group consisting of L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. [The present invention 1069] 9. The method of any of claims 1063 to 1068, wherein the favorable response to said quinazolinamine derivative TKI alone or in combination with an anti-cancer therapy comprises a decrease in tumor size or tumor burden, an inhibition of tumor growth, a reduction in tumor-associated pain, a reduction in cancer-associated pathology, a reduction in cancer-associated symptoms, non-progression of cancer, an increase in disease-free interval, an increase in time to progression, induction of remission, a reduction in metastasis, or an increase in patient survival. [The present invention 1070] The method of any of claims 1063 to 1069, further comprising administering said quinazolinamine derivative TKI alone or in combination with a second anti-cancer therapy to said patient predicted to have a favorable response. [This invention 1071] The method of any one of claims 1063 to 1070, wherein the quinazolinamine derivative TKI is administered orally. [This invention 1072] The method of any of claims 1063 to 1071, wherein the quinazolinamine derivative TKI is administered at a dose of 5 to 25 mg. [This invention 1073] The method of any one of claims 1063 to 1072, wherein the quinazolinamine derivative TKI is formulated as a tablet. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description herein. [Brief explanation of the drawings]

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

[0020] [Figure 1]Figures 1A-1D: Second-generation quinazoline TKIs are more potent and selective for atypical EGFR mutations in vitro than third-generation TKIs such as osimertinib. (Figure 1A) Heat map of log IC50 values ​​for Ba / F3 cells expressing primary atypical mutations spanning exons 18-21 and treated for 72 hours with the indicated inhibitors. Mutations are arranged top to bottom from most resistant to most sensitive, and drugs are arranged left to right from lowest IC50 value to highest IC50 value. (Figure 1B) Heat map of the ratio of IC50 values ​​for Ba / F3 cells expressing primary atypical mutations spanning exons 18-21 divided by IC50 values ​​for Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF) and treated for 72 hours with the indicated inhibitors. The order of mutations is maintained the same as in panel A, but the drugs are rearranged from left to right from most selective to least selective. (Figure 1C) Bar graph of mean ± SEM IC50 values ​​for Ba / F3 cells expressing atypical mutations and separated by drug class. N = 40 cell lines, and symbols represent individual cell lines. Statistical significance was determined by ANOVA. (Figure 1D) Bar graph of mean ± SEM mutant / WT EGFR ratios for Ba / F3 cells expressing atypical mutations and separated by drug class. N = 40 cell lines, and symbols represent individual cell lines. Statistical significance was determined by ANOVA. [Figure 2]Figures 2A-2D: In vivo, P-loop exon 18 mutations confer primary resistance to osimertinib but not other EGFR TKIs. (Figure 2A) Tumor growth curves for NSCLC PDX models harboring an EGFR exon 18 P-loop mutation (G719A) treated with the indicated inhibitors for 28 days. (Figure 2B) Bar graphs of the mean ± SEM percent change in G719A tumor volume at the end of the 28-day experiment after treatment with the indicated inhibitors. Symbols represent individual mice. Significant differences were determined by ANOVA and Tukey's test for multiple comparisons. (Figure 2C) Tumor growth curves for NSCLC PDX models harboring non-P-loop exon 18 EGFR mutations (E709K L858R) treated with the indicated inhibitors for 28 days. (Figure 2D) Bar graph of the mean ± SEM percent change in E709K / L858R tumor volume at the end of the 28-day experiment after treatment with the indicated inhibitors. Symbols represent individual mice. Significant differences were determined by ANOVA and Tukey's test for multiple comparisons. [Figure 3]Figures 3A-3D: Acquired atypical mutations confer resistance to osimertinib but are sensitive to quinazoline TKIs, and the drug sensitivity / resistance profile of co-occurring mutations may be driven by primary mutations. (Figure 3A) Heatmap of log IC50 values ​​for Ba / F3 cells expressing acquired atypical mutations spanning exons 18-21 and treated with the indicated inhibitors for 72 hours. Mutations are ordered from top to bottom from most resistant to most sensitive, and drugs are ordered from left to right from lowest IC50 value to highest IC50 value. (Figure 3B) Heatmap of the ratio of IC50 values ​​for Ba / F3 cells expressing acquired atypical mutations spanning exons 18-21 divided by the IC50 values ​​for Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF) and treated with the indicated inhibitors for 72 hours. The order of mutations is maintained the same as in panel A, but the drugs are rearranged from left to right from most selective to least selective. (Figures 3C-3D) Bar graphs of mutant / WT ratios for the Ex19del primary mutation (Figure 3C) and the L858R primary mutation (Figure 3D) by drug class, with and without the primary mutation. For primary mutations alone (open bars), symbols represent the average mutant / WT ratio for the individual drug, and for primary mutations + atypical mutations (filled bars), symbols represent the average mutant / WT ratio for all drugs in the listed drug class for each mutation. Due to unequal sample sizes, statistical differences were determined using a nonparametric Student's t-test. [Figure 4]Figures 4A-4D: The T790M mutation, whether primary or tertiary, confer resistance to first- and second-generation drugs. However, unique third-generation TKIs and drug repurposing can overcome tertiary T790M-positive drug resistance. (Figure 4A) Heatmap of log IC50 values ​​for Ba / F3 cells expressing EGFR mutations co-occurring with the T790M mutation and treated for 72 hours with the listed classical EGFR inhibitors. Mutations are arranged top to bottom from most resistant to most sensitive, and drugs are arranged left to right from lowest to highest IC50 values. (Figure 4B) Heatmap of the ratio of IC50 values ​​for Ba / F3 cells expressing EGFR mutations co-occurring with the T790M mutation divided by IC50 values ​​for Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF) and treated with the indicated classical EGFR inhibitors for 72 hours. The order of mutations is maintained the same as in panel A, but the drugs are rearranged from left to right from most selective to least selective for the indicated classical EGFR inhibitors. (Figure 4C) Heatmap of the log IC50 values ​​for Ba / F3 cells expressing EGFR mutations co-occurring with the T790M mutation and treated with the indicated repurposed inhibitors for 72 hours. The order of mutations is maintained the same as in panel A, and the drugs are rearranged from left to right from lowest IC50 value to highest IC50 value. (Figure 4D) Heatmap of the ratio of IC50 values ​​for Ba / F3 cells expressing EGFR mutations co-occurring with the T790M mutation divided by the IC50 values ​​for Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF) and treated with the indicated repurposed inhibitors for 72 hours. The order of mutations is kept the same as in panel A, but the drugs are rearranged from left to right from most selective to least selective. DETAILED DESCRIPTION OF THE INVENTION

[0021] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This study identified osimertinib-resistant EGFR mutations in various malignancies, including NSCLC. The drug sensitivity of these resistant mutations was systematically evaluated across multiple TKIs. The resistant EGFR mutations were found to be sensitive to second-generation quinazolinamine derivative TKIs.

[0022] Therefore, certain embodiments of the present disclosure provide a method for treating cancer patients with osimertinib-resistant EGFR mutations.In particular, the method comprises administering a second-generation quinazolinamine derivative TKI to patients identified as having one or more osimertinib-resistant EGFR mutations (e.g., mutations in exons 18, 19, 20, or 21, such as acquired atypical mutations and / or classic mutations).The size and flexibility of the second-generation quinazolinamine derivative TKI overcome steric hindrance and inhibit EGFR mutants at low nanomolar concentrations.Therefore, the second-generation quinazolinamine derivative TKI is a potent EGFR inhibitor that can be used to target osimertinib-resistant EGFR mutations.

[0023] I. Definition As used herein, "a" or "an" may mean one or more. When used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0024] Use of the term "or" in the claims is used to mean "and / or" unless it is expressly specified to refer to alternatives only or the alternatives are mutually exclusive, but the present disclosure supports a definition that refers to alternatives only, and "and / or." As used herein, "another" can mean at least a second, or more.

[0025] As used herein, " essentially free " in relation to a specific component means that the specific component is not intentionally formulated in the composition, and / or the specific component is present only as a contaminant or only in trace amounts.The total amount of the specific component resulting from any unintentional contaminants of the composition is typically less than 1% of the composition, more preferably less than 0.1%, and even more preferably less than 0.01%.Most preferred is a composition in which measurable amounts of the specific component cannot be detected using standard analytical methods.

[0026] The term "essentially" is understood to mean that the method or composition includes the specified steps or materials, and only those steps or materials that do not materially affect the basic and novel characteristics of those methods and compositions.

[0027] The term "substantially free" is used for 98% of the listed component and less than 2% of the component that the composition or particle is substantially free of.

[0028] As used herein, the terms "substantially" or "approximately" may be applied to modify any quantitative comparison, value, measurement, or other representation that may vary to an acceptable extent without changing the basic function associated with it.

[0029] The term "about" generally means within the standard deviation of the specified value as determined using standard analytical techniques to measure the specified value. The term can also be used to refer to the specified value ±5%.

[0030] "Treatment" or "treating" includes (1) inhibiting disease (e.g., preventing further progression of the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease, (2) ameliorating disease (e.g., reversing the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease, and / or (3) affecting any measurable reduction in disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease. For example, treatment can include administering an effective amount of a second-generation quinazoline derivative TKI.

[0031] "Prophylactically treating" includes (1) reducing or alleviating the risk of developing a disease in a subject or patient who may be at risk and / or susceptible to the disease, but who has not yet experienced or displayed any or all of the disease's symptoms or symptomology, and / or (2) delaying the onset of disease symptoms or symptomology in a subject or patient who may be at risk and / or susceptible to the disease, but who has not yet experienced or displayed any or all of the disease's symptoms or symptomology.

[0032] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0033] The term "effective," as used herein and / or in the claims, means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means that the amount of the compound, when administered to a subject or patient for treating or preventing a disease, is sufficient to affect such treatment or prevention of the disease.

[0034] As used herein, the term "IC 50 " refers to an inhibitory dose, which is 50% of the maximal response obtained. This quantitative measurement indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit half of a particular biological, biochemical, or chemical process (or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0035] An "anti-cancer" agent can negatively affect cancer cells / tumors in a subject by, for example, promoting the killing of cancer cells, inducing apoptosis in cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the survival of a subject with cancer.

[0036] The term "insertion" or "insertion mutation" refers to the addition of one or more nucleotide base pairs into a DNA sequence.

[0037] "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 nucleic acids of interest 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.

[0038] "Probe" refers to a polynucleotide at least 8 nucleotides in length that forms a hybrid structure with a target sequence due to complementarity between at least one sequence in the probe and a sequence in the target region. The polynucleotide may be composed of DNA and / or RNA. In certain embodiments, the probe is detectably labeled. Probes can vary greatly in size. Typically, probes are at least 8-15 nucleotides in length, for example. Other probes are at least 20, 30, or 40 nucleotides in length, for example. Still other probes are somewhat longer, for example, at least 50, 60, 70, 80, or 90 nucleotides in length. Probes can also be of any particular length within the above ranges. Preferably, the probe does not contain a sequence complementary to a sequence used to prime the target sequence during polymerase chain reaction.

[0039] "Oligonucleotide" or "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.

[0040] "Modified ribonucleotide" or deoxyribonucleotide refers to molecules that can be used in place of natural bases in nucleic acids, 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, methylphosphonamidites, methylphosphonamidites, 5'-β-cyanoethylphosphonamidites, methylenephosphonates, phosphorodithioates, peptide nucleic acids, achiral, and neutral internucleotide linkages.

[0041] "Variant" refers to a polynucleotide or polypeptide that differs from the wild type or from the type most prevalent in a population of individuals due to one or more substitutions, deletions, or insertions of nucleotides or amino acids, respectively. The number of substituted, deleted, or inserted nucleotides or amino acids can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, e.g., 25, 30, 35, 40, 45, or 50.

[0042] A "primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) and primes a nucleic acid synthesis reaction. A primer may be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. A primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of primer length are determined empirically. The lower limit of primer length is the minimum length required to form a stable duplex upon hybridization with the target nucleic acid under the conditions of the nucleic acid amplification reaction. Very short primers (usually less than 3 to 4 nucleotides in length) do not form a thermodynamically stable duplex with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of forming a duplex in a region of the target nucleic acid other than the predetermined nucleic acid sequence. Generally, suitable primer lengths range from about 10 to about 40 nucleotides in length. In certain embodiments, for example, primers may be 10 to 40, 15 to 30, or 10 to 20 nucleotides in length. A primer, when placed under appropriate conditions, is capable of acting as a point of initiation of synthesis on a polynucleotide sequence.

[0043] "Detection," "detectable," and their grammatical equivalents refer to a method of determining the presence, and / or quantity, and / or homology of a target nucleic acid sequence. In some embodiments, detection results in amplification of the target nucleic acid sequence. In other embodiments, sequencing of a target nucleic acid can be characterized as "detecting" the target nucleic acid. The label attached to the probe can include any of a wide variety of labels known in the art that are detectable, for example, by chemical or physical means. Labels that can be attached to the probe include, for example, fluorescent and luminescent materials.

[0044] "Amplify," "amplification," and their grammatical equivalents refer to any method that replicates at least a portion of a target nucleic acid sequence in a template-dependent manner, including, but not limited to, a wide variety of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for carrying out the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, Cambridge, UK), and self-sustained sequence replication (3SR), as well as multiplexed 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 multiplex chain reaction - CCR), etc. Descriptions of such techniques can be found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[0045] Generally, as used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals, with a reasonable benefit / risk ratio and without undue toxicity, irritation, allergic response, or other problem or complication.

[0046] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable as described above and has the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanesulfonic acid, and the like. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as pionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not hazardous, so long as the salt, as a whole, is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their methods of preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0047] II. Resistant 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 exons 18, 19, 20, or 21). The subject may have two, three, four, or more EGFR exon 20 mutations. The 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. The one or more EGFR mutations may be G719X, E709X, G724S, L718X, L861Q, T854I, V843I, C797S, and / or L792X, where X is any amino acid. Mutation detection methods are known in the art and include PCR analysis and nucleic acid sequencing, as well as FISH and CGH. In certain aspects, EGFR mutations are detected by DNA sequencing, for example, from tumor or plasma-derived circulating free DNA.

[0048] The EGFR exon 18 mutations may include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) between amino acids 688 and 728, in-frame deletions between amino acids in exon 18. The 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. The one or more EGFR exon 18 mutations may include E709A, L718Q, L718V, G719A, G719S, S720P, and G724S.

[0049] The EGFR exon 19 mutations may include one or more point mutations, insertions, and / or deletions (3-18 nucleotides) between amino acids 729-761, in-frame deletions between amino acids of exon 19. The one or more EGFR exon 19 mutations may be located at one or more residues selected from the group consisting of I744, L747, L747, A755, K757, and / or D761. The one or more EGFR exon 19 mutations may include I744V, I744T, L747S, L747FS, A755T, K757R, and / or D761N.

[0050] The EGFR exon 20 mutations may include one or more point mutations, insertions, and / or deletions (3 to 18 nucleotides) at amino acids 763 to 823. The one or more EGFR exon 20 mutations may be located at one or more residues selected from the group consisting of G724, A763, S768, V769, H773, V774, C775, S784, G796, C797, S811, and R776. The one or more EGFR exon 20 mutations may include S784F, R776C, S768I, V774M, S768I, H773insAH, V774A, V769M, S768dupSVD, A763insLQEA, G796D, S784F, C775Y, and / or S811F.

[0051] The EGFR exon 21 mutations may include one or more point mutations, insertions, and / or deletions (3-18 nucleotides) between amino acids 824-875, in-frame deletions between amino acids of exon 21. The one or more EGFR exon 19 mutations may be located at one or more residues selected from the group consisting of S784, G796, C797, S811, L833, G836, V843, T854, L861, L861, L862, L844, and L858. The one or more EGFR exon 21 mutations may include L858R, L833F, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and / or T854I.

[0052] In some aspects, the subject may have or develop a mutation at the C797 residue of EGFR, which may cause resistance to TKI (for example, second-generation quinazolinamine derivative TKI).Therefore, in certain aspects, the subject is determined not to have a mutation at C797 and / or T790 of EGFR (for example, C797S and / or T790M).In some aspects, the subject with T790 mutation (for example, T790M) can be administered osimertinib, and the subject with C797 mutation (for example, C797S) can be administered chemotherapy and / or radiation therapy.For example, if C797S is acquired in association with classic EGFR mutation (for example, L858R or exon 19 deletion) and T790M is absent, these mutations can be sensitive to quinazolinamine TKI. However, when the C797S mutation is acquired together with the T790M mutation or exon 20 insertion mutation, these mutations may be resistant to quinazolinamine TKIs. Furthermore, when the T790M mutation is acquired together with a classic mutation (e.g., L858R or exon 19 deletion), these mutations may be resistant to quinazolinamine TKIs, but may not be resistant to osimertinib. Also, when the T790M mutation is acquired together with a point mutation in exon 18 (G719X / T790M) in vitro, these mutations appear to remain sensitive to quinazolinamine TKIs. In some aspects, the L858R / C797S, Ex19del / C797S, or G719X / T790M mutation is sensitive to quinazolinamine TKIs. However, in certain aspects, L858R / T790M / C797S, exon 19 deletion / T790M / C797s, and exon 20 insertion + C797S or T790M mutants are resistant to EGFR TKIs.

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

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

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

[0056] The presence or absence of the resistant EGFR mutations described herein can be determined using methods well known in the art. For example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and / or arrays can be used to detect the presence or absence of insertion mutations. If desired, nucleic acid amplification can be achieved 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. Insertion mutations can be detected by any method described herein, for example, by sequencing or by hybridization to a nucleic acid probe of a gene in genomic DNA, RNA, or cDNA, for example, a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to specifically or preferentially hybridize with a particular mutation.

[0057] A probe set typically refers to a set of primers (usually a primer pair) and / or a detectably labeled probe used to detect a target gene mutation (e.g., an EGFR mutation) used in the present disclosure's recommended treatment. The primer pair is used in an amplification reaction to define an amplicon spanning the region for the target gene mutation for each of the genes. The amplicon set is detected by a matched probe set. In an exemplary embodiment, the method can use TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assay to detect a set of target gene mutations (e.g., an EGFR mutation). In one embodiment, the probe set is a set of primers used to generate amplicons that are detected by a nucleic acid sequencing reaction, such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) technology can be utilized.

[0058] 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 electrophoresis analysis.Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., 1992), solid-phase sequencing (Zimmerman et al., 1992), sequencing with mass spectrometry, such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., 1998), and hybridization sequencing (Chee et al., 1996; Drmanac et al., 1993; Drmanac et al., 1998).Non-limiting examples of electrophoresis analysis include slab gel electrophoresis, for example, agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing can be performed using commercially available kits and equipment from companies such as the Life Technologies / Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche / 454 Next Generation Sequencing System.

[0059] Other methods of nucleic acid analysis include direct manual sequencing (Church and Gilbert, 1988; Sanger et al., 1977; U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-strand conformation polymorphism assay (SSCP) (Schafer et al., 1995); clumped 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); and infrared matrix-assisted laser. These methods may include infrared / ionization melting (IR-MALDI) mass spectrometry (WO 99 / 57318); mobility shift analysis (Orita et al., 1989); restriction enzyme analysis (Flavell et al., 1978; Geever et al., 1981); quantitative real-time PCR (Raca et al., 2004); heteroduplex analysis; chemical mismatch resolution (CMC) (Cotton et al., 1985); RNase protection assay (Myers et al., 1985); the use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR, as well as combinations of such methods. See, e.g., U.S. Patent Publication No. 2004 / 0014095, which is incorporated herein by reference in its entirety.

[0060] In one example, a method for identifying an EGFR mutation in a sample comprises contacting nucleic acid from such sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding a mutated EGFR protein, or a fragment thereof containing the mutation, and detecting said hybridization. In a particular embodiment, such a probe is capable of specifically hybridizing to a nucleic acid encoding a mutated EGFR protein, or a fragment thereof containing the mutation, and detecting said hybridization. In a particular embodiment, such a probe is capable of specifically hybridizing to a nucleic acid encoding a mutated EGFR protein, or a fragment thereof containing the mutation, for example, a radioisotope ( 3 H, 32 P, or 33The probe is detectably labeled using a fluorescent agent (rhodamine or fluorescein), a fluorescent agent (P), or a coloring agent. In a specific embodiment, the probe is an antisense oligomer, such as a PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be about 8 nucleotides to about 100 nucleotides, or about 10 to about 75 nucleotides, or about 15 to about 50 nucleotides, or about 20 to about 30 nucleotides. In another aspect, such a probe of the present disclosure is provided in a kit for identifying EGFR mutations in a sample, the kit comprising an oligonucleotide that specifically hybridizes to or is adjacent to a mutation site in the EGFR gene. The kit further comprises instructions for treating a patient having a tumor containing an EGFR mutation with a second-generation quinazolinamine derivative TKI based on the results of a hybridization test using the kit.

[0061] In another aspect, a method for detecting an EGFR mutation in a sample comprises amplifying a nucleic acid sample corresponding to the EGFR gene or a fragment thereof suspected of containing a mutation, and comparing the electrophoretic mobility of the amplified nucleic acid with that of the corresponding wild-type EGFR gene or a fragment thereof. A difference in mobility indicates the presence of a mutation in the amplified nucleic acid sequence. Electrophoretic mobility can be measured on a polyacrylamide gel.

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

[0063] III. Treatment method Further provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering an effective amount of a second-generation quinazolinamine derivative TKI or a structurally similar inhibitor to the individual, the subject being determined to have a resistant EGFR mutation. The subject may have one or more EGFR mutations.

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

[0065] In some embodiments, the subject is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein). In one embodiment, the subject is in need of an enhanced immune response. In certain embodiments, the subject is or is at risk of being susceptible to infection. For example, the subject is undergoing or has undergone chemotherapeutic treatment and / or radiation therapy. Alternatively, or in combination, the subject is or is at risk of being susceptible to infection as a result of infection.

[0066] A specific embodiment relates to the administration of a second-generation quinazolinamine derivative TKI to a subject determined to have an osimertinib-resistant EGFR mutation. The second-generation quinazolinamine derivative TKI can be orally administered, for example, as a tablet. The second-generation quinazolinamine derivative TKI can be administered at a dose of 4 to 25 mg, for example, at a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg. Dosing can be daily, every other day, every three days, or once a week. Dosing can be on a continuous schedule (e.g., a 28-day cycle).

[0067] Osimertinib, chemotherapy, and / or radiation can be administered alone or in combination with a second-generation quinazolinamine derivative TKI. Osimertinib can be administered at a dose of 25 to 100 mg, for example, at a dose of about 40 or 80 mg. Dosage can be daily, every other day, every two days, every three days, or once a week. Osimertinib can be administered orally, for example, as a tablet.

[0068] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising a second-generation quinazolinamine derivative TKI and a pharmaceutically acceptable carrier for subjects 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 one or more optional pharmaceutically acceptable carriers (see Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers can be prepared by mixing with a suitable carrier such as PEG-1000 or PEG-10 ... , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., 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, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0070] B. Combination Therapy In certain embodiments, the compositions and methods of this embodiment comprise a second-generation quinazolinamine derivative TKI in combination with at least one additional therapy.The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof.The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0071] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of an agent with reduced side effects (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the therapy, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0072] The second-generation quinazolinamine derivative TKI can be administered before, during, after, or in various combinations with an additional cancer therapy, such as immune checkpoint therapy. Administration can range from simultaneous administration to minutes apart, days apart, or weeks apart. In embodiments in which the second-generation quinazolinamine derivative TKI is provided to a patient separately from the additional therapeutic agent, it is generally ensured that there is no significant time between each delivery so that the two compounds can still provide a beneficial combined effect to the patient. In such cases, it is contemplated that the antibody therapy and anticancer therapy may be provided to a patient within about 12 to 24 or 72 hours of each other, more particularly within about 6 to 12 hours of each other. In some situations, it may be desirable to provide an interval of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration, significantly extending the period for treatment.

[0073] Various combinations may be utilized. In the examples below, the second generation quinazolinamine derivative TKI is "A" and the anti-cancer therapy is "B." TIFF0007822597000001.tif18128

[0074] Administration of any compound or treatment of the present embodiments to a patient follows standard protocols for the administration of such compounds, taking into account the toxicity of the agent, if any. Thus, in some embodiments, there is a step of monitoring toxicity resulting from the combination therapy.

[0075] 1.Chemotherapy A variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to compounds or compositions administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within cells, for example, whether they affect the cell cycle and which stage of the cell cycle they affect. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intervene in DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

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

[0077] 2. Radiation therapy Other agents that cause DNA damage and are widely used include gamma rays, also known as X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA-damaging agents are also contemplated, including microwave ovens, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0078] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapies can be used in combination or in conjunction with the method of this embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. An antibody alone can be used as an effector of therapy, or the antibody may recruit other cells that actually affect 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 a lymphocyte bearing a surface molecule that directly or indirectly interacts with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0079] Antibody-drug conjugates have emerged as a groundbreaking approach in the development of cancer treatments. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver payloads (drugs) to tumor cells along with abundant levels of antigen. Targeted delivery of drugs also minimizes drug exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, validates this approach. Currently, more than 30 ADC drug candidates are in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs heavily depends on the identification and validation of novel targets suitable for this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high level expression in tumor cells and robust internalization.

[0080] In one aspect of immunotherapy, tumor cells must have some markers that are suitable for targeting (i.e., not present in the majority of other cells). Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory 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. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1999); 8; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapy described herein.

[0082] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints strengthen or weaken signals (e.g., costimulatory 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 inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 system and / or CTLA-4.

[0083] Immune checkpoint inhibitors can be drugs, e.g., small molecules, recombinant forms of ligands or receptors, or, in particular, antibodies, e.g., human antibodies (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized, or human forms of antibodies. Those skilled in the art will recognize that alternative and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this specification. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0084] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 system antagonists for use in the methods provided herein are known in the art and are described, for example, in U.S. Patent Publication 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., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[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 costimulatory protein, CD28; 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 CTLA-4, an inhibitory receptor for 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] Suitable anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) for use in this method can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129; International Patent Publication Nos. WO01 / 14424, WO98 / 42752, and WO00 / 37504 (CP675,206, also known as tremelimumab; formerly known as ticilimumab); U.S. Patent No. 6,207,156; Hurwitz et al., 1998; Camacho et al., 2004; and Mokyr et al., 1998 can be used in the method disclosed herein. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications WO2001 / 014424 and WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0089] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody binds to and / or competes for binding to the same epitope on CTLA-4 as the above-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. Pat. Nos. 5,844,905, 5,885,796, and International Patent Applications WO1995 / 001994 and WO1998 / 042752; all incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, incorporated herein by reference.

[0091] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic or staging, therapeutic, and palliative surgery. Therapeutic surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be combined with other therapies, such as the treatment of this embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0092] In the removal of part or all of the cancer cells, tissue, or tumor, a cavity may be formed in the body.Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments can also be varied in dosage.

[0093] 5. Other drugs It is contemplated that other agents may be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that act on the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intracellular signaling by increasing the number of GAP junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy.

[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 to osimertinib-resistant EGFR mutations. The kit may further include instructions for using the primers to detect the presence or absence of the specific osimertinib-resistant EGFR mutations described herein. The kit may further include instructions for diagnostic purposes indicating that positive identification of the osimertinib-resistant EGFR mutations described herein in a sample from a cancer patient is indicative of sensitivity to a second-generation quinazolinamine derivative TKI or a structurally similar inhibitor. The kit may further include instructions indicating that positive identification of the osimertinib-resistant EGFR mutations described herein in a sample from a cancer patient indicates that the patient should be treated with a second-generation quinazolinamine derivative TKI or a structurally similar inhibitor. [Example]

[0095] V. Working Example The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow are techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed herein and still obtain like or similar results without departing from the spirit and scope of the invention.

[0096] Example 1 - Identification of drugs for cancer cells with osimertinib-resistant EGFR mutations A panel of Ba / F3 cell lines expressing osimertinib- or erlotinib-resistant mutations, including atypical EGFR mutations spanning exons 18-21 and classic EGFR mutations, was generated. The transforming potential of the mutations was then assessed by the persistence of cell viability after IL-3 withdrawal. Activating EGFR mutant Ba / F3 cells were then screened against EGFR TKIs. Cell viability was determined by the Cell Titer Glo assay.

[0097] Second-generation quinazolinamine derivative TKIs inhibited the proliferation of Ba / F3 cell lines expressing atypical mutations (e.g., L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S) with IC50 values ​​of less than 3 nM.

[0098] In another study, NSCLC PDX models harboring an EGFR exon 18 P-loop mutation (G719A) were treated with the described inhibitors for 28 days. It was further found that in vivo, the P-loop exon 18 mutation conferred primary resistance to osimertinib, but not to other EGFR TKIs (Figure 2).

[0099] Ba / F3 cells expressing acquired atypical mutations spanning exons 18-21 were treated with the inhibitor for 72 hours. We observed that the acquired atypical mutations confer resistance to osimertinib but remain sensitive to quinazoline TKIs, and that the drug sensitivity / resistance profile of the co-occurring mutations may be conferred by the primary mutation (Figure 3). We also demonstrated that the T790M mutation, whether primary or tertiary, confer resistance to first- and second-generation drugs, but that unique third-generation TKIs and drug repurposing can overcome tertiary T790M-positive drug resistance.

[0100] (Table 1) IC50 values ​​for primary atypical EGFR mutations. TIFF0007822597000002.tif255155TIFF0007822597000003.tif25559

[0101] (Table 2) Acquired osimertinib resistance mutations (T790M negative). TIFF0007822597000004.tif25590

[0102] (Table 3) Acquired resistance mutation (T790M positive) – conventional EGFR TKI. TIFF0007822597000005.tif25395

[0103] (Table 4) Acquired resistance mutations (T790M positive) – non-conventional / repurposed EGFR TKIs. TIFF0007822597000006.tif211101

[0104] (Table 5) EGFR mutant vectors. TIFF0007822597000007.tif214151TIFF0007822597000008.tif62151

[0105] Therefore, second-generation quinazolinamine derivative TKIs are effective inhibitors for NSCLC harboring both de novo and acquired atypical osimertinib-resistant EGFR mutations, including L861Q, G719S, L858R / L792H, L858R / C797S, and Ex19del / C797S. This study demonstrated that second-generation TKIs overcame osimertinib resistance in NSCLC harboring atypical EGFR mutations.

[0106] Example 2 – Materials and Methods Generation of Ba / F3 cell line and IL-3 withdrawal: The Ba / F3 cell line was established as previously described (Robichaux et al., 2018). Briefly, stable Ba / F3 cell lines were generated by retroviral transduction of the Ba / F3 cell line for 12 hours. Retroviruses were generated by transfecting Phoenix 293T-ampho cells (Orbigen) with pBabe-Puro-based vectors (Addgene and Bioinnovatise) summarized in Table 1 using Lipofectamine 2000 (Invitrogen). Three days after transduction, 2 μg / ml puromycin (Invitrogen) was added to the RPMI medium. The cell lines were then grown for two weeks in the absence of IL-3, and cell viability was assessed every three days using the Cell Titer Glo assay (Progema). The resulting stable cell lines were maintained in RPMI-1640 medium containing 10% FBS and without IL-3.

[0107] Cell viability assay and IC 50Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described (Robichaux et al., 2018). Briefly, 2,000–3,000 cells per well were plated in 384-well plates (Greiner Bio-One) with three technical replicates. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone in a final volume of 40 μL per well. After 3 days, 11 μL of Cell Titer Glo was added to each well. 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 DMSO-treated cells, and normalized values ​​were plotted in GraphPad Prism using a nonlinear regression fit to the normalized data with a variable slope. IC 50 Values ​​were calculated by GraphPad Prism as 50% inhibition.

[0108] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods described herein, and to the steps or order of steps of the methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein, while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0109] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007822597000009.tif194158TIFF0007822597000010.tif224115TIFF0007822597000011.tif215107TIFF0007822597000012.tif55128

Claims

1. 1. A pharmaceutical composition for treating cancer in a subject, comprising a quinazolinamine derivative tyrosine kinase inhibitor (TKI), the subject is determined to have one or more epidermal growth factor receptor (EGFR) TKI resistance mutations selected from the group consisting of L718Q, S720P, G724S, T725M, I740dupIPVAK, L747P, K754E, K757R, V769L, R776C, R776H, S784F, S811F, L833F, L833V, L858R / L718Q, L858R / L792H, L858R / L718V, and L858R / V834L; The quinazolinamine derivative TKI is selected from the group consisting of afatinib, dacomitinib, and sapatinib. Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1, wherein the EGFR TKI resistance mutation is an atypical EGFR mutation.

3. 2. The pharmaceutical composition of claim 1, wherein the EGFR TKI resistance mutation is an acquired atypical EGFR mutation.

4. 2. The pharmaceutical composition of claim 1, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of L718Q, S720P, and G724S.

5. 2. The pharmaceutical composition of claim 1, wherein the one or more EGFR TKI resistance mutations is K757R.

6. 2. The pharmaceutical composition of claim 1, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of S784F, R776C, and S811F.

7. 2. The pharmaceutical composition of claim 1, wherein the one or more EGFR TKI resistance mutations is L833F.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the subject is resistant to a previously administered TKI, and the TKI is erlotinib or osimertinib.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the subject has been determined to not have a mutation at residues C797 and / or T790.

10. 2. The pharmaceutical composition of claim 1, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of G724S, and L718Q.

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

12. The pharmaceutical composition of any one of claims 1 to 10, wherein the cancer is non-small cell lung cancer.

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